Desiccation and Survival in Plants, Drying Without Dying

Desiccation prels 19/3/02 1:42 pm Page i
Desiccation and Survival in Plants
Drying Without Dying

Desiccation prels 19/3/02 1:42 pm Page ii
Desiccation prels 19/3/02 1:42 pm Page iii
Desiccation and Survival in Plants
Edited by
M. Black
King’s College
University of London
UK
and
H.W. Pritchard
Royal Botanic Gardens, Kew

Wakehurst Place
UK
CABI Publishing
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CABI Publishing is a division of CAB International

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Library of Congress Cataloging-in-Publication Data

Desiccation and survival in plants : drying without dying / edited by M. Black and H.W. Pritchard.
p. cm.
Includes bibliographical references (p. ).
ISBN 0-85199-534-9 (alk. paper)
1. Plants--Drying. 2. Plant-water relationships. 3. Plants--Adaptation. I. Black,
Michael. II. Pritchard, H. W.
QK870 .D57 2002
581.4--dc21
2001043835
ISBN 0 85199 534 9
Typeset in Melior by Columns Design Ltd, Reading

Printed and bound in the UK by Biddles Ltd, Guildford and King’s Lynn
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Contents
Contributors vii
Preface ix
PART I. INTRODUCTION 1
1 Drying Without Dying 3
Peter Alpert and Melvin J. Oliver
PART II. METHODOLOGY 45
2 Methods for the Study of Water Relations Under Desiccation Stress 47
Wendell Q. Sun
3 Experimental Aspects of Drying and Recovery 93
Norman W. Pammenter, Patricia Berjak, James Wesley-Smith and
Clare Vander Willigen
4 Biochemical and Biophysical Methods for Quantifying Desiccation
Phenomena in Seeds and Vegetative Tissues 111
Olivier Leprince and Elena A. Golovina
PART III. BIOLOGY OF DEHYDRATION 147
5 Desiccation Sensitivity in Orthodox and Recalcitrant Seeds in Relation to
Development 149
Allison R. Kermode and Bill E. Finch-Savage
6 Pollen and Spores: Desiccation Tolerance in Pollen and the Spores of Lower
Plants and Fungi 185
Folkert A. Hoekstra
7 Vegetative Tissues: Bryophytes, Vascular Resurrection Plants and Vegetative
Propagules 207
Michael C.F. Proctor and Valerie C. Pence
8 Systematic and Evolutionary Aspects of Desiccation Tolerance in Seeds 239
John B. Dickie and Hugh W. Pritchard
v
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vi Contents
PART IV. MECHANISMS OF DAMAGE AND TOLERANCE 261

9 Desiccation Stress and Damage 263
Christina Walters, Jill M. Farrant, Norman W. Pammenter and Patricia Berjak
10 Biochemistry and Biophysics of Tolerance Systems 293
Julia Buitink, Folkert A. Hoekstra and Olivier Leprince
11 Molecular Genetics of Desiccation and Tolerant Systems 319
Jonathan R. Phillips, Melvin J. Oliver and Dorothea Bartels
12 Rehydration of Dried Systems: Membranes and the Nuclear Genome 343
Daphne J. Osborne, Ivan Boubriak and Olivier Leprince
PART V. RETROSPECT AND PROSPECT 365
13 Damage and Tolerance in Retrospect and Prospect 367
Michael Black, Ralph L. Obendorf and Hugh W. Pritchard
Glossary 373
Taxonomic Index 383
Subject Index 401
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Contributors
Peter Alpert, Biology Department, University of Massachusetts, Amherst, Massachusetts

01003-5810, USA. palpert@bio.umass.edu
Dorothea Bartels, Institute of Botany, University of Bonn, Kirschallee 1, D-53115 Bonn,
Germany. bartels@mpiz-koeln.mpg.de
Patricia Berjak, School of Life and Environmental Sciences, University of Natal, Durban
4041, South Africa. berjak@biology.und.ac.za
Michael Black, Division of Life Sciences, King’s College, Franklin Wilkins Building, 150
Stamford Street, London SE1 6NN, UK. michael.black@kcl.ac.uk
Ivan Boubriak, The Oxford Research Unit, Open University, Foxcombe Hall, Boars Hill
OX1 5HR, UK. I.Boubriak@open.ac.uk
Julia Buitink, UMR Physiologie Moléculaire des Semences, Institut National
d’Horticulture, 16 Bd Lavoisier, F49045 Angers, France. julia.buitink@inh.fr
John B. Dickie, Seed Conservation Department, Royal Botanic Gardens Kew, Wakehurst
Place, Ardingly, West Sussex RH17 6TN, UK. J.Dickie@rbgkew.org.uk
Jill M. Farrant, Department of Molecular and Cellular Biology, University of Cape Town,
7700, South Africa. farrant@molbiol.uct.ac.za
Bill E. Finch-Savage, Horticulture Research International, Wellesbourne, Warwick CV35
9EF, UK. bill.finch-savage@hri.ac.uk
Elena A. Golovina, Laboratory of Plant Physiology, Department of Plant Sciences,
University of Wageningen, Arboretumlaan 4, 6703 BD Wageningen, The Netherlands
and Timiryazev Institute of Plant Physiology, Botanicheskaya 35, Moscow 127276,
Russia. e.golovina@pph.dpw.wau.nl
Folkert A. Hoekstra, Laboratory of Plant Physiology, Department of Plant Sciences,
University of Wageningen, Arboretumlaan 4, 6703 BD Wageningen, The Netherlands.
Folkert. Hoekstra@pph.dpw.wau.nl
Allison R. Kermode, Department of Biological Sciences, Simon Fraser University,
Burnaby, BC, V5A 1S6, Canada. allison_kermode@sfa.ca
Olivier Leprince, UMR Physiologie Moléculaire des Semences, Institut National
d’Horticulture, 16 Bd Lavoisier, F49045 Angers, France. olivier.leprince@inh.fr
Ralph L. Obendorf, Seed Biology, Department of Crop and Soil Sciences, Cornell
University, Ithaca, New York, USA. rlo1@cornell.edu
Melvin J. Oliver, USDA-ARS Plant Stress and Germplasm Development Unit, 3810 4th
Street, Lubbock, Texas 79415, USA. moliver@lbk.ars.usda.gov
vii
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viii Contributors
Daphne J. Osborne, The Oxford Research Unit, Open University, Foxcombe Hall, Boars
Hill OX1 5HR, UK. D.J.Osborne@open.ac.uk
Norman W. Pammenter, School of Life and Environmental Sciences, University of Natal,
Durban 4041, South Africa. pammente@biology.und.ac.za
Valerie C. Pence, CREW, Cincinnati Zoo and Botanical Garden, 3400 Vine Street,
Cincinnati, OH 45220, USA. VCPence@aol.com
Jonathan R. Phillips, Max-Planck-Institute for Plant Breeding Research, Carl-von-Linné-
Weg 10, D-550829 Köln, Germany. phillips@jupiter.mpiz-koeln.mpg.de
Hugh W. Pritchard, Seed Conservation Department, Royal Botanic Gardens Kew,
Wakehurst Place, Ardingly, West Sussex RH17 6TN, UK. H.Pritchard@rbgkew.org.uk
Michael C.F. Proctor, School of Biological Sciences, University of Exeter, Washington
Singer Laboratories, Perry Road, Exeter EX4 4QG, UK. M.C.F.Proctor@exeter.ac.uk
Wendell Q. Sun, Department of Biological Sciences, National University of Singapore,
Kent Ridge Crescent, Singapore 119260. dbssunwq@nus.edu.sg
Clare Vander Willigen, Department of Botany, University of Capetown, Private Bag,
Rondebosch 7701, South Africa. willigen@molbiol.uct.ac.za
Christina Walters, USDA-ARS National Seed Storage Laboratory, 1111 South Mason
Street, Fort Collins, CO 80521, USA. chrisv@lamar.colostate.edu
James Wesley-Smith, School of Life and Environmental Sciences, University of Natal,
Durban 4041, South Africa. wesleysm@biology.und.ac.za
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Preface
Plant survival of desiccation as sporophytic and gametophytic tissues was last

reviewed in detail in two books published in 1980. The first, by J. Levitt, on
Responses of Plants to Environmental Stresses, Volume II, Water, Radiation, Salt
and Other Stresses is a classic. The topic of plant water stress consumes about
200 pages and is set mainly at the introductory level but is still of sufficient
detail to stimulate post-graduate researchers. Interestingly, there is no mention
in Levitt’s book of desiccation sensitivity in seeds. However, this latter topic was
specifically covered in another book by H.F. Chin and E.H. Roberts (Recalcitrant
Seeds). At that time recalcitrant seed behaviour was something of a novelty and
the book deals mainly with descriptions of germination, a listing of species with
such seeds and an indication of how best to store the material in the short term.
Aspects of plant desiccation have been considered in other publications dealing
with the biology and biophysics of dehydration and in contributions to general
works on seeds but a comprehensive treatment of desiccation and plant survival
is not yet available.
Since 1980 there has been a revolution in plant science as new methods of
cell and molecular biology and biophysics have been applied to environmental
stress, particularly in relation to desiccation tolerance. The basic level of under-
standing of how plant cells cope with extreme water stress has increased
tremendously and considerable effort has been made in the last 10 years to
develop diagnostic markers for desiccation tolerance. At the physiological level,
studies have often focused on seed material and on the responses of resurrection
plants. At a more mechanistic level, model membrane systems have been used
extensively, and exploration of the molecular genetics of desiccation tolerance
has begun on developmental mutants, especially of seeds of crop species.
These progressive but fundamental changes in approach to investigating the
basis of survival of plant tissues under desiccation since the 1980s have meant
that our perceptions of this subject have altered significantly. It seems particular
appropriate now to take stock of these recent developments, to assess critically
the importance of the experimental systems available for investigation and to
ix
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x Preface
consider possible foci for future research work. This book sets out to address
these issues. The Introduction surveys the topic of desiccation, and the remain-
der of the book is divided into four parts, dealing with: (i) the technical back-
ground to desiccation tolerance studies; (ii) the frequency and levels of
dehydration stress tolerance in biological systems; (iii) mechanisms of damage
and tolerance; and (iv) a brief retrospect and prospect. It will not attempt to
address in detail plant drought stress (i.e. at relatively high water potentials).
This subject has been covered in detail in the last 10 years, for example in
Environmental Stress in Plants – Biochemical and Physiological Mechanisms
(Cherry. J.H. (ed.), Springer Verlag, 1989) and Plants Under Stress (Jones, H.G.,
Flowers, T.J. and Jones, M.B. (eds), SEB Seminar Series, Cambridge, 1989).
However, drought stress will be referred to in several places within this text.
In dealing with the different aspects of desiccation it is inevitable that certain
topics will receive consideration in more than one chapter. But the authors and
editors have attempted, as far as is possible, to avoid repetition of detail.
Extensive cross-referencing has been used, to aid the reader in identifying
where, within the special viewpoints of the treatments, similar subjects are
considered.
This comprehensive presentation on desiccation and survival in plants
will be of value to all researchers in the field, both beginners and the more
experienced, and to those with interests in basic and applied plant sciences –
physiology, ecology, conservation biology, agriculture and horticulture.
M. Black
H.W. Pritchard
01 Desiccation -Chap 1 18/3/02 1:53 pm Page 1
Part I
Introduction
1 Drying Without Dying
Peter Alpert1 and Melvin J. Oliver2

1BiologyDepartment, University of Massachusetts, Amherst, Massachusetts
01003-5810, USA; 2Plant Stress and Water Conservation Laboratory,
Agricultural Research Service, US Department of Agriculture, 3810 4th Street,
Lubbock, Texas 79415, USA
1.1. Introduction 4
1.2. Defining and Measuring Desiccation Tolerance 4
1.2.1. Operational and conceptual definitions 4
1.2.2. Measuring tolerance 6
1.3. A Brief History of Research on Desiccation Tolerance 6
1.3.1. Early work (1702–1860) on the question of whether life can
stand still 6
1.3.2. The next step: establishing records 7
1.4. The Occurrence of Desiccation Tolerance in Plants: Rarity and Ubiquity 8
1.4.1. Seeds, pollen and spores 8
1.4.2. Vegetative tissues 9
1.5. The Ecology of Desiccation Tolerance in Plants: a Diversity of Cycles in
Marginal Habitats 13
1.5.1. Habitats 17
1.5.2. Cycles 17
1.5.3. Hypotheses 19
1.6. Mechanisms of Desiccation Tolerance 20
1.6.1. Damage 21
1.6.1.1. Damage during desiccation 21
1.6.1.2. Damage during rehydration 22
1.6.1.3. Poikilochlorophylly 23
1.6.2. Protection 24
1.6.2.1. Proteins 24
1.6.2.2. Sugars 26
1.6.3. Repair 28
1.7. Future Prospects and Agricultural Significance 30
1.8. References 31
© CAB International 2002. Desiccation and Survival in Plants: Drying Without Dying
(eds M. Black and H.W. Pritchard) 3
4 P. Alpert and M.J. Oliver
1.1. Introduction Though some desiccation-tolerant plants

can survive droughts more intense and pro-
Water is a universal requirement for life as longed than any that occur almost any-
we know it. Water is the most abundant where on earth, tolerant plants are in the
compound in all active cells, it is essential minority. Desiccation-sensitive plants dom-
for metabolism and all organisms must take inate the world’s vegetation. The rarity of
in water to survive. Living things therefore the apparently excellent ability to tolerate
face a major problem whenever they desiccation raises a second, cautionary
emerge above ground on land: the air is question about desiccation tolerance: How
almost always drier than they are and takes does surviving desiccation affect plant sur-
water from them. This is a life and death vival? These two questions, one largely
problem for most organisms in most habi- genetic and biochemical and the other
tats, because the air is at least sometimes mainly physiological and ecological, frame
deadly dry. For example, when the relative the topic of desiccation and plant survival.
humidity is about 50% and the tempera- The purpose of this introductory chap-
ture 28°C, a plant cell that dries to equilib- ter is to summarize some of the current
rium will drop to a water potential of about answers to these questions and lead into
100 MPa (Gaff, 1997). This kills over 99% the more detailed reviews of questions and
of flowering plants. answers about desiccation and plant sur-
Terrestrial plants appear to have vival in the chapters that follow. We begin
evolved two solutions to the problem of with some terms and techniques that pro-
maintaining an aqueous self in a withering vide concepts and methods for research on
world. The majority solution, at least at the desiccation tolerance in plants, and a brief
present evolutionary time, is never to dry summary of the surprisingly lively history
out – to maintain a chronic disequilibrium of research on desiccation tolerance. We
between wet cells and dry air. Some of the then give an overview of the range and
most universal features of plant form, such ecology of desiccation tolerance in plants,
as waxy coatings on shoots, and pores that subjects that bear on how surviving desic-
can open and close on leaves, seem largely cation affects plant survival. Last, we dis-
designed to conserve water. cuss mechanisms of desiccation tolerance
The minority solution is to dry up but in plants, the keys to understanding how
not die – to desiccate during drought and plants survive desiccation, and consider
rehydrate and resume growth when the potential for breeding crops that can
drought ends. About 300 species of flower- dry without dying. We will sometimes
ing plants, or perhaps 0.1% of those named, abbreviate desiccation tolerance to ‘toler-
are known to tolerate desiccation ance’, and we will call plants that cannot
(Porembski and Barthlott, 2000). Some of tolerate desiccation ‘desiccation-sensitive’
these species can lose all of the free water or ‘sensitive’. We will consider desiccation
in their cells or remain dry for up to 5 years tolerance in plants and in some organisms
and still recover (Gaff, 1977). These prodi- that are not in the plant kingdom, mainly
gious abilities raise the first and fundamen- cyanobacteria, algae and fungi.
tal question about desiccation tolerance:
How do plants survive desiccation? Most
recent research on desiccation tolerance has 1.2. Defining and Measuring
focused on discovering the mechanisms of Desiccation Tolerance
desiccation tolerance, partly in the hopes of
some day engineering tolerance in econom- 1.2.1. Operational and conceptual definitions
ically important species and banishing the
spectre of famine from drought. Desiccation tolerance can be operationally
However, the ability to survive desicca- defined as the ability to dry to equilibrium
tion may not always increase the ability of with moderately dry air and then resume
plants to survive in natural systems. normal function when rehydrated, where
Drying Without Dying 5
‘moderately dry’ means 50–70% relative 2000). Some tolerant species are more dam-
humidity at 20–30°C . This definition is aged by being held at intermediate water
workable because there seems to be a wide contents than at full hydration or complete
gap between the maximum tolerance of desiccation (Gaff, 1997), and one advantage
sensitive plants and the minimum toler- of rapid desiccation may be to minimize
ance of tolerant ones (Gaff, 1997). Almost time spent at intermediate levels of hydra-
all species known to recover from complete tion (Kappen and Valladares, 1999; Proctor,
drying at 80% relative humidity also 2000; Chapters 3 and 5). Second, cells must
recover from drying at 50% (but see preserve enough cellular organization and
Bochicchio et al., 1998). functional enzymes so that metabolism can
The reason why a gap exists between the resume after rewetting. Preserving a skele-
ranges of tolerance of drying in desiccation- tal machinery for metabolism must involve
sensitive and desiccation-tolerant plants both protection and repair (Section 1.6).
may be that desiccation tolerance depends Enzymes and membranes must be pro-
on the ability to reversibly cease metab- tected from loss of configuration and orga-
olism as cells dry out. There may not be nization, and the damage that accumulates
very many marginally desiccation-tolerant from degradative non-metabolic reactions
plants because, once metabolism has while plants are inactive must be repaired.
stopped, it cannot be stopped further. Clegg Differences in effectiveness of protec-
(1973) argued on biochemical grounds that tion may explain much of why desiccation-
metabolism, defined as ‘systematically con- tolerant plants do differ in the intensity
trolled pathways of enzymatic reactions’ (minimum water content or water poten-
(Clegg, 2001), cannot take place at a cell tial) of desiccation that they can stand. For
water content of less than 0.1 g H2O g1 instance, tolerant angiosperms tend to sur-
dry mass because not enough water vive equilibration with lower relative
remains to hydrate intracellular proteins. humidities than do tolerant pteridophytes
Organisms this dry do show chemical in South Africa (Gaff, 1977). Species with a
activity. For instance, dried pollen can greater degree of protection of molecular
incorporate water vapour into organic com- configuration and cellular organization
pounds (Wilson et al., 1979). However, may survive with smaller fractions of
chemical reactions, even some characteris- water. Differences in effectiveness of repair
tic of living things such as oxygen uptake, may help explain why species also differ in
do not necessarily require metabolism: iron the duration of desiccation (length of time
rusts (Clegg, 1986). We propose that desic- in the dried state) that they can stand (e.g.
cation tolerance can be conceptually Sagot and Rochefort, 1996). Those with
defined as reversible cessation of metab- more effective repair mechanisms may be
olism in response to water loss. better able to undo non-metabolic degrada-
This suggests that the mechanisms of tion suffered while dry.
desiccation tolerance must involve at least There has been some confusion about
two key elements (Section 1.6). First, there the difference between ‘desiccation toler-
must be an orderly shutdown of metabo- ance’ and ‘drought tolerance’. We would
lism during desiccation. Different meta- like to propose that desiccation tolerance is
bolic pathways must slow at compatible one form of drought tolerance. Drought
rates to avoid fatal accumulations of inter- may be defined as any level of water avail-
mediates and generation of free radicals. ability that is low enough to reduce plant
Oxidation is a major hazard of desiccation performance. ‘Drought tolerance’ is most
(e.g. Smirnoff, 1993), and the advantages of often used to refer to tolerance of water
minimizing photo-oxidation may explain availabilities that are suboptimal but not
why some desiccation-tolerant plants cease low enough to cause complete drying to
photosynthesis at relatively high water equilibrium with the air, i.e. desiccation.
contents during drying (e.g. Sherwin and Mechanisms of drought tolerance include
Farrant, 1998; Tuba et al., 1998; Farrant, ways of maintaining cell water content,
such as osmotic regulation and stomatal undergo cycles of drying and wetting.
closure, whereas desiccation tolerance con- Chapter 4 reviews the rapidly expanding
sists of ways of surviving the nearly com- range of non-invasive techniques available
plete loss of water. Some papers on to study the diffusion of water, the configu-
intertidal algae maintain the confusion by rations and interactions of macromole-
using ‘desiccation’ to refer to any amount of cules, metabolism, thermal events
water loss (e.g. Leuschner et al., 1998; Bjork associated with membrane phase transi-
et al., 1999). They are probably wrong, tions, ultrastructure, oxidative stress, fer-
since the Oxford English Dictionary (1989) mentation and the physical properties of
defines ‘to desiccate’ as ‘to make quite dry; membranes, cytoplasm and protein com-
to deprive thoroughly of moisture’. plexes during desiccation and rehydration.
Chapter 7 summarizes some of the techni-
cal developments in infrared gas analysis
1.2.2. Measuring tolerance and fluoroscopy that have improved our
capacity to quantify responses to desicca-
Techniques for quantifying the degree of tion on the physiological level.
desiccation tolerance in different species
are reviewed in Chapters 2–4 and 7.
Chapter 2 discusses the advantages and 1.3. A Brief History of Research on
limitations of different measures of water Desiccation Tolerance
content and techniques for distinguishing
water properties in plant cells. Chapter 3 The 300-year history of the science of desic-
notes how the survival and recovery rates cation tolerance began with a lengthy
of seeds and vegetative tissues vary with period of discovery and doubt. In the
rate of drying, light conditions during dry- course of discovering desiccation tolerance,
ing, storage conditions and length of time scientists confronted the nature of life. The
in the dehydrated state. In general, highly next step was to enumerate the organisms
desiccation-tolerant bryophytes can sur- that tolerate desiccation and test the limits
vive rapid drying but tolerant angiosperms of their tolerance. In the 1960s, researchers
cannot; this seems to be related to differ- started to investigate the physiological ecol-
ences in their mechanisms of tolerance ogy of desiccation tolerance in plants, espe-
(Section 1.6). A few species appear insensi- cially the cycles of wetting and drying and
tive to rate of drying, but most probably their effects on carbon uptake in bryophytes
have an optimal rate or optimal range of and lichens. Since the 1980s, emphasis has
drying rates. For instance, desiccation in shifted to the biochemistry and molecular
less than 6 hours or over more than 7 days biology of desiccation tolerance. We now
kills the otherwise highly tolerant pterido- know more about how plants survive desic-
phyte Selaginella lepidophylla (Eickmeier, cation than about how tolerating desicca-
1983). Rates and final levels of recovery tion affects plant survival.
can decrease with increasing intensity or
duration of desiccation (e.g. Gaff, 1977;
Alpert and Oechel, 1987; Davey, 1997). 1.3.1. Early work (1702–1860) and the
Quantifying desiccation tolerance therefore question of whether life can stand still
also requires techniques for imposing
known rates, intensities and durations of It took scientists one and a half centuries to
desiccation, and for measuring rates and establish that desiccation tolerance exists
final levels of recovery (Chapter 3). Rate of (Keilin, 1959). At the end of an often ran-
drying is particularly hard to standardize corous debate, the nature of life had been
across species. called into question: Can life stop, be con-
Investigating the mechanisms of desic- tained in a static array of molecules and
cation requires techniques for measuring restart? Anthony von Leeuwenhoek was
processes and states in cells as they apparently the first to glimpse desiccation
tolerance, soon after he invented the micro- can be cooled to within 0.5°C of absolute
scope. In 1702, he wrote to a friend (see zero, absolute zero being the temperature at
Schierbeek, 1959): which all molecular motion is believed to
stop, and then revive when rewarmed and
The following day the sky was very hot and
rehydrated (Becquerel, 1951). The discov-
dry and, about nine in the morning, I took
some of the sediment which has been in the
ery of desiccation tolerance has shown us
leaden gutter … and poured on it a small that living things can come to exist in three
quantity of rain-water taken out of my stone states: alive, dead and still (Clegg, 2001).
cistern … so that if there were still any living
animalcules in it they might issue forth;
though I confess I never thought that there 1.3.2. The next step: establishing records
could be any living creatures in a substance
so dried as this was. The scientific battle over whether living
I was, however, mistaken; for scarce an things could dry without dying was fought
hour had elapsed, when I saw at least a
over animals. Starting in the 1960s, toler-
hundred of the animalcules before described.
ance was identified in the larvae of at least
These animals were rotifers. By the mid- one insect and of some other arthropods
1800s, others had seen desiccation toler- (Hinton, 1968; Crowe et al., 1992) but has
ance in two more phyla of animals, never been found in any life stage of any
nematodes and tardigrades (Keilin, 1959; vertebrate or in the adults of any animals
Alpert, 2000). However, others still flatly except microscopic rotifers, nematodes and
denied it was possible to survive drying tardigrades. In contrast, tolerance was
out. A French biologist, Felix Pouchet found to be widespread in plants. Tolerant
(1859), wrote that: ‘Dry and completely bryophytes were reported by 1886, fern
mummified animals cannot be resuscitated gametophytes by 1914, fern sporophytes by
by hydration. Rational beliefs, observation, 1931 and angiosperms by 1921 (tables and
and experiment unite to demonstrate it.’ citations in Kappen and Valladares, 1999;
The Société de Biologie in Paris convened Alpert, 2000; Chapter 7).
a special commission and conducted its The intensity and duration of desicca-
own tests on rotifers. Its report effectively tion that plants and plant-like organisms
settled the matter: ‘[organisms] reaching could survive were shown to be remark-
the most complete degree of desiccation able. Like tardigrades (Doyère, 1842), cer-
that can be realized … may yet retain the tain lichens, bryophytes, pteridophytes and
ability to revive in water’ (Broca, 1860). angiosperms survived equilibration with
However, the commission was silent on air of nearly 0% relative humidity, in
the deeper question of whether this means closed volumes over concentrated H2SO4
that life can stop and restart. As phrased by or P2O5 (e.g. Lange, 1953; Hosokawa and
Pouchet’s main scientific opponent Kubota, 1957; Gaff, 1977). The liverwort
(Doyère, 1842), ‘Has there been a mere Riccia macrocarpa produced new apical
slowing down of the vital phenomena, … cells after 23 years of air-dryness (Breuil-
or truly an absolute destruction that one Sée, 1993); the moss Grimmia laevigata
could compare to death itself?’ grew when rehydrated after 10 years in a
The modern consensus on this question herbarium (Keever, 1957); lichens survived
seems to be that some organisms can slow 10 years of being desiccated and frozen
their metabolism at least to the point at (Larson, 1988); and leaves of ferns and
which it cannot be detected against the flowering plants took up neutral red dye or
background of physical chemical reactions excluded Evans blue dye after 5 years of
and then resume normal metabolism air-dryness (Gaff, 1977). Some desiccation-
(Keilin, 1959; Hinton, 1968; Clegg, 2001). tolerant plants clearly survive longer and
The most convincing evidence may be that more intense drought than ever occurs
some desiccated tardigrades, rotifers, where they grow, raising the question of
seeds, spores, algae, lichens and mosses what has selected for such tolerance.
Dessicated plants were also shown to Porembski and co-workers (e.g. Porembski
tolerate other extreme stresses. Various taxa and Barthlott, 2000) are probably the clos-
survived extreme cold (Becquerel, 1951; est approaches to surveys for whole plants.
Pence, 2000), and some mosses survived The overall pattern one sees is taxonomic
heating to 100°C (Glime and Carr, 1974; and geographic breadth contrasted with
Norr, 1974). Eickmeier (1986) found that ecological narrowness.
more desiccation-tolerant populations of
Selaginella were also more heat-tolerant.
The fungus Schizophyllum commune pro- 1.4. The Occurrence of Desiccation
duced hyphae after 34 years in a vacuum of Tolerance in Plants: Rarity and Ubiquity
less than 0.01 mm Hg (Bisby, 1945), show-
ing long-term tolerance of both desiccation Part of the puzzle of desiccation tolerance in
and lack of oxygen. Takács et al. (1999) cor- plants is that it is both very uncommon and
related desiccation and UV-B tolerance in a nearly universal (Alpert, 2000). The relative
set of bryophyte species. biomass of desiccation-tolerant plants in all
The correlation between tolerance of des- but the most arid or frigid habitats is very
iccation and tolerance of cold, heat and low, and fewer than one in a thousand
anoxia has suggested that there may be species of flowering plants is known to toler-
some basic properties or mechanisms that ate desiccation. At the same time, desicca-
confer ‘broad-spectrum’ tolerance. Since tion-tolerant species are found on all
freezing often dehydrates cells, cold and continents, in all major plant groups except
desiccation stress have an obvious func- gymnosperms, and among species of all
tional link. Another parallel between desic- growth forms except trees; and the great
cation and cold tolerance is that both can be majority of flowering plants and also gym-
‘softened’ by periods of low stress and nosperms have desiccation-tolerant seeds or
‘hardened’ by ones of moderate stress. pollen or both. Desiccation tolerance appears
Plants may lose some of their desiccation to be a universal evolutionary potential of
tolerance after prolonged periods of full plant cells that has been little selected for
hydration (e.g. Gaff, 1977; Schonbeck and except in resting stages of the life cycle and
Bewley, 1981; Kappen and Valladares, in organisms that have not evolved effective
1999). Desiccation tolerance can vary sea- ways of avoiding desiccation.
sonally (Dilks and Proctor, 1976; Gaff, 1980) Detailed reviews of the occurrence of
and increase in winter (Kappen, 1964). desiccation tolerance in seeds, pollen and
However, the correlation between tolerance other spores, and vegetative tissues are
of desiccation and other stresses is not given in Chapters 5, 6, 7 and 8. Other recent
absolute. Wood and Gaff (1989) saw no cor- reviews of the occurrence of tolerance in
relation between desiccation and salinity adult plants and non-plant non-animals
tolerance in species of the grass Sporobolus. include those of Kappen and Valladares
As records of desiccation tolerance (1999), Alpert (2000), and Porembski and
accumulated, pictures emerged of the taxo- Barthlott (2000). In this section, some of the
nomic and geographic ranges of desicca- main points in these reviews will be dis-
tion tolerance in plants. These pictures cussed, a few of the examples they give will
remain somewhat haphazard because there be mentioned and some additional exam-
have been few systematic surveys for desic- ples and points will be presented.
cation tolerance within taxa or habitats.
Relatively extensive lists exist for seeds
(Chapter 8). A survey of all the soil algae at 1.4.1. Seeds, pollen and spores
one site was published by Evans (1959).
The lists of tolerant vascular plants from As in adult plants, the desiccation toler-
different regions published by Gaff and co- ance of seeds can vary greatly between
workers (e.g. Gaff, 1977, 1986; Gaff and species within genera, between individuals
Latz, 1978) and from rock outcrops by within species and between tissues within
individuals; and there is a continuum of 1.4.2. Vegetative tissues

degree of tolerance across species
(Chapters 5 and 8). However, whereas des- Desiccation tolerance appears common
iccation tolerance is rare in adult flowering though not universal in bryophytes (e.g.
plants, it is so much the rule in their seeds Richardson, 1981; Proctor, 1990), common
that tolerant seeds are traditionally known in lichens (Kappen and Valladares, 1999),
as ‘orthodox’ and desiccation-sensitive uncommon in pteridophytes and rare in
seeds as ‘recalcitrant’. Desiccation sensitiv- angiosperms (Chapter 7). No gymnosperms
ity may be a derived character in seeds, are known to tolerate desiccation (Gaff,
evolved through neoteny, and is probably 1980; Chapter 7), even though gym-
associated with large seeds and trees nosperms may have desiccation-tolerant
(Chapter 8). Another difference between seeds or pollen (Chapters 5 and 6).
desiccation tolerance in adult plants and Desiccation tolerance occurs in non-lich-
seeds is that tolerance and desiccation are enized fungi, cyanobacteria and algae (Ried,
environmentally induced in adults but may 1960; Mazur, 1968; Bertsch, 1970;
be developmentally programmed in seeds. Schonbeck and Norton, 1978; Potts, 1994,
Seeds become tolerant as part of develop- 1999; Dodds et al., 1995) but little is known
ment and dry because the parent withholds about its extent. It must be very common in
or withdraws water from them. Once they free-living algae and bacteria that grow on
germinate, the seedlings of desiccation-sen- the surface of plants or soil, where they are
sitive species with desiccation-tolerant very probably subject to desiccation.
seeds lose their tolerance within hours. Different vegetative parts of a plant
The obvious ecological advantage of ortho- may have different degrees of tolerance.
doxy is that seeds can survive periods of There seem to be two main patterns. First,
drought and disperse the offspring of a in some species only the perennating
plant more widely in space and time, structures survive desiccation, such as
although orthodoxy is not a prerequisite for corms in Limosella grandiflora (Gaff and
dormancy (Chapter 5). Two advantages of Giess, 1986) or special dry-season organs
being recalcitrant are that seeds need never in the small shrub Satureja gilliesii
stop growing and may germinate more (Montenegro et al., 1979). As in plants
rapidly – as in whole plants, there may be that are desiccation-sensitive but have
a trade-off between desiccation tolerance desiccation-tolerant seeds, tolerance in
and productivity in seeds. these species is confined to relatively
Desiccation tolerance is probably also inactive plant parts. Second, leaves may
the rule rather than the exception in pollen be more desiccation-tolerant when
and spores, and tolerance and desiccation younger. Younger leaves are more tolerant
are developmentally programmed in spores than older ones in Chamaegigas intre-
as in seeds (Chapter 6). However, there are pidus (Gaff and Giess, 1986) and some
at least three differences between tolerance species of Borya (Gaff, 1989). In the leaves
in seeds and in spores. Tolerant pollen has of some grasses, only the basal meristem-
no dormancy, it survives no more than a atic zone tolerates drying (Gaff and
few months of dry storage at room tempera- Sutaryono, 1991). This suggests that some
ture, and spores of some pteridophytes can tissues may lose tolerance as they differ-
survive cycles of drying and wetting. entiate or age; the processes involved
Desiccation-sensitive pollen is relatively could conceivably parallel those that
common in species of Poaceae, cause loss of tolerance after germination
Cucurbitaceae and Araceae (Chapter 6), of seeds. In all these examples of differen-
and may be associated with hot, humid tial tolerance in leaves, there are con-
habitats. The prevalence of desiccation tol- geners whose leaves remain tolerant as
erance in seeds and spores is one reason to they mature, offering inviting systems for
believe that the genetic potential to tolerate comparative studies of the ecology and
desiccation exists in all plants. mechanisms of desiccation tolerance.
No one appears to have assessed the rel- and Valladares, 1999; Porembski and
ative prevalence of desiccation tolerance in Barthlott, 2000). Desiccation-tolerant mono-
different taxa of bacteria, cyanobacteria, cotyledons outnumber tolerant dicotyle-
fungi and algae. Acinetobacter radioresis- dons. The monocotyledonous family
tans survives 150 days at 31% relative Velloziaceae may have over 200 tolerant
humidity, which helps make it a persistent species (Kubitzki, 1998). At least 39 species
source of infection in hospitals (Jawad et of Poaceae tolerate desiccation (Gaff, 1997).
al., 1998). At least 400 species of algae and One very small family of angiosperms, the
cyanobacteria tolerate desiccation (e.g. Myrothamnaceae, is entirely desiccation-
Davis, 1972; Potts, 1994, 1999; Trainor and tolerant (Porembski and Barthlott, 2000). At
Gladych, 1995). Evans (1959) found that the other extreme, some species, such as
many but not all of the freshwater algae in Borya nitida (Liliaceae), contain both toler-
pond mud survived desiccation in the ant and sensitive individuals (Gaff, 1981).
field; at least two species survived 69 days Phylogenetic analysis suggests that desicca-
of desiccation in the laboratory without tion tolerance in active phases of the life
forming resting stages. Two interesting phe- cycle has evolved at least eight separate
nomena that have been reported from some times in vascular plants (Oliver et al., 2000).
green algae but apparently not from other Desiccation-tolerant angiosperms are
groups are dependence of tolerance on also widely but unevenly geographically
nutrient availability (McLean, 1967, cited distributed. They occur on all continents
in Chandler and Bartels, 1999) and loss of except Antarctica, but very few species are
capacity to reproduce after desiccation known from Europe or North America. The
(Hsu and Hsu, 1998). We know of few European species are all in two genera from
reports of desiccation tolerance in non- one family (Ramondia and Haberlea in the
lichenized fungi (Bisby, 1945; Gesneriaceae) (Muller et al., 1997; Drazic
Zimmermann and Butin, 1973), but there is et al., 1999). The North American species
an extensive literature on tolerance in include three grasses (Iturriaga et al., 2000).
lichens, at least 50 species of which have The greatest concentrations of known desic-
been shown to tolerate desiccation cation-tolerant angiosperms are in southern
(Kappen and Valledares, 1999). Africa, western Australia and eastern South
Desiccation tolerance is broadly but America (Figs 1.1 and 1.2; Gaff, 1977, 1987;
unevenly distributed among taxa in plants. Gaff and Latz, 1978; Porembski and
Most of the 25,000–30,000 species of Barthlott, 2000). Different taxa predominate
bryophytes probably tolerate at least brief in each of these three areas.
desiccation of low intensity (Chapter 7); Desiccation-tolerant plants have a wide
the proportion of desiccation-tolerant range of morphological and physiological
species appears to differ between orders of characteristics (Porembski and Barthlott,
mosses and to be higher in mosses than in 2000). There are desiccation-tolerant annuals
liverworts. There are also desiccation-toler- and perennials, graminoids and forbs, and
ant hornworts (Oliver et al., 2000). herbs, shrubs and arborescent rosette plants.
Porembski and Barthlott (2000) estimated Tolerant species may be caespitose, stolonif-
that there are 275–325 desiccation-tolerant erous or rhizomatous. Some species are xero-
species of vascular plants. At least nine fam- morphic, such as B. nitida (Gaff and
ilies of pteridophytes and seven families of Churchill, 1976); others are not, such as Boea
angiosperms contain desiccation-tolerant hygroscopica (Gaff, 1981). A few desiccation-
sporophytes (Chapter 7). Some fern gameto- tolerant species, like C. intrepidus, have mor-
phytes also tolerate desiccation (e.g. Pence, phological features typical of aquatic plants
2000). Groups of ferns and allies that seem (Gaff and Giess, 1986), and at least one
to be relatively rich in desiccation-tolerant species is succulent (Barthlott and
species include the family Pteridaceae and Porembski, 1996). Desiccation-tolerant
the genera Cheilanthes and Selaginella angiosperms can have crassulacean acid
(Gaff, 1977; Gaff and Latz, 1978; Kappen metabolism (Barthlott and Porembski, 1996;
(a)
(b)
01 Desiccation -Chap 1
18/3/02
1:53 pm
(c) (d)
Page 11
Fig. 1.1. Southern Africa is a centre of diversity for desiccation-tolerant angiosperms, including (a) Craterostigma wilmsii, (b) Xerophyta viscosa, (c) Xerophyta
11
retinervis, and (d) Myrothamnus flabellifolius. Each is shown in its desiccated (left) and hydrated (right) state. (Photos by J. M. Farrant.)
(a)
(b) (c)
(d) (e)
Fig. 1.2. The large, isolated, granitic or gneissic outcrops known as inselbergs are a major habitat for desiccation-
tolerant vascular plants in Australia, Brazil and Africa. (a) An inselberg in the Mata Atlantica of Brazil; (b) a mat of
the pteridophyte Selaginella sellowii on a Brazilian inselberg; (c) an arborescent Brazilian monocot (Velloziaceae);
(d) a species of Borya (Boryaceae, shown desiccated) in Australia; (e) Afrotrilepis pilosa (Cyperaceae, shown
desiccated), a dominant, mat-forming species on inselbergs in West Africa. (Photos by S. Porembski.)
Markovska et al., 1997) and probably C4 pho- The wide distribution of desiccation tol-
tosynthesis (Lazarides, 1992). However, no erance in plants has suggested to some
plants more than 3 m tall and hence no trees authors that the basic mechanism of toler-
are known to tolerate desiccation, possibly ance must be simple (Chandler and Bartels,
because they cannot re-establish upward 1999). According to the ‘water replacement
movement of water once the xylem cavitates hypothesis’ of Crowe et al. (1998a), the
during desiccation (e.g. Sherwin et al., 1998). evolution of desiccation tolerance in all
organisms depends on the selection and habitats where desiccation-sensitive plants

synthesis of sufficient concentrations of do not live (Fig. 1.3). In habitats where
molecular substitutes for water (Clegg, water availability and temperature are
2001). Under certain circumstances, tre- moderate and sensitive plants are abun-
halose may even induce desiccation toler- dant, desiccation-tolerant vascular plants
ance in human cells (Guo et al., 2000). grow mostly on outcrops of bare rock
However, tolerance in plants also involves (Porembski and Barthlott, 2000). In the
other mechanisms (Section 1.6), and the driest and coldest habitats, especially
ecology of desiccation-tolerant plants sug- where dew and fog are major water sources,
gests that the evolution of tolerance in desiccation-tolerant bryophytes, lichens,
plants is constrained by its consequences algae or cyanobacteria may form the only
for growth and competition. vegetation (e.g. Thompson and Iltis, 1968;
Friedmann and Galun, 1974; Davey, 1997).
Despite the ability of some of these species
1.5. The Ecology of Desiccation to tolerate a drought that is longer and
Tolerance in Plants: a Diversity of Cycles more intense than occurs in these habitats,
in Marginal Habitats the most xeric microsites are often still
bare (e.g. Alpert, 1985). On rocks and soil
Desiccation-tolerant plants grow mainly in in the desert, small differences in exposure
the interstices and on the margins of the to the sun may determine whether a patch
world’s vegetation, in microhabitats and of soil or stone is colonized or not.
(a) (b)
(c) (d)
Fig. 1.3. (a) Exposed surfaces of granitic boulders in the western foothills of the Cuyamaca Mountains in
southern California are colonized mainly by an assemblage of desiccation-tolerant lichens and bryophytes.
(b) Two of the most common mosses are Grimmia laevigata (left) and Grimmia apocarpa (right), shown
hydrated. Crevices support desiccation-tolerant pteridophytes such as Pentagramma triangularis (gold-back
fern), shown (c) desiccated and (d) hydrated. (Photos by P. Alpert.)
(a) (b)
(c)
Fig. 1.4. Desiccation-tolerant bryophytes are common even in cool, moist climates. The mosses (a)
Orthotrichum anomalum, (b) Anomodon viticulosus, and (c) Tortula latifolia all occur in the UK. Each is
shown in its desiccated and its rehydrated state. (Photos by M.C.F. Proctor.)
These patterns appear tied to the differ- dry out again in hours. Desiccation-tolerant
ent sources of water that different desicca- vascular plants are only known to rewet
tion-tolerant plants can use to rehydrate, from rain; they recover in hours to days
the rates at which they rewet and dry out, and dry out in days to weeks. The cumula-
and their ability to recover after desicca- tive effect of repeated cycles of desiccation
tion and achieve a cumulative net gain of on net photosynthesis and growth may
resources. Lichens and bryophytes may explain why desiccation-tolerant plants fail
rewet from dew, recover in minutes and to survive in the most exposed microsites.
(a)
(b)
(c)
Fig. 1.5. The most intensively studied desiccation-tolerant bryophyte is (a) Tortula ruralis, shown in the fully
hydrated state (top), after slow drying (lower right), and after rehydration for 2 min (lower left). T. ruralis is
common in dry habitats in North America, as shown on rocks at Mesa Verde National Park, USA (b). One of the
most studied desiccation-tolerant angiosperms is the grass Sporobolus stapfianus (c), shown after drying in a pot
for 14 days (left) and after subsequent immersion in water for 24 h (right). (Photos by M.J. Oliver and B. Mishler.)
(a) (b)
(c)
Fig. 1.6. Effects of desiccation and rehydration on ultrastructure in leaves of the moss Tortula ruralis. The
transmission electron micrographs (after Bewley and Pacey, 1978) show papillose cells in (a) the fully
hydrated state (note chloroplast (C) with grana stacks (g), starch grains (s), and plastoglobuli (p, labelled in
(b)); a vesicle (V) and rough (RER) and smooth endoplasmic reticulum (SER); a mitochondrion (m) with
prominent internal membranes or cristae; and electron-dense bodies (E); and (b) after desiccated plants had
been rehydrated for 5 min (note that the chloroplasts are swollen but the nucleus (N) is not). The
freeze–fracture micrograph (c) (from Platt et al., 1994) shows a portion of a cell from a slowly dried leaf
(note the large, tightly appressed grana stacks (G) in the portion of the chloroplast visible and the
mitochondrion (M) outside the chloroplast.)
Trade-offs between tolerance and growth pensation point for photosynthesis

and competition with sensitive plants may (Schipperges and Rydin, 1998). In warm
explain why desiccation-tolerant plants, and cold deserts, tolerant algae and lichens
though they can rise again from ‘apparent grow inside or on the underside of translu-
death’ (Doyère, 1842), have not dominated cent rocks (Friedmann and Galun, 1974;
the earth. Kappen, 1993; Nienow and Friedmann,
1993). Species of Nostoc, Anacystis and
other cyanobacteria form desiccation-
1.5.1. Habitats tolerant crusts on bare walls and rocks
from the tropics to the boreal zone (Potts,
In contrast to the wide taxonomic and 1994; Lüttge, 1997). Algae, lichens and
geographical ranges and the broad mor- bryophytes join cyanobacteria to form
phological diversity of desiccation-tolerant crusts on desert soils, which are important
vascular plants, their ecological range is in nitrogen cycling (e.g. Nash and Moser,
narrowly confined to chronically or sea- 1982; Lange et al., 1994, 1997). Since
sonally dry habitats or microhabitats lichens and bryophytes dry so rapidly, they
where desiccation-sensitive plants are may be active mostly when conditions are
sparse or absent. Porembski and Barthlott effectively mesic and function as ‘shade
(2000) estimated that 90% of desiccation- plants’, even in exposed, xeric habitats
tolerant vascular plants are associated (Green and Lange, 1994; Proctor, 2000).
with rock outcrops, mainly in tropical to Degree of desiccation tolerance seems to
lower temperate latitudes. Some species explain some of the relative ability of toler-
grow on exposed rock surfaces, while oth- ant species to occupy xeric microsites or
ers are associated with crevices (Nobel, habitats (e.g. Hernandez-Garcia et al., 1999;
1978; Gildner and Larson, 1992). Franks and Bergstrom, 2000). For instance,
Ephemeral pools on rock outcrops in ability to tolerate desiccation (Mitchell et
Africa harbour a set of aquatic, desicca- al., 1999), to maintain photosynthesis dur-
tion-tolerant vascular plants (e.g. Volk, ing desiccation (Robinson et al., 2000) and
1984; Gaff and Giess, 1986). Tolerant to recover photosynthesis after repeated
angiosperms and pteridophytes also grow cycles of desiccation (Davey, 1997) are
in semiarid or desert grasslands, especially associated with occurrence of bryophytes
(Eickmeier, 1983; Gaff, 1987; Kappen and in relatively dry sites in Antarctica. The
Valladares, 1999) though not invariably ability to tolerate prolonged desiccation
(Gaff and Sutaryono, 1991), on shallow and to recover quickly upon rehydration
soils. There are exceptions to this narrow- appeared necessary but not sufficient to
ness of ecological range. A few tolerant allow mosses to colonize highly insolated
vascular species, such as Boeah hygro- surfaces on boulders in chaparral in
scopica (Gaff, 1981) and Pentagramma California (Alpert, 1985; Alpert and
triangularis (P. Alpert, personal obser- Oechel, 1987). A species of Selaginella
vation), occur in forest understoreys. from dry habitats recovered net photosyn-
Desiccation-tolerant bryophytes, lichens thesis faster than one from moister habitats
and algae occupy a much wider ecological (Eickmeier, 1980). Shirazi et al. (1996)
range than do tolerant vascular plants, reported differences in desiccation toler-
including both less and more arid sites ance between populations of lichens from
(Fig. 1.4). For example, tolerant bryophytes different habitats.
and lichens are common on rocks, trunks
and soil in moderately moist forests. They
may be common in tundra, although the 1.5.2. Cycles
Sphagnum species characteristic of tundra
do not necessarily recover net photosyn- Desiccation-tolerant plants vary greatly in
thesis after losing more than about 10% the rates at which they dry out, rehydrate
of the water content they hold at the com- and recover upon rehydration and there-
fore in the rhythms of desiccation and Xerophyta scabrida began to respire within
growth they experience in nature (Tuba et 20 min after rehydration, reached full rates
al., 1998; Kappen and Valladares, 1999; of respiration within 6 h, began to synthe-
Chapter 7). In general, bryophytes and size chlorophyll after 12 h and did not
lichens dry out in hours in the sun, complete synthesis until 36 h (Tuba et al.,
whereas ferns and angiosperms take a day 1994). Poikilochlorophylly appears to be a
or more. Minimum times to net photosyn- programmed rather than a pathological
thesis after rehydration range from minutes response to desiccation. For instance, it is a
in lichens and mosses rewetted with liquid necessary component of tolerance in the
water, to hours in lichens and mosses rehy- leaves of some species: when these leaves
drated with water vapour and in some vas- are detached before drying, they stay green
cular plants rewetted with liquid, to days as they dry but they die (Gaff, 1981).
in other vascular plants (Fig. 1.5; Lange, Natural cycles of wetting and drying
1969; Lange and Kilian, 1985; Gaff and have been followed for a number of
Giess, 1986; Reynolds and Bewley, 1993a; mosses and lichens (e.g. Kappen et al.,
Scott and Oliver, 1994; Scheidegger et al., 1979; Lange et al., 1994; Sancho et al.,
1997; Tuba et al., 1998). 1997) but very few desiccation-tolerant
Desiccated lichens can resume net pho- vascular plants (Nobel, 1978; Gaff and
tosynthesis by taking up water vapour Giess, 1986). During a year in the Negev
(Hahn et al., 1993; Schroeter et al., 1994), Desert, thalli of the lichen Ramalina maci-
but only if the phycobiont is a green alga formis underwent a cycle of wetting and
rather than a cyanobacterium (Kappen and drying almost daily, mostly from dew
Valladares, 1999). A few mosses can (Kappen et al., 1979). Some bryophytes in
recover at least very slow rates of net pho- semiarid grasslands can likewise experi-
tosynthesis by taking up water vapour after ence diurnal desiccation cycles driven by
desiccation (Lange, 1969; Rundel and dew during dry seasons (Csintalan et al.,
Lange, 1980). Both bryophytes and lichens 2000). At the other extreme, some
can rehydrate with dew (Lange et al., 1994; angiosperms may undergo a single period
Csintalan et al., 2000). Despite the lack of a of desiccation per year, with a cycle of
cuticle, differences in thallus, leaf and activity almost like that of an annual plant
shoot morphology and packing produce (e.g. Gratani et al., 1998).
several-fold differences in drying rates Three factors that determine how cycles
between different species of bryophytes of desiccation translate into growth are
and lichens (e.g. Gimingham and Smith, light damage, nutrient relations and carbon
1971; Proctor, 1982; Scott, 1982; balance. Photodamage can occur as plants
Valladares, 1994); differences in morpho- dry or while they are desiccated, due at
logical control of water loss may help least in part to light absorption without
explain differences in ability to colonize energy transfer to photosynthesis (e.g. Seel
xeric microhabitats. et al., 1992; Gauslaa and Solhaug, 1996).
Desiccation-tolerant angiosperms are Desiccation-tolerant plants show a variety
known to rehydrate in nature only after of mechanisms likely to reduce photodam-
rain. Woody angiosperms may take longer age, including leaf curling, accumulation of
to desiccate and rehydrate than herbaceous anthocyanin and carotenoids, and xantho-
ones (Sherwin and Farrant, 1996; Farrant et phyll metabolism (e.g. Muslin and
al., 1999). The slowest to recover from des- Homann, 1992; Eickmeier et al., 1993;
iccation are the poikilochlorophyllous des- Lebkeucher and Eickmeier, 1993;
iccation-tolerant plants, monocots that Calatayud et al., 1997; Deltoro et al., 1998;
dismantle their photosynthetic machinery Beckett et al., 2000; Farrant, 2000).
when they dry and reassemble it again Antarctic mosses, which could be subject
when they rehydrate (Sherwin and Farrant, to photodamage during freezing, show
1996; Tuba et al., 1998). In one desiccation reversible photoinhibition and zeaxanthin
study, the poikilochlorophyllous species activity (Lovelock et al., 1995).
Little is known about the interaction These factors are most important in des-
between desiccation tolerance and nutrient iccation-tolerant plants that tend to have
relations. Uptake and metabolism of min- short periods of hydration or frequent
eral nutrients must be interrupted from cycles of desiccation, such as lichens and
some point during drying to some point mosses in arid habitats, and are probably
during rehydration and recovery. In one reason why these species grow so
mosses, leakage of solutes during rehydra- slowly (Stark, 1997; Kappen and
tion could also reduce net nutrient uptake. Valladares, 1999; Badacsonyi et al., 2000).
Increased frequency of desiccation cycles Since brief periods of hydration can result
decreases potassium content but not cumu- in net carbon loss (Lange et al., 1994;
lative phosphorus uptake in Tortula ruralis Csintalan et al., 2000), it is possible that
(Badacsonyi et al., 2000). Activity of nitrate some desiccation-tolerant plants may have
reductase decreases rapidly during desicca- been selected for traits that help prevent
tion in T. ruralis (Mahan et al., 1998; rewetting by small amounts of water. Water
Badacsonyi et al., 2000); activity can repellence in epilithic lichens (Bertsch,
recover in less than 8 h after rehydration if 1966) and hair points on some epilithic
the moss has dried slowly, but may take mosses (P. Alpert, unpublished data) could
24 h after rapid drying (Mahan et al., be examples.
1998), and may decrease during the first
hour of rehydration (Marschall, 1998).
However, Bates (1997) found that weekly, 1.5.3. Hypotheses
24 h desiccation did not decrease uptake of
N, P or K in two mosses compared to The rarity of desiccation-tolerant vascular
uptake during continuous hydration, and plants in habitats where other vascular
Badacsonyi et al. (2000) saw no difference plants are abundant suggests that surviving
between the effect of low water potential desiccation may have negative as well as
on nitrate reductase activity in desiccation- positive effects on survival overall. One
tolerant and sensitive mosses. hypothesis is that there is a trade-off
Cycles of desiccation tend to reduce net between tolerance and growth, and that tol-
carbon gain by favouring respiration over erant plants are out-competed by sensitive
photosynthesis and by decreasing the ones where the latter can survive, because
amount of time that plants are active. the latter grow faster and larger. This
Desiccation increases the ratio of respira- should cause selection against tolerance in
tion to photosynthesis because: (i) photo- habitats where plants can acquire and con-
synthesis ceases before respiration during serve enough water to avoid desiccation.
drying and resumes after respiration during An alternative possibility is that tolerance
rehydration; (ii) respiration in some species is merely lost in plants that are not
increases above normal levels during recov- exposed to desiccation, due to lack of
ery from desiccation; and (iii) plants tend to selection pressure to maintain tolerance.
stay hydrated at night when they cannot Does desiccation tolerance entail a
photosynthesize but do respire, and to des- reduction in growth rate or maximum
iccate most rapidly when light levels are size? There are a number of reasons to
high (e.g. Alpert, 1979; Proctor, 1982; Lange suppose this, but little direct evidence.
et al., 1994; Tuba et al., 1998). Tuba et al. Kappen and Valladares (1999) proposed
(1999) examined the hypothesis that an that some morphological features that pro-
increase in atmospheric CO2 might improve mote tolerance also conflict with produc-
carbon balance during cycles of desicca- tivity. In angiosperms, hairs or scales that
tion. Elevated CO2 does prolong photosyn- reduce water loss and can thus prolong
thesis during drying in X. scabrida, but the periods of net photosynthesis also inhibit
authors concluded that this aspect of global rehydration (Kappen and Valladares,
change was unlikely to favour desiccation- 1999). In lichens and bryophytes, having
tolerant over sensitive species. high maximum water content tends to
prolong hydration but inhibits photosyn- tats? This hypothesis has been partially
thesis, since much of the water is typi- tested in bryophytes and lichens (e.g.
cally held externally or in upper layers of Alpert, 1990; Pintado et al., 1997; Williams
the lichen thallus and so slows gas diffu- and Flanagan, 1998; Kappen and
sion (Green and Lange, 1994; Valladares, Valladares, 1999). For example, during a
1994; Lange et al., 1996; Tuba et al., morning after nocturnal rain or dew,
1996a; but see Sojo et al., 1997). bryophytes and lichens growing on sloped
Populations of Ramalina capitata in dry, surfaces in north temperate latitudes tend
bright sites tend to have greater capacity to dry more rapidly if they are on surfaces
to store water and slower gas diffusion that face south or east than if they are on
than populations in more shaded sites surfaces that face north or west (Kappen et
(Pintado et al., 1997), suggesting that al., 1980; Alpert and Oechel, 1985). Those
selection favours water storage more when on north- and west-facing surfaces are
light is less limiting. Proctor (2000) pro- more likely to recoup the respiratory losses
posed that bryophytes have been selected incurred during the night before desicca-
for rapid desiccation to minimize time tion arrests photosynthesis in the morning.
spent at intermediate water contents, This probably at least partly explains why
which most dispose plants to damage. mosses are ‘more common on the north side
Rapid desiccation would also reduce time of the tree’. There appear to be no studies
available for photosynthesis and growth. on the effect of microsite on carbon balance
Cellular mechanisms of tolerance such as in desiccation-tolerant vascular plants.
sugar and protein synthesis (Section 1.6) Oliver et al. (2000) hypothesized that
seem likely to impose metabolic costs and desiccation tolerance was once the major-
thus reduce growth. If cavitation during ity solution to the problem of living in dry
desiccation precludes desiccation-tolerant air. They suggested that tolerance is a prim-
plants from exceeding 3 m in height itive characteristic in green plants that
(Sherwin and Farrant, 1998), then they allowed them to colonize the land. Once
will be overtopped wherever trees can plants evolved vascular tissues and effi-
grow. Some comparative studies on cient internal water transport, they lost
mosses (Bates, 1997; Arscott et al., 2000) their tolerance of desiccation except in
and anecdotal reports on grasses (Gaff, parts that had to be cut off from water
1989) have found that more productive transport – their spores, seeds and pollen.
species are less desiccation-tolerant. The Tolerance in adult plants then re-evolved
long-standing hypothesis (Grime, 1979) several times in different lineages.
that stress tolerance conflicts with pro- Porembski and Barthlott (2000) proposed
ductivity is intuitively appealing but that this re-evolution occurred as
mechanically elusive. Further compara- angiosperms colonized the bare rock out-
tive studies on desiccation-tolerant plants crops where their desiccation-tolerant
could help reveal mechanisms that dictate species are now most diverse. If this sce-
trade-offs between tolerance and growth. nario is correct, then desiccation tolerance
The absence of desiccation-tolerant in plants has evolved not just as a way of
plants in some highly xeric habitats where surviving in marginal habitats, but as a way
no other plants occur suggests that surviv- of colonizing frontiers, first from water on
ing desiccation may not assure survival, to land and then from soil on to stone.
even where competition is not a factor. One
hypothesis to explain why tolerant plants
are not more abundant in barren habitats is 1.6. Mechanisms of Desiccation
that the plants cannot maintain a cumula- Tolerance
tive positive carbon balance under certain
regimes of water availability (Ried, 1960). Until the mid-1970s, it was generally
Does carbon balance limit the survival of believed that the mechanisms of desicca-
desiccation-tolerant plants in xeric habi- tion tolerance in plants were mechanical
(see reviews by Bewley, 1979; Oliver and 1.6.1. Damage

Bewley, 1984). Structural features such as
flexible cell walls, small vacuoles and Before discussing what is known of the
lack of plasmodesmata were suggested as cellular protection and repair mecha-
key elements in tolerance (Gaff, 1980; nisms of desiccation tolerance, it is worth
Bewley and Krochko, 1982; Oliver and reviewing the effects of desiccation and
Bewley, 1984). In a landmark paper, rehydration on cellular integrity in desic-
Bewley (1979) articulated the alternative cation-tolerant plants. The critical ques-
view that desiccation tolerance is primar- tion, when deciding what type of
ily protoplasmic in nature. This theory mechanisms desiccation-tolerant plants
argues that certain plants and plant tis- employ, is when might damage occur? Is
sues achieve desiccation tolerance as a it during the drying process or upon rehy-
result of the inherent properties of their dration? For instance, if damage does not
cellular contents (protoplasm). Most evi- actually occur during desiccation, then
dence now supports this view, though there is good reason to believe that pro-
structural features are clearly important tective mechanisms are in place. If dam-
in desiccation tolerance in some cases age occurs upon rehydration, and the cell
(Sherwin and Farrant, 1996; Farrant et subsequently recovers, repair mecha-
al., 1999). nisms are probably operative. In addition,
Bewley (1979) further defined three the amount of damage and the rate at
critical features of desiccation tolerance which cells return to a normal status
based on the observation that many desic- measure the effectiveness of protective
cation-tolerant plants exhibit cellular and repair processes and the overall level
changes, some of which can be described of desiccation tolerance.
as extensive damage, during and follow-
ing desiccation. The plant or tissue must:
1.6.1.1. Damage during desiccation
(i) limit damage to a repairable level; (ii)
maintain its physiological integrity in the The timing of damage is still controversial,
dried state (perhaps for extended periods but a consensus is building that little dam-
of time); and (iii) mobilize mechanisms age occurs during drying in desiccation-
upon rehydration that repair damage suf- tolerant tissues. Much of the work in this
fered during desiccation and rehydration. area has focused on the plasma membrane.
These criteria laid the experimental foun- All desiccation-tolerant tissues leak
dation for the field from the 1980s solutes during rehydration (Simon, 1978;
onwards and continue to influence the Bewley, 1979; Bewley and Krochko, 1982),
way we think about how plants survive indicating that the cell membrane has been
desiccation. In particular, it is now compromised. Early electron microscopy
widely accepted that the cells of desicca- of seeds (Webster and Leopold, 1977;
tion-tolerant plants employ mechanisms Morrison-Baird et al., 1979) and bryophyte
that protect them from the rigours of tissues (reviewed by Oliver and Bewley,
extensive water loss and also mecha- 1984) suggested that membranes in dried
nisms, at least in the case of vegetative plant cells were completely disorganized.
cells, that repair damage suffered during With the advent of more sophisticated
desiccation or rehydration (Bewley and technologies, these observations were
Oliver, 1992). This introductory overview determined to be artefacts of sample
of the mechanisms of desiccation toler- preparation and chemical fixation (Bewley,
ance will therefore concentrate on cellu- 1979; Thompson, 1979; Bewley and
lar features (the so-called ‘inherent Krochko, 1982; Oliver and Bewley, 1984).
properties’ of desiccation-tolerant cells) The use of non-aqueous fixatives elimi-
that have been suggested to play a major nated some of these artefacts but the heavy
role in protection and repair. Details are use of chemical treatments still made
covered in subsequent chapters. interpretation difficult (Thompson, 1979;
Öpik 1980, 1985; Tiwari et al., 1990; 1.6.1.2. Damage during rehydration
Smith, 1991). Freeze–fracture electron
microscopy, however, has yielded the most As noted above, all plant tissues leak
reliable data. Dried tissues are eminently solutes when rehydrated following a dry-
suited for freeze–fracture preparation ing event. In desiccation-tolerant tissues,
because their low water content virtually however, this is a transient event (Simon,
eliminates the formation of ice crystals, 1978; Bewley, 1979; Bewley and Krochko,
which make high-quality replicas difficult 1982). Several hypotheses have been
to obtain. Freeze–fracture studies clearly offered to explain imbibitional (or rehydra-
demonstrated that the membranes of seeds tive) leakage (Simon, 1974; Senaratna and
(Thompson and Platt-Aloia, 1982; Bliss et McKersie, 1983a,b; Crowe et al., 1989,
al., 1984) and pollen (Platt-Aloia et al., 1992; Hoekstra et al., 1992). The prevailing
1986) could retain normal bilayer organi- hypothesis is that imbibitional leakage is
zation and dispersal patterns of intramem- the result of lipid-phase transitions occur-
branous particles at water contents as low ring in the plasma membrane as a result of
as 0.08 g H2O g1 dry mass. The plasma dehydration and rehydration (Crowe et al.,
and organelle membranes of vegetative 1992). During drying, membranes pass
cells of the desiccation-tolerant pterido- from the liquid crystalline to the gel phase,
phyte Selaginella lepidophylla and the and they return to the liquid crystalline
moss Tortula ruralis also retain normal phase during rehydration. In artificial
organization and dispersal patterns in the membranes, this transition can lead to a
dried state (Platt et al., 1994; Fig. 1.6). transient leakage event (Hammoudah et al.,
The effects of desiccation on cellular 1981), and, since phase transitions have
components that cannot be observed by been demonstrated in drying and rehydrat-
freeze–fracture microscopy are more diffi- ing desiccation-tolerant cells (Crowe et al.,
cult to evaluate, largely due to the likeli- 1989; Hoekstra et al., 1992), it has been
hood of partial rehydration and the generally accepted that phase transition is
production of artefacts during chemical the basis of imbibitional leakage in most
fixation. In seeds, the uncertainty is com- desiccation-tolerant tissues. In seeds, how-
pounded by the fact that the tissues ever, it is thought that membrane-phase
are part of a developing system. changes do not occur because of the pres-
Nevertheless, observations tend to sug- ence of a seed coat, which impedes the
gest that desiccation of tolerant plants passage of water to the dried cells.
generates an ordered ‘collapse’ of the Hoekstra et al. (1999) suggested that the
cellular milieu that results in little ultra- slow rate of penetration of water may set
structural damage (Oliver and Bewley, up a ‘pre-hydration’ state where the mem-
1984; Gaff, 1989; Goldsworthy and branes are in a liquid crystalline state
Drennan, 1991; Sherwin and Farrant, before liquid water surrounds the rehydrat-
1996; Farrant et al., 1999). If desiccation- ing cells. Since leakage does occur during
tolerant plants successfully avoid damage the rehydration of these tissues (Hoekstra
during the dehydration process, as it et al., 1992; Tetteroo et al., 1996), it has
appears they do, is there any consequence been concluded that leakage must occur
at all of desiccation in these plants? The through an intact lipid bilayer, as suggested
answer appears to be yes. All desiccation- by Senaratna and McKersie (1983b).
tolerant plants and plant tissues show Recently, a new hypothesis has emerged
signs of cellular damage when the dried from some exciting new studies on dehy-
tissue is rehydrated. It is, however, debat- drating and rehydrating pollen (Hoekstra et
able whether or not the damage occurs al., 1997, 1999; Golovina et al., 1998;
during the drying process (but is not Buitink et al., 2000; Chapter 10). This body of
observable at an ultrastructural level) or work using amphiphilic spin probes demon-
as the result of the inrush of water into strates that during dehydration endogenous
the cells during rehydration. amphiphilic substances partition from the
aqueous cytoplasm into pollen membranes. Within minutes after rehydration, the
Using data obtained from liposome-based ex- chloroplasts of the green gametophytic tis-
periments, Golovina et al. (1998) suggested sues of desiccation-tolerant bryophytes
that it is the presence of these amphiphiles appear swollen and globular. Their outer
in the membrane that causes imbibitional membranes are folded and separated from
leakage and that as the pollen rehydrates the the thylakoids, which are no longer com-
amphiphilic substances move out of the pacted (Oliver and Bewley, 1984). The
membranes and leakage stops. This hypo- extent of thylakoid disruption increases
thesis could explain how transient leakage with the rate of prior desiccation. The
can occur through an intact membrane. In a chloroplasts of desiccation-tolerant
more recent study, Buitink et al. (2000) angiosperms tend to be more resistant to
demonstrated that the movement of disruption than those of bryophytes,
amphiphilic compounds into membranes although vesicularization within the
also occurs in imbibing radicles of peas and chloroplast internal membranes is common
cucumbers. This study, using electron para- (Gaff and Hallam, 1974; Gaff et al., 1976;
magnetic resonance (EPR) spectroscopy and Sherwin and Farrant, 1996). In all desicca-
inserted nitroxide spin probes, demon- tion-tolerant plants, mitochondria swell
strated a difference in partitioning behav- and exhibit disruption of the cristae
iour between desiccation-tolerant and (reviewed by Bewley and Krochko, 1982).
sensitive tissues. Spin probes partitioned Swelling and disruption of mitochondria
into the membranes at higher water content are not affected by rate of desiccation. In
in desiccation-sensitive tissues than in toler- all cases, organelles regain normal struc-
ant tissues. These authors suggest, from in ture within 24 h.
vitro portioning experiments, that it is the
microviscosity of the cytoplasm that con-
1.6.1.3. Poikilochlorophylly
trols portioning of amphiphilic compounds
into the plasma membrane. What remains to At least eight genera of desiccation-tolerant
be determined is the role of the native monocots are ‘poikilochlorophyllous’, i.e.
amphiphilic compounds in membrane dam- they reversibly lose their chlorophyll and
age and, if they are important in desiccation dismantle their chloroplasts during desic-
tolerance, the role they play in the long-term cation (Gaff, 1989; Tuba et al., 1998). The
stability of membranes in the dried state. thylakoid system within desiccated chloro-
Golovina et al. (1998) speculated that plasts is completely replaced by small
amphiphiles may have antioxidant proper- groups of plastoglobuli and by osmophilic,
ties that protect membranes from damage stretched lipid material, which appears to
by free radicals generated during desicca- occupy the positions previously occupied
tion and rehydration. If so, imbibitional by the thylakoids (Hallam and Luff, 1980;
leakage may be a necessary trade-off for Tuba et al., 1993a,b; Sherwin and Farrant,
protection. Much work will be required 1996). After 10–12 h rehydration, when
before the importance of native full turgor and maximum leaf water con-
amphiphilic compounds in desiccation tol- tent are reached, synthesis of chlorophylls
erance can be determined, but amphiphiles and carotenoids and the reassembly of thy-
are an intriguing new development in our lakoids begin. Early in reassembly, sets of
understanding of desiccation tolerance. two primary thylakoids stack to form
Rehydration-induced damage other than grana. Within 72 h the chloroplasts appear
leakage is difficult to distinguish from nor- normal and full photosynthetic capacity is
mal development in seeds and pollen but restored (Tuba et al., 1993b, 1994). From
is clearly evident in the tissues of most these studies and later physiological in-
desiccation-tolerant vegetative tissues, vestigations (Tuba et al., 1997), it appears
especially in organelles (reviewed by Bewley that these changes can be classified as
and Krochko, 1982; Oliver and Bewley, genetically programmed responses to des-
1984; Gaff, 1989; Oliver and Wood, 1997). iccation rather than damage.
1.6.2. Protection reach a maximum about three days before

the seed begins to desiccate (Galau and
Much of what we know of the cellular pro- Hughes, 1987; Galau et al., 1987). The
tection mechanisms involved in desiccation other class contains 12 transcripts, which
tolerance in plants comes from studies of appear late in maturation and achieve max-
orthodox seeds (Bewley and Black, 1994; imum expression just before and during
Chapter 5) and, to a slightly lesser extent, desiccation. LEA proteins make up 30% of
pollen (Crowe et al., 1992; Hoekstra et al., the non-storage protein and 2% of the total
1992). The ability of seeds to withstand soluble protein in the mature cotton
desiccation is acquired during their devel- embryos and are uniformly localized
opment. This acquisition is usually sub- throughout the cytoplasm (Roberts et al.,
stantially earlier than the culmination of 1993). LEA proteins and the acquisition of
the drying event itself, which is the termi- desiccation tolerance during seed matura-
nal event in orthodox seed maturation. tion have been linked in other dicots (e.g.
Seeds of some species can withstand pre- soybean: Blackman et al., 1995) and in
mature desiccation well before the mid- monocots (e.g. maize: Mao et al., 1995;
point of their development (Bewley and Wolkers et al., 1998).
Black, 1994; Chapter 5). Among the meta- A set of LEA proteins arises in develop-
bolic changes that take place just prior to or ing barley and maize embryos at the time
during drying is the synthesis of proteins that tolerance of desiccation is acquired. A
and sugars, which have long been postu- small subset of these proteins is induced
lated to form the basis of a series of overlap- when barley embryos at the intolerant stage
ping protective mechanisms that limit are cultured in abscisic acid (ABA) (Bartels
damage to cellular constituents (Bewley, et al., 1988; Bochicchio et al., 1991), and a
1979; Leprince et al., 1993; Oliver and causal relationship between ABA and lea
Bewley, 1997). These two components have gene expression has been suggested.
since been widely implicated as being criti- Evidence for, and against, this relationship
cal for desiccation tolerance in all plant exists in the literature. In cotton embryos,
cells including vegetative cells (Ingram and high expression of the first class of lea
Bartels, 1996; Oliver and Bewley, 1997; genes occurs as ABA content increases.
Scott, 2000). Over the years it has also High expression of the second set of lea
become clear that the synthesis of antioxi- genes, however, occurs at the start of, and
dants and enzymes involved in oxidative during, maturation drying, when the
metabolism also play a critical role in cellu- endogenous ABA content is low. There are
lar protection and desiccation tolerance explanations for this lack of correlation,
(Chapter 10). However, this aspect of pro- e.g. there is an early-regulated, ABA-con-
tection will not be addressed here. trolled mechanism, which operates only
later when drying commences. On the
other hand, an ABA-independent pathway
1.6.2.1. Proteins
may be involved in the synthesis of the
Only one subset of proteins that accumu- second group of LEA proteins.
late at the time of the acquisition of desic- LEA proteins have been identified in the
cation tolerance has been extensively vegetative tissues of all desiccation-tolerant
investigated, the late embryogenesis abun- plants studied so far (Ingram and Bartels,
dant (LEA) proteins, first described in cot- 1996; Oliver and Bewley, 1997; Blomstedt
ton (Galau and Hughes, 1987; Galau et al., et al., 1998) and proteins related to some of
1987, 1991; Chapter 5). The genes that the LEA proteins, e.g. dehydrins (see
encode LEA proteins in developing cotton- below), have been associated with the
seeds are comprised of two distinct classes response of non-tolerant plants to water
whose regulation is coordinated. One class stress (Skriver and Mundy, 1990; Bray,
contains six different lea transcripts, which 1997). In nearly all instances, the induction
appear relatively early in development and of LEA protein synthesis in vegetative tis-
sues can be elicited by exogenous ABA LEA protein synthesis is also highly
application (Ingram and Bartels, 1996; induced in the vegetative tissues of desic-
Campalans et al., 1999). cation-tolerant angiosperms during drying
LEA proteins fall into five groups by (Bartels et al., 1993; Blomstedt et al., 1998;
virtue of sequence similarities (Dure et al., Bartels, 1999). Callus derived from vegeta-
1989; Ingram and Bartels, 1996; Cuming, tive tissue of the desiccation-tolerant plant
1999). All are highly hydrophilic and all are Craterostigma plantagineum is not inher-
very stable, as evidenced by their resistance ently tolerant but can be made so by the
to the denaturing effects of boiling (with the application of ABA (Bartels et al., 1990).
exception of Group 5 LEA proteins). Group The application of ABA to this tissue
1 LEA proteins are characterized by a 20- results in the synthesis of novel proteins,
amino acid motif and are represented by the some of which are LEA proteins including
wheat Em protein, the first LEA protein the Group 2 LEA proteins, the dehydrins
identified (Cuming and Lane, 1979). Group (Bartels et al., 1993). The desiccation-toler-
2 LEA proteins are characterized by a 15- ant moss T. ruralis utilizes a more primi-
amino acid motif, the K-segment, a stretch tive mechanism of desiccation tolerance
of serine residues and a conserved motif (Oliver et al., 2000), which involves a con-
near the N-terminus of the protein (Close, stitutive cellular protection strategy, and in
1997). This group of proteins is also called this plant, unlike others, dehydrins are not
the dehydrins and these are the most wide- induced by dehydration or by ABA but are
spread and most studied of the LEA pro- constitutively expressed (Bewley et al.,
teins. Group 3 LEA proteins share a 1993). Dessication-sensitive species
characteristic 11-amino acid repeat motif exposed to sub-lethal dehydration stress
(Dure et al., 1989), which is predicted to also respond by synthesizing LEA proteins
form an amphipathic -helix. These amphi- and LEA-like proteins, in particular dehy-
pathic helices are postulated to form intra- drins (Close, 1997). These examples and
and intermolecular interactions that may many more all point to the importance of
have important consequences for their func- LEA proteins in dehydration responses and
tion (Baker et al., 1988; Dure, 1993a). The desiccation tolerance.
least studied of the LEA proteins are those in The most convincing pieces of evidence
Groups 4 and 5, which are somewhat to suggest that LEA proteins have an
atypical (Dure, 1993b; Galau et al., 1993). important role in cellular protection come
Group 5 LEA proteins are more hydrophobic from transgenic studies using a barley
than other LEA proteins and are not resistant Group 3 lea gene, HVA1. This gene, when
to high temperature. Most of the LEA protein expressed in a constitutive fashion in
groups have been identified in many differ- transgenic rice, increased its tolerance to
ent plants. All groups are thought to play a water and salt stress (Xu et al., 1996).
role in desiccation tolerance, and the evi- HVA1 overexpression in wheat, driven by a
dence for this viewpoint is growing. maize ubiquitin promoter, resulted in
The evidence for the involvement of transgenic lines that performed in a supe-
LEA proteins in desiccation tolerance is rior fashion under soil-water deficits
circumstantial but compelling. LEA protein (Sivamani et al., 2000).
synthesis in seeds, as mentioned above, is There are a variety of suggested mecha-
associated with both the acquisition of des- nisms by which LEA proteins might pro-
iccation tolerance and the final stage of tect cellular components. Many LEA
seed maturation just prior to desiccation. proteins have extensive regions of random
In addition, ABA-deficient (aba) and ABA- coiling, which has been postulated to pro-
insensitive (abi3) double-mutants of mote the binding of water, helping to main-
Arabidopsis seeds do not dry on the parent tain a minimum water requirement (Ingram
plant, do not tolerate desiccation and lack and Bartels, 1996). For instance, the Em
several LEA proteins (Koorneef et al., 1989; protein of wheat is considerably more
Meurs et al., 1992). hydrated than most common proteins, and
over 70% of the Em protein is configured in accumulation during desiccation.

as random coils (McCubbin et al., 1985). Constitutive expression of HSPs is unusual
Baker et al. (1988) suggested that the ran- in vegetative tissues and resembles the
dom coil nature of some LEA proteins may expression pattern of these proteins in
allow them to conform to the shape of cel- seeds. In addition, exogenous ABA
lular constituents and thus, by virtue of induced both the expression of HSPs and
their hydroxyl groups, help to maintain the acquisition of desiccation tolerance in
their solvation state when water is C. plantagineum callus tissues (Alamillo et
removed. These authors also suggested that al., 1995). Finally, a LEA-like HSP, HSP-12,
the Group 2 LEA proteins (dehydrins), by from yeast was shown to be capable of pro-
virtue of their amphipathic helical repeats, tecting liposomal membranes from the
provide surfaces when bundled together damaging effects of desiccation in a way
that would sequester ions. This may be similar to that seen with the sugar tre-
crucial as the increasing ionic strength dur- halose (Sales et al., 2000). Thus it appears
ing drying could cause irreversible damage that small HSPs may also play a role in cel-
to cellular proteins and structural compo- lular protection during desiccation: per-
nents. Recently, Velten and Oliver (2001) haps this capability is related to their
described an LEA-like protein from T. chaperonin-like activities, which may help
ruralis that contains 15 15-amino-acid maintain protein structure under denatur-
repeats predicted to form amphipathic ing conditions. Other proteins whose tran-
helices. This protein appears to be synthe- scripts accumulate during the dehydration
sized during the rehydration event and phases of vegetative desiccation-tolerant
may serve to trap valuable ions that would angiosperms have been identified but little
otherwise be lost. Studies using individual has been done to confirm their roles in des-
LEA proteins in in vitro assays also add to iccation tolerance (Kuang et al., 1995;
the possible mechanisms by which these Ingram and Bartels, 1996; Blomstedt et al.,
proteins exert protection of cellular compo- 1998; Bockel et al., 1998; Neale et al.,
nents. Wolkers (1998) suggested from data 2000). See Chapters 5 and 11 for further
obtained from the study of a pollen Group discussion of all these proteins.
3 LEA protein and its effect on sucrose
glass formation that LEA proteins may act
1.6.2.2. Sugars
as anchors in a structural network that sta-
bilizes cytoplasmic components during The accumulation of soluble sugars is also
drying and in the dried state. strongly correlated to the acquisition of
At this point it seems likely that each desiccation tolerance in plants and other
individual group of LEA proteins may have organisms (for reviews see Crowe et al.,
different, complementary effects. Most des- 1992; Leprince et al., 1993; Vertucci and
iccation-tolerant tissues contain a represen- Farrant, 1995; Chapters 5 and 10). Soluble
tative of most, if not all, of the different sugars, especially sucrose, accumulate in
groups of LEA proteins, and it is also likely seeds (Leprince et al., 1993), pollen
that all are needed to achieve the highest (Hoekstra et al., 1992) and in desiccation-
degree of desiccation tolerance. tolerant vegetative tissues (Bewley and
There is mounting evidence that another Krochko, 1982; Ingram and Bartels, 1996;
class of proteins, the small heat-shock pro- Oliver and Bewley, 1997). In Craterostigma
teins (HSPs), may play a role in cellular plantagineum, 2-octulose stored in the
protection during desiccation. Small HSPs hydrated leaves is converted to sucrose
accumulate in maturing seeds of many during drying to such an extent that in the
plant species (Vierling, 1991; Wehmeyer et dried state it comprises about 40% of the
al., 1996) prior to desiccation. Alamillo et dry weight (Bianchi et al., 1991).
al. (1995) reported that small HSPs are Sucrose is the only free sugar available
expressed constitutively in the vegetative for cellular protection in desiccation-toler-
tissues of C. plantagineum and increased ant mosses, including Tortula ruraliformis
and T. ruralis (Bewley et al., 1978; Cytoplasmic glass formation has also
Smirnoff, 1992). The amount of this sugar been postulated to maintain the structural
in gametophytic cells of T. ruralis is and functional integrity of macromolecules
approximately 10% of dry mass, which is (Sun and Leopold, 1997; Crowe et al.,
sufficient to offer membrane protection 1998b), which has been well demonstrated
during drying, at least in vitro (Strauss and with in vitro models (Roos, 1995).
Hauser, 1986). Moreover, neither drying Intracellular glasses, by virtue of their high
nor rehydration in the dark or light results viscosity, drastically reduce molecular
in a change in sucrose concentration, sug- movement and impede diffusion of reac-
gesting that it is important for cells to tive compounds in the cell. It is by this
maintain sufficient amounts of this sugar property that glasses are thought to prolong
(Bewley et al., 1978). The lack of an the longevity of desiccated tissues by slow-
increase in soluble sugars during drying ing down degradative processes during
appears to be a common feature of desicca- storage. Buitink et al. (1998) recently
tion-tolerant mosses (Smirnoff, 1992). demonstrated a strong relationship
It is thought that sugars protect the cells between molecular mobility and storage
during desiccation by two mechanisms. longevity in both pollen and pea seeds.
First, the hydroxyl groups of sugars may Thus, although glass formation may not be
substitute for water to maintain important in the initial acquisition of des-
hydrophilic interactions in membranes and iccation tolerance, it may be crucial for sur-
proteins during dehydration (Crowe et al., vival of the dried state (as suggested by
1992). This has so far only been demon- Buitink, 2000; Chapter 10).
strated in vivo, using liposomes and iso- Other carbohydrates besides sucrose
lated proteins (Crowe et al., 1992). accumulate in desiccation-tolerant tissues,
Secondly, sugars are a major contributing the principal ones being the oligosaccha-
factor to vitrification, the formation of a rides stachyose and raffinose (Horbowicz
biological glass, of the cytoplasm of dry and Obendorf, 1994), and have been postu-
cells (Leopold et al., 1994; Chapter 10). lated to play a part in desiccation toler-
This mechanism has been the subject of ance. The presence of these compounds
intense research over the last 15 years. has also been correlated with seed
Vertucci and Leopold (1986) suggested longevity (Hoekstra et al., 1994;
that desiccation tolerance in seeds had to Horbowicz and Obendorf, 1994), which
be associated with some feature or solute has linked them to a possible role in the
combination that would avoid crystalliza- stabilization of intracellular glasses
tion of the cytoplasm as dehydration pro- (Leopold et al., 1994; Bernal-Lugo and
gressed. Burke (1986) proposed that high Leopold, 1995; Sun, 1997). However,
concentrations of sugars lead to vitrification Buitink et al. (2000) demonstrated that the
of the cytoplasm during desiccation and reduction in oligosaccharides in primed
thus prevent crystallization. Glass forma- seeds did not alter Tg (the glass-to-liquid
tion has since been demonstrated in seeds transition temperature) or viscosity and
(Williams and Leopold, 1989; Leopold et thus they contended that oligosaccharides
al., 1994; Leprince and Walters-Vertucci, do not affect the stability of intracellular
1995), pollen (Buitink et al., 1996) and in glasses. These results support the earlier
leaf tissues of C. plantagineum (Wolkers et studies of Black et al. (1999), which had
al., 1998). Walters (1998) went as far as to shown a lack of a temporal correlation
say that glass formation is an intrinsic prop- between the induction of desiccation toler-
erty of any complex system that can survive ance by a mild dehydration treatment and
desiccation. However, glass formation may the appearance of raffinose in wheat
not be sufficient to confer desiccation toler- embryos. These studies cast doubt on the
ance since desiccation-sensitive tissues are role of oligosaccharides in the acquisition
capable of forming cytoplasmic glasses of tolerance and the maintenance of viabil-
(Sun et al., 1994; Buitink et al., 1996). ity in the dried state.
1.6.3. Repair In the desiccation-tolerant fern

Polypodium virginianum rehydrating tis-
The repair processes associated with des- sues do accumulate novel transcripts but
iccation tolerance have been difficult to their identities have not been investigated
detail and characterize. In seeds, repair (Reynolds and Bewley, 1993b). Recent
mechanisms are difficult to separate from work with the desiccation-tolerant grass
events that are associated with germina- Sporobolus stapfianus has identified two
tion and early seedling growth, but evi- transcripts that accumulate during the
dence for repair does exist. In vegetative early phases of dehydration but also accu-
angiosperms, the major emphasis appears mulate during rehydration. The first of
to be on effective cellular protection and these is a transcript coding for a plant
much of the research in angiosperms has Rab2, a small GTP-binding protein that in
focused on this component. The most other systems is an important protein in
promising models for investigating a direct the targeting of membrane vesicles in
role for cellular repair in desiccation toler- vesicular trafficking pathways and a path-
ance appear to be the highly desiccation- way directly involved in membrane con-
tolerant bryophytes. struction (O’Mahony and Oliver, 1999a).
In seeds, most of the evidence for cellu- The second transcript encoded a polyubi-
lar repair derives from investigations into quitin, a protein involved in protein
the causal relationship between cellular turnover (O’Mahony and Oliver, 1999b). In
damage and loss of viability during storage C. plantagineum very few transcripts were
(Bewley and Black, 1994). Consequently, identified as being specific for the rehydra-
one has to keep in mind that the repair tion process. Those that were appeared to
processes that have been identified may be involved in the metabolism of sugars
not play a major role in desiccation toler- that is required to re-establish the pools of
ance per se but rather in the ability to sur- octulose required for the generation of
vive long term in the dried state. There are sucrose during dehydration (Bernacchia et
two reports, however, that indicate the al., 1996).
repair of cellular components, proteins and Cellular repair, as a component of desic-
DNA, which may directly affect desicca- cation tolerance mechanisms, is more easily
tion tolerance as well as storage longevity. defined in desiccation-tolerant bryophytes.
Mudgett et al. (1997) demonstrated that Desiccation-tolerant bryophytes are thought
proteins containing abnormal L-isoaspartyl to employ a mechanism for desiccation tol-
residues could be repaired in aged barley erance that represents the most primitive
seeds by the activity of the enzyme L-isoas- form expressed in land plants (Oliver et al.,
partyl methyltransferase. These authors 2000). Unlike the acquisition of desiccation
argue that this type of repair is particularly tolerance in seeds, which may be develop-
important during dehydration where pro- mentally programmed, and in desiccation-
tein turnover rates are slow. Boubriak et al. tolerant angiosperms, which is
(1997) demonstrated that one of the earliest environmentally induced by drying, desic-
activities seen in imbibing cereal grains is cation tolerance in most bryophytes
the repair of damage to genomic DNA appears to be constitutive (Oliver and
incurred whilst the seeds were dry and in Bewley, 1997). The difference in mecha-
storage. If the repair processes were nisms of tolerance in these systems is
blocked during imbibition then DNA reflected in their biology. Desiccation-toler-
degradation became severe. If universal, ant angiosperms have morphological and
DNA repair would certainly qualify as a physiological adaptations in place that can
key process in the mechanism of desicca- retard the loss of water. The mechanism of
tion tolerance in seeds (Chapter 12). desiccation tolerance that has evolved in
The identification of repair processes in these plants takes advantage of these adap-
vegetative tissues of desiccation-tolerant tations by being inducible. As the rate of
tracheophytes has received little attention. water loss is relatively slow, there is time
to establish the protective measures bolism when rehydrated. During the first 2
required, and the plant can thus survive a h following rehydration of dried T. ruralis,
drying event. If water loss is too rapid, the synthesis of 25 proteins (termed
these plants succumb to the damaging hydrins) is terminated or substantially
effect of water loss and die (Gaff, 1989; decreased, and the synthesis of 74 proteins
Bewley and Oliver, 1992; Oliver and (termed rehydrins) is initiated or substan-
Bewley, 1997). Bryophytes, on the other tially increased (Oliver, 1991). Controls
hand, have little in the way of adaptations over changes in synthesis of these two
to retain water within the plant and, as a groups of proteins are not mechanistically
result, the internal water content of these linked. It takes a certain amount of prior
plants rapidly equilibrates to the water water loss to fully activate the synthesis of
potential of the environment (Proctor et al., rehydrins upon rehydration. Perhaps this
1998). A consequence of this is that many is a strategy that has evolved to link the
bryophytes experience drying rates that are amount of energy expended in repair to
extreme and therefore have insufficient the amount of damage potentiated by dif-
time to induce and set in place protective fering degrees of drying.
measures. Thus, it appears that bryophytes In T. ruralis, there also appears the
have evolved a constitutive mechanism for capability of preparing the cell for a rapid
desiccation tolerance, one that has protec- recovery if drying rates are sufficiently
tive measures that are always in place. This slow (4–6 h). Using cDNA clones corre-
conclusion is supported by the observa- sponding to T. ruralis transcripts that are
tions that both sucrose and LEA proteins preferentially translated during rehydra-
are maintained at constant levels in desic- tion (Scott and Oliver, 1994), it was deter-
cation-tolerant bryophytes during drying mined that several ‘recovery’ transcripts
and rehydration (see above). The constitu- accumulate during slow drying (Oliver
tive protection mechanism appears to be and Wood, 1997; Wood and Oliver, 1999).
particularly effective in preventing damage Recent studies clearly demonstrate that
to the photosynthetic apparatus, as evi- these transcripts are sequestered in the
denced by the very rapid recovery of pho- dried gametophytes in messenger ribo-
tosystem II activity (Tuba et al., 1996b; nucleoprotein (mRNP) particles (Wood
Csintalan et al., 1999; Proctor and and Oliver, 1999). Of 18 rehydrin cDNAs
Smirnoff, 2001). isolated (Scott and Oliver, 1994) and
How does this relate to cellular repair sequenced (Oliver et al., 1997; Wood et
and the uniqueness of bryophytes for al., 1999) in T. ruralis, only three exhibit
studying this aspect of desiccation toler- significant sequence homology to known
ance? It appears that the level of protec- genes in the Genbank databases. Tr155 has
tion that bryophytes are capable of a strong sequence similarity to an alkyl
maintaining is not sufficient to completely hydroperoxidase linked to seed dormancy
prevent damage, especially to membranes, in barley (Aalen et al., 1994) and
during rehydration. To achieve desicca- Arabidopsis embryos (Haslekas et al.,
tion tolerance, bryophytes thus rely heav- 1998), and in rehydrated but dormant
ily on repair mechanisms induced during Bromus secalinas seeds (Goldmark et al.,
the initial phases of hydration following 1992). Tr213 exhibits a high degree of sim-
rewetting (Oliver and Bewley, 1997; ilarity to polyubiquitins from several
Oliver et al., 1998). plant sources, suggesting that protein
Most work on repair in mosses has cen- turnover may be an important part of
tred on the proteins whose synthesis is repair in mosses as well as in
induced immediately upon rehydration of angiosperms. Tr288 encodes an LEA-like
desiccated gametophytic tissue. Early protein (see above), which suggests that
work (see Bewley, 1979, for review) estab- LEA proteins may have a protective role
lished the ability of T. ruralis and other during rehydration and a role in cellular
mosses to rapidly recover synthetic meta- repair (Velten and Oliver, 2001).
1.7. Future Prospects and Agricultural tolerance and thus the genetic information
Significance necessary for expanding their drought tol-
erance may not be exploitable or indeed
Our present agricultural system is almost present. In contrast, more may be gained by
totally dependent upon the ability of ortho- understanding how stress-tolerant plants or
dox seeds to tolerate desiccation. The iden- plant structures accomplish tolerance, and
tification and functional analysis of genes from such sources genes that contribute
involved in the developmentally pro- directly to tolerance can be identified. As
grammed desiccation tolerance stage of pointed out by Bartels and Nelson (1994),
seeds is a vital step in understanding this the limiting factor for the improvement of
complex trait. The knowledge gained from abiotic stress tolerance in crops is ‘the
such studies will impact on many diverse availability of structural genes and regula-
areas of agricultural concerns such as tory elements which positively contribute
germplasm preservation, seed production to stress tolerance improvement’. If our
and seedling establishment. Apart from efforts to utilize our understanding of the
genetic considerations, the ongoing studies underlying mechanisms of desiccation tol-
on the role of biological glasses in desicca- erance are to bear fruit, we must discover
tion tolerance and the determination of and identify the genes that are central to
which proteins and sugars are important in this trait.
their stability will affect our ability to pre- Cushman and Bohnert (2000) describe a
serve viable germplasm for longer periods strategy for cataloguing genes central to
of time, preserving genetic diversity for particular traits in their discussion on
future breeding needs. This goal will also genomic approaches to plant stress. The
be benefited by our continued progress in initial phase of gene discovery is the large-
understanding of how desiccation-tolerant scale sequencing of randomly selected
tissues deal with oxidative stress. cDNA clones, termed Expressed Sequence
Over 35% of the world’s land surface is Tags (ESTs), which are synthesized from
considered to be arid or semiarid, experi- mRNA pools representing a specific devel-
encing precipitation that is inadequate for opmental stage or response state. EST col-
most agricultural uses. Ramanathan (1988) lections that will enhance gene discovery
has argued, based on predictions of global in desiccation tolerance have been started,
environmental changes, that developing the most extensive of which is that for
crops that are more tolerant to water developing seeds of Arabidopsis thaliana
deficits while maintaining productivity (Girke et al., 2000). Smaller EST collec-
will become a critical requirement in the tions have been made for C. plantagineum
early part of this century. Understanding leaves that had been dried for an hour or
how plant cells tolerate water loss is a vital fully dried (Bockel et al., 1998) and S. stap-
prerequisite for developing strategies that fianus leaves during dehydration
can influence agricultural and horticultural (Blomstedt et al., 1998; Neale et al., 2000).
crop productivity and survival under these Wood et al. (1999) have established a lim-
conditions of decreasing water availability. ited sample of ESTs (152) from a cDNA
Much has been accomplished in the dis- library developed from the mRNP fraction
covery and characterization of those genes of slowly-dried T. ruralis gametophytes. In
that are expressed during the response of the EST collections from vegetative desic-
crop and model plants to water deficit or cation-tolerant plant tissues, many of the
salt stress. From this work, our knowledge sequenced clones were of unknown iden-
of stress tolerance has improved tity (71% for Tortula) and/or not previ-
immensely and some success has been ously associated with water stress.
achieved, but these traits are very complex Cushman and Bohnert (2000) suggested
and breeding progress has been slow. This that this may indicate, as suggested above,
approach is also restricted in that most that these plants may possess ‘unique gene
crops have a limited capacity for drought complements or regulatory processes that
contribute to desiccation stress’ and, by The next step in the search for insight
inference, novel genes that may prove use- into gene function and regulatory controls
ful in breeding for drought stress tolerance. in desiccation tolerance will come from the
The cataloguing of gene products that are use of expression profiling with cDNA
expressed during the acquisition and estab- microarrays. Such work is under way but
lishment of desiccation tolerance is only the little has been reported as yet. The first
first step. The ultimate goal is to determine information is likely to come from the
which genes are central to desiccation toler- analysis of Arabidopsis EST microarrays in
ance and what functions they perform. The extensions of the studies reported by Girke
major approach used to gain a functional et al. (2000), which may pinpoint tran-
understanding of individual gene products scripts to the exact time of the acquisition
has been the overexpression of genes in of desiccation tolerance in developing
transgenic plants. We have already men- seeds. Direct approaches to elucidate func-
tioned the studies of Xu et al. (1996) and tionality of individual genes or gene fami-
Sivamani et al. (2000) concerning the over- lies will be slower to develop. The vast
expression of HVA1, a Group 3 lea gene, and array of genetic tools, such as transgenic
their success in improving drought toler- capability, mutant generation and screen-
ance in rice and wheat. Two other groups ing tools, T-DNA and transposon-tagged
have attempted to modify sugar metabolism knockouts, and map-based cloning tech-
to improve tolerance by engineering tre- nologies, will make Arabidopsis and seed
halose 6-phosphate synthetase genes from desiccation tolerance the initial foci of
non-plant sources, from yeast (Holmström et functional studies. Nevertheless, tools are
al., 1996) and from bacteria (Pilon-Smits et becoming available for vegetative desicca-
al., 1998) into tobacco. The aim was to pro- tion-tolerant model plants and these will
mote trehalose accumulation in leaf cells, play an important role in evaluating target
and both groups were successful and genes. An example of this has been the
achieved greater tolerance to water deficits exciting use of activation tagging, by trans-
in tobacco. The exciting conclusion from genic random insertion of a highly active
these studies, and those with the Group 3 foreign promoter to ‘activate’ native genes,
LEA protein, is that the engineering of a sin- by Furini et al. (1997) to isolate a gene
gle gene can achieve results that affect such (cDT-1) involved in regulation of the
a complex trait as drought tolerance. It will response of C. plantagineum callus to des-
be interesting to see how these results will iccation. Advances in the fields of molecu-
translate into an advancement in our under- lar biology, genetics, genomics and
standing of how these gene products func- biophysics have put us on the threshold of
tion to achieve greater tolerance and if these a new era in our quest to understand one of
plants will have an impact on drought- the most complex and important traits in
tolerance breeding efforts. plant biology: desiccation tolerance.
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02 Dessication - Chap 2 18/3/02 1:53 pm Page 45
Part II
Methodology
2 Methods for the Study of Water Relations

Under Desiccation Stress
Wendell Q. Sun
Department of Biological Sciences, National University of Singapore,
Kent Ridge Crescent, Singapore 119260
2.1. Introduction 48
2.2. Expression of Water Status 48
2.2.1. Mass-based measures for tissue hydration 48
2.2.1.1. Water content 48
2.2.1.2. Relative water content 49
2.2.2. Thermodynamic measures for tissue hydration 50
2.2.2.1. Water activity 50
2.2.2.2. Chemical potential of water and water potential 51
2.3. Measurement of Tissue Water Potential 53
2.3.1. Psychrometric and hydrometric methods 53
2.3.2. Osmometric or cryoscopic method 54
2.3.3. Isothermal equilibrium method 55
2.4. Water Relations – the Thermodynamic Approach 55
2.4.1. The Höfler diagram and pressure–volume curve 55
2.4.1.1. Change of cell turgor pressure during desiccation 55
2.4.1.2. Change of osmotic potential during desiccation 57
2.4.1.3. The volume of water in symplast, apoplast and
intercellular spaces 57
2.4.1.4. Volumetric elasticity of the cell wall 59
2.4.2. Analysis of water sorption isotherms 60
2.4.2.1. Theoretical models 60
2.4.2.2. Temperature dependency of water sorption 62
2.4.2.3. Monolayer hydration and water-clustering function 65
2.4.2.4. Occupancy of water-binding sites 66
2.5. Measurement of Drying Rate and Desiccation Stress 68
2.5.1. Driving force for water loss and expression of drying rate 68
2.5.2. Quantification of desiccation stress 68
2.6. Water Relations – the Kinetic and Functional Approach 70
2.6.1. General considerations 72
48 W.Q. Sun
2.6.1.1. Time scale 72

2.6.1.2. Structural complexity and dynamics of
molecular ordering 72
2.6.1.3. The model-dependent interpretation: the pitfalls 73
2.6.2. Biophysical techniques 74
2.6.2.1. Differential scanning calorimetry 74
2.6.2.2. Thermally stimulated current (TSC) method 75
2.6.2.3. Nuclear magnetic resonance (NMR) 76
2.6.2.4. Electron spin resonance 77
2.7. Concluding Remarks 78
2.8. References 79
Appendix 84
2.1. Introduction of water relations that are directly related to

desiccation tolerance of plant tissues will be
In hydrated plant cells, water is the main introduced. The strengths and limitations of
constituent. The organization of cellular various methods or techniques of measure-
structures (both supramolecular assemblies ment of water relations during desiccation
and micromolecular structures) and the over- will be discussed. An effort will be made to
all biochemistry (the thermodynamics of bio- give a basic understanding of terms and
logical processes and their rate parameters) of concepts concerning cellular water status
an organism depend on water. Water is a sol- and the expression of dehydration stress.
vent and a medium in which diffusion of
solutes and biochemical reactions take place
in plant cells. It is often a participant and/or 2.2. Expression of Water Status
a product of various biochemical reactions.
In low-moisture systems such as naturally The most important quantity that has to be
dried pollen grains and plant seeds, cellular measured in all studies of desiccation toler-
water also plays an important role as a plasti- ance is the degree of dehydration stress. So
cizer, influencing the translational or rota- far, there is no agreed parameter of dehydra-
tional motions of entire molecules, or tion stress measurement. The change in
segments of macromolecules and intramolec- water content of plant tissues and organs is
ular motions. Water is involved in virtually often used as an indicator of dehydration.
every dynamic process in a living cell. However, insufficient attention has been
The loss of water from plant cells is an paid to problems commonly associated with
important environmental stress. Changes in the use of water content as an indicator of
the aqueous environment influence the dehydration stress. For example, different
complex thermodynamics and kinetics of concepts and approaches are currently used
structural stability and all aspects of biologi- by research groups working on biological
cal functions. The accurate measurement of systems, ranging from bacteria and fungal
the status of cellular water is essential for spores to microscopic animals, pollen
the study of both desiccation stress in plants grains, large seeds and resurrection plants.
and the mechanisms of plant desiccation
tolerance. The method of quantification and
2.2.1. Mass-based measures for tissue
interpretation must be applicable not just to
hydration
the narrowly defined desiccation condi-
tions, but also to all other types of physio-
2.2.1.1. Water content
logical stresses with a dehydration
component, such as freezing and salinity. In Water content on a wet-weight basis (WC, %
this chapter, several fundamental principles w.b.) is widely used in the literature of desic-
Methods for Studying Water Relations Under Stress 49
cation studies, and is adopted by the WC (g g1 dw) = WC (% w.b.)/[100 – WC

International Seed Testing Association (% w.b.)] (3)
(ISTA, 1993) for the expression of seed water
content. WC (% w.b.) is the percentage mass 2.2.1.2. Relative water content
fraction of water of the total tissue mass:
Relative water content (RWC) is another
WC (% w.b.) = (fresh weight dry
mass-based parameter. RWC is widely used
weight)/fresh weight 100 (1)
in the pressure–volume analysis of plant
WC (% w.b.) is not a linear expression of tissue water stress. RWC is a simple and
water content in tissues, because fresh useful measure of the extent to which a tis-
weight appears in both the numerator term sue is in water deficit. It is related to tissue
and the denominator term in Equation (1). water content at full turgor (WCF). During a
When WC (% w.b.) is used to monitor the dehydration experiment, RWC is calcu-
loss of water during desiccation, the lated by dividing water content at a given
decrease of WC (% w.b.) does not necessar- time by water content at full turgor, and
ily reflect the exact extent of dehydration expressed as a fraction value or as a per-
stress. The change of WC (% w.b.) during centage. If water content in the tissue is
drying is, in fact, related to the change of determined as WC (g g1 dw), the calcula-
the reciprocal of tissue fresh weight. For tion of RWC is straightforward, being
example, when the tissue of 80% WC (% WC/WCF. But, if water content is deter-
w.b.) is dried to 70% and 60% water con- mined as WC (% w.b.), RWC is calculated
tent, the tissue actually loses 41.7% and by the equation:
62.5% of its initial water quantity, respec-
RWC = [WC (100 – WCF)]/[WCF
tively, not just 12.5% and 25% reduction as
(100 – WC)] 100 (4)
implied by the values of WC (% w.b.). The
quantity of water lost during dehydration RWC is a linear expression of moisture
from 80% to 70% WC (% w.b.) is twice as condition. The change in RWC over time
much as water loss during dehydration serves as a good indicator for the rate of
from 70% to 60% WC (% w.b.). dehydration. Physiological responses of
Water content on a dry-weight basis mea- plant water deficit are highly correlated
sures the mass ratio between water and the with RWC (Sinclair and Ludlow, 1985). The
dry mass in tissues, and is often expressed use of RWC is particularly advantageous for
by g water per g dry weight (i.e. g g1 dw): comparative studies, in which initial water
content at full turgor or full hydration
WC (g g1 dw) = (fresh weight dry
varies considerably among different
weight)/dry weight (2)
species, different tissues of the same
WC (g g1 dw) is a linear expression of species or the same tissue at different
water content, and the change of WC (g g1 developmental stages, such as seeds. In cer-
dw) during drying is proportional to the tain cases, it may be even preferred over
loss of water in a tissue. On the mass basis, water potential, because RWC also accounts
a tissue with a WC of 0.20 g g1 dw is for the effect of osmotic adjustment in
hydrated exactly twice as much as the tis- affecting plant water status. For example,
sue with a WC of 0.10 g g1 dw, and four- two plants with the same leaf water poten-
fold as much as the sample with a WC of tial can have different RWCs if they differ
0.05 g g1 dw. For this reason, some in their ability for osmotic adjustment.
researchers have argued that WC (g g1 dw) In many desiccation studies on higher
is a more sensible expression than WC (% plants, water content of a tissue at full tur-
w.b.) for the measurement of dehydration. gor was not specifically determined, and
At WC < 15%, the difference between WC instead water content after full hydration in
(% w.b.) and WC (g g1 dw) is fairly small. water was used. Typically, leaf samples (e.g.
WC (% w.b.) is converted to WC (g g1 dw) discs or sections) of higher plant species
using the following equation: are taken and weighed immediately. The
50 W.Q. Sun
samples are then hydrated in distilled based parameters for the expression of
water for 4–6 h, after which they are dehydration stress has been shown by a
weighed again and their water contents are number of studies. The critical onset water
determined by drying the samples in an potential of Quercus rubra (Pritchard, 1991)
oven. In higher plants, the amount of inter- and Quercus robur (Pritchard and Manger,
cellular water is small or non-existent 1998) is about –3 MPa, but the correspond-
(Oertli, 1989). However, it should be noted ing mass water contents vary substantially
that some plant tissues do contain intercel- due to different seed oil content. Sun and
lular water, which is held in spaces Gouk (1999) studied the water relation
between cells of a tissue at relatively high responses of three recalcitrant (desiccation-
water potential (near zero). Therefore, water sensitive) seeds (Aesculus hippocastanum,
content at full turgor has different physio- Andira inermis, Q. rubra) during controlled
logical meaning from water content of the dehydration. The critical water potentials
tissue at full hydration when the tissue confor seeds are quite similar for all three
tains intercellular water. If intercellular species (7 to 8 MPa), but their corre-
water is present, water content of the tissue sponding critical water contents are 0.45,
at full turgor has to be estimated from the 1.10 and 0.35 g g1 dw for A. hippocas-
plot of water potential on water content (g tanum, A. inermis and Q. rubra, respec-
g1 dw). To determine the water content of tively. If the critical water content were
the tissue at full turgor, two linear regres- used to express the relative desiccation tol-
sion lines can be fitted, respectively, with erance, one would conclude incorrectly
data points where water potential remains that seeds of A. hippocastanum and Q.
almost unchanged during initial water loss rubra are much more desiccation-tolerant
and the next few points where water poten- than seeds of A. inermis. Therefore, a mass-
tial starts to fall. The intercept of these two based parameter of water loss may not be a
regression lines gives the water content at reliable indicator for the degree of desicca-
full turgor. Beckett (1997) reported that the tion stress in plant tissues.
amount of intercellular water varied greatly
among species of bryophytes. If intercellu-
lar water exists in a tissue, correction needs 2.2.2. Thermodynamic measures for tissue
to be made to the raw RWC readings, which hydration
are calculated according to the water con-
tent at full hydration. The method used to The response of plant tissues to desiccation
correct the raw RWC readings was is related to the thermodynamic and
described in detail by Beckett (1997). kinetic status of tissue water, rather than to
There are shortcomings in using mass- actual water content. The water status of
based parameters for the expression of plant tissues can be expressed in terms of
water content. Plant tissues are heteroge- energy status of water molecules, i.e. the
neous, complex biological systems, in partial molar Gibbs’ free energy or water
which carbohydrates, proteins and lipids potential. This thermodynamic approach is
and other components have different hydra- preferred to the mass-based expression of
tion properties. As a consequence, when tissue hydration, because thermodynamic
plant tissues of various species are equili- parameters (i.e. energy status) are related
brated under given conditions of tempera- directly to the numerous biophysical and
ture and relative humidity, equilibrium physiological events that contribute to des-
water content varies considerably among iccation stresses and desiccation tolerance
species. For example, seeds with large lipid of plant tissues.
reserves equilibrate to lower water contents
than starchy seeds, even though the chemi-
2.2.2.1. Water activity
cal potential of water molecules is the same
for all tissues when equilibrium is Water activity (aw) is used to describe
achieved. The disadvantage of using mass- water status in the studies of desiccation
tolerance and storage survival for spores, In such cases, the measured vapour pres-
pollen, seeds and resurrection plants (Ellis sure of water may not be the equilibrium
et al., 1990, 1991; Berjak and Pammenter, vapour pressure, but the vapour pressure of
1994; Vertucci et al., 1994, 1995; Walters, a ‘stationary’ state that is time-dependent.
1998a). Water activity is measured as the However, studies in food sciences have
ratio of the vapour pressure of water in a suggested that water activities measured
system to the vapour pressure of pure are likely to be close to equilibrium and the
water at the same temperature. It is related differences should be within the uncer-
to the equilibrium relative humidity (RH) tainty associated with the experimental
of the air surrounding the system (i.e. RH = determination (Chirife and Buera, 1996).
aw 100). Water activity can be viewed as The usefulness of the water activity con-
the ‘effective’ water content, which is ther- cept in seed storage stability has been dis-
modynamically available to various physi- cussed by Walters (1998b).
ological processes in cells. For the survival
of organisms under water stress, the ‘effec-
2.2.2.2. Chemical potential of water and
tive’ water is more important than the total
water potential
amount of water present in the tissue.
Water activity of fresh plant tissues may The quantity of free energy of a component
vary only between 0.980 and 0.996. Within (µj) in a system is measured by its chemical
this narrow range, it is not useful for the potential. The chemical potential of water
expression of dehydration stress or tissue (µw) in a system is defined by:
water status. However, for the studies of –
µw = µ*
w + RT ln aw + VwP + zwFE + mwgh (5)
severe water stress and extreme desicca-
tion, water activity has several advantages where µ*w is the chemical potential of pure
over water content, including its concep- water at ideal reference conditions. The
tual simplicity, measurability, easy experi- second term RT ln aw is for water activity. R
mental manipulation, and its applicability is the gas constant (8.314 103 kJ mol1
to both simple and complex systems. A K1), T is the absolute temperature (K, in
number of physiological processes that are kelvin), and aw is water activity (RH/100).
–
relevant to desiccation tolerance or damage Vw is the partial molal volume of water (i.e.
have been shown to occur at specific water the differential increase or decrease in vol-
activities, and some of those are presented ume when a differential amount of water is
–
in Fig. 2.1. added or removed). Vw is influenced by the
Water activity in plant tissues can be presence of solutes and is also tempera-
determined using the hygrometric method ture-dependent (1.805 105 m3 mol1 at
and the isothermal equilibrium sorption 20°C). P is the hydrostatic pressure on
method. The hygrometric instrument water in excess of atmospheric pressure
–
method directly measures the equilibrium (MPa, 1 MPa = 103 kJ m3). The term VwP
RH of plant tissues in a closed chamber. represents the effect of pressure on the
With the equilibrium sorption method, chemical potential of water and is
samples of plant tissues are equilibrated to expressed in energy per mole (kJ mol1).
a series of known water activities at a spec- zwFE is the electrochemical potential of
ified temperature. The relationship water, which equals zero because water is
between water content and water activity uncharged (zw = 0). The last term mwgh is
upon equilibrium (i.e. the sorption the gravitational term, representing the
isotherm curve) is then used to calculate work needed to move 1 mole of water to a
water activity of plant tissues at different given height. Practically, mwgh will remain
water contents. Water activity is defined at constant in most circumstances of desicca-
equilibrium. However, plant tissues at low tion studies.
and intermediate moisture levels may not The water potential is proportional to
be in a true state of equilibrium at all, but the chemical potential of water (µw µ*w) in
in an amorphous metastable state instead. a system as described in Equation (5).
52 W.Q. Sun
Therefore, water potential is actually the osmotic potential and h is the gravita-
potential energy of water per unit mass. tional potential. The total water potential is
While water content tells how much water the sum of hydrostatic, osmotic and gravita-
is in a sample, water potential tells you tional components. The gravitational term
how available that water is. By convention, (h) depends on the position of water in a
water potential is defined as follows: gravitational field and is not relevant to
= P + π + h (6)
most desiccation studies. Osmotic potential
depends on the concentration of dissolved
where p = P and is hydrostatic pressure substances in water. Osmotic potential is
on water as defined in Equation (5), π is related to water activity by the equation:
Coffea species
Mean of minimum value for bacterial growth

Cell respiration starts to cease
Lysozyme activity stops
at lower aw
colonies in situ
Minimum required for photosynthesis

Nucleic acids and proteins fully hydrated
Water vapour above saturated NaCl solution
–50
Water potential of water vapour (MPa)
seeds typically survive at lower aw
saturated CaCl2 solution
Desiccation tolerance of embryos of

DNA disordered and damaged
–100
Typical exposure of Nostoc
–150
Water vapour above
–200
Orthodox
–250
20 40 60 80 100
Relative humidity (%)
Fig. 2.1. Water activities (relative humidities) that limit physiological activities and cell growth. Physical
parameters and physiological processes are drawn with data from Wolfe and Leopold (1986), Potts (1994)
and Sun and Gouk (1999). The relationship between relative humidity and water potential is calculated
according to Equation (7) at 25°C. A similar diagram that is specific to a plant tissue can be established.
Such a diagram would serve as a valuable reference for experimental design and data interpretation, since it
gives a clear concept about the possible sequence of potential physiological and biochemical events and
their interactions as the tissue loses water.
–
Vw π = RT ln aw (7) face. This technique, however, is unsuitable
for many desiccation-tolerant plant tissues,
During dehydration, the water content in
e.g. lichens, bryophytes, spores, pollen grain
seed tissues is reduced, resulting in an
and seeds. This is because the pressure
increase in the concentration of solutes and
chamber method measures the xylem ten-
thus a decrease in water activity and
sion, which is broadly equal to the leaf
osmotic potential (i.e. π becomes more
water potential. Water potential of plant
negative). A similar situation occurs during
parts that do not have vascular systems can-
freezing. The formation of ice leads to the
not be measured with the pressure chamber
dehydration of the protoplast and the con-
method. However, water potential of plant
centration of solutes.
tissues can be measured by a number of
The interactions of water with biological
other techniques. These techniques use
surfaces and interfaces are of great impor-
either the relationship of the sample water
tance to desiccation tolerance of plant tis-
potential to the equilibrium vapour pressure
sues, especially at low moisture levels. The
immediately around the sample or the prin-
influence of such interactions on water
ciple of the freezing-point depression in the
potential in a tissue is commonly called
liquid solution.
‘matric’ potential. Rapid water uptake by
dry seeds during the early stage of germina-
tion is mainly attributed to large matric
2.3.1. Psychrometric and hydrometric
potentials. Another example is the reduced
methods
rate of water loss as the tissue is dried to
lower water content. Matric potential
Both methods are widely used for the mea-
depends on the adsorptive forces that bind
surement of tissue water potential. The mea-
water to a matrix. The amount of matrix-
surement of water potential by a
bound water in recalcitrant Q. robur
psychrometer and a hydrometer is called
embryonic axes is as high as 0.25–0.30 g
the wet-bulb depression method and the
g1 dw (Pritchard and Manger, 1998).
dew-point depression method, respectively.
However, the forces of such water–matrix
A psychrometer measures water potential of
interactions are adequately represented by
samples (placed in closed chambers)
their contributions to hydrostatic pressure
through its ability to determine the RH of
(P) and osmotic potential (π). For exam-
the closed environment. The instrument
ple, the presence of aqueous interfaces in
uses high-sensitivity thermometers to mea-
cells lowers water activity through interfa-
sure temperature reduction resulting from
cial attractions and binding of water near
the heat of vaporization of water in a sample
their surfaces, which has already been
relative to pure water. It can measure water
included in the osmotic component in
potential of solid tissue materials and
Equation (7). Therefore, matric potential
droplets of solutions. The sample is first
does not represent additional new forces.
sealed in a small chamber containing a ther-
mocouple. After an equilibration period, a
cooling current is applied to the thermocou-
2.3. Measurement of Tissue Water ple in order to condense water on the ther-
Potential mocouple junction. The amount of
condensed water is proportional to the
A pressure chamber (pressure bomb) is com- water potential of the tissue. The water is
monly used to measure directly leaf water allowed to evaporate, causing a change in
potential of higher plants. The detached leaf the thermocouple output, and the output is
is sealed in a steel chamber with the cut calibrated for water potential, using stan-
petiole protruding out. Pressure that is dard salt solutions. On the other hand, a
applied to the chamber is taken as the hydrometer maintains the dew-point
xylem (leaf water) potential when the sap depression temperature during the measure-
meniscus appears at the petiole xylem sur- ment using a thermocouple. The dew-point
54 W.Q. Sun
depression temperature is the temperature osmolal aqueous solution, the osmotic

to which the air in the sealed chamber must potential at 0°C is ideally 2.271 MPa, and
be reduced so that the air becomes saturated the freezing-point depression is 1.86°C.
with water vapour. Psychrometric and (An osmole is the mass of a substance that
hydrometric methods can be used to mea- when dissolved in 1 kg water generates an
sure both water potential and osmotic osmotic pressure equivalent to that pro-
potential of plant tissues. To measure duced by 1 mole of an ideal solute dis-
osmotic potential, a sample has to undergo solved in 1 kg water. After dissolving, an
the freeze–thaw cycles to disrupt the cellu- ideal solute gives 6.023 1023 osmotically
lar structures before the measurement, active particles.) Theoretically, the osmotic
whereas the water potential is measured potential of an unknown sample can be
using undisrupted tissues. estimated from the depression of its freez-
A psychrometer is very sensitive to tem- ing point by the following relationship:
perature change because it measures very
small temperature differences. A change in −2.271 MPa
Ψπ = ∆T = −1.221∆T (8)
water potential of 1.0 MPa is reflected by a 1.86°C
change in wet-bulb temperature depression where T is the depression of the freezing
of only approximately 0.085°C. A hydro- point. The effect of osmotic potential on
meter is less affected by the changes in freezing-point depression also holds for
ambient temperature during the measure- non-ideal solutions such as plant saps.
ment compared with a psychrometer. However, freezing-point depression is non-
Psychrometric and hydrometric methods linear with concentration changes during
are suitable only for plant tissues of high dehydration. Water potential (MPa at 0°C)
water content. At low water content, the can be derived by the empirical equation
equilibrium may take several hours to (Crafts et al., 1949):
achieve. The nominal range of the Peltier
Ψ = 1.206T + 0.0021T 2 (9)
thermocouple measurement is limited from
0 to 6.0 MPa for these two methods. Yet With the osmometric method, a sample
many desiccation-tolerant plant tissues can is usually supercooled a few degrees below
survive far below 6.0 MPa. Even if one its freezing point to induce immediate
uses the Richards thermocouple, it extends crystallization. As the heat of fusion is
only to –25 MPa and the accuracy released, the sample temperature rises to
decreases to –0.1 MPa at –10 MPa its freezing point, and its equilibrium tem-
(Decagon Devices Inc., Pullman, perature is measured. Alternatively, the
Washington, USA), corresponding to an RH temperature at which ice crystals start
of ~ 84% at 25°C. melting can be measured and taken as the
equilibrium freezing temperature (i.e.
Ramsay’s method). The applicability of
2.3.2. Osmometric or cryoscopic method Equations (8) and (9) to the measurement
of water potential or osmotic potential in
A freezing-point osmometer measures the plant tissues was examined by Sun and
osmotic concentration of a biological liquid Gouk (1999), using seed tissues that were
using the principle of the freezing-point pre-equilibrated with saturated salt solu-
depression. The freezing-point depression tions (from 3 to 35 MPa). The freezing-
is one of the four colligative properties of a point depression was determined with a
solution. The freezing point is the unique differential scanning calorimeter, using the
temperature at which the ice phase and the onset temperature for the exotherm on
liquid phase can coexist in equilibrium at cooling. Calculated water potentials were
standard pressure. When a solute is dis- found to be very close to the pre-freezing
solved in the water, the freezing point of water potentials of seed tissues, with
the water is lowered in proportion to the Equation (9) fitting the data slightly better
osmotic potential of the solution. For a 1.0 than Equation (8).
2.3.3. Isothermal equilibration method cessfully in seed desiccation studies by a

number of workers (Pritchard, 1991; Poulsen
It is more difficult to measure directly and Eriksen, 1992; Vertucci et al., 1994; Sun
water potential of low-moisture systems. et al., 1997; Tompsett and Pritchard, 1998).
Perhaps the convenient, yet accurate and
reliable method is first to establish the
empirical relationship between water con- 2.4. Water Relations – the
tent and water potential for a particular Thermodynamic Approach
plant tissue. Samples of plant tissues are
equilibrated over different salt solutions 2.4.1. The Höfler diagram and the
that would maintain a series of constant pressure–volume curve
water vapour pressures (i.e. RH) in closed
containers. Upon equilibrium, the water Water relations parameters of plant tissues
contents of tissue samples are determined can be presented by the Höfler diagram and
gravimetrically, and their water potential at the pressure–volume curve (PV curve). The
equilibrium is then the same as the water Höfler diagram shows the relationship
potential of the air in the closed containers, between water potential and relative water
which in turn equals the osmotic potential content (Fig. 2.2a). The PV curve is a plot
of the salt solutions used. Therefore, water between the reciprocal of water potential
potential of tissue samples is calculated by (1/) and RWC or water loss (1 – RWC)
the equation: during desiccation (Fig. 2.2b). Both the
Höfler diagram and the PV curve are
= RT %RH
– ln (10) widely used to characterize water relations
Vw 100
of plant tissues. To construct a Höfler dia-
where R is the gas constant, T is the gram or a PV curve, the changes in water
–
absolute temperature (kelvin), Vw is the par- potential and RWC are monitored as the
tial molal volume of water, and %RH is the tissue is dehydrating. Several important
percentage relative humidity inside the parameters can be obtained by analysing
containers. (Note that Equation (10) is the components of cell water potential,
essentially the same as Equation (7).) The including the osmotic potentials at full tur-
empirical relationship between water con- gor and at the partially dehydrated state,
tent and water potential can be described the apoplastic and symplastic water vol-
by exponential and polynomial (Poulsen ume in tissues, a plot of turgor pressure
and Eriksen, 1992) or other functions (Sun (i.e. hydrostatic water potential in Equation
and Gouk, 1999). The derived mathematical (6)) as a function of RWC, and the tissue
expression is then used to calculate water bulk modulus of elasticity. Without know-
potential of plant tissues at any water con- ing these biophysical metrics, it would be
tent within the limit of experimental range. impossible to identify different kinds of
This method is particularly useful in moni- cellular stresses associated with the loss of
toring the change of tissue water potential water in the tissue and to examine the sig-
during desiccation. Water potential of dehy- nificance of an array of biochemical and
drating tissues can be calculated immedi- physiological responses during desicca-
ately from the data of water loss. tion. Moreover, valid comparisons of the
Constant RH can be achieved using satu- response of cell function to water stress
rated or non-saturated salt solutions, poly- among different organisms cannot be made
ethylene glycol solutions and glycerol without such knowledge.
solutions. Physico-chemical data of various
salts and their solutions are presented in the
2.4.1.1. Change of cell turgor pressure during
Appendix. This technique does not need spe-
desiccation
cial instruments to measure water potential,
and can avoid the difficulty in measuring RH In fully turgid cells, turgor pressure is equal
accurately. This method has been used suc- to the osmotic potential (with opposite
56 W.Q. Sun
(a) 2
p
0
Water potential (MPa)

–2 External
water
–4
–6
0.0 0.2 0.4 0.6 0.8 1.0 1.2
Relative water content (RWC)
(b)
p
Reciprocal of water potential
–1/ (MPa–1)
0
p
0.7 1.0 1.3
Incipient RWC
plasmolysis
(p = 0)
–1/
0.0
–0.2 0.0 0.2 0.4 0.6 0.8 1.0

1 – RWC
Fig. 2.2. Cellular water relations. (a) Höfler diagram showing the components of cell water potential.
Intercellular or external water (RWC > 1.0) in many plant tissues is held at near-zero water potential and,
during the initial dehydration, cell water potential () and turgor pressure (p) do not change significantly
(the horizontal dashed line). Maximum osmotic potential is found at the point of full turgor (RWC = 1.0),
where p = π. As the plant tissue loses water, turgor pressure decreases, and at the turgor-loss point
(RWC = ~0.8), = π (the vertical dashed line). At RWC < ~0.8, the relationship between RWC and π
follows a rectangular hyperbola (RWC = a + b/π). Osmotic potential at RWC = ~0.8–1.0 is extrapolated
from the rectangular hyperbola relationship. Turgor pressure is the difference between the measured water
potential and the extrapolated osmotic potential. (b) The pressure–volume curve showing the relationships
between , p and π during dehydration. The reciprocal of water potential is plotted against (1 RWC).
Beyond the turgor-loss point (incipient plasmolysis), the relationship between (1 RWC) and 1/ (or 1/π)
is linear. The extrapolation of this linear relationship toward the y-axis intercept gives osmotic potential (the
dashed line) of the tissue when the tissue is still turgid. The difference between the measured water potential
and the extrapolated osmotic potential is turgor pressure (inset).
signs). During dehydration, the PV curve of cation of negative turgor pressure. It is con-
a plant tissue initially displays a concave ceivable that the development of negative
region, beyond which the curve is linear turgor pressure may reduce mechanical
(Fig. 2.2b). The loss of turgor is marked by damage on cellular structures by preventing
the point at which the relation of 1/ to collapse of the cells.
(1 RWC) deviates away from linearity.
Turgor pressure (p) is calculated as the
2.4.1.2. Change of osmotic potential during
difference between the extrapolated linear
desiccation
portion of the PV curve and the water
potential actually measured, and is often When cell turgor pressure falls to zero dur-
plotted as a function of RWC. The relation- ing desiccation, the water potential of the
ship between turgor pressure and RWC can cell is equal to the osmotic potential (see
be described sufficiently by a quadratic or Equation (6)). As desiccation continues,
cubic function. osmotic potential and cell water potential
Certain plant tissues might develop nega- are equal and inversely proportional to the
tive turgor pressure before the cells collapse volume of osmotically active water. The rela-
and p can become zero under severe water tionship between RWC and the reciprocal of
stress. When negative p develops, the PV osmotic potential is a straight line. The
curve would fall below the extrapolated lin- osmotic potential at full turgor is calculated
ear portion of the graph (Fig. 2.3a). If the from the extrapolation of the linear portion
cells are sufficiently strong, do not collapse of the PV curve to the RWC at full turgor (i.e.
and the plasma membrane remains firmly RWC = 1.0 in Fig. 2.2b). In the Höfler dia-
attached to the cell wall, the formation of an gram, the relationship between osmotic
intracellular gas bubble will increasingly potential and RWC is represented by a rec-
become possible (cavitation). The develop- tangular hyperbolic function to the data
ment of negative turgor pressure and intra- points corresponding to the linear part of the
cellular cavitation appear to play some roles PV curve (dashed part of the π in Fig. 2.2a).
in desiccation tolerance of certain cells. A
good example of a cell surviving large nega-
2.4.1.3. The volume of water in symplast,
tive turgor pressure and cavitation is the
apoplast and intercellular spaces
ascospore of Sordaria (Milburn, 1970). The
volume of Sordaria ascospores changes very In hydrated plant tissues, water may exist
little, and the protoplast remains in contact in the symplast, in the apoplast (i.e. the
with the spore wall at all times. Under porous spaces in the cell wall) and in the
water stress (by air-drying or in osmotic intercellular spaces (large voids) as dis-
solution), these cells might generate nega- cussed before. Intercellular water, also
tive p as much as –4 MPa. Beyond this called ‘external’ cell water by some work-
negative turgor pressure, a small bubble ers, may account for up to 35% of total
appears inside the protoplasm suddenly, water in certain plant tissues, such as
which increases slowly in size and lichens, liverworts, mosses and fern fronds
approaches the walls quite closely without (Beckett, 1997; Proctor, 1999) and develop-
losing its spherical appearance. Honegger ing embryos of higher plants (W.Q. Sun,
(1995) and Scheidegger et al. (1995) also unpublished data). During desiccation,
showed that ascomycetous lichen myco- water potential and turgor potential do not
bionts form large intracellular gas bubbles fall with initial water loss at RWC > 1.0
when desiccated. More recently, the PV (Fig. 2.2a and b inset). The volume of water
analysis by Beckett (1997) suggested the that is lost before turgor pressure starts to
existence of negative turgor pressure in veg- fall is assumed to be intercellular water.
etative cells of several desiccation-tolerant The volume of symplastic water represents
(poikilohydric) plants (e.g. Dumortiera hir- the amount of osmotically active water in
suta and Myrothamnus flabellifolia). PV the tissue, and is obtained by subtracting
curves of most plants do not show any indi- the apoplastic water volume from the water
58 W.Q. Sun
(a)
1
p
2
0
Reciprocal of water potential
–1/ (MPa–1)
1 0.6 0.8 1.0

RWC
2
0.0
–0.2 0.0 0.2 0.4 0.6 0.8 1.0

1 – RWC
(b) 1000
100
Water potential (–MPa)
10
1.0
0.1
0.0 0.2 0.4 0.6 0.8 1.0

Relative water content (RWC)
Fig. 2.3. (a) The pressure–volume curves of plant tissues that develop negative turgor pressure (curve 1) and
intracellular cavitation (curve 2) during desiccation. The inset in (a) shows the change of cell turgor pressure
(p) during the early stage of drying. When intracellular cavitation occurs, the p suddenly changes to zero
(curve 2, inset), and is equal to π (curve 2). If intracellular cavitation does not occur, the cell wall will
collapse or deform when the p develops beyond the threshold to which the cell wall can resist (i.e. (1
RWC) > 0.15). The collapse or deformation of the cell wall will lead to a gradual increase in (curve 1)
and p (curve 1, inset). (b) The semi-logarithmic plot between RWC and tissue water potential. The high
RWC break point corresponds to the turgor-loss point, whereas the low RWC break point corresponds to the
volume of apoplastic water. Drawn with data from Quercus rubra seeds (Sun, 1999).
content at full turgor. Symplastic water Apoplastic or osmotically inactive water

generally declines over a range of water is present in very small pores and strong
potential from about 0.5 to 10 MPa, in water-binding sites of biological surfaces in
line with that of osmotic potential. plant tissues. This fraction of water is held
by matric and molecular forces, and lost tion of cellular membrane and molecular
only when plant tissues are desiccated to a assemblies. So far, workers have paid little
water potential less than 15 MPa attention to the location of water in plant
(Meidner and Sheriff, 1976). The loss of tissues. The difference in the relative vol-
apoplastic water in some species extends ume of external, symplastic, and apoplastic
to approximately 800 MPa. The amount water should be taken into account in the
of such matrix-bound water in plant tissues comparative studies on mechanisms of des-
can be as high as 0.1–0.2 RWC or up to iccation tolerance among cells, tissues or
0.25–0.35 g g1 dw. This fraction of water plants. A similar analysis of water relations
does not generally act as a solvent in cells, was found to be very useful in developing a
and is not readily freezable. From the mechanistic understanding of the role of
Höfler diagram, the apoplastic volume is dehydration in freezing tolerance in earth-
estimated from the fitted hyperbolic func- worms (Holmstrup and Zachariassen, 1996).
tion. From the PV curve, the volume of
apoplastic water is commonly estimated by
extrapolation of the linear relationship 2.4.1.4. Volumetric elasticity of the cell wall
between RWC and the reciprocal of The cell wall may undergo elastic expan-
osmotic potential to the (1 RWC) axis sion or contraction. Elastic (mechanical)
after the loss of turgor pressure. However, properties of cell walls play an important
the simple extrapolation from the PV curve role in cell water relations. For example,
is not a reliable method of estimating the the negative turgor pressure that can
apoplastic volume, and in some cases gives develop in a cell largely depends on the
negative values (Proctor et al., 1998). mechanical properties of the cell wall. The
The apoplastic volume of water should elasticity of the cell wall is represented by
be derived with data from the isothermal the volumetric elasticity module , where
sorption study at low water potentials depends on both p (turgor pressure) and
(water activity), rather than the extrapola- V (cell volume) and is defined as:
tion method, because the linear relation-
ship between RWC and the reciprocal of p
= V (11)
osmotic potential does not hold for the V
apoplastic volume of water (which is where V is volume change caused by a
osmotically inactive). Compared to the given pressure change p. Equation (11)
removal of osmotically active (symplastic) indicates that a high value of implies a
water, the measured osmotic potential rigid cell wall, whereas a low value implies
(including the term of matric potential) a more elastic cell wall. The value can be
declines much more rapidly when the calculated from the relationship between
apoplastic water is removed. Therefore, the p and RWC (Steudle et al., 1977;
volume of apoplastic water is marked by Stadelmann, 1984). The change of as a
the point at low water content at which the function of RWC is given by the first deriv-
relationship of 1/ to (1 – RWC) again ative of the quadratic or cubic function of
deviates away from linearity (Fig. 2.3b). turgor pressure on RWC. The value of the
The volume of apoplastic water roughly p/RWC derivative curve at RWC = 1.0 is
corresponds to the primary hydration in usually taken as the bulk modulus of elas-
tissues (including both strong and weak ticity and used for purposes of comparison.
water-binding sites). A pressure probe technique can be used
One can expect that plant tissues would directly to determine the turgor pressure
respond differently to the loss of external, and the for individual plant cells. This
symplastic and apoplastic water. The loss of technique is useful for continuous mea-
symplastic water can cause osmotic pertur- surement of cell turgor pressure, cell wall
bation of physiological and biochemical elasticity and hydraulic conductivity of the
processes, whereas the loss of apoplastic cell membrane in single cells (Hüsken et
water may disrupt the structure and func- al., 1978). The intracellular hydrostatic
60 W.Q. Sun
pressure is transmitted to the pressure rated salt solutions in closed desiccators

transducer via an oil-filled microcapillary until constant weights are achieved,
introduced into the cell, which transforms whereas an adsorption isotherm is devel-
into a proportional voltage. This technique oped by rehydrating dried tissues over satu-
permits volume changes and turgor pres- rated salt solutions. A desorption curve can
sure changes to be determined with an also be developed during drying of tissues
accuracy of 105–106 µl and 3–5 103 in any atmospheric condition by measur-
MPa, respectively. ing, at various points in time, the water
At present, very little information is content of the tissue and the equilibrium
available on cell wall properties of desicca- RH of its surrounding air in a closed con-
tion-tolerant plant tissues. Proctor (1999) tainer. Similarly, the dry tissue can be rehy-
found that two highly desiccation-tolerant drate with a given quantity of water to raise
liverworts have low values of bulk elastic the water content and equilibrium RH.
modulus. He thought that extensible cell Sophisticated instruments such as con-
walls might be a part of structural adapta- trolled atmosphere microbalance and
tion to rapid changes of cell volume in dynamic vapour sorption systems (Surface
their intermittently desiccated habitats. Measurement Systems, London, UK) use
Ultrastructural studies on dry mesophyll the latter methods. Desorption and adsorp-
cells of desiccation-tolerant Selaginella tion isotherms are used, respectively, to
lepidophylla by Thomson and Platt (1997) study the properties of dehydration and
showed highly folded cell walls and con- rehydration of plant tissues. Desorption and
tinuous apposition of plasmalemma to the adsorption curves are rarely the same: the
walls. Vicre et al. (1999) studied the cell desorption curve usually gives a higher
wall architecture of leaf tissues of water content than the adsorption isotherm.
Craterostigma wilmsii (a resurrection The difference in the equilibrium water
plant), and also observed extensive folding content between two curves is called hys-
of the cell wall during desiccation. The teresis. Hysteresis is evidence of the irre-
folding of the cell wall allows the plasma versibility of the sorption process, and
membrane to remain firmly attached to the therefore indicates the limited validity of
wall as the cell loses water. Biochemical the equilibrium thermodynamic approach
modifications of the cell wall were to investigate the dehydration–rehydration
observed during desiccation and rehydra- properties of plant tissues. Hysteresis might
tion, leading to the change in its tensile be an important issue when considering
strength that may prevent the total collapse critical water activities for desiccation
of the walls in the dry tissue and avoid stress during dehydration–rehydration
rapid expansion upon rehydration. The cycles and when investigating storage stabil-
change in cell wall elasticity during desic- ity after manipulation of moisture content of
cation can be determined easily by taking seeds and pollen.
the first derivative of the function of turgor
pressure on RWC.
2.4.2.1. Theoretical models
Plant tissues show a sigmoid sorption
2.4.2. Analysis of water sorption isotherms isotherm (Fig. 2.4a). The inflection point of
the isotherm is believed to indicate either a
The water sorption isotherm is the depen- change of water-binding capacity and/or
dence of water content on water activity of the relative amount of ‘bound’ or ‘free’
the surrounding environment at a given water. Water sorption data are normally
temperature. There are two types of sorp- analysed using theoretical models, from
tion isotherms: desorption isotherm and which useful biophysical parameters of
adsorption isotherm (Fig. 2.4a). water relations are derived. Commonly
Conventionally, a desorption isotherm is used models include the Brunauer–
developed by drying fresh tissues over satu- Emmett–Teller (BET) model, the
Guggenheim–Anderson–de Boer (GAB) aw C − 1  1

model and the D’Arcy–Watt model. =  aw + (12)
M w (1 − aw )  M m  M mC
The BET model (Brunauer et al., 1938)
is derived from statistical and thermody- where aw is the water activity, Mw is equi-
namic considerations. The equation can be librium water content in the tissue, Mm is
written as: the BET monolayer (water content corre-
(a)
I II III
Water content
Desorption
Adsorption
Water activity
(b)
III
II
I
Enthalpy
Sorption enthalpy
Free energy
Entropy
Water content
Fig. 2.4. The analysis of water sorption isotherms. (a) The typical shape of desorption curves and adsorption
curves of plant tissues. The difference between these two curves shows hysteresis, which indicates the
irreversibility of water sorption in the tissues during dehydration and rehydration. The sigmoid shape of
sorption curves is presumably due to the existence of three types of water-binding sites in tissues (strong (I),
weak (II) and multilayer molecular sorption sites (III)). (b) Differential enthalpy (H), free energy (G) and
entropy (S) of hydration. Desorption curves can be used to calculate H and S of tissue hydration. See
text for detailed discussion.
62 W.Q. Sun
sponding to the monolayer hydration) and amount of water in those three regions can
C is temperature dependence for sorption be estimated. K is the number of strong
excess enthalpy (Brunauer et al., 1938, water-binding sites, multiplied by the mole-
1940). BET equation parameters, Mm and C, cular weight of water and divided by
can be calculated by plotting aw/[Mw (1 Avogadro’s number (6.023 1023); K is the
aw)] against aw. The y-axis intercept of the strength of the attraction of the strong water-
straight line is equal to 1/(MmC) and the binding sites for water; c is a measure (lin-
slope is equal to (C 1)/Mm. The BET is ear approximation) of the affinity and the
valid only for aw < 0.5, thus data points number of weak water-binding sites; k
within that range are used to estimate the relates to the number of multimolecular
monolayer value (Mm). The BET model is water sorption sites; and k relates to the
an effective method for estimating the activity of water (D’Arcy and Watt, 1970).
amount of water bound specifically to The number of water-binding sites in tissues
polar sites (monolayer), but cannot be used can be calculated from the derived equation
to give a complete estimation of specific coefficients. The number of strong, weak
hydration parameters. and multimolecular water-binding sites are
The GAB model is an extension of the KN/M, cN/(Mo), and kN/M, respectively,
BET model, taking into consideration the where N is Avogadro’s number, M is the
modified properties of the sorbing materi- molecular weight of water and o is the satu-
als in the multilayer region and the bulk rated vapour pressure of pure water.
liquid properties through the introduction The D’Arcy–Watt model has been used
of a third constant, K. The GAB equation is extensively for the analysis of desiccation-
written as: tolerant and desiccation-intolerant plant
tissues (Vertucci and Leopold, 1986, 1987a,
MmCKaw
Mw = (13) b; Sun et al., 1997). Both the GAB and the
(1 Kaw)(1 Kaw + CKaw) D’Arcy–Watt models are valid over a wider
range of water activities for plant tissues.
where C and K are temperature-dependent The GAB model has some advantages over
coefficients. Constants, Mm, C and K are the D’Arcy–Watt model, which assumes the
estimated via the curve fitting of sorption three types of water-binding sites. The GAB
data. In the field of food sciences, the GAB model does not have such an assumption.
model is the most widely accepted due to For biological systems it is more reasonable
its accuracy and its validity over a wide to assume that the number of water-binding
range of water activities from 0.05 to 0.9 sites is changing continually along with the
(Rahman and Labuza, 1999). binding energies. Moreover, the GAB model
The D’Arcy–Watt model was developed can be more easily applied to other thermal
for the analysis of sorption isotherms of analyses (e.g. water-clustering function).
non-homogeneous materials (D’Arcy and
Watt, 1970). This model assumes that there
is a fixed number of water-binding sites 2.4.2.2. Temperature dependency of water
with different discrete binding energies. The sorption
D’Arcy–Watt equation can be written as: Desiccation involves the transfer of liquid
K Kaw kkaw
water in plant tissues into the vapour
Mw = + caw + (14) phase. Temperature influences evaporation
1 + Kaw 1 − κaw
rate through the heat supply as well as
where K, K, c, k and k are equation coeffi- through its effect on the partial water
cients (adjustable parameters). The equation vapour pressure in air and the energy sta-
has three terms, which represent the tus of water in plant tissues. In isothermal
amounts of water that are strongly bound, conditions, air acts as an osmotic mem-
weakly bound and sorbed in multimolecular brane and equilibrium is often slow and
water clusters, respectively. For a tissue that dependent on temperature. An increase in
is in equilibrium with a given aw, the temperature generally results in a decrease
in equilibrium water content of plant tis- event. A high negative H value at low
sues at a given RH (i.e. water activity) or an water content suggests the strong affinity of
increase in equilibrium water activity at adjacent water molecules toward ionic sites
constant tissue water content. The shift of and/or other polar sites of the substrate. As
water activity at the constant water content water content increases, the H becomes
by temperature is mainly due to the change less negative (Fig. 2.4b). The primary hydra-
in water binding, dissociation of water, tion process (i.e. strong and weak binding
physical state of water or increase in solu- sites) is considered to be completed when
bility of solute in water. Tensile strength of the differential enthalpy of hydration (H)
water, the pressure holding molecules approaches zero (Luscher-Mattli and Ruegg,
together, increases by 81.6 mbars on aver- 1982; Rupley et al., 1983; Bruni and
age for a reduction of 1°C. Temperature Leopold, 1991). The change of S reflects
dependence of isotherm shift is described the relative degree of order, and the S peak
by the Clausius–Clapeyron equation: is presumably associated with the saturation
1
of all primary hydration sites. It should be
aw2 q +
w 1
ln = =  −  (15) clearly noted that the relationships of
aw1 R T
 2 T1
H/WC, G/WC and S/WC describe ther-
where q is the excess heat of sorption;
w is modynamic interactions between water and
the latent heat of vaporization for water biomaterials, but not necessarily the func-
(44.0 kJ kg1 at 25°C); R is the gas constant; tions of water and biological structures in
aw1 and aw2 are water activities for a given physiological processes. A possible associa-
equilibrium water content at temperature tion between water sorption behaviours and
T1 and T2, respectively. The plot of ln aw desiccation tolerance of plant tissues was
against 1/T at any given tissue water con- discussed in a number of studies (Vertucci
tent is a straight line and its slope gives (q and Leopold, 1987b; Farrant et al., 1988;

w)/R, from which the excess heat of Pritchard, 1991; Sakurai et al., 1995; Eira et
sorption, q, can be derived (Fig. 2.5a). al., 1999; Sun, 2000). No consistent differ-
In practice, some thermodynamic quan- ence in water sorption characteristics has
tities of tissue hydration can be calculated been found between desiccation-sensitive
according to isotherms at two different (recalcitrant) and desiccation-tolerant
temperatures. The aw1 and aw2 for a given (orthodox) seed tissues (Sun, 2000).
equilibrium water content at two tempera- The van’t Hoff relationship provides
tures can be taken from water sorption another convenient means to analyse tem-
curves or calculated from fitted sorption perature dependence of sorption isotherm.
equations (Fig. 2.5b and Fig. 2.7a). The van’t Hoff equation and the
Differential enthalpy of hydration (H, Clausius–Clapeyron equation are essen-
including q and
w), differential free tially the same in theory, but different in
energy of hydration (G) and differential their mathematical treatment of experimen-
entropy of hydration (S) are given by: tal data. The Clausius–Clapeyron equation
handles two temperature points, whereas

RT1T2 aw1
H ln (16) the van’t Hoff equation can handle a series
T2 T1 aw2
of temperature points at once. The van’t
G RT ln (aw) (17) Hoff equation expresses the relationship of
the equilibrium water activity (aw) for a
S H G (18)
T given water content against the tempera-
ture (1/T) (Fig. 2.5b), and is written in its
These thermodynamic quantities are the differential mathematical form as:
functions of water content in tissues. The
H
relationships of H/WC, G/WC and ∂ ln aw R ∂(1/T) (19)
S/WC provide important information with
regard to the hydration properties of tissues where T is absolute temperature in kelvin,
(Fig. 2.4b). Water sorption is an exothermic and R is the gas constant. The H is the
64 W.Q. Sun
differential enthalpy of water sorption. It is require the storage of desiccation-sensitive

important to note that the relationship seeds and other tissues in a refrigerated
between ln(aw) and (1/T) is not necessarily condition or at liquid nitrogen tempera-
a straight line. Within a relatively narrow ture. When the extrapolation is used, the
range of temperature, linear approximation non-linear nature of the relationship
may be used to calculate H accurately. between ln(aw) and (1/T) needs to be taken
However, there is considerable interest in into consideration. The study on tempera-
studying water sorption properties of bio- ture dependence of water sorption using
logical tissues at a much wider range of the van’t Hoff equation (Fig. 2.5a and b) is
temperature. For example, long-term used to establish the theoretic framework
preservation of genetic resources may for the optimization of germplasm preser-
(a) 0.0
0.09 g g–1 dw
–1.0
ln (aw or RH/100)
–2.0 0.04 g g–1 dw
–3.0
–4.0 0.02 g g–1 dw
3.3 3.4 3.5 3.6

1/Temperature (K, 10–3)
(b)
0.14
Water content (g g–1 dw)
0.12
0.10 50% RH
0.08
0.06
0.04
10% RH
0.02
0 5 10 15 20 25
Temperature ( C)
Fig. 2.5. (a) Temperature dependence of water sorption for the same seed material at different water
contents (i.e. the van’t Hoff plot). Drawn with data from Eira et al. (1999). (b) Equilibrium water content at
specific water activities as a function of temperature for whole seeds of Coffea arabica cv. Mundo Nova.
This relationship is called ‘isopleth’.
vation protocols (Vertucci et al., 1994, polar hydration sites. A recent study using
1995; Eira et al., 1999). the D’Arcy–Watt model suggested that
water redistribution among different types
of hydration sites might be related to the
2.4.2.3. Monolayer hydration and water-
rapid loss of seed viability during storage
clustering function
after osmotic priming and drying back (Sun
The monolayer hydration values of plant et al., 1997).
tissues, the amount of water bound to spe- Water clustering in binding sites is
cific polar sites, can be easily determined, another important hydration event that is
using BET or GAB isotherm models. For of significance to desiccation tolerance of
most plant tissues and their major chemi- plant tissues and the survival of tissue in
cal components, the monolayer value at the dried state. Clustering formation is
ambient temperature is estimated to be related to a number of transport phenom-
between 0.04 and 0.09 g g1 dw using the ena. For example, clustering reduces the
BET or GAB model (Rahman and Labuza, effective mobility of water by increasing
1999). The BET monolayer value of many the size of the diffusing molecular group or
orthodox seeds was also found to be in this by increasing the tortuosity of diffusion
range (Vertucci and Leopold, 1987a,b; paths (Stannett et al., 1982). The range of
Bruni and Leopold, 1991; Vertucci and water activity where the self-association of
Roos, 1993; Sun et al., 1997). The mono- water takes place can be examined by the
layer value of Typha pollen was much less clustering function (Lugue et al., 1995;
than that of orthodox seeds (Buitink et al., Dominguez and Heredia, 1999). The clus-
1998b). The monolayer hydration is gener- tering function is written as:
ally complete at a water activity of
G11/V1 V2[(aw/V1)/aw] 1 (20)
0.20–0.30 (i.e. 150 to 250 MPa). It is
important to note that the monolayer value where G11/V1 is the clustering function, V1
decreases rapidly as temperature increases, is the volume fraction of water, V2 is the
and increases as temperature declines. The volume fraction of biopolymers, and aw is
monolayer water is of great importance for water activity (Zimm and Lundberg, 1956).
the survival of many dry organisms (e.g. The subscript ‘11’ in G11/V1 denotes the
spores, pollen grains and seeds) during water–water interaction as a function of
storage. In food science, the water activity water content (component 1). The cluster-
at the monolayer value is defined as the ing function can be applied to an isotherm
critical water activity. At a water activity sorption model such as the GAB equation
above 0.20–0.30, the rate of chemical reac- with some modifications. The GAB equa-
tions begins to increase significantly tion needs to be rewritten in terms of vol-
because of the greater solubility and mobil- ume fraction instead of weight fraction.
ity of the reactants. At water contents The GAB equation can be rewritten as:
below the monolayer value, the rate of MmCKaw
lipid oxidation and associated free radical MwV1p1/V2p2 (21)
(1Kaw)(1KawCKaw)
damage increases. The presence of mono-
layer water inhibits the undesirable inter-
or
actions between polar groups on adjacent
carbohydrate or protein molecules, thereby (1Kaw)(1KawCKaw)
preserving their rehydration ability and aw/V1 (22)
MmCKp2V2
biological functions (Rahman and Labuza,
1999). where p1 and p2 are the density of water
There is no defined monolayer parame- and biopolymers. The density of sorbed
ter in the D’Arcy–Watt model. However, water is assumed to be equal to 1.0 g cm3.
the first term of the D’Arcy–Watt equation Substituting aw/V1 in Equation (20) with
may be used as an approximation, as it rep- Equation (22), the clustering function can
resents water that is bound strongly to be expressed as:
66 W.Q. Sun
(2KCKMmCK)(2CK22K2)aw expressed as the percentage of the corre-

G11/V1 (23)
MmCKp2 sponding maximum value in the fully
hydrated tissues (Fig. 2.7b). Therefore, the
According to Equation (23), G11/V1 is pro-
occupancy relationship indicates the
portionally related to aw and the reciprocal
degree of hydration for different types of
of polymer density (p2) function. G11/V1
hydration sites during desiccation. Figure
can be solved easily by the substitution of
2.7b shows that the occupancy for three
Mm, C and K constants from the GAB equa-
types of hydration sites changes as the
tion. Figure 2.6 shows a plot of the water-
water content of Q. rubra seed tissues
clustering function of soybean axes. The
decreases during desiccation. Desiccation
clustering plot is basically a straight line
of seed tissues to 0.30 g g1 dw (the critical
against water activity. When G11/V1 is
water content) removed about 90% of mul-
greater than 1, water is expected to clus-
tilayer molecular sorption water, but only
ter (Zimm and Lundberg, 1956). The auto-
about 10% of water molecules attached to
association (clustering) of water in a few
the weak hydration sites in seed tissues.
desiccation-tolerant seeds is observed to
The removal of water from weak hydration
occur at water activity ranging from 0.55 to
sites appears to be related to desiccation
0.60 (W.Q. Sun, unpublished data).
damage in Q. rubra seeds (Sun, 1999). The
critical water content of Q. robur axes also
2.4.2.4. Occupancy of water-binding sites corresponds to the amount of matrix-bound
water (Pritchard and Manger, 1998).
The D’Arcy–Watt model can be used to However, the question of whether the
examine the occupancy of water-binding water-binding or sorption behaviour in
sites as a function of water content accord- seed tissues is related to their desiccation
ing to Luscher-Mattli and Ruegg (1982). tolerance remains unresolved. The loss of
The occupancy represents the amount of viability in many recalcitrant seeds occurs
water attached to certain hydration sites, at a water content that is much higher than
4
Clustering function
–4
–8
–12
0.0 0.2 0.4 0.6 0.8 1.0

Water activity
Fig. 2.6. Water-clustering function showing the waterwater association in soybean seed axes as a function
of equilibrium water activity. Apparent water clusters first appear at a water activity of 0.58 (arrow). The
water-clustering function Equation (23) was solved through the study on biopolymer volumetric change
during hydration [i.e. P2= f (V1)] by applying water sorption analysis. See text for further explanation.
that of ‘bound’ water (Pammenter et al., recently released manual by Bell and
1991; Berjak et al., 1992). Clearly, more Labuza (2000). This book generally dis-
comprehensive studies are needed. cusses water activity in food materials, but
Readers who wish to know more about the principles are also applicable to plant
water sorption analysis may refer to a desiccation tolerance studies. Practical
(a) 0.8
5 C
4.974 / 0.0219 /
0.6 WC = + 0.0673 / +
1 + 90.2 / 1 – 0.990 /
25 C
1.279 / 0.026 /
0.4 WC = + 0.0373 / +
1 + 27.4 / 1 – 0.986 /
0.2
0.0
0.0 0.2 0.4 0.6 0.8 1.0

Water activity
(b)
100
80 5 C
Sorption sites occupied (%)
25 C
60
Strong binding site
40 Weak binding site
Multilayer sorption
20
0
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7
Fig. 2.7. (a) The interpretation of desorption isotherms of Quercus rubra cotyledonary tissues, using the
D’Arcy–Watt model. Equation coefficients are derived though curve-fitting of experimental data ( / o = aw).
See text for further explanation. (b) The occupancy for three types of hydration sites in Q. rubra
cotyledonary tissues at different water contents. The occupancy is based on the percentage of the
corresponding maximum values in the fully hydrated state (i.e. full turgor). The change of occupancy reveals
how and when water is removed in different types of hydration sites during dehydration.
68 W.Q. Sun
examples are provided to elucidate how to curves are biphasic. During the first drying
solve many equations. phase, the loss of water follows a simple
exponential function. During the second
phase, water content does not decrease
2.5. Measurement of Drying Rate and much because the tissue is very much
Desiccation Stress closer to achieving equilibrium with the
air. Because water loss during the first
2.5.1. Driving force for water loss and phase is described by an exponential func-
expression of drying rate tion, the rate constant () of water loss can
be used as an expression of drying rate.
The loss of water from tissues depends on
two factors: the gradient in water potential
between tissue surface and external air or 2.5.2. Quantification of desiccation stress
solution, and the hydraulic conductivity of
the tissue. The volume flow of water from The response of plant tissues to desiccation
the tissue to air can be described by: is significantly affected by dehydration con-
ditions, such as drying rate (see Chapter 3).
Vw ALp (o i) (24)
Under slow-drying conditions, plant tissues
where Vw is the volume flow of water per stay longer at intermediate water contents.
unit time (m3 s1), A is the surface area of Fast drying is often reported to improve
the tissue (m2) and Lp is the hydraulic con- desiccation tolerance of recalcitrant plant
ductivity of the tissue (m s1 Pa1). The o seeds (reviewed by Pammenter and Berjak,
and i are water potentials of external air 1999). There is no doubt that the level of
and the tissue, respectively. The difference desiccation stress would vary with drying
in water potential (o i) is a measure of rate, and the questions are: (i) how desicca-
the driving force for dehydration. i is a tion stress can be quantified; and (ii) how
function of time that describes the decrease drying rate affects the level of desiccation
in tissue water potential during drying. The stress. The change in chemical potential of
hydraulic conductivity of the tissue (Lp) is a cellular water is a good measure for the
measure of the diffusional resistance of the degree of desiccation stress. When the
water transport pathway within the tissue. chemical potential of water is compared,
A good measure for the drying rate is *w and mwgh in Equation (5) cancel out.
essential for comparative studies on desic- The difference in chemical potential of cel-
cation tolerance. According to Equation lular water between the dehydrated state
(24), the rate of water loss from the tissue is (Dw) and the hydrated state (Hw) is:
time-dependent. Under constant RH and –
HwDwRT(ln aHwln aDw)Vw(PHwPDw) (26)
temperature, the water content of the tissue
is expected to decrease exponentially over According to Equation (26), the degree of
time until i reaches o (Fig. 2.8a). The desiccation stress is proportional to
curve of water loss can be described by: changes in osmotic potential and hydrosta-
tic pressure (P) in cells. Therefore, the
WC = exp(t) (25)
change of water potential, d/dt, can be
where is the initial water content, is the used to quantify the level of desiccation
rate constant of water loss, and t is time of stress. Figure 2.8b shows the plots of tissue
drying. This relationship was first used by water potential against drying time. Under
Tompsett and Pritchard (1998) to compare the conditions of constant temperature and
the dehydration rate of A. hippocastanum relative humidity, such plots are straight
seeds. Drying curves of other seed tissues lines down to the fraction of apoplastic
have been examined under a wide range of water. Water potential of the tissue
desiccation conditions, and they conform decreases faster and deviates away from
to Equation (25) (Li and Sun, 1999; Liang the straight line when the apoplastic water
and Sun, 2000). Typically, water content is lost (see Fig. 2.3b, the low-RWC break
point). The slope of each straight line por- mathematical evaluation of this linear rela-
tion (d/dt) represents the degree of direct tionship will not be presented here.
physical stress under different desiccation If the plant tissue is viewed as a viscoelas-
conditions. The relationship between tic system, the mechanical stress caused by
d/dt and the rate constant () of water water loss can be considered as a simple
loss (drying rate) is linear (Fig. 2.9). A stress–strain response. The physico-chemical
(a)
3.2
33% RH
88% RH
2.4
94% RH
1.6
0.8 = 0.00502
= 0.0211
0.0 = 0.103
0 80 160 240 320 400

Drying time (h)
(b)
0
33% RH
88% RH
–5 94% RH
–10
d/dt = –0.032
–15
d/dt = –0.160
d/dt = –0.689
–20
0 80 160 240 320 400

Drying time (h)
Fig. 2.8. Measurement of drying rate and quantification of desiccation stress for Theobroma cacao axes. (a)
Drying curves of isolated axes in three constant relative humidities (RHs). The data are fitted with
exponential functions (WC = exp(t)), and the rate constants of water loss, , are shown near each
curve. (b) Plots of tissue water potential against drying time. The mechanical stress on tissue caused by the
water loss can be considered as a simple stress–strain response. The slope of /time plot, d/dt, is directly
related to the intensity of desiccation stress. Data from Liang and Sun (2000).
70 W.Q. Sun
1.6
1.2
Dehydration rate (–d/dt)
0.8
0.4
0.0
0.00 0.06 0.12 0.18 0.24 0.30

Rate constant of water loss ()
Fig. 2.9. The relationship between the rate constant of water loss () in Equation (25) and dehydration rate
(d/dt) for Theobroma cacao (cocoa) axes. Isolated axes were dehydrated at 16°C under constant relative
humidities ranging from 6 to 94% to achieve different drying rates and stress conditions. Drawn using data
from Liang and Sun (2000).
aspect of desiccation stress can be assessed by treats biological systems as fully reversible
integrating the function of tissue water poten- ones and does not give much considera-
tial over time (Fig. 2.8b). Figure 2.10a and b tion to the term time, one of the most
shows an example of the quantitative analysis important factors in any biological
of desiccation stress. The cumulative water response. This limitation is particularly
stress during desiccation at three different RHs relevant to the study of desiccation. The
was plotted against drying time and water application of thermodynamics is gener-
content, respectively. Under the slow-drying ally sufficient in many cases for fully
condition (94% RH), the mechanical stress hydrated tissues. However, at intermediate
(d/dt) is small (Fig. 2.8b); however, the or low moisture levels, the non-equilib-
cumulative physico-chemical stress is remark- rium, kinetic principles play a more
ably high because the time to dry to the same important role. During desiccation, the
water content increases exponentially as the biological system basically shifts from a
drying rate decreases (Fig. 2.10a and b). The thermodynamic state to a non-equilibrium
quantitative analysis of mechanical and kinetic state (Leopold et al., 1994; Sun et
physico-chemical aspects of desiccation stress al., 1994; Sun, 1997, 1998). The thermo-
has led to an understanding of the physiologi- dynamic approach does not sufficiently
cal basis of the optimal drying rate to achieve address the kinetics of various reactions
the maximum desiccation tolerance of and processes in intermediate- to low-
Theobroma cacao axes (Liang and Sun, 2000). moisture systems.
The kinetic and functional approach to
cellular water relations focuses on how the
2.6. Water Relations – the Kinetic and interactions between water and other cellu-
Functional Approach lar components can influence the struc-
tures and biological properties of each
The thermodynamic approach to water other. In this chapter, principles of the
relations has its limitations, because it kinetic approach and the interactions
(a)
2000
Cumulative water stress (MPa h)

1500
1000
33% RH
500 88% RH
94% RH
0 80 160 240 320 400

Drying time (h)
(b)
2000
33% RH
Cumulative water stress (MPa h)
88% RH
1500
94% RH
1000
500
0.0 0.6 1.2 1.8 2.4 3.0

Fig. 2.10. (a) Cumulative water stress during desiccation as the function of drying time. The cumulative water
stress is calculated by integrating the /time function (see text for details). (b) Cumulative water stress as the
function of tissue water content under different desiccation conditions. Cumulative stress is much higher in
slow-drying conditions because the dehydration time increases exponentially as drying rate decreases.
between water and many other biomole- Instead, a general account will be offered,
cules will not be discussed in detail. These so that readers can be confident about
topics will be covered in other chapters on choosing the appropriate method to quan-
desiccation damage and mechanisms of tify particular water properties in studies
desiccation tolerance (see Chapters 9–12). on desiccation tolerance.
72 W.Q. Sun
2.6.1. General considerations the study on water in skeletal muscle as an

example, fast techniques (e.g. laser Raman
2.6.1.1. Time scale spectroscopy, infrared spectroscopy and
dielectric relaxation) could not find any
Any study on the state of water by a bio- intramolecular differences in hydrogen
physical technique involves measuring bond lengths, angles or strength between
parameters of time scale directly or indi- muscle water and pure water (Beall, 1981).
rectly. Biophysical techniques often use the However, slower techniques, such as
diffusional correlation time as the time nuclear magnetic resonance (NMR) (Fung
scale to make comparisons of the measure- and McGaughy, 1974), electron paramag-
ments on water. Many of the physical prop- netic resonance (EPR) (Belagyi, 1975) (see
erties of water are theoretically related to Chapter 4), fluorescence polarization
the diffusional correlation time (D) of (Knight and Wiggins, 1979) and freezing
water, the average time between jumps in behaviour (Rustgi et al., 1978), showed a
position for water molecules in the system. restricted motion of at least a portion of cell
Two critical numbers on this time scale (D) water. This situation is identical to pho-
are 105 s and 1011 s. The D of water in ice tographing moving objects with different
is 105 s and in pure liquid is 1011 s, one shutter speeds. If the shutter speed is very
million times faster. Under normal condi- high relative to the velocities of two moving
tions, water in biological systems exists in a objects (e.g. 1/800 s), the photo will proba-
state somewhere between the solid state of bly not record any information as to
crystalline ice and the liquid hydrogen- whether one object is moving faster than
bonded lattice of pure water. In low- the other. On the other hand, if the shutter
moisture systems, however, the D of water speed is too slow (e.g. 1/2 s), the images of
molecules in the intracellular glasses would both moving objects will be blurred and no
be much slower than 105 s. Readers may meaningful information can be obtained
refer to a series of studies on molecular from such a photo. Only with a proper
mobility in seeds and pollen by Buitink et shutter speed can the photo reflect the dif-
al. (1998a, 1999, 2000a,b,c; Chapter 10 of ference in the velocity between the two
this volume). moving objects.
The ability of a biophysical technique to
yield useful information about the physical
2.6.1.2. Structural complexity and dynamics
state of water largely depends on how fast a
of molecular ordering
measurement can be made. Slow tech-
niques which require measurement times Hydration of protein is a good example,
greater than the diffusional correlation time illustrating the complexity of structures
yield an average over all molecules in the and functions for water in biological sys-
population with a kinetic contribution from tems. When a mole of lysozyme is hydrated
diffusion, whereas fast techniques can yield by 60 moles of water (~ 0.07 g g1 dry pro-
instantaneous information about intramole- tein), water is primarily located to charged
cular factors such as H–O bond lengths and groups, and its mobility is reduced at least
hydrogen bond angles (geometric factors). by 100 times relative to pure water. At this
To choose the appropriate technique, one low hydration level, no structural differ-
has to bear in mind that the type of prop- ence is observed in the protein. As hydra-
erty or structure that a technique can probe tion increases to 220 moles of water per
is related to the time scale. Generally speak- mole of protein (~ 0.25 g g1 dry protein),
ing, fast techniques would probably pro- water begins to form clusters of various
duce data of instantaneous structures of sizes and arrangements around the charged
water and other biomolecules, but slow and polar sites of the protein. Internal pro-
techniques would provide more informa- tein motion (H exchange) increases by 1000
tion about the interactions between water times to be comparable to that of solution,
molecules and their environment. Taking while the protein sample is still a solid. At
a hydration level of 300 moles of water per tions that a model allows, have to be taken
mole of protein (~ 0.38 g g1 dry protein), into consideration for experimental design
enzymatic activity, mobility of bound lig- and implementation. If possible, additional
and and fast water motion become easily experiments should be conducted to con-
detectable. Dielectric relaxation measure- firm the results and to examine whether the
ments show two water relaxation times, assumptions are satisfied. Unfortunately,
one of 2 1011 s, close to that of bulky biophysical models or equations have fre-
pure water, and the other of 109 s, indicat- quently been used to analyse the actual
ing the heterogeneous nature of water data without checking their assumptions.
behaviours and functions (Rupley et al., Worse still, sometimes a model was
1983). At the hydration level of 0.38 g g1 selected after the entire experiment was
dry protein, each water molecule covers, on completed (Beall, 1983).
average, 20 Å2 of protein surface, which is Conceptual models have been used for
twice the effective area of a water molecule. the interpretation of the data on water in
Yet several populations of water molecules biological systems, with many pitfalls. For
are observed at such low hydration. example, the ‘two-fraction fast-exchange
The surfaces of membranes, proteins model’ (Zimmermann and Brittin, 1957)
and other macromolecules impose geomet- assumes that there is a small fraction of
ric limitations on the possible arrange- highly immobilized cell water on the sur-
ments of hydration water. Interfacial water face of macromolecules (ice-like) and a
molecules, being part of the network of bio- large fraction of cell water that behaves like
logical interfaces, are dynamically oriented bulk water. Rapid exchange between the
and exhibit restricted motion (i.e. are two populations yields reduced average
‘bound’). The ordering of molecules on var- properties. This two-fraction model can be
ious biological surfaces is strictly local, written as:
and may fluctuate rapidly between possible 1 X (1 X)
arrangements. Ideally, a biophysical tech- (27)
T * Tslow TH2O
nique used to study the state of water in
biological systems should have the resolu- where T * is measured (average) relaxation
tion to differentiate closely related struc- time; X and (1X) are the fraction of
tures or populations. However, as discussed immobilized water and the fraction of cell
earlier, kinetic measurements reflect only water that is like bulk water, respectively;
average or time-average properties over all Tslow and TH2O are the relaxation times of
molecules in the system and, in many the slow fraction and bulk water. In this
cases, do not provide definitive answers to equation, there is only one measured para-
the questions of interest. To interpret the meter (T *), but three unknown quantities
data of kinetic measurements, the investiga- (X, Tslow and TH2O). To estimate X, Tslow
tor must impose a conceptual model, which and TH2O must be arbitrarily assigned. This
may be controversial (Beall, 1983). Such model represents a simplistic view on the
studies are often misunderstood and misin- dynamics of water in biological systems,
terpreted by readers who are less familiar which is still in use by some workers. If
with the biophysical techniques used. one intends to solve Tslow, then X must be
estimated through other methods. When X
is equated to the ‘non-freezable fraction’ or
2.6.1.3. The model-dependent interpretation:
‘osmotically inactive fraction’, additional
the pitfalls
assumptions are made. By redefining X as
The selection of a theoretic model or the an adjustable parameter in different sys-
development of a new model is an impor- tems, a new model is established (Beall,
tant step in any kinetic study, which 1983). This example clearly shows the
should be done before actual measure- uncertainty of biophysical interpretation.
ments are made. The assumptions that a Simply because the model is easy to use
model contains, and the specific predic- and fits the data well it does not necessarily
74 W.Q. Sun
mean that it represents the true state of measurements is briefly summarized in

water in a system. Of course, all models are Table 2.1. Readers are advised to consult
open to interpretation. other references, including those cited
However, it is possible to measure above, and Chapter 4 in this volume. In this
changes in the properties of water in living chapter, only a brief introduction will be
systems that correlate with physiological provided on several techniques that have
functions (Clegg et al., 1982; Clegg, 1986; been increasingly used in recent years.
Bruni et al., 1989). Changes in dynamic
properties of water at different hydration
2.6.2.1. Differential scanning calorimetry
levels indicate the existence of different
fractions of water, which may vary in struc- Differential scanning calorimetry (DSC) is
ture and property and presumably play dif- probably the most commonly used thermal
ferent biological roles. Studies have analysis technique. It has been used by a
identified the existence of at least four or number of workers to study the possible
five fractions of water, presumably relating relationship between freezing, desiccation
to different interactions between water and tolerance and water properties in plant tis-
cellular constituents (Clegg, 1986; sues (Williams and Leopold, 1989;
Ratkovic, 1987; Vertucci, 1990; Pissis et al., Vertucci, 1990; Pammenter et al., 1991;
1996; Sun, 2000). Hydration levels corre- Berjak et al., 1992; Sun et al., 1994; Vertucci
sponding to these fractions of cellular et al., 1994, 1995; Buitink et al., 1998b;
water are associated with the onset of vari- Pritchard and Manger, 1998; Sun and
ous metabolic activities in organisms Davidson, 1998; Sun, 1999). DSC measures
(Clegg, 1986). the heat flow of plant tissues associated
with various thermal events during cooling
and/or heating scans. Such thermal events
2.6.2. Biophysical techniques include phase transitions (e.g. freezing,
(see also Chapter 4) melting, glass transition, etc.), polymor-
phism, thermochemistry and the kinetics
Kinetic properties and functions of water for a variety of complex reactions (e.g. in
have been studied, using calorimetry vivo protein denaturation). The key idea
(Ruegg et al., 1975; Bakradze and Balla, involved in DSC measurement of water sta-
1983; Vertucci, 1990; Sun, 1999), infrared tus is that thermal changes of water and
(IR) and Raman spectroscopy (Careri et al., their corresponding quantities of energy
1979; Cameron et al., 1988), NMR spec- are greatly affected by the presence of other
troscopy (Fung and McGaughy, 1974; biomaterials in plant tissues, and that ther-
Mathur-de Vre, 1979; Seewaldt et al., 1981; mal behaviours of other biomaterials in
Rorschach and Hazlewood, 1986; Ratkovic, plant tissues are affected by water content.
1987), quasi-elastic neutron-scattering For example, as water content decreases,
spectroscopy (Lehmann, 1984; Trantham et the onset freezing and melting temperature
al., 1984) and dielectric relaxation tech- of water decreases due to solute concentra-
niques (Harvey and Hoekstra, 1972; tion, while at the same time glass transition
Kamiyoshi and Kudo, 1978; Clegg et al., temperature of the tissue increases due to
1982; Pissis et al., 1987, 1996; Bruni and the reduced plasticization effect by water.
Leopold, 1992). These techniques differ By analysing thermal behaviours of water
greatly in how and what they measure with and biomaterials as a function of water
respect to the dynamic properties and content, temperature and time, the status of
structures of water and other biomolecules. water in plant tissues can be studied and
A great deal of confusion over the physical the water status correlated to its biological
state of water in biological systems has functions.
resulted from the separation of information Technically, DSC is really a quite simple
obtained with diverse techniques applied method. There are two cells in the DSC
to similar systems. The nature of different detector, one reference cell and one sample
cell. An empty crucible is placed into the 2.6.2.2. Thermally stimulated current (TSC)
reference cell and a crucible containing the method
tissue sample is placed into the sample
Different dielectric relaxation techniques
cell. During a DSC experiment, both refer-
had been used previously to study the
ence cell and sample cell are cooled and/or
properties of water in biological systems
heated at a constant rate over a range of
(Harvey and Hoekstra, 1972; Kamiyoshi
temperatures. When a thermal event occurs
and Kudo, 1978; Clegg et al., 1982; Careri
in the tissue, it releases or absorbs heat and Giansanti, 1984). More recently, the
energy (i.e. heat flow). A plot of heat flow TSC technique has been employed to study
as a function of temperature is called a the mode of hydration and water organiza-
thermogram, from which the thermal tion in plant tissues (Pissis et al., 1987,
behaviour of the tissue can be deduced. 1996; Bruni and Leopold, 1992; Sun et al.,
Glass transition is usually marked by a 1994; Sun, 2000). This technique is capable
stepwise shift in the baseline of a thermo- of providing information concerning the
gram. This distinguishes it from freezing mobility and rotational freedom of hydra-
and melting transitions, which produce tion water, hydration sites and mechanisms
peaks. A melting event is accompanied by (Mascarenhas, 1980; Pissis et al., 1987,
an endothermic peak and a freezing event 1996; Pissis, 1990; Bruni and Leopold,
is accompanied by an exothermic peak. 1992). The TSC technique is based upon:
The area under the particular peak repre- (i) the dependence of the microdynamics of
sents the total heat energy or enthalpy water dielectric relaxation on their sur-
change (H) for the event. DSC is a highly roundings resulting in different dielectric
informative tool for analysing biological relaxation times for water in different frac-
materials. Other information such as transitions; and (ii) the influence of water on the
tion temperature and heat capacity change dielectric relaxation mechanisms of other
(Cp) of the tissue upon cooling or heating biomolecules (similar to those used for
can also be calculated from a thermogram. DSC measurements).
The difficulty in analysing DSC thermo- The TSC method measures the tiny
grams lies in the correct identification of current generated by the thermally acti-
origin for thermal events in heterogeneous vated release of stored dielectric polariza-
biological samples. For example, lipid tran- tion during controlled heating and
sition in seeds can mask the actual glass basically consists of three steps: (i) the
transition (Williams and Leopold, 1989) polarization of a sample by a strong d.c.
and interfere with the accurate calculation electric field at a particular temperature;
of freezing and melting enthalpies (Sun, (ii) ‘freeze-in’ the polarization by cooling
1999). down to a sufficiently low temperature
Table 2.1. Biophysical techniques used to study the dynamic and structural properties of water and
macromolecules in biological systems.
Type of information Information about
Time scale Time-
Techniques (s) average Dynamic Structural Water Macromolecule
Thermal analysis (DSC, DTA) 101 ~103 + +
X-ray diffraction 101 ~102 + + + +
Spectroscopy (NMR, EPR) 104 ~100 + (+) (+) +
Relaxation (NMR, EPR, dielectric) 1011 ~100 + + (+)
Ultrasonic absorption 1010 ~105 + + +
Quasi-elastic neutron scattering 1013 ~107 + + +
Infrared and Raman spectroscopy 1016 ~1012 + + + +
DSC, differential scanning calorimetry; DTA, differential thermal analysis; NMR, nuclear magnetic resonance;
EPR, electron paragmagnetic resonance.
76 W.Q. Sun
(e.g. liquid nitrogen temperature) while magnetic field at the nucleus.) Since the
the field is still on; and (iii) the measure- electron density around each nucleus in a
ment of the TSC spectrum during heating molecule varies according to the type of
after the d.c. field is disconnected (Bruni nuclei and its molecular environment, the
and Leopold, 1992). When a polarized tis- opposing field and thus the effective field
sue reaches a temperature at which dipole at each nucleus will differ, which is called
molecules (such as water) relax (lose their ‘chemical shift’. The chemical shift of a
fixed orientation), a tiny current is gener- nucleus is the difference between the reso-
ated and recorded. From a TSC spectrum, nance frequency of the nucleus and a stan-
several important physical parameters can dard (relative to the standard, expressed in
be obtained, including the intensity of p.p.m., ). The chemical shift is a very pre-
depolarization charge (peak size, related cise measure of the chemical environment
to the size of the water pool), depolariza- around a nucleus.
tion temperature, its activation energy and The major frustration for many biolo-
static permittivity. The measurement of gists wishing to understand and to use
dielectric relaxation properties of water NMR is the complexity of the subject.
and water-plasticized biomolecules offers However, as with other physical tech-
valuable insight into the organization of niques used in studies of biological sys-
water in plant tissues and the molecular tems, NMR may be used in an ‘empirical’
interactions between water and other bio- mode, simply examining the variation of
molecules during desiccation (Bruni and an NMR parameter with the change of
Leopold, 1992; Sun, 2000). experimental variable (e.g. water content)
(James, 1993). Figure 2.11 shows 1H-NMR
spectra of mung bean seeds at three water
2.6.2.3. Nuclear magnetic resonance (NMR)
contents. The 1H-NMR spectra were broad,
NMR spectroscopy studies the interaction with the line width in the order of 103 Hz.
of electromagnetic radiation with matter. It Two NMR peaks were easily identifiable.
is a powerful tool for the studies of kinetic The peak of water in the immobile fraction
motion of water in tissues and of macro- had a peak maximum (chemical shift)
molecule/water or membrane/water inter- value of 4.3 p.p.m. relative to the proton in
actions. Solid-state NMR can be used to D2O, which was used as a standard refer-
determine the molecular structure of solid ence. The peak of water in the mobile frac-
tissue samples. Solid-state H-NMR is often tion had a peak maximum value at the
used to investigate the relaxation character- same place as the proton in D2O (i.e. = 0
istics of the protons of water molecules in p.p.m.). At a water content of 0.07 g g1
low-moisture biological systems (Mathur- dw, water appeared to exist primarily in
de Vre, 1979; Seewaldt et al., 1981; the immobile fraction. The very small pro-
Rorschach and Hazlewood, 1986; Ratkovic, portion of water in the mobile fraction
1987; Chapter 4). The basic principle of H- appeared as a shoulder in the spectrum.
NMR is that each of two hydrogen nuclei The amount of water in the mobile fraction
in a water molecule possesses a single spin increased rapidly as water content
proton, which will cause the nucleus to increased. At 0.24 g g1 dw, two peaks
produce an NMR signal. When an atom is merged almost completely as one peak,
placed in a magnetic field, the spin of its centred at = 0 p.p.m. The relative propor-
electrons will orient toward the direction tion of the immobile and mobile water frac-
of the applied magnetic field. This orienta- tions may be estimated using the standard
tion produces a small local magnetic field signal processing techniques.
at the nucleus that opposes the externally Two important spin relaxation parame-
applied field, resulting in a smaller mag- ters are T1, the spin–lattice relaxation, and
netic field (i.e. effective field) at the T2, the spin–spin relaxation time. The
nucleus than the applied field. (Note that spin–lattice relaxation (T1) involves the
in some cases it might also enhance the exchange of energy with the environment
(the lattice), and is caused by fluctuating come into resonance with monochromatic
local magnetic fields arising from the radiation. The magnetic field of most com-
motion of the molecules. The spin–spin mercial ESR spectrometers is about 0.3 T,
relaxation (T2) characterizes interactions corresponding to resonance with an elec-
between spins and is related to the width tromagnetic frequency of ~10 GHz and
of the NMR peak (Fig. 2.11). T1 and T2 can wavelength of ~3 cm. Therefore, the range
be used to study chemical kinetics and of applicability of ESR is narrower than
rotational and conformational motion of that of NMR. ESR is basically a microwave
molecules. technique, and is one of the fastest-growing
areas in analytical instrumentation because
of recent and remarkable achievements in
2.6.2.4. Electron spin resonance
microwave technology. ESR consists of a
(see also Chapter 4)
microwave source, a cavity, a microwave
Electron spin resonance (ESR) or electron detector and an electromagnet. The sample
paramagnetic resonance (EPR) is related to is placed in a glass or quartz tube, which is
NMR. It is the study of molecules with inserted into the cavity. The ESR spectrum
unpaired electrons (free radicals, transition is obtained by measuring the microwave
metal complexes, triplet states, etc.) by absorption as the magnetic field strength is
observing the magnetic fields at which they continuously changed. This method
2
Signal amplitude
0.24 g g–1
0.14 g g–1
0.07 g g–1
D2O
–16 –8 0 8 16 24
Chemical shift (, p.p.m.)
Fig. 2.11. The 1H-NMR spectra of water in mung bean seeds at three water contents. The width of all
spectra was 4000 Hz. D2O was used as a standard reference. The inset shows the assignment of 1H-NMR
signal into two different water fractions: mobile water (fraction 1) and immobile water (fraction 2). The
spectrum was recorded with FX90QNMR (JEOL Ltd, Japan). The powdered sample, weighing approx.
1–2 g, was loaded into the standard 5 mm NMR tube. A 90° 36-µs electromagnetic pulse was applied to
the sample. A total of eight scans were used to improve the resolution. A repeat time was 20 s to re-establish
the equilibrium via spin–lattice and spin–spin relaxation before the next scan (W.Q. Sun, unpublished data).
78 W.Q. Sun
detects the number of ‘unpaired spins’ of cals) is commercially available. By incor-

electronic charges. The ‘strange’ ESR spec- porating some probes such as nitroxide
trum is the first derivative of the derivatives into tissues before drying,
microwave energy absorption (Fig. 2.12). detailed studies may be undertaken on the
The hyperfine structure (splitting of indi- changes in the aqueous and non-aqueous
vidual resonance lines into components) of intracellular environments upon desicca-
an ESR spectrum is a fingerprint that helps tion. The ESR technique provides a fairly
to identify free radical species in the sam- direct measurement of the change in cyto-
ple and characterize their environments. plasmic viscosity, which probably plays
The ESR technique does not directly an important role in metabolic down-
measure water properties in tissues; how- regulation in desiccation tolerance (see
ever, it can be used to study many ques- Chapter 10).
tions that are related to desiccation. Using
an ESR spin-labelling technique, Belagyi
(1975) reported that a portion of cell water 2.7. Concluding Remarks
in muscles exhibited a restricted motion. In
recent years, Hoekstra and his co-workers Comparative studies play a key role in
have used this technique to study mem- understanding the mechanisms or strate-
brane behaviours, molecular mobility, cyto- gies of various organisms in the survival of
plasmic viscosity and partitioning of desiccation. The water status of tissues in
amphiphilic molecules of desiccation-tol- desiccation tolerance studies should be
erant and desiccation-intolerant plant tis- expressed precisely by preferred thermody-
sues upon desiccation (Golovina et al., namic parameters to permit the compari-
1998; Buitink et al., 1999, 2000a,b,c; son of data from different biological
Leprince et al., 1999; Chaper 4). A variety systems. The commonly used parameter,
of molecular spin probes (stable free radi- water content, is not adequate for the
Microwave absorption (A)
(a)
(b)
First derivative (dA/dB)
Magnetic field (B)
Fig. 2.12. (a) Microwave energy absorption. (b) The peculiar appearance of the electron spin resonance
(ESR) spectrum. The ESR spectrum is the first derivative signal of microwave energy absorption. The peak of
absorption corresponds to the point where the first derivative passes through zero (dashed lines). (See
Figures in Chapter 4.)
expression of tissue water status in most of desiccation stresses would certainly

cases. Whenever possible, the researcher improve the mechanistic studies of desic-
should first study the components of the cation tolerance. Water plays an important
water relations of cells or tissues and role in maintaining the structural integrity
obtain important reference parameters of biological systems. Although the kinetic
about their water status. The Höfler dia- properties of water in many biological sys-
gram and PV curve can be applied to most tems have been extensively studied, the
well-hydrated plant tissues, whereas the organization of cellular water and its rela-
isothermal sorption study can be applied to tion to desiccation tolerance or desiccation
intermediate- to low-moisture systems. damage are not fully understood. Further
Drying rate and desiccation stress can be studies on the interactions between water
quantified by introducing thermodynamic and macromolecular structures by biophys-
concepts into the study of water-loss ical techniques are essential to identify
dynamics during desiccation (drying fundamental cellular or metabolic compo-
curve). The quantitative (instead of qualita- nents that are associated with desiccation
tive) analysis of physico-chemical aspects damage or desiccation tolerance.
2.8. References
Bakradze, N.G. and Balla, Y.I. (1983) Crystallization of intracellular water in plant tissues. Biophysics
28, 125–128.
Beall, P.T. (1981) The water for life. The Sciences January 1, 6–29.
Beall, P.T. (1983) States of water in biological systems. Cryobiology 20, 324–334.
Beckett, R.P. (1997) Pressure–volume analysis of a range of poikilohydric plants implies the exis-
tence of negative turgor in vegetative cells. Annals of Botany 79, 145–152.
Belagyi, J. (1975) Water structure in striated muscle by spin labeling techniques. Acta Biochimica et
Biophysica; Academiae Scientiarum Hungaricae 10, 63–70.
Bell, L.N. and Labuza, T.P. (2000) Moisture Sorption: Practical Aspects of Isotherm Measurement and
Use. Eagan Press, Eagan, Minnesota, 123 pp.
Berjak, P. and Pammenter, N.W. (1994) Recalcitrance is not an all-or-nothing situation. Seed Science
Research 4, 263–264.
Berjak, P., Pammenter, N.W. and Vertucci, C. (1992) Homoiohydrous (recalcitrant) seeds: develop-
mental status, desiccation sensitivity and the state of water in axes of Landolphia kirkii Dyer.
Planta 186, 249–261.
Brunauer, S., Emmett, P.H. and Teller, E. (1938) Adsorption of gases in multimolecular layers. Journal
of American Chemical Society 60, 309–319.
Brunauer, S., Deming, L.S., Deming, W.E. and Teller, E. (1940) On a theory of the van der Waals
absorption of gasses. Journal of American Chemical Society 62, 1723.
Bruni, F. and Leopold, A.C. (1991) Hydration, protons and onset of physiological activities in maize
seeds. Physiologia Plantarum 81, 359–366.
Bruni, F. and Leopold, A.C. (1992) Pools of water in anhydrobiotic organisms: a thermally stimulated
depolarization current study. Biophysical Journal 63, 663–672.
Bruni, F., Careri, G. and Clegg, J.S. (1989) Dielectric properties of Artemia cysts at low water con-
tents: evidence for a percolative transition. Biophysical Journal 55, 331–338.
Buitink, J., Claessens, M.M.A.E., Hemminga, M.A. and Hoekstra, F.A. (1998a) Influence of water con-
tent and temperature on molecular mobility and intracellular glasses in seed and pollen. Plant
Buitink, J., Walters, C., Hoekstra, F.A. and Crane, J. (1998b) Storage behaviour of Typha latifolia
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Appendix: Solutions for Controlled are generally accurate within 2%. RHs of
Dehydration and Rehydration commonly used salt solutions between 5
and 40°C are compiled in Table A2.2. RHs
Saturated salt solutions of additional salt solutions at 25°C are
given in Table A2.3. These data are taken
In a closed container, a saturated salt solu- from earlier works of Rockland (1960),
tion (with excess salt present) produces a Winston and Bates (1960) and Young
constant water vapour pressure at a given (1967), tested and corrected by the author.
temperature. Relative humidity (RH) in the Users are advised to avoid salts that release
container is calculated by: salt vapours into the atmosphere. When
autoclaving is required, the decomposition
RH A exp(B/T) (1)
temperature of a salt should be checked.
where A and B are constants, and T is the After autoclaving, the closed container
temperature in kelvin (Wexler, 1997). The should be allowed sufficient time to equili-
values of A and B as well as the valid tem- brate (i.e. avoiding condensation and
perature range are given in Table A2.1 for supersaturation). The supersaturation in
various salts. For example, RH of saturated the liquid phase and the condensation of
KCl solution at 25°C is equal to 49.38 water on the wall in the container affect
exp(159/298) = 84.2 (%). Calculated values RH significantly.
Table A2.1. Commonly used salts, their vapour pressure constants A and B, and the valid temperature
range. Data are taken from Wexler (1997).
Temperature range Relative humidity
Compound (°C) at 25°C A B
NaOH.H2O 15–60 6 5.48 27
LiBr.2H2O 10–30 6 0.23 996
ZnBr2.2H2O 5–30 8 1.69 455
KOH.2H2O 5–30 9 0.014 1924
LiCl.H2O 20–65 11 14.53 75
CaBr2.6H2O 11–22 16 0.17 1360
LiI.3H2O 15–65 18 0.15 1424
CaCl2.6H2O 15–25 29 0.11 1653
MgCl2.6H2O 5–45 33 29.26 34
NaI.2H2O 5–45 38 3.62 702
Ca(NO3) 2.4H2O 10–30 51 1.89 981
Mg(NO3) 2.6H22O 5–35 53 25.28 220
NaBr.2H2O 0–35 58 20.49 308
NH4NO3 10–40 62 3.54 853
KI 5–30 69 29.35 254
SrCl2.6H2O 5 –30 71 31.58 241
NaNO3 10–40 74 26.94 302
NaCl 10–40 75 69.20 25
NH4Cl 10–40 79 35.67 235
KBr 5–25 81 40.98 203
(NH4) 2SO4 10–40 81 62.06 79
KCl 5–25 84 49.38 159
Sr(NO3) 2.4H2O 5–25 85 28.34 328
BaCl2.2H2O 5–25 90 69.99 75
CsI 5–25 91 70.77 75
KNO3 0–50 92 43.22 225
K2SO4 10–50 97 86.75 34
Table A2.2. Equilibrium relative humidities of saturated salt solutions at different temperatures. Data are
taken from Rockland (1960), Winston and Bates (1960) and Young (1967).
Temperature
Saturated
salt solution 5°C 10°C 15°C 20°C 25°C 30°C 35°C 40°C
H2SO4 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0
ZnCl2 5.5 – 5.5 – 5.5 – 5.5 –
NaOH 6.0 – 6.0 6.0 7.2 – 7.5 –
LiBr 9.0 – 8.0 – 7.0 – 7.0 –
KOH 13.0 – 9.0 – 8.0 – 8.0 –
LiCl.H2O 14.0 13.5 13.0 12.5 12.0 11.5 11.5 11.0
CaBr2 23.0 – 20.0 18.5 16.5 – 15.0 –
KAc 24.8 24.0 23.5 23.0 23.0 23.0 22.0 23.0
MgBr2 32.0 31.0 31.5 31.0 30.5 30.0 30.0 30.0
MgCl2 34.0 33.5 33.5 33.0 32.5 32.0 32.5 32.0
CaCl2 40.0 38.0 35.0 32.5 30.0 – 30.0 –
K2CO3 43.0 47.0 44.0 44.0 43.0 42.0 41.5 40.0
NaI 43.5 – 38.0 38.5 38.0 36.0 34.0 32.5
Zn(NO3) 2 45.0 43.0 40.7 40.0 32.5 24.0 21.0 19.0
KCNS 54.0 52.0 50.0 47.0 46.5 43.5 41.5 41.0
Mg(NO3) 2 55.0 53.0 53.7 53.0 52.5 52.0 50.5 51.0
Na2Cr2O7.H2O 59.5 60.0 56.5 54.5 53.0 52.5 51.0 50.0
NaBr 60.5 58.0 58.0 57.8 57.2 57.0 57.0 57.0
Ca(NO3) 2 61.0 66.0 58.0 56.0 52.2 51.0 45.5 46.0
NaBr.2H2O 63.0 61.0 59.0 57.5 56.0 54.5 53.0 51.5
CuCl2 66.7 68.0 68.0 68.5 67.0 67.0 67.0 67.0
LiAc 72.0 72.0 71.0 70.0 68.0 66.0 65.0 64.0
NH4NO3 73.0 75.0 70.0 65.5 62.5 59.5 56.8 53.0
NaCl 76.0 75.8 75.5 75.3 75.1 75.0 75.0 75.0
NaNO3 79.0 77.5 76.5 76.0 74.0 72.5 71.0 70.5
(NH4) 2SO4 81.7 81.2 80.0 79.8 79.7 79.5 79.2 79.0
NH4Cl 82.0 79.0 79.5 79.0 78.0 77.5 75.5 74.0
Li2SO4 84.0 84.0 84.0 85.0 85.0 85.0 85.0 81.0
KBr 85.0 86.0 85.0 83.5 83.0 82.0 81.0 80.0
KCl 87.8 86.7 86.0 85.3 85.0 83.5 83.0 83.0
K2CrO4 89.0 89.0 88.0 88.0 87.0 86.0 84.0 82.0
BaCl2 95.0 93.0 92.0 90.7 90.0 89.0 88.0 87.0
ZnSO4 95.0 93.0 92.0 90.0 88.0 86.0 85.0 84.0
KNO3 96.5 95.5 95.0 94.0 92.5 91.5 89.5 88.5
K2SO4 98.0 97.0 97.0 97.0 97.0 97.0 96.0 96.0
Na2HPO4 98.0 98.0 98.0 98.0 97.0 96.0 93.0 91.0
Pb(NO3) 3 99.0 98.5 98.5 98.5 96.2 95.5 95.2 94.7
Non-saturated salt solutions during equilibration, because the equilibra-

tion between the tissue, vapour phase and
Non-saturated salt solutions can also be solution phase results in a slight decrease or
used. This method allows for the creation of increase in salt concentration. This problem
a precise and evenly graded series of RHs (or can be minimized using high mass ratios
water potentials) with the same salt. The dis- between the solution and the sample. A
advantage of using non-saturated salt solu- mass ratio of 150–200 (i.e. 150–200 g solu-
tions is that they have limited buffering tion g1 tissue) is sufficient. Water potentials
capacity relative to saturated salt solutions. of NaCl solutions at different concentrations
RHs inside the container are not constant and temperatures between 5 and 40°C are
86 W.Q. Sun
Table A2.3. A list of saturated salt solutions (with the presence of excess salt) and their equilibrium
relative humidities (RHs) at 25°C.
Saturated salt solution RH (%) Saturated salt solution RH (%)
ZnBr2 8.6 SrBr2.6H2O 58.5
H3PO4 9.0 FeCl2.4H2O 60.0
CaAc2.H2O + sucrose 13.0 NaMnO4.3H2O 61.5
CaAc2.H2O 17.0 NH4NO3+ AgNO3 61.5
Ca(CNS) 2.3H2O 17.5 CuBr2 62.5
LiI.3H2O 18.0 CoCl2 64.0
KHCO2 (Formate) 21.5 NaNO2 64.2
KAc.1.5H2O 22.2 K2S2O3 66.0
NiBr2.3H2O 27.0 CuCl2.2H2O 67.7
MgBr2.6H2O 31.5 NaCl + NaNO3 69.0
Sr(CNS) 2.3H2O 31.5 SrCl2.6H2O 71.0
SrI2.6H2O 33.0 SrCl2 71.0
MnBr2.6H2O 34.5 NH4Cl + KNO3 71.2
Cu(NO3) 2.6H2O 35.0 NaCl + KCl 71.5
Ca(MnO4) 2.4H2O 37.5 NaAc.3H2O 73.0
FeBr2.6H2O 39.0 NaCl + Na2SO4.7H2O 74.0
NaI.2H2O 39.2 BaBr2 74.5
Mg(ClO4) 2.6H2O 41.0 K tartrate 75.0
CoBr2 41.5 NH4Br2 75.0
CrCl3 42.5 Zn(CNS) 2 80.5
BaI2.2H2O 43.0 NaH2PO4 81.0
K2CO3.2H2O 43.0 AgNO3 82.0
CeCl3 45.5 KCl + KClO3 85.0
LiNO3.3H2O 47.0 KNa tartrate 87.0
Mg(CNS) 2 47.5 Na2CO3.10H2O 87.0
KNO2 48.1 MgSO4.7H2O 89.0
K4P4O7.3H2O 49.5 BaCl2.2H2O 90.3
Co(NO3) 2.6H2O 49.8 Na tartrate 92.0
NH4NO3 + NaNO3 50.0 (NH4)H2PO4 92.7
KBr + urea 51.0 NH4HPO4 93.0
Zn(MnO4) 2.6H2O 51.0 CaH4(PO4) 2.H2O 96.0
NiCl2.6H2O 53.0 KH2PO4 96.0
Na2Cr2O7.2H2O 53.7 CaHPO4.2H2O 97.0
Ba(CNS) 2.2H2O 54.5 CuSO4.5H2O 97.2
Pb(NO3) 2 + NH4NO3 55.0 K2Cr2O7 98.0
MnCl2.4H2O 56.0 KClO3 98.0
given in Table A2.4. Besides NaCl solutions, tial or RH are often not available. PEG is
CaCl2 and KCl solutions offer good RH con- inexpensive and not corrosive, whereas
trol. Water potentials of CaCl2 and KCl solu- many salt solutions are corrosive and
tions are listed in Table A2.5. volatile. Effects of PEG concentration and
temperature on water potential were studied
by Michel and Kaufmann (1973). An empiri-
Polyethylene glycol (PEG) solutions cal equation has been derived to calculate
the water potential of PEG solutions at given
PEG solutions are widely used in controlled concentrations and temperatures:
dehydration and rehydration. PEG solutions
have several advantages over salt solutions. (1.18 103C) – (1.18 105 C2)
Salt solutions at high specific water poten- (2.67 105CT) (8.39 108C 2T) (2)
Table A2.4. Water potentials of sodium chloride (NaCl) solutions at different concentrations and
temperatures. Mass (%); g solute per 100 g solution.
Molality Mass
(mol kg1) (%) 5°C 10°C 15°C 20°C 25°C 30°C 35°C 40°C
0.05 0.29 0.22 0.22 0.23 0.23 0.23 0.24 0.24 0.25
0.1 0.58 0.43 0.44 0.45 0.45 0.46 0.47 0.48 0.49
0.2 1.16 0.85 0.87 0.88 0.90 0.92 0.93 0.95 0.96
0.3 1.72 1.27 1.30 1.32 1.34 1.37 1.39 1.42 1.44
0.4 2.28 1.69 1.73 1.76 1.79 1.82 1.86 1.89 1.92
0.5 2.84 2.12 2.16 2.20 2.24 2.28 2.32 2.36 2.40
0.6 3.39 2.54 2.59 2.64 2.69 2.74 2.79 2.84 2.89
0.7 3.93 2.97 3.03 3.09 3.15 3.21 3.27 3.33 3.39
0.8 4.47 3.40 3.47 3.54 3.61 3.68 3.75 3.82 3.89
0.9 5.00 3.83 3.92 4.00 4.08 4.16 4.23 4.31 4.39
1.0 5.52 4.27 4.37 4.46 4.55 4.64 4.73 4.82 4.90
1.1 6.04 4.71 4.82 4.92 5.03 5.13 5.23 5.32 5.42
1.2 6.55 5.16 5.28 5.39 5.51 5.62 5.73 5.84 5.94
1.3 7.06 5.61 5.74 5.87 5.99 6.12 6.24 6.35 6.47
1.4 7.56 6.07 6.21 6.35 6.49 6.62 6.75 6.88 7.01
1.5 8.06 6.53 6.68 6.84 6.99 7.13 7.28 7.41 7.55
1.6 8.55 7.00 7.16 7.33 7.49 7.65 7.81 7.95 8.11
1.7 9.04 7.46 7.64 7.82 8.00 8.17 8.33 8.49 8.65
1.8 9.52 7.94 8.13 8.33 8.52 8.70 8.88 9.04 9.21
1.9 9.99 8.43 8.63 8.84 9.04 9.24 9.43 9.60 9.78
2.0 10.46 8.92 9.13 9.36 9.57 9.78 9.98 10.16 10.35
where C is the concentration of PEG (molec- water potential is calculated with Equation
ular weight 6000) in g kg1 water and T is (2). The PEG solution can be dried at 105°C
temperature (°C). Water potential of PEG in an oven to a constant dry weight. The
solutions is curvilinearly related to the con- problem with submerging the tissue in a
centration and increases linearly with tem- PEG solution is that PEG can enter the inter-
perature. The error of calculated water cellular spaces. PEG is considered a non-
potential is generally within 0.03 MPa in penetrating polymer because it does not
comparison with the psychrometric mea- cross the membrane! Our calorimetric study
surements. Water potentials of PEG-6000 reveals a PEG melting peak in submerged
solutions at concentrations ranging from 10 tissues, even after extensive washing.
to 400 g kg1 water and at temperatures
between 5 and 40°C are given in Table A2.6.
PEG solutions are very viscous, especially at Glycerol solutions
high concentrations; hence it takes a longer
time to achieve the equilibrium between the Glycerol can also be used to create a pre-
PEG solution and the tissue. Caution is cise and evenly graded series of RHs
needed to prevent bacterial and fungal cont- between 30 and 98%. Glycerol solutions
amination during the study. The equilibra- are safe to use and less corrosive (except
tion can be accelerated by placing closed for tin) than salt solutions. Microbial
containers in a gently shaking incubator and growth can be effectively inhibited by
in some cases submerging the tissue in solu- adding four drops of saturated CuSO4 solu-
tion. The change in PEG concentration after tion to each 100 ml of glycerol solution
the experiment can be determined by the (ASTM, 1983). The CuSO4-treated solution
gravimetric method, and the equilibrium can be used repeatedly after the required
88 W.Q. Sun
Table A2.5. Water potentials of potassium chloride (KCl) and calcium

chloride (CaCl2) solutions at 20 and 25°C. Mass (%): g solute per 100
g solution.
KCl CaCl2
Mass (%) 20°Ca 25°Cb 20°Ca 25°Cb
0.5 0.28 0.31 0.27 0.28
1 0.56 0.61 0.54 0.57
2 1.12 1.22 1.07 1.16
3 1.68 1.85 1.62 1.81
4 2.26 2.48 2.22 2.50
5 2.83 3.13 2.87 3.24
6 3.42 3.80 3.58 4.02
7 4.02 4.48 4.36 4.89
8 4.64 5.18 5.23 5.77
9 5.25 5.90 6.15 6.73
10 5.87 6.65 7.16 7.76
12 7.18 8.21 9.40 10.09
14 9.89 12.00 12.85
16 11.68 14.99 16.13
18 13.62 18.45 20.02
20 15.69 22.34 24.60
22 26.50
24 20.34 30.89 36.20
26 36.26
28 42.37 51.67
30 50.06
32 60.68 71.78
36 97.33
40 129.12
a Derived from Handbook of Chemistry and Physics, 78th edn
(Lide, 1997). Water potential is calculated according to the

freezing-point depression.
b Data were derived from Robinson and Stokes (1959).
concentration adjustment. The composi- The ASTM’s method uses the refractive
tion of glycerol solutions can be accurately index at 25°C to express glycerol concen-
determined using specific gravity or the tration. The relationship between RH,
refractive index. Equilibrium RHs of glyc- refractive index and temperature is
erol solutions at 24°C were reported by described by the following equations:
Braun and Braun (1958). Using the same
(R0 A)2 (100 A)2 A2 (RH A)2 (3)
set of data, Forney and Brandl (1992)
derived an empirical equation to calculate A 25.6 0.195T 0.0008T 2 (4)
equilibrium RHs of glycerol solutions.
R 1.3333 (1.398 103 R0) (5)
Equilibrium RHs and water potentials of
glycerol solutions at 20°C were derived where RH is the desired relative humidity
according to the freezing-point depression. (%), R is the refractive index of the glycerol
These data are listed in Table A2.7. One solution, and T is temperature (°C). The
can calculate the required concentration of value of A is calculated using Equation (4).
a glycerol solution for a desired RH at a Ro is calculated by substituting A and RH
given temperature, using the ASTM’s stan- in Equation (3). The refractive index at
dard recommended practice (ASTM, 1983). 25°C is calculated using Equation (5). For a
glycerol solution of the known refractive where A is defined by Equation (4), and Ro
index (R), equilibrium RH at different tem- is calculated from Equation (5). The mea-
peratures can be calculated by rearranging surement of refractive index is quite
Equations (3), (4) and (5) (Sun and Gouk, tedious. The concentration (mass %) of the
1999). The relationship is: desired glycerol solution has been derived
from concentrative properties of aqueous
RH = (100 + A)2 + A2 − (R0 + A)2 − A (6) glycerol solutions (Lide, 1997) by the
Table A2.6. Water potentials of polyethylene glycol (MW 6000) solutions. Data were derived according
to Michel and Kaufmann (1973).
Water potential (MPa) at different temperatures
PEG
(g kg1 H2O) 5°C 10°C 15°C 20°C 25°C 30°C 35°C 40°C
10 0.012 0.010 0.009 0.007 0.006 0.005 0.003 0.002
20 0.025 0.023 0.020 0.017 0.014 0.011 0.008 0.006
30 0.042 0.037 0.033 0.028 0.024 0.020 0.015 0.011
40 0.060 0.054 0.048 0.042 0.036 0.030 0.024 0.018
50 0.081 0.073 0.065 0.058 0.050 0.042 0.034 0.027
60 0.104 0.094 0.085 0.075 0.066 0.056 0.047 0.037
70 0.129 0.118 0.106 0.095 0.083 0.072 0.061 0.049
80 0.157 0.143 0.130 0.116 0.103 0.090 0.076 0.063
90 0.186 0.171 0.156 0.140 0.125 0.109 0.094 0.078
100 0.22 0.20 0.183 0.166 0.148 0.131 0.113 0.096
110 0.25 0.23 0.21 0.194 0.174 0.154 0.134 0.114
120 0.29 0.27 0.25 0.22 0.20 0.179 0.157 0.135
130 0.33 0.30 0.28 0.26 0.23 0.21 0.182 0.157
140 0.37 0.34 0.32 0.29 0.26 0.24 0.21 0.181
150 0.41 0.38 0.35 0.32 0.30 0.27 0.24 0.21
160 0.46 0.43 0.39 0.36 0.33 0.30 0.27 0.23
170 0.51 0.47 0.44 0.40 0.37 0.33 0.30 0.26
180 0.56 0.52 0.48 0.44 0.41 0.37 0.33 0.29
190 0.61 0.57 0.53 0.49 0.45 0.41 0.37 0.33
200 0.66 0.62 0.58 0.53 0.49 0.45 0.40 0.36
210 0.72 0.68 0.63 0.58 0.54 0.49 0.44 0.40
220 0.78 0.73 0.68 0.63 0.58 0.53 0.48 0.43
230 0.84 0.79 0.74 0.68 0.63 0.58 0.53 0.47
240 0.91 0.85 0.79 0.74 0.68 0.63 0.57 0.51
250 0.97 0.91 0.85 0.79 0.73 0.67 0.62 0.56
260 1.04 0.98 0.92 0.85 0.79 0.73 0.66 0.60
270 1.11 1.05 0.98 0.91 0.85 0.78 0.71 0.65
280 1.19 1.11 1.04 0.97 0.90 0.83 0.76 0.69
290 1.26 1.19 1.11 1.04 0.96 0.89 0.82 0.74
300 1.34 1.26 1.18 1.10 1.03 0.95 0.87 0.79
310 1.42 1.34 1.25 1.17 1.09 1.01 0.93 0.85
320 1.50 1.41 1.33 1.24 1.16 1.07 0.99 0.90
330 1.58 1.49 1.41 1.32 1.23 1.14 1.05 0.96
340 1.67 1.58 1.48 1.39 1.30 1.20 1.11 1.01
350 1.76 1.66 1.56 1.47 1.37 1.27 1.17 1.07
360 1.85 1.75 1.65 1.54 1.44 1.34 1.24 1.13
370 1.95 1.84 1.73 1.62 1.52 1.41 1.30 1.20
380 2.04 1.93 1.82 1.71 1.60 1.48 1.37 1.26
390 2.14 2.02 1.91 1.79 1.68 1.56 1.44 1.33
400 2.24 2.12 2.00 1.88 1.76 1.64 1.52 1.40
90 W.Q. Sun
Table A2.7. Equilibrium relative humidity (RH) and water potential of glycerol solutions at 20°C and
24°C. Mass (%): g glycerol per 100 g solution.
20°C 24°C
Mass (%) Specific gravity a % RH b b (MPa) % RH c (MPa)
10 1.0215 97.9 2.79 98.0 2.77
12 1.0262 97.4 3.45 97.5 3.47
14 1.0311 96.9 4.16 97.0 4.18
16 1.0360 96.4 4.89 96.5 4.89
18 1.0409 95.8 5.69 95.9 5.74
20 1.0459 95.2 6.52 95.4 6.46
24 1.0561 93.9 8.35 94.1 8.34
28 1.0664 92.4 10.42 92.6 10.55
32 1.0770 90.7 12.71 91.0 12.94
36 1.0876 88.9 15.28 89.2 15.68
40 1.0984 86.9 18.19 87.2 18.79
44 1.1092 84.9 22.46
48 1.1200 82.5 26.39
52 1.1308 79.7 31.13
56 1.1419 76.6 36.57
60 1.1530 73.2 42.78
64 1.1643 69.4 50.11
68 1.1755 65.2 58.68
72 1.1866 60.6 68.71
76 1.1976 55.5 80.77
80 1.2085 49.8 95.64
84 1.2192 43.6 113.88
88 1.2299 36.7 137.51
a Taken from Handbook of Chemistry and Physics, 78th edn (Lide, 1997).
b Calculated according to the freezing-point depression.
c The relationship between concentration and equilibrium RH was reported by Braun and Braun (1958).
The equation derived by Forney and Brandl (1992) was used to determine equilibrium RH of solutions
at other concentrations.
author. RHs of glycerol solutions at concen- ASTM’s method is 0.2% at 25°C, and
trations between 10 and 92% (mass %) and increases as temperature deviates from
at temperatures between 5 and 35°C has 25°C (ASTM, 1983). Data in Table A2.8 are
been calculated according to the ASTM’s consistent with those in Table A2.7, which
method (Table A2.8). The accuracy of were derived by different methods.
Table A2.8. Equilibrium relative humidity (RH) of glycerol solutions at different

temperatures. Values were derived, using the refractive index of the solution at 25°C.
Mass (%): g glycerol per 100 g solution.
RH (%) at different temperatures
Mass (%) 5°C 10°C 15°C 20°C 25°C 30°C 35°C
10 98.1 98.1 98.2 98.2 98.3 98.3 98.3
12 97.6 97.7 97.7 97.8 97.8 97.9 97.9
14 97.1 97.2 97.2 97.3 97.4 97.4 97.5
16 96.6 96.6 96.7 96.8 96.9 96.9 97.0
18 96.0 96.1 96.1 96.2 96.3 96.4 96.4
20 95.4 95.5 95.5 95.6 95.7 95.8 95.9
24 94.0 94.1 94.2 94.3 94.4 94.5 94.6
28 92.5 92.6 92.8 92.9 93.0 93.1 93.2
32 90.8 91.0 91.1 91.3 91.4 91.5 91.7
36 88.9 89.1 89.3 89.4 89.6 89.7 89.9
40 86.8 87.0 87.2 87.4 87.6 87.7 87.9
44 84.5 84.7 84.9 85.1 85.3 85.5 85.7
48 82.0 82.2 82.5 82.7 82.9 83.1 83.3
52 79.2 79.5 79.7 80.0 80.2 80.4 80.6
56 76.2 76.5 76.7 77.0 77.2 77.4 77.7
60 72.8 73.0 73.3 73.6 73.8 74.1 74.3
64 69.0 69.3 69.6 69.9 70.1 70.4 70.6
68 64.9 65.2 65.5 65.8 66.1 66.3 66.6
72 60.3 60.6 60.9 61.2 61.5 61.8 62.1
76 55.2 55.6 55.9 56.2 56.5 56.8 57.1
80 49.5 49.8 50.2 50.5 50.8 51.1 51.4
84 43.0 43.4 43.7 44.0 44.4 44.7 45.0
88 35.5 35.9 36.2 36.5 36.9 37.2 37.5
References
ASTM (1983) Maintaining constant relative humidity by means of aqueous solutions. In: 1983
Annual Book of ASTM Standards (Standard E104). American Society for Testing and Materials,
Philadelphia, pp. 572–575.
Braun, J.V. and Braun, J.D. (1958) A simplified method of preparing solutions of glycerol and water
for humidity control. Corrosion 14, 117–118.
Forney, C.F. and Brandl, D.G. (1992) Control of humidity in small controlled environment chambers
using glycerol–water solutions. HortTechnology 2, 52–54.
Lide, D.R. (ed.) (1997) Handbook of Chemistry and Physics, 78th edn. CRC Press, New York, pp. 8,
57–81.
Michel, B.E. and Kaufmann, M.R. (1973) The osmotic potential of polyethylene glycol 6000. Plant
Robinson, R.A. and Stokes, R.H. (1959) Electrolyte Solutions, 2nd edn. Butterworths Scientific
Publications, London, 559 pp.
Rockland, L.B. (1960) Saturated salt solutions for static control of relative humidity between 5 and
40°C. Analytical Chemistry 32, 1375–1376.
Sun, W.Q. and Gouk, S.S. (1999) Preferred parameters and methods for studying moisture content of
recalcitrant seeds. In: Marzalina, M., Khoo, K.C., Jayanti, N., Tsan, F.Y. and Krishnapillay, B.
(eds) Recalcitrant Seeds. Proceedings of IUFRO Seed Symposium 1998. Forest Research Institute
Malaysia, Kuala Lumpur, pp. 404–430.
Wexler, A. (1997) Constant humidity solutions. In: Lide, D.R. (ed.) Handbook of Chemistry and
Physics, 78th edn. CRC Press, New York, pp. 15, 24–25.
Winston, P.W. and Bates, D.H. (1960) Saturated solutions for the control of humidity in biological
research. Ecology 41, 232–237.
Young, J.F. (1967) Humidity control in the laboratory using salt solutions – a review. Journal of
Applied Chemistry 17, 241–245.
Dessication - Chap 03 18/3/02 1:55 pm Page 93
3 Experimental Aspects of Drying and

Recovery
Norman W. Pammenter,1 Patricia Berjak,1 James Wesley-Smith1 and

Clare Vander Willigen2
1School of Life and Environmental Sciences, University of Natal, Durban 4041,
South Africa; 2Department of Botany, University of Cape Town, Private Bag,
Rondebosch 7701, South Africa
3.2. Drying Rate 94
3.2.1. Commonly employed drying techniques 94
3.2.1.1. Seed material 95
3.2.1.2. Vegetative tissue 97
3.2.2. Quantification and modelling of drying rates 98
3.2.3. Effects of different drying rates 99
3.2.3.1 Desiccation-sensitive tissue 99
3.2.3.2. Desiccation-tolerant tissue 100
3.2.4. ‘Ultradrying’ of desiccation-tolerant material 101
3.3. Influence of Rehydration Technique 102
3.4. Length of Time in the Partially Dehydrated State 102
3.5. Methods of Assessing Response to Rehydration 103
3.5.1. ‘Germination’ 103
3.5.2. Resurrection plants 104
3.5.3. Electrolyte leakage 104
3.5.4. Tetrazolium test 104
3.5.5. Other responses 104
3.6. Expression of Water Content Data 105
3.6.1. Mass basis 105
3.6.2. Water potential 105
3.6.3. Relative water content 106
3.7. Conclusion 106
3.8. References 106
94 N.W. Pammenter et al.
3.1. Introduction dration may have adverse metabolic and

other deleterious consequences unrelated
When plant material is subjected to dehy- to drying.
dration, the observed response (in terms of 3. The rate of air movement across the tis-
damage accumulation and survival) can sue; this affects the thickness of the bound-
vary with the techniques used to assess the ary layer and the ability of water vapour to
response. This has the potential to cause diffuse through it. Forced ventilation will
confusion in the interpretation of experi- lead to faster drying than will still air.
mental data. It is the objective of this chap- 4. The size and shape of the tissue; this
ter to outline the range of techniques that affects the surface area-to-volume ratio and
have been used, to note the effects each can the distance water must diffuse from the
have on the observed response and, as far interior of the tissue to the surface. Small
as possible, to suggest underlying causes of tissue pieces will dry faster than large
these effects. ones, and ‘flat’ tissue will dry faster than
Experimental or technical aspects that ‘bulky’ tissue.
can influence the response to dehydration 5. The chemical and physical composition
include drying rate, the length of time the of the outer layer of the tissue; this will
tissue is maintained in the (partially) dry affect the permeability of the tissue to
state, the rehydration and recovery tech- water, in either the liquid or vapour form.
niques, and the method used to assess the Tissues with lignified, suberized or waxy
response. The developmental status of the outer layers will dry more slowly than
tissue may also influence the response to those without such layers.
drying. The information presented in this 6. The amount of material to be dried; this
chapter is derived almost exclusively from will affect factors such as surface area-to-
studies on seeds and vegetative tissue (vas- volume ratios and boundary layers.
cular and non-vascular species). The dis- Generally, small quantities of material will
cussion has not been extended to pollen dry faster than will a large mass.
and spores, largely because their size is
These factors are, to a greater or lesser
such that they normally dehydrate rapidly
extent, under the control of the experi-
and so effects of drying conditions are less
menter, who can thus alter (but not control
frequently studied (see Chapter 6).
with precision) the rate at which the mater-
ial dries. It should be noted that the terms
‘rapid’, ‘intermediate’ and ‘slow’ are com-
3.2. Drying Rate parative only within a single study; there
are no absolute boundaries to these terms.
During drying, water molecules diffuse A drying rate that in one study might be
from the tissue to the surrounding air. described as ‘rapid’ might be considered to
There are a number of factors that can be ‘slow’ in another.
affect the rate at which this diffusion
occurs and hence the rate at which the tis-
sue will dry. Some of these factors are:
3.2.1. Commonly employed drying
1. The vapour pressure of the surrounding techniques
air; this affects the difference in free energy
of water between the tissue and the air, and Commonly used drying techniques vary in
hence the drying rate. Tissue will obvi- the degree to which temperature and
ously dehydrate faster in dry than in humidity are controlled and the extent of
humid air. air movement across the material being
2. Temperature; this will also influence the dried. In the case of seeds they also vary in
water free energy difference, which will be whether or not outer coverings are
greater at higher temperatures. However, removed. The choice of method is often
use of elevated temperatures during dehy- influenced by the amount of material that
Experimental Aspects of Drying and Recovery 95
is being dried, the facilities available and will vary amongst species. Cromarty et al.
the reason for which the material is being (1985) have given details of the design of
dried. Some of the methods are specific for seed bank facilities for orthodox seeds,
seeds, and some for resurrection plants, but including detailed consideration of drying
many can be used for any small piece or protocols. Drying times using these proto-
pieces of tissue. Commonly used tech- cols are of the order of days.
niques are summarized in Table 3.1. As 4. Air flow in a laminar flow cabinet
drying conditions can have marked effects (Grout et al., 1983; Normah et al., 1986;
on the response of tissue to drying (Section Pence, 1992; Chandel et al., 1995). This
3.2.3), it is critical that the drying condi- technique can be used for small seeds and
tions (e.g. temperature, relative humidity parts of seeds (excised embryos or embry-
(RH), light conditions, air flow) used in any onic axes) and has the advantages that it
investigation are reported. introduces an air flow over the material
(forced ventilation) and reduces the intro-
duction of further microbial contaminants.
3.2.1.1. Seed material However, the temperature and RH of the air
Techniques that have been used to dry seed are not controlled, and will vary with
material are outlined below: locality and between seasons, but drying
rates of the order of hours can be achieved
1. Air drying of seeds or fruits in sun or with small tissue pieces.
shade (e.g. Albrecht, 1993). Of all the tech- 5. Drying seeds by burying them in acti-
niques used this one offers the least control vated silica gel (Pammenter et al., 1998).
of the various factors affecting drying, but This will yield the most rapid rate of dry-
it is commonly used in the field where ing in the absence of forced ventilation,
facilities are limited, samples are large and and has been adopted as the standard tech-
drying shortly after collection is desired. nique by the IPGRI–Danida Forest Seed
When drying seeds, the usual practice is to Centre sponsored project on the handling
spread them in an even layer, but, if the and storage of recalcitrant tropical tree
layer is more than one seed deep, the sam- seeds (IPGRI/DFSC, 1996).
ple must be turned frequently to prevent 6. Rapid air flow over material in a small
uneven drying. A similar practice is chamber, i.e. flash drying. A common prac-
employed for the whole fruits, for particu- tice is to place the material on a grid in a
lar species. Drying using this technique can small container and pass air into the bot-
take several days. tom, over the sample, and vent from the
2. Drying under ambient laboratory condi- top of the chamber (Berjak et al., 1990;
tions (e.g. Wu et al., 1998) has the same Pammenter et al., 1991). The air may or
disadvantages as the first method, except may not be dried. If air from a gas cylinder
that ambient conditions probably vary less or a compressor is used, it should be dry.
in the laboratory than outside. Drying (As a word of warning, not all compressors
times will be similar to those achieved in are properly maintained, and a compressed
the first method. air line to a laboratory bench may have
3. Walk-in chambers with temperature droplets of water in it and is obviously
and RH control. These facilities are nor- unsuitable.) An improved system designed
mally available only in large-scale com- by one of us (J.W.-S.) in which air is circu-
mercial or research organizations and are lated through silica gel and over excised
generally used to dry substantial quanti- embryonic axes is illustrated in Fig. 3.1;
ties of material. Recommended practice is this equipment yields the fastest drying
to dry at 10–25°C and 10–15% RH rates of all the techniques we have used
(International Plant Genetic Resources (several minutes to hours).
Institute (IPGRI), 1994). This approach 7. Drying excised axes under partial vac-
permits standard and repeatable drying uum (Fu et al., 1993). Although this tech-
conditions, but the drying rate achieved nique yielded rapid drying (faster than
96
Dessication - Chap 03
Table 3.1. Summary of drying methods and approximate drying times used in studies on responses to desiccation.
Quantity of Drying
18/3/02
Drying method material time Reference

Seeds Sun drying Kilograms Days Albrecht, 1993
Still air at controlled temperature and RH Days to weeks Cromarty et al., 1985; IPGRI, 1994;
Wu et al., 1998
1:55 pm
Forced ventilation at controlled temperature and RH Monolayer Hours to days Ntuli et al., 1997
Buried in silica gel Tens to hundreds Grout et al., 1983; Pammenter et al.,
of seeds 1998
Excised axes Laminar flow hood Monolayer Hours Grout et al., 1983; Normah et al., 1986
Page 96
Flash-drying – forced ventilation with or without Tens of minutes Pammenter et al., 1991
silica gel to hours
Vegetative tissue Withdrawal of watering from whole plant Whole tracheophytes Days Gaff et al., 1992; Vander Willigen, et al.,
N.W. Pammenter et al.
2001
Excised plant parts or moss clumps on bench top Segments of leaves, Hours Bartels et al., 1990; Farrant et al., 1999
twigs, moss clumps
Small chambers at controlled temperature and RH Hetherington and Smillie, 1982;
Seel et al., 1992
Forced ventilation in laminar flow hood or small Bochicchio et al., 1998; Farrant et al.,
chambers 1999
storage above silica gel), none of the axes axes excised from seeds (Grout et al., 1983;
survived the treatment. Consequently, this Normah et al., 1986; Pritchard and
technique is not recommended without Prendergast, 1986; Berjak et al., 1990). This
further investigation. has the advantage of permitting drying
8. Drying to equilibrium at constant RH. rates of the order of tens of minutes to a
This is generally done by placing the mate- few hours, but can be very laborious if
rial over saturated solutions of salts, within large numbers of axes are required. If this
a small chamber. The air can be still or may approach is adopted, it is important to
be stirred by a fan in the chamber (Ntuli et minimize damage whilst excising the axes.
al., 1997). The advantage of this technique
is that the equivalent water potential 3.2.1.2. Vegetative tissue
at equilibrium is known ( =
(RT/V)*ln(RH/100), where R is the univer- A similar wide range of drying techniques
sal gas constant, T the absolute tempera- has been applied to vegetative material,
ture, V molal volume of water and RH although in this case the drying is gener-
relative humidity). However, under differ- ally for experimental purposes and only
ent RH conditions the material will dry at small quantities are dried. One of the few
different rates and will reach equilibrium examples of drying large quantities con-
cerns the alga Porphyra (Ooshua, 1993). As
after different times, ranging up to several
is the case with seeds, the various tech-
days.
niques differ in the degree of control and
Whatever drying method is adopted, the the drying rates achieved.
rate of drying is often ultimately deter- In experiments on tracheophytes, a com-
mined by the size of the tissue; large seeds mon method is to induce desiccation by the
will always dry more slowly than small simple expedient of withholding water (e.g.
ones. To overcome this limitation, it has Gaff et al., 1992; Reynolds and Bewley,
become common practice to dry embryonic 1993; Quartacci et al., 1997; Vander
Embryonic axes
Plastic mesh
Fan
Silica gel
Fig. 3.1. The improved apparatus for flash-drying of excised embryonic axes. A computer fan circulates air
through the silica gel and over the axes.
Willigen et al., 2001). This technique mim- 3.2.2. Quantification and modelling of
ics conditions and drying rates (usually drying rates
days to weeks) occurring in the natural
habitat of the plants, most drying appearing The factors determining drying rate are
to occur only after the soil has lost virtually manifold, many of them are difficult to
all its water (Sherwin et al., 1998; Norwood quantify and they change during the drying
et al., 1999). The size and nature of the process. Consequently, mechanistic model-
material (younger leaves enclosed or cov- ling of the drying process is extremely diffi-
ered by older ones) often results in uneven cult. During the drying process, water is
drying within a single plant. converted from the liquid form in the tissue
Faster drying rates (hours to days) have to the vapour phase in the surrounding air.
been achieved by using excised tissues The driving force is the difference in the
(twigs, leaves or callus) on the laboratory free energy of water between the tissue and
bench top, with forced ventilation in a lam- the air. As the tissue dries the free energy of
inar flow cabinet, or rapid air flow in a the water decreases, leading to a decrease in
small chamber (e.g. Bartels et al., 1990; the free energy differential and so to non-
Reynolds and Bewley, 1993; Quartacci et linear drying kinetics. There are a number
al., 1997; Farrant et al., 1999). Drying in of resistances retarding the diffusion of
small chambers over silica gel or saturated water from the tissue to the air. These
salt solutions, with or without stirring the include the boundary layer (the layer of still
air, has also been employed (Hetherington air in immediate contact with the tissue),
and Smillie, 1982; Bochicchio et al., 1998; the outer layer of the tissue (the nature and
Farrant et al., 1999). Non-vascular plants permeability of which vary) and the resis-
dry in the field within a few hours tance to the movement of water from the
(although this rate could be retarded by the interior of the tissue to the surface. The site
clumped nature of the growth form). of evaporation is not known, and so it is
Laboratory drying techniques are often unclear whether water moves from the inte-
chosen to simulate natural rates, within rior to the tissue surface in the liquid or
small enclosed chambers at RHs ranging vapour phase. The boundary layer will be
from 50 to 85% at constant temperature influenced by the speed of the air moving
(e.g. Seel et al., 1992; Oliver et al., 1993; over the tissue and by the size and shape of
Tuba et al., 1996). Very rapid drying has the tissue. With forced ventilation, the air
been achieved by the use of silica gel or of flow is probably turbulent, rather than lami-
a lyophilizer (Oliver and Bewley, 1984; nar, and so difficult to model. The size and
Oliver et al., 1998). shape of seeds and excised axes are often
An extremely important experimental irregular and variable, and so their effect on
aspect of drying vegetative tissue is the the boundary layer is also difficult to model.
light conditions during dehydration, as During the drying process, as the tissue
photo-oxidation can be an important com- dehydrates, the resistance to the transfer of
ponent of desiccation damage in photosyn- water from the interior to the surface will
thetic tissue (Seel et al., 1992; Sherwin and probably change, complicating the analysis.
Farrant, 1998; Tuba et al., 1998). Although All this complexity means that mechanistic
many seeds have green cotyledons that pre- modelling of drying rates must, by necessity,
sumably contain chlorophyll, the light con- be based on simplifying assumptions.
ditions during drying have not attracted Cromarty et al. (1985) have suggested a
the attention of seed biologists. It is also model for use in seed banks, based on satu-
possible that temperature could affect the ration vapour pressure at seed temperature,
response; seeds of Zizania palustris are velocity of air over the seed lot, seed mass
more tolerant of desiccation when dried at and oil content. The model assumes a thin
25°C than at lower (Kovach and Bradford, layer of seeds and a spherical shape.
1992; Berjak et al., 1994; Ntuli et al., 1997) Empirical models of drying rates are also
or higher (Ntuli et al., 1997) temperatures. not always successful. Depending on the
methods used, and the drying rates heterophyllus (J. Wesley-Smith, unpub-
obtained, different factors could be deter- lished data), C. australe (Govindasamy,
mining the rate of drying, and the rate-deter- 1997) and T. dregeana (Govindasamy,
mining factor could be changing during the 1997; Pammenter et al., 1999) were dried at
drying process. Although exponential rela- 96% RH (slowly), drying was exponential;
tionships sometimes can be fitted to the data when axes of these species were dried over
from a drying time course, particularly for silica gel (rapidly), the initial drying was
seeds or excised axes, it is our experience faster than predicted by an exponential
that in many cases the same form of equa- rate. Similarly, when seeds of Ekebergia
tion cannot be used to describe the data for capensis were dried slowly (seeds with
different drying rates (e.g. Pammenter et al., endocarp buried in silica gel; dehydration
1998). As a generalization, if tissue is dried over 10 days), drying was exponential;
relatively slowly, the relationship between when seeds were dried more rapidly (seeds
water content and drying time is exponen- without endocarp buried in silica gel;
tial. However, for material dried rapidly, the dehydration over 1 day), initial drying was
initial water loss is considerably faster than faster than predicted by an exponential
that predicted by an exponential relation- relationship (Fig. 3.2; Pammenter et al.,
ship. It must be re-emphasized that the 1998). These authors suggested that the
terms ‘slowly’ and ‘rapidly’ are relative. drying kinetics indicated that uneven dry-
There is no drying rate common across ing of the tissue might be occurring under
species where drying changes from faster rapidly dehydrating conditions. It does
than exponential to exponential; a fast dry- appear, however, that excised axes of
ing rate in one experiment could be the Theobroma cacao show exponential drying
equivalent of slow in another. over a range of drying rates (see Chapter 2).
For example, during ‘slow’ drying of
whole seeds of Landolphia kirkii
(Pammenter et al., 1991), and of Camellia 3.2.3. Effects of different drying rates
sinensis (Berjak et al., 1993), the water con-
tent of the axes within the seeds followed It is beyond the scope of this chapter to
an exponential relationship with time, but discuss in detail the consequences of dehy-
‘rapid’ drying of excised axes did not drating plant tissue. However, reference
(curve-fitting exercises were not reported must be made to the subject to understand
in the original publications; the data have the influence of drying rate on the response
been re-analysed). Similarly, initial faster- to dehydration. The effect appears to
than-exponential drying rates have been depend on whether the tissue is inherently
observed in rapidly dried excised axes of desiccation-sensitive or -tolerant.
Syzigium guiniense, Castanospermum aus-
trale, Trichilia dregeana, Artocarpus het-
3.2.3.1. Desiccation-sensitive tissue
erophyllus, Azadirachta indica, and the
radicle tips of axes of Podocarpus henkelii. Certainly with desiccation-sensitive (recal-
By way of contrast, axes of Avicennia citrant) seeds, or embryonic axes excised
marina and entire axes of P. henkelii (the from these seeds, material that is dried
embryonic axes of both species are rela- rapidly (of the order of tens of minutes to
tively large) show exponential drying hours) can survive to lower water contents
(N.W. Pammenter, P. Berjak and J. Wesley- before viability is lost than material that is
Smith, unpublished observations). It might dried slowly (over a period of days)
be argued that the same relationship can- (Normah et al., 1986; Pritchard and
not be used to describe ‘slow’ and ‘rapid’ Prendergast, 1986; Farrant et al., 1989;
drying because different material is often Pammenter et al., 1991, 1998; Pritchard,
used to obtain different drying rates: seeds 1991; Berjak et al., 1993; Pritchard and
for slow drying and excised axes for rapid Manger, 1998). The rapid drying is not
drying. However, when excised axes of A. actually increasing desiccation tolerance as
Axis water content (g water g–1 dry mass)
2.5
(a) (b)
2.0
1.5
1.0
0.5
0.0
0 2 4 6 8 10 0 10 20 30 40 50
Time (days) Time (h)
Fig. 3.2. Data illustrating that although the rate of water loss of slowly dried seeds (a) can be described by
an exponential equation, that of rapidly dried seeds (b) is not exponential. In (a) the line is an exponential fit
to the data; in (b) the dotted line is an exponential fit, the solid line is drawn by eye. Note the different time
scales. Data from Pammenter et al. (1998).
such; it is simply that, if the tissue is dried state, it rapidly loses viability (Walters et
fast enough, low water contents can be al., 2001).
achieved before sufficient time elapses for Studies on the effects of dehydration of
it to die. It has been suggested that some of desiccation-sensitive vegetative tissue are
the processes leading to viability loss are more limited. However, if much of the vol-
aqueous-based and so occur at relatively ume of the cells is occupied by fluid-filled
high (intermediate) water contents, of the vacuoles, mechanical damage associated
order of 1.0–0.3 g water g1 dry mass, cor- with volume reduction consequent upon
responding to water potentials of about drying might be important (Iljin, 1957;
1.5 to 14 MPa (Vertucci and Farrant, Vertucci and Farrant, 1995; Farrant et al.,
1995; Farrant et al., 1997; Pammenter and 1997), in which case drying rate might
Berjak, 1999; Walters et al., 2001). Material have little effect.
that is dried very rapidly passes through
these intermediate water contents so fast
3.2.3.2. Desiccation-tolerant tissue
that the damage caused by the deleterious
processes does not have time to accumu- In tissue that is, or becomes, desiccation-
late; thus viability loss does not occur as a tolerant, the effect of drying rate depends
consequence (Pammenter et al., 1998; upon the stage of development and/or the
Pammenter and Berjak, 1999). Although nature of the tolerance mechanisms.
rapid drying does permit viability retention Developing orthodox seeds start to
to low water contents, those tolerated by acquire desiccation tolerance concomitant
desiccation-sensitive seeds or axes (mini- with, or slightly preceding, reserve accu-
mum of about 0.2 g water g1 dry mass) are mulation, and the population as a whole
never as low as water contents usually is generally tolerant by the end of this
occurring naturally in dry desiccation-tol- stage (e.g. Bewley and Black, 1994). The
erant (orthodox) seeds (< 0.05 g water g1 response to rate of drying of orthodox
dry mass). Also, survival of these low seeds that are still in the desiccation-sen-
water contents by embryonic axes of recal- sitive stage of development is in almost
citrant seeds is apparent only if the mater- direct contrast to the response of recalci-
ial is assessed for survival immediately trant seeds. If, after histodifferentiation,
after drying. If the material is maintained prior to the acquisition of desiccation tol-
(at room temperature) in the partially dry erance, a developing orthodox seed is
dried rapidly, it will not survive; if it is chlorophyll during drying, whereas

dried slowly, it will (Kermode and Bewley, species that are poikilochlorophyllous lose
1985; Bewley and Black, 1994). This is chlorophyll and dismantle thylakoid mem-
probably because, during slow drying, suf- branes (Hetherington and Smillie, 1982;
ficient time elapses for the development of Sherwin and Farrant, 1998), although it is
the tolerance mechanisms. possible that some of the observed abnor-
The response of desiccation-tolerant malities are a consequence of the fixation
vegetative tissue (‘resurrection’ plants) to method (see Platt et al., 1997).
drying rate appears to vary with the Poikilochlorophylly appears to preclude
nature of the tolerance mechanism rapid drying rates and, although some
(reviewed by Oliver and Bewley, 1997). In homoiochlorophyllous plants can survive
non-vascular resurrection plants, toler- faster drying rates, there are other species
ance seems to be achieved predominantly that cannot (Farrant et al., 1999).
by an ability to repair damage caused by Excluding old leaves, which would natu-
desiccation, and is thus primarily based rally senesce, all tissues of most resurrec-
on constitutive mechanisms (Oliver and tion angiosperms are tolerant; however,
Bewley, 1997). Drying rate appears to have there are species in which only the young,
little influence on ultimate survival, but immature tissues are tolerant (Gaff and
recovery takes longer in rapidly dried tis- Ellis, 1974; Vander Willigen et al., 2001),
sue, perhaps suggesting the existence of suggesting that developmental stage is
some inducible protection mechanisms another compounding factor. Leaf tissues
(Schonbeck and Bewley, 1981a). It has been of some species show tolerance whether
observed that non-vascular resurrection attached or detached from the parent
plants tend to become less desiccation- plant, whereas others survive only
tolerant if kept in the hydrated state for attached, or after an initial drying phase
extended periods compared with daily on the parent plant, during which they
dehydration/rehydration ‘hardening’ cycles presumably acquire the necessary signals
(Schonbeck and Norton, 1979; Schonbeck for tolerance (Gaff and Loveys, 1992). A
and Bewley, 1981b). detailed discussion of these responses,
Resurrection tracheophytes have been and their underlying causes, is beyond the
classified as ‘modified desiccation-tolerant scope of this chapter. Suffice it to say that
plants’, in comparison with non-vascular these complexities must be borne in mind
resurrection plants (‘fully desiccation-tol- by the investigator when designing and
erant plants’), because their ability to sur- interpreting the results of experiments.
vive desiccation is rate-dependent (Oliver
and Bewley, 1997). The responses of resur-
rection angiosperms to drying technique 3.2.4. ‘Ultradrying’ of desiccation-tolerant
appears to be complex. Rapid desiccation material
is generally lethal, although Bochicchio et
al. (1998) have shown that it is water con- There has been recent interest in, and
tent, rather than drying rate, that affects lively debate on, the effects of storing
survival of detached leaves of the resurrec- orthodox seeds at water contents below
tion plant Boea hygroscopica. The general those normally used in gene banks. It is not
effect of drying rate on the response of res- intended to review that debate here, but
urrection tracheophytes is a consequence simply to point out that, even in desicca-
of an inducible desiccation tolerance, this tion-tolerant tissue, the experimental tech-
tolerance being based on protection during nique – the extent to which the material is
desiccation, rather than repair on rehydra- dried – may have an influence on the
tion. This group can be further subdivided response to drying (and subsequent stor-
into two, based on the strategy to prevent age). For more information, the reader is
light-associated damage on drying. The referred to Ellis (1998), Walters (1998) and
homoiochlorophyllous species retain Walters and Engels (1998).
3.3. Influence of Rehydration Technique or more slowly in misting chambers (Seel

et al., 1992, Tuba et al., 1996). As with
It is well known that if dry biological mate- dehydration, lighting and temperature
rial is immersed in water a number of sub- must be carefully controlled.
stances of low molecular weight will leak
from the tissue (discussed by Hoekstra et
al., 1999). If the tissue is desiccation-toler- 3.4. Length of Time in the Partially
ant, this leakage will subside with rehydra- Dehydrated State
tion, although, if the tissue is initially very
dry, leakage can be extensive and could be When desiccation-sensitive tissue is dehy-
damaging, this damage being exacerbated drated, it is subjected to a number of
by imbibition at low temperatures (Pollock, stresses as it dries. The type of damage that
1969; Hobbs and Obendorf, 1972). Because potentially can occur will change as the
of this, it is common in studies on seeds or water content decreases (see Chapter 9); as
excised axes to ‘prehumidify’ tissue by the intensity of the stress increases, the
maintaining it in a saturated atmosphere or effect on the tissue generally becomes more
placing on damp paper, before immersion severe. However, the effect of a stress, par-
in water. However, many recalcitrant seeds ticularly a mild stress, is not instantaneous.
are damaged at water contents far in excess If a stress induces a metabolic disorder, it
of those at which ‘imbibitional damage’ takes time for the damage consequent upon
occurs, and there is little evidence to sug- that disorder to accumulate. Thus, the
gest that such damage upon imbibition is effect of a stress depends not only on its
an important factor in desiccation-sensitive intensity, but also on the time for which the
seed material. Kovach and Bradford (1992) stress is applied. It is this concept of ‘inten-
initially ascribed the response to desicca- sity’ versus ‘duration’ of a stress that under-
tion of seeds of Z. palustris to imbibitional lies the confusion that has obscured the
damage, although Vertucci et al. (1995) interpretation of the effects of drying rates
suggested that the damage was a direct on desiccation-sensitive seed material.
result of desiccation, rather than imbibi- To unravel the confounding issues of
tion. Sacandé et al. (1998) have demon- water content and time in drying experi-
strated imbibitional damge exacerbated by ments, it is necessary to dry tissue almost
low-temperature imbibition and increased instantaneously to a range of water con-
storage time in seeds of neem (A. indica) at tents and then to maintain the material at
water contents < 8% (fresh mass basis), but these water contents. In practice, this is not
this is a water content considerably lower a simple experimental achievement. Most
than most recalcitrant seeds will survive. seeds are too large to dry rapidly and, if
In vegetative tissues, rehydration tech- isolated embryonic axes are dried, it is dif-
niques are generally even less well ficult to ‘store’ them in the partially dehy-
described and assessed than are the dehydrated state without some other deleterious
dration techniques, and consequently the conditions (such as anoxia or microbial
effects of various methods of rehydration proliferation) occurring. Maintaining vege-
are relatively unknown. In the tracheo- tative tissue in the partially dehydrated
phytes, whole plants are generally rewa- state is probably even more difficult and no
tered to field capacity with (Norwood et experiments are known where this has
al., 1999) and with or without (Sherwin been attempted.
and Farrant, 1998) additional aerial spray- Despite these difficulties, some informa-
ing. Dehydrated excised leaves are rehy- tion is available. Walters et al. (2001) have
drated by floating on or in water (Gaff and shown that partially dehydrated embryonic
Loveys, 1992; Reynolds and Bewley, 1993; axes of tea lose viability within a few days,
Bochicchio et al., 1998; Dace et al., 1998). and that the rate at which viability is lost
Mosses are rehydrated by immersion in depends on the water content. Similarly,
distilled water (Oliver and Bewley, 1984) isolated axes of T. dregeana dried over sil-
ica gel lost viability as the water content 3.5.1. ’Germination’

reached the level in equilibrium with the
desiccant, whilst axes dried at 96% RH lost With seeds, the most common assessment
viability some days after the tissue had method is to set them out to germinate.
reached equilibrium (Pammenter et al., However, as pointed out by Hong and Ellis
1999). Similarly, in the relatively long-lived (1996), it is possible that the treatment may
seeds of Araucaria huntsteinii, longevity at induce a dormancy and that seeds that do
6°C was reduced as water content was not germinate may not actually be dead.
reduced below about 45% (Pritchard et al., Those authors recommended that the dura-
1995). As an aside, this raises questions tion of germination tests be extended until
concerning the concept of ‘degree of recal- all non-germinated seeds have been posi-
citrance’. Would this be assessed on the tively identified as dead by the fact that they
basis of the minimum water content to rot. Another problem is that a seed may pro-
which the seed (or embryonic axis) can be duce a radicle, and so be scored as having
dried without loss of viability, or on the germinated, but be so damaged as to be
time for which it survives in equilibrium unable to establish a viable seedling (Fu et
with some predetermined water potential? al., 1993; Berjak et al., 1999). The precision
The concept of ‘intensity’ versus ‘duration’ of a germination test can be increased by fol-
of a stress has practical applications. It has lowing the time course of germination. An
been suggested that, in the case of recalci- increased lag before the first seed germi-
trant seeds that germinate in storage, partial nates or a decrease in the rate of germina-
dehydration may prevent this and so tion (increased time to 50% germination)
increase storage life span (Hong and Ellis, may indicate damage that is repaired during
1996). However, it appears that even a mild the lag phase. A simple assessment of final
water stress applied to seeds of the tropical germination would not reveal this damage.
species T. dregeana is deleterious (Drew et A number of suggestions have been made
al., 2000), and relatively mild partial drying concerning fitting equations to germination
of the temperate seeds of A. huntsteinii data (Brown and Mayer, 1988), but they gen-
(Pritchard et al., 1995) and Aesculus hip- erally require more samples than are often
pocastanum (Tompsett and Pritchard, 1998) available when undertaking studies on
can reduce longevity. Thus ‘sub-imbibed recalcitrant seeds from wild species.
storage’ should be approached with caution When experiments are conducted on
as it can actually reduce life span. excised embryonic axes, ‘germination’ can
In passing, it should be noted that even be assessed by placing the axes on damp
desiccation-tolerant organisms do not have paper in an enclosed chamber (such as a
an infinite life span in the dehydrated Petri dish), although it is common to use in
state; they accumulate damage in this state, vitro growth media. As many recalcitrant
and so desiccation could be considered to seeds, particularly from the tropics, har-
be a stress, even in tolerant tissues. bour fungal propagules (Berjak, 1996;
Calistru et al., 2000), their removal by suit-
able pretreatment is essential under these
3.5. Methods of Assessing Response to conditions. As with seeds, care must be
Dehydration taken in assessing ‘germination’. Swelling
and/or greening of an axis suggests that it
A variety of techniques have been used to is not dead (although a dehydrated axis
assess damage in response to drying. will swell on rehydration), but it does not
Different techniques measure different phe- necessarily imply that it is capable of pro-
nomena (membrane characterisics, respira- ducing an independent plantlet.
tory competence, photosynthetic activity), Assessment can also be complicated by
but the ultimate test is whether an indepen- choice of the medium, as this may have an
dent functioning organ(ism) can be re-estab- influence on the ‘growth’ of the tissue (as it
lished after dehydration and rehydration. does in normal in vitro multiplication and
propagation). Material that has been dam- damage associated with imbibition. With
aged, but not killed, by the dehydration seeds or excised axes there is generally
treatment may take a considerable time to good agreement between leakage character-
show signs of growth, and so care should istics and other signs of damage such as
be taken not to discard it too early. loss of vigour or viability (McKersie and
Tomes, 1980; Pammenter et al., 1991;
Berjak et al., 1992, 1993). Older techniques
3.5.2. Resurrection plants to assess membrane integrity involve the
use of vital dyes, which leak from damaged
With whole resurrection plants, the criteria cells (Gaff and Loveys, 1992).
for determining whether the organism is
‘functional’ may vary. This may simply be
on physical appearance (particularly the 3.5.4. Tetrazolium test
greening of poikilochlorophyllous tissue),
measurement of chlorophyll fluorescence The reduction of colourless tetrazolium
characteristics (Fv/Fm), which indicates the chloride (2,3,5-triphenyl tetrazolium chlo-
photochemical efficiency of photosystem ride (TTZ)) to a pink/red formazan dye is
II), or assessment of the ability to assimi- taken as a measure of respiratory activity, as
late carbon dioxide photosynthetically, or TTZ is reduced by components of the mito-
to respire. chondrial electron transport chain.
Although the tetrazolium test is used exten-
sively to assess the quality of orthodox
3.5.3. Electrolyte leakage seeds, its use in the study of desiccation
response has been limited. Ntuli et al.
A technique that is commonly employed (1997) showed that considerable differences
with small pieces of tissue (small seeds, occurred between the ability to germinate
excised axes, leaves of resurrection tra- and apparent viability as a result of tetra-
cheophytes, ‘pieces’ of non-vascular zolium tests, when investigating the desic-
plants) is to measure leakage of electrolytes cation response of Z. palustris, indicating
or of specific ions such as K+. An advan- that the two tests were not equivalent or not
tage of this technique is its simplicity and necessarily measuring the same thing. A
rapidity, especially using multiple-cell further caveat in using the TTZ test as a via-
electrical conductivity meters, which are bility assay is that a dead seed supporting a
commercially available. The extent of elec- vigorous mycelium internally will test posi-
trolyte leakage is considered to assess the tive as a result of fungal respiration.
degree of membrane damage (Bramlage et
al., 1978; McKersie and Tomes, 1980). As
the rate of leakage over the first few min- 3.5.5. Other responses
utes is often higher than the steady rate
established later, the steady-state rate of A number of biochemical and biophysical
leakage is often taken as an indication of responses to dehydration have been
membrane damage (McKersie and Stinson, assessed by a variety of workers. Examples
1980). An alternative approach is to assess include the activities of antioxidants
leakage (or rate of leakage) after a given (Tommasi et al., 1999), accumulation of late
time as a proportion of total leakage (or embryogenesis abundant proteins (Finch-
maximum rate), which occurs when all Savage et al., 1994; Gee et al., 1994), ethyl-
membranes are fully disrupted by treat- ene production, respiration and protein
ments such as homogenizing, boiling, auto- synthesis (Salmen Espindola et al., 1994),
claving or repeated freeze/thaw cycles. It is partitioning of amphipathic molecules
common practice to prehumidify tissue in between cytosol and membranes (Golovina
a saturated atmosphere or on damp filter et al., 1998) and changes in cytoplasmic vis-
paper prior to immersion to reduce any cosity (Leprince and Hoekstra, 1998). These
are not covered here because often it was change in ‘water content’ reflects the pro-
the details of these responses that were the portional change in the amount of water in
objectives of the investigations, and so they the tissue; if the water content changes
do not fall within the scope of a discussion from 1.0 to 0.5 g water g1 dry weight, the
of general experimental approaches. tissue has lost half its water. If the data are
expressed on a fresh mass basis, for tissue
at 1.0 g water g1 dry mass that loses half
3.6. Expression of Water Content Data its water, water content on a fresh mass
(see also Chapter 2) basis changes from 50% to 33.3%.
3.6.1. Mass basis

3.6.2. Water potential
The amount of water in plant tissue has
been expressed in a number of different The responses of tissue to drying are deter-
ways, but generally most commonly on mined by the processes that occur during
some form of ‘mass’ basis. This is probably drying. The processes that occur at any
because it is the simplest measure to water content are influenced by the free
obtain; the hydrated/partially hydrated tis- energy status of the water, and so it is bio-
sue is weighed, dried for some predeter- logically more meaningful to express tissue
mined time, and reweighed. The water in terms of water potential rather
International Seed Testing Association rec- than water content. Water potential of
ommends drying at 103°C for 17 h (ISTA, small pieces of tissue can be measured by
1999); an alternative approach, particularly thermocouple psychrometry, but this is
if using small amounts of tissue, is to dry at limited to higher potentials (above about
a lower temperature (to reduce loss of non- 5 MPa). However, recent technical
water volatile material) to constant weight. advances permit dewpoint psychrometric
The data can then be expressed on a fresh measurements to much lower water poten-
mass basis (mass of water per unit fresh tials. Sorption isotherms (equilibrating tis-
mass, often presented as a percentage); this sue at known fixed RH to constant water
indicates the proportion of the hydrated or content) can be used to establish the rela-
partially dehydrated tissue that is water. tionship between water content and water
Alternatively, the data can be expressed on potential (Vertucci et al., 1994), although
a dry mass basis (mass of water per unit this is difficult at high water potentials
dry mass). Strictly speaking, when express- because the relationship between RH and
ing the data on a mass basis, the term water potential changes rapidly in this
‘water content’ is incorrect; this should be region. Soaking tissue in solutions of
reserved for expressing the absolute known concentrations (and hence known
amount of water in the tissue, irrespective water potentials) of non-penetrating solutes
of the quantity of tissue. What is generally such as polyethylene glycol (PEG) 8000 has
described as ‘water content’ is actually a been used to assess the water
‘water concentration’ (the amount of water content/water potential relationship at
per unit amount of fresh or dry tissue). high water potentials (Vertucci et al., 1994;
Thus, water content on a dry mass basis Pritchard et al., 1995; Tompsett and
should be termed ‘dry mass-specific water Pritchard, 1998). A complication of estab-
concentration’. However, use of the term lishing sorption isotherms is the phenome-
‘water content’ to describe a ‘water concen- non of hysteresis; the equilibrium water
tration’ is so deeply entrenched that it is content at any RH depends on whether dry
unlikely to change. We prefer data to be tissue is being hydrated or hydrated tissue
expressed on a dry mass basis. In this case, is losing water (for a discussion, see Eira et
the basis to which values are being normal- al., 1999). There is an additional problem
ized does not change as the amount of when working with recalcitrant seeds or
water changes, and the proportional axes of tropical species. This material gen-
erally harbours very high levels of fungal complication when calculating RWC is the
propagules, and fungal proliferation almost estimate of water content at ‘full turgor’. If
invariably accompanies attempts to equili- recalcitrant seeds are immersed in water,
brate tissue with atmospheres of RH above they will take up water as they germinate,
about 80%; it is therefore impossible to and so full turgor cannot be equated with
discriminate between the contribution of water content of tissue imbibed in water.
the seed material and the fungal mycelium Data could be expressed relative to the
to the derived isotherm. Under these con- water content at shedding, but this can be
ditions, use of concentrated solutions of very variable among seeds within a harvest,
PEG 8000 is advised. as well as between collections. With vegeta-
tive tissue it is possible to over-hydrate tis-
sue such that liquid water occupies some of
3.6.3. Relative water content the intercellular air spaces in leaves, or
exists as intercellular or surface water in
Relative water content, RWC (amount of non-vascular plants (Beckett, 1997). These
water in the tissue/amount of water at full effects lead to overestimates of water con-
turgor), has also been used to express the tent at full turgor.
water status of tissue (see Grange and Finch-
Savage (1992) for seed tissue, and Vander
Willigen et al. (2001) for vegetative tissue). 3.7. Conclusion
It is more meaningful than simple water
contents, although relative cell volume When investigating the response of tissue
(which is based on symplastic water only) is to dehydration, a range of techniques are
probably a better measure of direct stress to available. However, the observed response
which the tissue is subjected. To assess the is likely to depend on factors such as the
proportions of apoplastic and symplastic drying method, and possibly the rehydra-
water in tissue requires the construction of tion technique and method of assessment.
pressure–volume curves. Not only is this The techniques adopted will depend on
difficult (because of the difficulty of measur- the size or amount of tissue being dried,
ing water potential at low water contents), the facilities available and, importantly, the
but it is possible that the assumptions purpose of the investigation. The investiga-
underlying the analysis of pressure–volume tor should be aware of the technical com-
curves (Tyree and Hammel, 1972) may not plications when assessing and interpreting
hold at low water contents. An additional the data obtained.
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4 Biochemical and Biophysical Methods for

Quantifying Desiccation Phenomena in Seeds
and Vegetative Tissues
Olivier Leprince1 and Elena A. Golovina2,3

1UMR Physiologie Moléculaire des Semences, Institut National d’Horticulture, 16 Bd
Lavoisier, F49045, Angers, France; 2Laboratory of Plant Physiology, Department of
Plant Sciences, University of Wageningen, Arboretumlaan 4, 6703 BD Wageningen,
The Netherlands; 3Timiryazev Institute of Plant Physiology, Botanicheskaya 35,
Moscow 127276, Russia

4.2. Caveats: the Consequences of Being Dry 112
4.3. How to Study Biochemical Responses to Drying 114
4.3.1. Responses of gas exchange and volatile emission to drying 114
4.3.1.1. Headspace analysis 114
4.3.1.2. Laser photoacoustic spectroscopy (PA) 114
4.3.2. NMR applications to measure steady-state concentrations
and to assess metabolic responses to drying 115
4.3.3. Photosynthesis studies 116
4.3.4. Oxidative stress and anhydrobiosis 116
4.4. Spectroscopy Techniques 119
4.4.1. Electron paramagnetic resonance (EPR) 119
4.4.1.1. General description 119
4.4.1.2. Applications of EPR methods 120
4.4.2. Nuclear magnetic resonance 127
4.4.2.1. General description 127
4.4.2.2. The NMR study of water in living systems 128
4.4.2.3. NMR imaging 130
4.4.2.4. High-resolution multinuclear NMR spectroscopy 131
4.4.2.5. Structure and dynamics of cellular membranes 133
4.4.3. Fourier transform infrared (FTIR) spectroscopy 134
4.4.3.1. General description of infrared spectroscopy 134
4.4.3.2. Biological applications 134
4.5. Additional Techniques to Study Biochemical and Biophysical
Aspects of Desiccation Tolerance 136
4.5.1. Differential scanning calorimetry (DSC) 136
4.5.2. Electron microscopy 136
4.6. Acknowledgements 137
4.7. References 137
112 O. Leprince and E.A. Golovina
4.1. Introduction resort to non-invasive techniques that

enable the investigator to assess any bio-
In recent years, the development of physi- chemical phenomenon in drying tissues
cal techniques has brought substantial without introducing water during the
insights into the physical state of water and analysis. The few techniques available are
cellular components of desiccated systems. briefly presented; and (ii) characterize
These techniques cover a large range of mutants and/or transgenic plants the phe-
spectroscopic techniques such as nuclear notypic traits of which can be associated
magnetic resonance (NMR), electron para- both with a particular biochemical path-
magnetic resonance (EPR, also referred to way and the level of desiccation tolerance
as electron spin resonance (ESR)), Fourier (Chapter 12; Wolkers et al., 1998a; Shiota
transform infrared (FTIR) spectroscopy as and Kamada, 2000; Weber et al., 2000;
well as dielectric measurements and Wehmeyer and Vierling, 2000). For exam-
calorimetry. The success of these tech- ple, the antisense inhibition of ADP-glu-
niques and spectroscopy in particular orig- cose pyrophosphorylase, a key enzyme in
inated in their versatility and in their starch synthesis, was found to increase the
ability to assess the physical state of desic- sucrose and nitrogen content of mature
cated systems by non-invasive means. seeds of transgenic Vicia narbonensis.
Below, the resourcefulness and limitations Also, the decrease in ADP-glucose
of the physical techniques are reviewed. pyrophosphorylase activity altered the cel-
In contrast to physical aspects, most of lular volume and water relations during
the fundamental questions pertaining to the seed-filling phase (Weber et al., 2000).
the biochemical aspects of desiccation These observations show that a specific
tolerance in anhydrobiotes remain to be alteration in carbon metabolism has
answered. The lack of non-destructive pleiotropic effects on seed development
and sensitive techniques has greatly and illustrate the potential of molecular
impeded our understanding of the role of biology to assess non-destructively the role
metabolism in desiccation tolerance. of various biochemical and biophysical phe-
Furthermore, all of our biochemical assays nomena related to desiccation tolerance.
and isolation of organelles have been set
up in dilute solutions using water or
organic compounds as solvents. However, 4.2. Caveats: the Consequences of Being
they have been indiscriminately applied to Dry
drying and desiccated specimens. Here it
is argued that the experimental approach Since water acts as a solvent and substrate
of grinding dry or nearly dried specimens in the cell in a variety of ways, its reduced
in aqueous buffers and measuring meta- availability in dried tissues will induce a
bolic activities and biological markers of set of physical and biochemical responses
oxidative stress in vitro in dilute solutions that may disappear during an invasive
is unlikely to reflect the in vivo situation. measurement, thereby confusing the inter-
This methodology has complicated the pretation of the data. Before ascertaining a
interpretation of data regarding biochemi- cause–effect relationship between desicca-
cal activities associated with different tion tolerance and a specific biochemical
hydration levels (Lynch and Clegg, 1986; and biophysical process, the following
Vertucci and Leopold, 1986). Hoekstra and remarks should be taken into account:
van Roekel (1983) have clearly illustrated
how isolation-inflicting injury of isolated 1. To adequately link the response of
mitochondria in germinating pollen can metabolism to drying with desiccation tol-
confuse the interpretation of results erance, it is necessary to map metabolic
obtained in vitro. To partially overcome activities as a function of water content or
this technical bottleneck, two strategies water potential of the drying cell and not
can be adopted: (i) whenever possible, as a function of time of drying (Vertucci
Methods for Quantifying Desiccation Phenomena 113
and Leopold, 1986; Leprince et al., 1999, oxidative reactions) occurring during nat-
2000). This necessity is more acute when ural ageing (i.e. below Tg) are compared
accumulation of dry matter occurs during with those occurring during accelerated
development. Often a sensitive microbal- ageing (between Tc and Tg (for example,
ance is needed to determine fresh and dry 75–85% relative humidity (RH) and tem-
weights of milligram quantities of samples. peratures between 35 and 50°C) or above
Furthermore, fast measuring techniques Tc (100% RH, 41°C)). This is illustrated in
should be preferred over time-consuming the study of Lievonen et al. (1998) on non-
assays so that the rates of water loss match enzymatic browning reaction rates around
the time necessary to acquire the data. the Tg of mixtures made of water, glycerol
Furthermore, Hendry (1993) argued that and maltodextrin. It is also clearly illus-
attempts to characterize desiccation phe- trated in the kinetics of seed and pollen
nomena in drying tissues should be set ageing (see, for example, Buitink et al.,
against a range of desiccation-induced 2000g and references therein).
damage and not only against percentages of 3. To be quantified and characterized,
survival after drying. metabolites, proteins, DNA, organelles,
2. It is important to recognize that during etc., must be extracted and purified
drying the cytoplasmic viscosity increases beforehand. This procedure strongly
dramatically until glass formation depends on the tissue water content. This
(Leprince and Hoekstra, 1998; Buitink et is valid for both water-soluble compounds
al., 2000e; Chapters 2 and 10). Unless using aqueous extraction and lipid-solu-
enzymatic activities are assessed in an ble compounds using organic solvents.
environment similar to that found in the For lipid extraction and separation using
drying cells, we do not know how the rise the Folch’s method, Hamilton et al. (1992)
in viscosity during drying will affect meta- recommended that the amount of water
bolic rates and/or pathways. We can present in the tissues should be calculated
already predict that O2-processing systems and taken into account during the differ-
will be altered by the loss of water since ent washing and partitioning procedures.
O2 solubility is known to decrease with For aqueous extraction, we do not know
rising viscosity (Gros et al., 1992; Leprince whether the extraction and purification of
and Hoekstra, 1998). Biochemical events water-soluble metabolites or proteins dif-
during drying should obey different laws fer quantitatively and qualitatively when
of diffusion since the cytoplasm will tissues are ground either fresh or dried.
undergo a physical transformation from a This point is particularly relevant for
liquid state to a solid-like state (i.e a glass). amphiphilic molecules such as phenolic
The moisture contents at which these compounds, which are likely to partition
changes in diffusion characteristics occur into the membranes and/or oil bodies dur-
during drying should preferably be deter- ing drying and vice versa during rehydra-
mined. It has been suggested that this tion (Chapter 10; Golovina et al., 1998;
moisture content corresponds to the glass Buitink et al., 2000e). Thus, the desicca-
formation that is measured by the tempera- tion-induced changes in metabolite con-
ture at which the drying cytoplasm forms a centrations and in protein conformation
glass (Tg). Below Tg, solid state should be interpreted with caution if these
physics/chemistry prevails (Chapter 10). changes are assessed from crude extracts.
However, Buitink et al. (2000f) suggested 4. Most of the methods used so far are
that the most important change in the averaging techniques. However, it is
physical properties of the cytoplasm likely that there is a gradient of water
occurs 50°C above Tg, at a temperature cor- within seed tissues during dehydration
responding to the so-called collapse tem- and rehydration. Therefore, a qualitative
perature (Tc). A distinction between liquid and quantitative gradient of responses
state and solid state should be made when within the tissues submitted to drying
chemical and biochemical events (such as might be expected.
4.3. How to Study Biochemical ter (Rogerson and Matthews, 1977; Vertucci
Responses to Drying and Leopold, 1986, 1987). In these tech-
niques, the gas to be analysed has to accu-
Two technically different strategies are mulate over time in a closed environment
available to probe the responses of metabo- before taking the measurement (i.e. static
lism to drying by non-invasive means: the headspace analysis). To reach a detectable
detection and analysis of gases that concentration, the gas accumulation may
emanate from, or are absorbed by, the dry- take some time, particularly in drying sam-
ing tissues (so-called headspace analysis) ples in which the metabolism and gas diffu-
and the resort to in vitro or in vivo NMR. In sion are greatly reduced by the lack of
photosynthetic anhydrobiotes, fluorescence water. Consequently, the assay may be too
spectroscopy is also a convenient tool to slow in comparison with the rate of water
characterize energy metabolism during dry- loss. Furthermore, an additional problem is
ing (see Chapter 7). The methods that reliably maintaining the specimen at the
assess free-radical-induced damage in des- same water content during the measure-
iccation tolerance are also reviewed. ment. Thus, unless the kinetics of water
loss matches the time frame needed to
assess the metabolic rates, the relation
4.3.1. Responses of gas exchange and between hydration levels and metabolic
volatile emission to drying activities in drying tissues may not be accu-
rate. Therefore, it is best to adapt a flow-
4.3.1.1. Headspace analysis through system coupled to an active
trapping system (i.e. dynamic headspace
A wide variety of gaseous metabolites can analysis). A flow of dry or humidified air
be studied in drying tissues: O2 and CO2 passes over the sample acting as both a gas
exchange as markers of respiration or photo- carrier and dehydrating agent. The volatiles
synthesis, ethanol and acetaldehyde as are then absorbed by a compound located
markers of fermentation, alkanes, alkones, in the exit flow and analysed by GC (Wilson
alkenes, aldehydes (volatile) as markers of and McDonald, 1986; Zhang et al., 1994).
oxidative stress. Several instruments such For dried tissues that are in a glassy state,
as infrared CO2 analysers, gas-phase O2 elec- the release of volatiles may take several
trodes or O2 analysers can detect and days or weeks because of the slow diffusion
analyse CO2 and O2 exchanges. These types of molecules. To overcome this problem,
of analysers have mainly been used to several authors have used a thermal desorp-
describe the effects of desiccation on photo- tion technique consisting of heating the dry
synthetic activities of resurrection plants specimens to at least 60°C. This procedure
(Schwab et al., 1989) and photosymbiotic is thought to purge the volatiles that are
lichens (Nash et al., 1990; Scheidegger et trapped in the glassy matrix (Wilson and
al., 1995). However, they are not always McDonald, 1986; Hailstones and Smith,
suited to assaying low respiration rates from 1989; Zhang et al., 1994; Degoussée et al.,
nearly dried material because of their low 1995). However, it is not always possible to
sensitivity (O. Leprince, unpublished data). determine whether the production of
Gas exchange rates and volatile produc- volatiles after desorption results from the
tion can also be measured by gas chro- heat treatment per se or not (Wilson and
matography (GC) (Kimmerer and McDonald, 1986).
Kozlowski, 1982; Gorecki et al., 1984; Klein
and Sachs, 1992; Leprince and Hoekstra,
4.3.1.2. Laser photoacoustic spectroscopy
1998; Leprince et al., 1999), gas chromatog-
(PA)
raphy–mass spectrometry (GC-MS) (Zhang
et al., 1994), high-pressure liquid chro- PA is an emerging technique that has over-
matography (HPLC) (Degoussée et al., 1995) come the problems associated with head-
or by using a Gilson differential respirome- space analysis. A PA set-up consists mainly
of two components: a line-tunable CO laser appears to be the most appropriate tech-

that excites gas molecules specifically nique for this purpose (Shachar-Hill and
according to their infrared fingerprint Pfeffer, 1996; Roberts, 2000). Several strate-
absorption, and parallel resonators. Each gies can be adopted using 13C-, 31P-, 14N- or
resonator is coupled to a sensitive micro- 15N-NMR, depending on the nature of the
phone in which the concentration of gases metabolite to be analysed. NMR studies

is sequentially measured based on an may or may not be destructive. The princi-
acoustic phenomenon (Harren and Reuss, ples of NMR spectroscopy and the advan-
1997; see Zuckerman et al., 1997, tages and disadvantages of in vivo
www.sci.kun.nl/tracegasfac/experime.htm applications as a non-invasive technique
for technical details of the experimental will be described below in Section 4.4 (see
set-up). This technique permits the probing also Shachar-Hill and Pfeffer, 1996).
of metabolic processes non-invasively that A first strategy is to monitor dynamic
result in the emission and/or absorption of changes of natural or enriched nuclei in the
ethylene, acetaldehyde, ethanol and CO2 samples over different intervals during dry-
from drying tissues without altering the ing. This approach may or may not be
water content (Leprince et al., 2000). destructive. In both cases, it yields dynamic
Furthermore, technical improvements are information about metabolic fluxes and fast
being made to allow the analysis of lipid responses of metabolism to physiological
peroxidation products such as ethene, perturbations. The natural abundance of 13C
ethane, pentane and hexane. PA techniques is only 1.1%. Thus 13C-NMR is not a sensi-
offer two important advantages over con- tive method and requires concentrations in
ventional headspace analysis: (i) biologi- the mM range. However, one can take
cally relevant gases can be detected with a advantage of this low sensitivity to trace
sensitivity limit of 100- to 1000-fold higher some metabolic changes by the detection of
than GC; and (ii) the time response is less compounds that accumulate to high levels.
than 1 min. The PA is set up as a flow- These metabolites include compatible
through system. The gas employed to dry solutes that accumulate in cyanobacteria
the tissues is also the carrier of the (Reed et al., 1985), sugars and oil in seeds
gas/volatile to be analysed. The disadvan- (Rutar, 1989; Ishida et al., 1990, 1996;
tages are: (i) to our knowledge, the access to Koizumi et al., 1995), and trehalose in fun-
PA is restricted to a handful of laboratories gal spores (Bécard et al., 1991). The non-
in The Netherlands (www.sci.kun.nl/ invasive character of NMR may allow the
tracegasfac/), Germany, Italy and the US; (ii) time-course of metabolic events to be
the equipment is not commercially avail- directly followed and the subcellular local-
able and the current prototypes require the ization of some metabolites to be deter-
assistance of physicists and engineers to mined, for instance, in maturing or
take and process the measurements; (iii) a germinating seeds (Colnago and Seidl,
limited range of biologically relevant gases 1983; Ishida et al., 1990, 1996).
can be measured; and (iv) water vapour Alternatively, since the natural abun-
strongly interferes with the measurement dance of 13C is low, it is possible to label
and must be totally removed from the gas. specific metabolites and monitor their fate
through the cellular network of metabolic
pathways in vivo or in vitro with crude
4.3.2. NMR applications to measure steady- extracts (Dieuaide-Noubhani et al., 1995;
state concentrations and to assess metabolic Roberts, 2000; Roscher et al., 2000).
responses to drying (see also Section 4.4.2) Commercially n-13C-labelled sugars are
available for almost every carbon position
To determine the effects of desiccation on of sucrose, glucose and fructose molecules.
the dynamics of metabolic pathways, the Similarly 31P- and 14N-labelled compounds
flux of metabolites through the different can be used to monitor the dynamics of
paths must be known. NMR spectroscopy phosphorylated metabolites and amino
acids, respectively. Whether in vivo NMR 4.3.4. Oxidative stress and anhydrobiosis
is applicable to drying tissues remains to
be ascertained. A particular problem is to Whether oxidative stress is a cause or an
maintain reliably the specimen in the same effect of desiccation sensitivity has yet to
functional state and with the same water be resolved due to a large body of conflict-
content during the NMR measurements. ing evidence in the literature. The core of
A second strategy, which is not mutu- the problem is two fold. From a physiologi-
ally exclusive to that above, is to screen cal point of view, Hendry (1993) argued
chemical fingerprints of crude extracts that attempts to correlate free radical
obtained from different hydration levels. processes with desiccation tolerance must
This application will yield analytical infor- be done in relation to the characterization
mation about metabolites in a steady state. of other desiccation-induced damage. This
By comparing spectra of crude extracts must be done in order to assess whether
obtained during drying, it should be possi- free radicals generated during drying are a
ble to pinpoint the metabolites, the concen- cause or an effect of the loss of viability.
trations of which are mostly sensitive to Echoing the earlier review of Gutteridge
changes in water content during drying and Halliwell (1990) and Leprince et al.
(Fan, 1996; Noteborn et al., 2000). Roughly (1990), he pointed out that free-radical-
0.5–1 g of fresh material is often required mediated injury can occur before or after
to take an NMR spectrum, which could be the time of death during drying (Hendry,
a limiting factor if the amount of biological 1993). Thus, great caution should be exer-
material is restricted. cised in ascertaining whether oxidative
stress plays a role in desiccation-induced
injury and loss of desiccation tolerance.
4.3.3. Photosynthesis studies Oxidative injury in both animals and plants
generally results from stress-induced meta-
The recent methods to assess photosyn- bolic disturbances, particularly in the elec-
thetic activities have become non-destruc- tron transport chains. Considering the
tive. They exploit the interactions between technical difficulties in estimating meta-
light, gas exchange, operation of the photo- bolic activities during drying (see Section
synthetic electron transport and ambient 4.3, p. 114), even greater care should then
conditions. For these reasons, they are be taken in attempts to link oxidative dam-
widely used to study the photosynthetic age to desiccation-induced metabolic per-
responses to environmental stresses turbation. Various pathologies and
(Bukhov et al., 1989; Foyer et al., 1994). degenerative diseases have been linked to
They have also been applied to compare the mitochondrial dysfunction and generation
photosynthetic responses of anhydrobiotes of reactive O2 species (ROS) in mammals
with those of desiccation-sensitive plants (Yates and van Houten, 1997; Esposito et
(Schwab and Heber, 1984; Schwab et al., al., 1999; Wallace, 1999). Thus, it should
1989; see Chapter 7). The most applied be interesting to see whether parallels exist
technique is chlorophyll a fluorescence, between animal and plant anhydrobiotes.
which assesses the efficiency of electron The second problem regarding the puta-
transport through photosystem II and non- tive role of oxidative stress in desiccation
photochemical quenching processes associ-
tolerance and ageing is the methodology
ated with it. Light-induced electron
that has been employed so far. A survey of
transport in photosystem II can be studied
methods employed to detect oxidative stress
using fluorescence induction kinetics
in seeds can be found in Benson (1990).
(Vertucci and Leopold, 1986). Light-scatter-
ing measurements at 535 nm are also useful 1. The array of techniques that have been
to gain insights into the physical and chem- employed to assess the role of free-radical-
ical events associated with the formation of induced injury in anhydrobiotes is very
a membrane potential in thylakoids. small. A survey of the literature on molecu-
lar markers used to estimate free-radical- function and cell death (Yates and van
induced damage during drying or in the Houten, 1997). Oxidation of proteins by
dry state shows that the results are based ROS can induce protein fragmentation or
mostly on a thiobarbituric acid-reactive enzyme inactivation, leading to the disrup-
substances (TBARS) assay and a non-inva- tion of glycolysis (Hyslop et al., 1988) and
sive EPR spectroscopy technique (Table the Calvin cycle (Kaiser, 1979). Protein oxi-
4.1). The former measures malonyldialde- dation has been linked to various patholo-
hyde (MDA) as a breakdown product of gies in humans (Dean et al., 1993; Berlet
lipid peroxidation using a simple and and Stadtman, 1997) and to seed ageing
rapid assay (Heath and Packer, 1968) and (Zhang et al., 1997).
the latter estimates a carbon free radical of 3. Analytical procedures to estimate lipid
unknown origin (Hendry, 1993). As dis- hydroperoxides in crude extracts are
cussed below, doubts have been cast as to fraught with potential artefacts (Gutteridge
whether these two assays are reliable and Halliwell, 1990; Hageman et al., 1992;
enough for all anhydrobiotic material. Meagher and Fitzgerald, 2000). The prob-
2. Table 4.1 also shows that, in 98% of lems are numerous, ranging from the pres-
these studies, lipid peroxidation was mea- ence of contaminants (metal ions) that
sured as a marker of free-radical-induced initiate lipid peroxidation during tissue
injury. It must be recognized that proteins grinding to instability of peroxidized lipids
(Dean et al., 1993; Berlet and Stadtman, during the extraction and lack of sensitiv-
1997) and DNA (von Sonntag and ity. Furthermore, the nature of peroxidative
Schuchmann, 1987; Hageman et al., 1992; damage depends on the type of free-radical
Wiseman and Halliwell, 1996) are also sen- initiator and the membrane or lipid compo-
sitive to free radicals, albeit less than fatty sition (McKersie et al., 1990). Thus, the
acids. Interestingly, mitochondrial DNA is lipid peroxidation assays currently used in
more sensitive to ROS than nuclear DNA. seed science cannot be indiscriminately
Free-radical-induced DNA damage can sig- applied to all seeds. Unfortunately, the
nificantly contribute to mitochondrial dys- studies surveyed in Table 4.1 did not
Table 4.1. Occurrence and types of methods employed to determine free-

radical damage in drying and/or ageing seeds, pollen and vegetative
tissues. Examples of free-radical damage specific to DNA are oxidized
nucleotides such as thymine glycol, 8-hydroxy-2-deoxyguanine and
methylguanine (Hageman et al., 1992). Examples of free-radical damage to
protein are iminopeptides, carbonyl content and glutamyl-semialdehyde
residues of oxidatively modified proteins (Berlet and Stadtman, 1997).
Number of studies
over the past three
Methods decades
MDA or TBARS determinationa 17
Determination of an organic free radical by
in vivo EPR spectroscopy 11
Free fatty acid determination 6
Chemical modifications of lipids (e.g. conjugated
dienes, fatty acid composition) 5
Other techniques (determination of breakdown
products resulting from lipid peroxidation,
chemiluminescence, fluorescent probes) 9
Free-radical damage specific to DNA 0
Free-radical damage specific to protein 2
aMDA, malonyldialdehyde; TBARS, thiobarbituric acid-reactive substances.
assess whether the marker used as an index known to accumulate in desiccation-toler-

of oxidative injury was sensitive or appro- ant systems. Anthocyanins accumulate to
priate for the biological material or experi- large concentrations in leaves of resurrec-
mental conditions. For example, in oily tion plants. Hodges et al. (1999) have intro-
seeds, lipid peroxidation is dependent on duced several modifications to overcome
the triacylglycerol composition and struc- this interference in leaf extracts. However,
ture of oil reserves (Neff et al., 1992). the suggested improvements did not
Furthermore, in an assay of peroxidized appear to be reliable for the results shown
lipids of a crude extract obtained from oily in Table 4.2.
seeds, the triacylglycerol fraction may 5. An organic stable free radical has been
mask more important and significant linked to respiration, oxidative stress and
changes occurring in membrane lipids. desiccation tolerance (Hendry et al., 1992;
Another example further illustrates the Hendry, 1993; Leprince et al., 1995).
point. Table 4.2 compares two methods However, measurements of this organic
estimating the level of peroxidized lipids radical using non-invasive EPR have gener-
in germinating pea axes in relation to the ated conflicting evidence concerning its
loss of desiccation tolerance. Using the link to desiccation tolerance (Hendry,
assay developed by Jiang et al. (1991), 1993). Its chemical nature and localization
results suggest that there is a link between should be identified to ascertain whether
an increase in oxidative damage following this is a reliable method to estimate oxida-
drying and the loss of desiccation toler- tive stress in seeds. The fact that the EPR
ance. In contrast, the TBARS assay pro- signal is sensitive to liquid water makes it
vides inconclusive evidence. difficult to use in drying tissues. Studying
frozen specimens could lessen the problem
4. The limitations of the TBARS test have
but this approach will not be able to tell
been known for several years (Gutteridge
whether the radical is generated by drying
and Halliwell, 1990; Hodges et al., 1999)
or by freezing.
and include a lack of sensitivity and speci-
ficity and a tendency to overestimate MDA From these remarks, we conclude that
contents. Recently, it has been shown that positive results from a lipid peroxidation
several compounds commonly found in assay will provide evidence that a free-radi-
plant extracts (e.g. sugars, oligosaccharides, cal reaction has occurred either during
anthocyanins) also react with thiobarbi- drying or during drying and extraction. A
turic acid, thereby interfering strongly with negative result will not provide any evi-
the peroxidized products (Table 4.3). Non- dence one way or the other. Therefore, it is
reducing sugars and oligosaccharides are suggested that a range of assays should be
Table 4.2. Comparison of two methods measuring lipid peroxidation as a marker of oxidative damage in
crude extracts of germinating axes of pea before and after fast drying. TBARS were measured as in
Leprince et al. (1990) and calculated as in Du and Bramlage (1992) to take into account interference by
sugars. Lipid hydroperoxide levels were measured using the xylenol orange/ammonium sulphate reagent
according to the method of Jiang et al. (1991) and quantified using H2O2 as a standard. Data are the
average (± SE) of 3–5 replicates (O. Leprince, J. Fajerman and F.A. Hoekstra, unpublished observations).
TBARSa Lipid peroxide
Sensitivity to drying Treatment (mol mg1 dw) (mol equiv. H2O2 mg1 dw)
Tolerant Fresh 2.76 ± 1.93 15 ± 3
Dried 9.89 ± 1.79 16 ± 2
Intolerant Fresh 33.05 ± 15.01 22 ± 2
Dried 27.12 ± 9.00 71 ± 15
aTBARS, thiobarbituric acid-reactive substances.
dw, dry weight.
tested as a necessary prerequisite to 4.4. Spectroscopy Techniques

address the role of free-radical processes
in desiccation tolerance and seed As follows from a consideration of quan-
longevity. To be validated, these assays tum mechanics, an atom or molecule has
should be carried out on material that has discrete energy states. Spectroscopy is the
been treated with agents to generate oxida- measurement of the energy differences
tive stress such as ultraviolet (UV) radia- between these states. The energy differ-
tion and free-radical generators such as ences E can be measured by the absorp-
paraquat or H2O2. tion spectra of electromagnetic radiation.
An increasing number of methodologies In conventional spectroscopy, the fre-
are currently being developed to analyse quency is varied and the frequency at
free-radical-induced damage in vitro and in which maximal absorption occurs reflects
vivo. Most of these methods are derived the difference between the states. The fre-
from studies on animals, humans or food- quencies vary from the MHz range for NMR
stuffs. The challenge will be to adapt these to the GHz (microwave) range for EPR
techniques to seed science and anhydrobi- spectroscopy. The frequencies for absorp-
ology, keeping in mind the caveats tion spectroscopy range from 1012 Hz for IR
described in this chapter and the guide- to 1016 Hz for UV light. The frequencies of
X-rays and -irradiation are 1019 Hz and
lines provided by studies aimed at unravel-
1021 Hz, respectively.
ling the free-radical chemistry occurring in
living tissues. Among emerging methodolo-
gies, PA (see above), the use of specific flu-
4.4.1. Electron paramagnetic resonance (EPR)
orescent probes (LeBel et al., 1992) and
(see also Chapter 2)
fluorescence spectroscopy, spin-trapping
techniques and non-invasive EPR spec-
4.4.1.1. General description
troscopy (see Section 4.4) and new spec-
troscopy assays (Jiang et al., 1991; LeBel et The energy differences that are studied
al., 1999; Junqua et al., 2000) warrant fur- with EPR spectroscopy are the result of the
ther investigation. interaction of unpaired electrons with a
Table 4.3. Interference of various compounds in the thiobarbituric acid-reactive

substances (TBARS) assay in crude extracts of plant and seed tissues. The TBARS
assay was performed using the procedure of Heath and Packer (1968) in the
presence of various amounts of sugars and anthocyanins. TBARS concentrations
were calculated from the difference between absorbance values at 532 and 600 nm
and expressed as a relative increase compared to extracts without interfering
compounds.
Absorbance peak
of the interfering Relative
Compound compound (nm) increase (%)
Apple peel extract +a
2.5 mM sucrose 440 + 9%
1 mM fructose 440 + 5%
Cabbage leaves extract +a
anthocyanins 540 + 272%
Germinating pea axes +b
0.3% (w:v) raffinose 441 + 35%
1% (w:v) raffinose + 79%
aData derived from Du and Bramlage (1992) and Hodges et al. (1999).
bUnpublished data of O. Leprince, J. Fajerman and F.A. Hoekstra.
static magnetic field (Zeeman splitting). In Sample preparation is relatively easy. The
EPR spectroscopy the electromagnetic fre- sample is incubated in an aqueous solution
quency is kept constant and the magnetic of spin-probe or spin-labelled molecules
field is scanned (due to limitations of for a short time and then transferred into
microwave electronics). There are four fre- the resonator of an EPR spectrometer for
quencies available in EPR spectrometers: spectra recording. Usually, neutral spin-
1.1 GHz (L-band), 3.0 GHz (S-band), 9.5 probe molecules can readily pass through
GHz (X-band) and 35 GHz (Q-band). membranes and partition within cells over
Among them, the X-band is the most com- the polar and apolar phases according to
monly used. The interaction between their partition coefficients. The EPR signal
nuclei and the electron (hyperfine interac- of the spin-probe molecules outside cells
tions) causes the hyperfine splitting of the can be eliminated by broadening agents via
EPR spectrum. The spectral shape can give spin–spin interaction. Paramagnetic metal
information about the sample under study. ion complexes such as chromium oxalate
Only systems that contain non-paired or ferricyanide are often used as broaden-
electrons will give an EPR signal. Pairing ing agents. Thus, the EPR spectrum of spin
gives zero net electron magnetic moment. probes in the sample in the presence of
Since a paired spin system is energetically broadening agents is exclusively derived
favourable, chemical bonding normally from the inside of cells. Different aspects of
results in molecules that have no unpaired desiccation tolerance can be studied by the
electrons and, hence, no EPR signal occurs. analysis of EPR spectra of spin probes
Exceptions to this rule are transition-metal introduced into the cells.
ions, free radicals and free electron centres Spin labels are stable free radicals. The
such as those produced by high-energy unpaired electron belongs to the nitroxide
irradiation of macromolecules. Free radi- group, which is flanked by quaternary car-
cals produced in biological systems usually bon atoms of methyl groups, protecting
cannot be detected by EPR because of their the radical from recombination and
short half-life times, resulting in low accounting for the high stability of the
steady-state concentrations. label. The EPR spectra of nitroxide spin
The introduction of spin-label/probe labels have a three-line nitrogen hyperfine
methods has considerably increased the structure and are environmentally sensi-
possibilities for the application of EPR in tive. The variety of spin probes of differ-
biological systems. The spin-label group ent properties and the possibility of the
that is almost exclusively used is the attachment of nitroxides to biological
nitroxide moiety synthesized by Rozantzev molecules of interest have created exten-
(1970) and introduced by McConnell sive applications of this method in biol-
(McConnell and McFarland, 1970). ogy (Marsh, 1981; Morse, 1985).
There is a limitation to the hydrated sam- A problem that often arises in experi-
ple size that can be used for spectra record- ments with biological samples is chemical
ing because of dielectric losses caused by reduction of spin labels and spin probes.
water. However, the high sensitivity of the Despite the high stability in aqueous and
method allows the use of very small sample other media, nitroxides are susceptible to
sizes of less than 1 mg. The relative simplic- (reversible) reduction by some biological
ity of sample preparation and spectra metabolites (such as ascorbic acid and thi-
recording enables the operator to handle ols), electron transport chains and other
large numbers of samples per day. redox systems, resulting in the disappear-
ance of paramagnetism. Ferricyanide is
4.4.1.2. Applications of EPR methods usually effective at limiting the rate of
reduction or reoxidizing reduced label
GENERAL REMARKS. The great variety of spin (Kaplan et al., 1973). Oxygenation, aeration
probes allows a multiplicity of information or the use of specific inhibitors can also be
about biological systems to be obtained. used to protect nitroxides against reduction
or to restore them from the reduced 4 daa wheat kernel

hydroxylamines (Marsh, 1981).
Another problem with the application of
the spin-label technique is the disturbance Dead
that might be caused by the guest molecules (a) tissue
themselves (e.g. membrane spin labels).
This is inherent in all methods using
reporter groups, in contrast to spectroscopic
methods that do not use guest molecules
(e.g. NMR, FTIR). Because of the high sensi-
tivity of the spin-label method, very low
concentrations of label can be used, which Difference (viable cells)
minimizes possible disturbance. However,
the possibility of obtaining information
about a specific environment of interest Oil
makes the EPR method attractive in the (b)
study of desiccation tolerance. In contrast,
NMR and FTIR give information that is
averaged along the sample, which can be
complementary to the information from
reporter group methods. Cytoplasm
CELLULAR VIABILITY BASED ON MEMBRANE INTEGRITY.

The principle of the EPR spin-probe Fig. 4.1. (a) The electron paramagnetic resonance
method for the estimation of the relative (EPR) spectrum of 4-oxo-2,2,6,6-tetramethyl-1-
amount of viable cells is based on the fact piperidinyloxy (TEMPONE) in developing wheat
kernel that was harvested at 4 days after anthesis (4
that membranes of viable cells are imper-
daa) and dried on the ears. The thick line represents
meable to some broadening agents,
the broad component of the spectrum and
whereas the membranes of damaged cells originates from TEMPONE in dead tissue; the thin
are not (Keith and Snipes, 1974). Thus, the line represents the total spectrum. (b) Spectrum
EPR signal of spin-probe molecules inside showing the difference between the total spectrum
cells with disrupted membranes is and broad component, representing TEMPONE
quenched by a broadening agent, and the located in viable cells. Peaks originating from
total amplitude of the EPR signal from the TEMPONE in the aqueous cytoplasm of viable cells
sample will correlate with the amount of and from oil bodies are indicated. Total scan width
viable cells in a sample (Dobrucki et al., is 100 gauss. Spectra are reproduced from Golovina
et al. (2001).
1990). Because of the high sensitivity of the
method, it is possible to determine small
amounts of viable cells in mostly dead tis-
sue. This approach has been applied in the Many of the anhydrobiotic systems con-
study of desiccation tolerance acquisition tain oil bodies as storage material. In this
of proembryonic cells in wheat kernels, case, amphiphilic spin-probe molecules
which were slowly dried on the ear at an will partition into lipid bodies as well, and
early stage when proembryos could not be EPR spectra are composed of two compo-
detected morphologically (Fig. 4.1) nents originating from spin-probe mole-
(Golovina et al., 2001). Such an approach cules in aqueous (cytoplasm) and
allows the developmental death of wheat hydrophobic (oil) environments. These
endosperm cells during kernel develop- spectra differ in the distance between lines
ment (Golovina et al., 2000) and the (the isotropic splitting constant aiso is
progress of cell death after cold (Fig. 4.2) or around 16 G for an aqueous environment
imbibitional stress in neem seeds to be fol- and around 14 G for a lipid environment)
lowed (Sacandé et al., 2001). and in the position of the central line
plasmic (hcyt) and oil peaks (hoil) can be

Axis of
neem used for the quantitative assessment of the
proportion of viable cells in a sample
(Golovina and Tikhonov, 1994; Golovina et
al., 1997a,b; Leprince et al., 1999).
Oil
(a) Control
PLASMA MEMBRANE PERMEABILITY. Changes in
plasma membrane permeability can be esti-
mated by using the water-soluble nitroxide
radicals in the presence of a broadening
Cytoplasm agent (Miller and Barran, 1977; Golovina et
al., 1998; Hoekstra et al., 1999). The
method is based on the presence of tempo-
(b) 7 days rary defects in membranes that allow ferri-
cyanide ions to penetrate into the cell and
broaden the signal arising from spin probes
located in the cytoplasm. The line-height
(c) 28 days ratio of the lipid peak to the water peak
(L/W) will correlate with the number of fer-
ricyanide ions that have penetrated the cell
through the plasma membrane and can be
Fig. 4.2. Electron paramagnetic resonance (EPR) used to characterize plasma membrane
study of chilling damage of neem (Azadirachta permeability (Fig. 4.3).
indica) seeds using 4-oxo-2,2,6,6-tetramethyl-1-
piperidinyloxy (TEMPONE) as a spin probe. CELL VOLUME AND OSMOTIC EFFECT. The height
Spectrum (a), mature, fresh axes used as control; ratio of cytoplasmic to lipid peaks in EPR
spectra (b) and (c), whole embryos after,
spectra can be used to determine cell vol-
respectively, 7 and 28 days of storage under humid
ume changes under osmotic stress. This
conditions at 5°C. Spectra are plotted in the same
scale to allow comparison. The oil and cytoplasmic follows from the fact that spin-probe mol-
peaks are indicated in the high-field region (right ecules are equally distributed inside and
side) of spectrum (a). Total scan width is 100 gauss. outside the cells. The total cellular vol-
Spectra are reproduced from Sacandé et al. (2001). ume that is not accessible to broadening
agents will determine the amount of spin
probe that is separated from the broaden-
(g-factor), and can be resolved in the high ing agent and, hence, the line-height of
field (right-side) part of the spectrum the cytoplasmic component in the EPR
(Marsh, 1981). In the case of loss of mem- spectrum. This total volume is the prod-
brane integrity, ferricyanide ions that have uct of the number and volume of viable
penetrated the cell only broaden the signal cells. Cell division and enlargement of
of TEMPONE (4-oxo-2,2,6,6-tetramethyl-1- cells during imbibition and germination
piperidinyloxy) molecules localized in the (Golovina et al., 2001) and osmotically
aqueous cytoplasm. The signal of TEM- induced changes in cell volume (Miller,
PONE from oil bodies remains unbroad- 1978) can cause changes in the line-
ened, because ferricyanide cannot partition height of the cytoplasmic component.
into the lipid phase. The intensity of this The ratio between the line-heights of
hydrophobic signal can then be used as a cytoplasmic and lipid peaks can be used
measure of the total amount of cells in the to quantify the osmotic effect. However,
sample, whereas the intensity of the polar when the amount of oil changes during
cytoplasmic component represents the seed development (Golovina et al., 2001)
amount of cells with intact membranes or during germination (Sacandé et al.,
(Golovina et al., 1997a,b). The ratio R 2001), the height of the cytoplasmic line
between the heights of the aqueous cyto- can be used instead.
Typha latifolia 1998). Line-height differences arising from

differentially broadened lines due to
(a) 0s
slowed motion of spin-probe molecules
(Fig. 4.4, spectra a and b) are used to esti-
(b) 7s
mate viscosity (Keith and Snipes, 1974).
The line-height ratio between lines can be
converted to viscosity. Based on such an
approach, the changes in cytoplasmic vis-
cosity with drying of desiccation-tolerant
(c) 30 s and sensitive samples (Leprince et al.,
1999) and with the acquisition of desicca-
tion tolerance during seed development
(Golovina et al., 2001) have been estab-
lished.
When spin-probe motion slows further,
L not only is a progressive increase in differ-
(d) 60 s ential broadening observed, but also a dis-
tortion of the line shape (Fig. 4.4, spectrum
W
c). Rigidly immobilized, randomly oriented
radicals give a powder spectrum (Marsh,
1981), which can be used to characterize
biological glasses (Buitink et al., 1998, 1999,
2000b,c,d,f). In this case, the viscosity must
Fig. 4.3. Electron paramagnetic resonance (EPR)
be estimated using saturation transfer EPR
study of imbibitional leakage of Typha latifolia
pollen using 4-oxo-2,2,6,6-tetramethyl-1-
or pulsed EPR methods (see below).
piperidinyloxy (TEMPONE) as a spin probe. Pollen
was either directly incubated in a solution of PARTITIONING OF AMPHIPHILES INTO THE LIPID PHASE
TEMPONE/ferricyanide (spectrum a) or after a WITH DRYING. The shape of spin-probe spec-
previous rehydration in liquid germination medium tra depends on properties of the environ-
for 7 s (spectrum b), 30 s (spectrum c) and 60 s ment; therefore amphiphilic spin probes
(spectrum d). All the spectra exhibit contributions can be used to follow their partitioning
from the aqueous cytoplasm (W) and from lipid (L) with drying (Golovina et al., 1998; Buitink
(oil bodies) environments, which are resolved in the et al., 2000e; Hoekstra and Golovina, 2000;
high-field region (right side). The spectra were
Golovina and Hoekstra, 2002). The samples
normalized to the height of the lipid (L) peak. The
ratio W/L was taken as a measure of plasma
are preloaded with spin probes and allowed
membrane permeability (see explanation in the text, to dry. The EPR spectra recorded from the
Section 4.4.1). Total scan width is 80 gauss. Spectra samples at different moisture content will
are based on data from Hoekstra et al. (1999). be composed of spectra originating from
spin-probe molecules at different locations
(Fig. 4.5). They can be decomposed, and the
CYTOPLASMIC VISCOSITY.
Because the shape of relative proportion of spin probes at the dif-
EPR spectra of spin probes is sensitive to ferent locations can be estimated (Hoekstra
molecular motion, cytoplasmic viscosity and Golovina, 2000; Golovina and
can be studied with spin-probe techniques Hoekstra, 2001).
(Keith and Snipes, 1974). To characterize
the shape of the spectrum originating from PHYSICAL PROPERTIES OF MEMBRANES. The small,
the cytoplasmic location of the spin probe, water-soluble spin probe TEMPO (2,2,6,6-
other components (lipid, starch-like) of the tetramethyl-1-piperidinyloxy) or spin-
spectrum have to be subtracted (Golovina labelled fatty acids, steroids or
et al., 2000, 2001). Alternatively, charged phospholipids are used to study the physi-
spin probes that do not partition into the cal properties of membranes (for refer-
lipid phase can be applied (Buitink et al., ences, see Berliner, 1976; Marsh, 1981;
(a) Cytoplasm
Root (a)
h0 h–1
(b) Oil bodies

h0/ h–1= 1.19
Axis (b)
Membrane
(c) surface
Sucrose
glass h0/ h–1= 1.75
(c) (d) Combined
2Amax
a:b:c = 5:10:85
Fig. 4.4. Electron paramagnetic resonance (EPR) Fig. 4.5. Typical electron paramagnetic resonance
spectra of 4-oxo-2,2,6,6-tetramethyl-1- (EPR) spectra of 4-oxo-2,2,6,6-tetramethyl-1-
piperidinyloxy (TEMPONE) in the cytoplasm of piperidinyloxy (TEMPONE) in aqueous cytoplasm
wheat seedling root (spectrum a) and hydrated (spectrum a), oil bodies (spectrum b), and of spin-
wheat axis (spectrum b). The ratio of the height of probe molecules immobilized at the membrane
the central line (h0) to the height of the high-field surface (spectrum c). Spectrum (d) is the sum of
line (h1) reflects cytoplasmic viscosity. The spectra (a), (b) and (c). Spectra were combined in
cytoplasmic viscosity is less in seedling root such a proportion that the relative amounts of spin
(h0/h1= 1.19) than that in hydrated wheat axis probe in spectra (a), (b) and (c) were 5%, 10% and
(h0/h1= 1.75). The EPR spectrum of TEMPONE in 85%, respectively. Spectrum (d) simulates the late
air-dried sucrose glass (spectrum c) is typical for stage of TEMPONE partitioning during drying. Total
rigidly immobilized, randomly oriented spin-probe scan width is 100 gauss. Spectra are based on data
molecules. The distance between the outer extremes from Golovina and Hoekstra (2001).
2Amax (in gauss) can be used to characterize the
slow motion of spin-probe molecules. Total scan
width is 100 gauss. Spectra are based on data from
Golovina and Hoekstra (2001). provides valuable information about mem-
brane dynamics, because the stable free-
radical doxyl group can be placed at
Morse, 1985). Isolated membranes can be different positions along the acyl chain.
labelled with TEMPO, which partitions Thus information can be obtained from dif-
between water and the membranes. The ferent depths in membranes, from the sur-
partitioning depends on membrane fluidity face to the core. Labelling model membranes
and aqueous-phase viscosity and can be poses no problem. The spin label is mixed at
used for the determination of the mem- 1 mol % with the lipids in organic solvent,
brane phase transition. However, the possi- which is subsequently removed by evapora-
ble partitioning of TEMPO into oil bodies tion. The anhydrous mixture is then dis-
complicates the in vivo membrane investi- persed in the appropriate amounts of
gations. The use of spin-labelled fatty acids aqueous phase. Labelling isolated biological
membranes poses more problems. Spin- teins and lipids in biological membranes
labelled fatty acids have first to be dis- (for references, see Marsh, 1981; Hemminga,
solved in ethanol and then added to the 1983) and glasses (Roozen et al., 1991). The
membranes in such a quantity that the final method has been successfully applied in
concentration of ethanol does not exceed 1 the study of biological glasses in anhydrobi-
mol %. Other ways of membrane labelling otic systems (Buitink et al., 1998, 1999;
have been reviewed by Marsh (1981). 2000b,c,d,f). This approach of measuring
Labelling membranes with doxyl stearates slow rotational motion has given stunning
in vivo following the same approach can be insight into the differences between biologi-
used but is more tricky than with isolated cal glasses and sugar or polymer glasses.
membranes. A small number of in vivo stud- For example, a remarkable observation orig-
ies have been published. Among them only inating from the ST-EPR measurements was
a few investigations have been conducted the occurrence of a second kinetic change
on desiccation-tolerant systems (Golovina in mobility at a definite temperature above
and Tikhonov, 1994; Vishnyakova et al., the glass transition temperature (Buitink et
2000; Golovina and Hoekstra, 2001). al., 2000f), which may have physiological
relevance for survival in the dry state (Fig.
ADDITIONAL EPR TECHNIQUES APPLIED TO DESICCATION-
10.3 in Chapter 10).
TOLERANT SYSTEMS. When conventional EPR Pulsed EPR. CW-EPR often cannot give
spectroscopy as described above yields the true values for the relaxation times of the
rigid-limit powder line shape (see Fig. 4.4, spin label, because of the inhomogeneous
spectrum c), it is insensitive to the rate of broadening of the lines. However, pulsed
molecular motion. The following two EPR EPR (electron spin echo technique) pro-
methods have been designed to overcome vides a direct method for the measurement
this problem and adapted to anhydrobiotic of relaxation times that give insight into
material. They are particularly suitable to the molecular dynamics of spin probes
characterize a glassy state. (Morse, 1985). This method has been used
to identify the glassy state in wheat seeds
Saturation-transfer EPR. Saturation-transfer
(Dzuba et al., 1993) and to characterize the
EPR (ST-EPR) allows the measurement of
motion of guest molecules in biological
very slow molecular motion with rotational
glasses of different moisture content
correlation times between 107 and 103
(Buitink et al., 2000a).
s. For comparison, conventional continuous
wave (CW) EPR enables rotational correla- EPR imaging (EPRI). The potential of using
tion times below 107 s to be resolved. In both NMR and EPR imaging was suggested
conventional EPR, motion averaging of the by Lauterbur (1973). However, while NMR
spectral anisotropy occurs within the time imaging (NMRI) has progressed into clinical
of spin–spin relaxation T2. In ST-EPR, this usage, the application of EPRI is restricted,
averaging occurs within the time of particularly in biological systems. This is
spin–lattice relaxation T1, which is 300 caused by the severe dielectric losses and
times longer than T2 for slow-moving consequent heating that occurs in aqueous
nitroxide molecules (Marsh, 1981). Thus, samples at conventional EPR frequencies (X-
ST-EPR extends the motional sensitivity of band). The dimension of the hydrated sam-
the spin-label technique to one that moni- ple that can be used for imaging is limited to
tors a 300-fold slower motion than with a few millimeters. This problem can be
conventional EPR. ST-EPR spectra of partly overcome by using low-frequency EPR
nitroxide spin probes can be analysed by (L-band) and a surface coil (Berliner and
independent line shape parameters. Using a Fujii, 1985). EPRI is used for visualizing
reference material of known viscosity, the paramagnetic centres in a sample. There are
molecular rotation can be calculated in an several biological applications in which
empirical way (Hemminga, 1983). ST-EPR EPRI has a significant advantage over NMRI:
has been used to study the motion of pro- the spatial distribution of O2 and redox
metabolism, mapping viable and non-viable this effect is proportional to the O2 concen-
cells, the diffusion of paramagnetically tration (Swartz, 1987).
labelled solutes, and mapping native free radi-
pH measurements. Spin-labelled amine
cals that are stable or trapped by incorporated
and carboxylic acids have been used to
spin traps at the sites of transient radical pro-
determine the pH in vesicles and cells
duction (Berliner and Fujii, 1986; Bacic et al.,
(Mehlhorn et al., 1982). The method is
1989; Dobrucki et al., 1990). In spite of the
based on the differential membrane perme-
potential advantage of EPRI, there are only a
ability for charged and neutral forms of
few cases in which the method has been
these spin probes. Because the equilibrium
applied to desiccation-tolerant systems. The
between charged and neutral amines and
pathways of bulk water penetration into wheat
acids depends on pH, the intracellular
kernels during imbibition have been studied
EPR signals of these spin probes can be
using the nitroxide radical TEMPOL (4-
used to calculate the intracellular pH.
hydroxy-2,2,6,6-tetramethyl-1-piperidinyloxy)
Unfortunately, such an approach can only
(Smirnov et al., 1988; Golovina et al., 1991).
be applied in the presence of a solution of
To avoid the problem of dielectric losses, the
a broadening agent. To study the changes
kernels were placed in liquid nitrogen.
in pH in a sample during drying, specific
Using perdeuterated 15N nitroxides as imag-
pH-sensitive spin probes can be applied.
ing substances, tens of micrometre resolu-
The reversible effect of pH on EPR spectra
tion can be achieved. Such resolution was
is associated with proton exchange in the
sufficient to obtain EPRI of viable cells in
radicals. Protonated and non-protonated
hydrated lettuce seeds in the presence of fer-
forms have different EPR parameters. The
ricyanide (Walczak et al., 1987). The image
protonable group in the radical structure
enabled contrast between embryo and stor-
has to be close to the unpaired electron.
age tissue to be observed. The EPRI of the
Iminonitroxides are the most promising in
penetration and distribution of natural spin
this respect (Khramtsov and Weiner, 1988).
probes (humic substances) in wheat kernels
has also been demonstrated (Smirnov et al., Spin trapping. The free radicals that are
1991). produced in anhydrobiotic organisms dur-
ing water loss (Section 4.3.4) cannot be
detected by EPR because of their short half-
POTENTIAL APPROACHES FOR STUDYING ANHYDRO-
life resulting in low steady-state concentra-
BIOSIS.Additional EPR methods have been
tion, or the short relaxation times leading to
designed to study several biochemical and
very broad lines. These radicals can be
biophysical aspects of biology. However,
trapped specifically by spin traps and
these methods have not yet been applied
detected by EPR in organic extracts or in
specifically to seeds or other types of anhy-
vivo (Knecht and Mason, 1993). Four differ-
drobiotic tissues.
ent traps are commonly used in biological
Intracellular O2 concentration. Intracellular systems: 2-nitrosopropane (MNP), phenyl-
O2 affects the shape of EPR spectra of spin N-tert-butylnitrone (PBN), -(4-pyridyl-1-
probes because, as a paramagnetic mole- oxide)-N-tert-butylnitrone (POBN), and
cule, it causes line broadening. Broadening 5,5-dimethyl-1-pyrroline-N-oxide (DMPO).
is proportional to O2 concentration, thereby The primary free radicals interact with the
allowing the intracellular concentration to double bond of diamagnetic spin-trap mole-
be calculated (Swartz, 1987). However, the cules and form radical adducts that are
application of this method may not be much more stable than the primary free
straightforward for drying biological mater- radicals. The radical adducts of these spin
ial because the rise of viscosity also intro- traps are nitroxide radicals. The primary
duces changes in line broadening. Another free radical can be identified either from the
approach is to use the effects of O2 on the spectra of radical adducts, or after purifica-
microwave power saturation: the presence tion of radical adducts and further identifi-
of O2 diminishes power saturation, and cation by mass spectroscopy. Various
shortcomings complicate the application of NMR signals can be characterized by

spin trapping in vivo in drying organisms: intensity, frequency, line shape and relax-
oxidation of the spin traps and reduction of ation times. All these characteristics are
the radical adducts, and amphiphilic affected by the physical and chemical envi-
behaviour which may relocate spin traps ronment of the magnetic nucleus and can
into the membranes (Knecht and Mason, be used to obtain information of biological
1993). interest such as the state of water, intracel-
lular pH and membrane dynamics. Signal
intensity is related to the number of mole-
4.4.2. Nuclear magnetic resonance cules that produce the signal. In relaxation
(see also Chapter 2) experiments, the intensity depends on the
time of signal registration and on the rate
4.4.2.1. General description of magnetization decay. For quantitative
estimation of peaks in NMR spectra, inte-
Analogous to EPR spectroscopy, NMR spec- gration of the lines should be used because
troscopy is based on the resonance absorp- of the different relaxation times of the sig-
tion of electromagnetic radiation by the nals. The limits of integration are deter-
system during the transition between two mined by the signal-to-noise ratio of the
discrete energy states. The energy differ- signal and the overlapping with other sig-
ences studied in NMR spectroscopy are due nals in a spectrum.
to the interaction of nuclear magnetic Local fields originating from the local
moments with the magnetic field (Zeeman electron density modify the external field
splitting for nuclei). The energy differences imposed on magnetic nuclei. As a result, the
are smaller than those in EPR because of resonance frequency of a nucleus depends
the smaller magnetic moment of nuclei. on its chemical environment, which is
This explains why electromagnetic radia- called chemical shift. Magnetization relaxes
tion in the radiofrequency range is required exponentially, and the faster the decay, the
to excite the transitions that produce the broader the line in the spectrum. Broad
NMR signal, whereas that in the microwave lines have lower amplitudes and overlap
range is used in EPR spectroscopy. with other lines, which leads to poorly
Because the energy differences in NMR resolved spectra. In living systems, the vari-
are small, the differences in number of ations of magnetic susceptibility across the
nuclei at different energy levels are also sample cause line broadening, which makes
small. As a consequence, the signal it difficult to record high-resolution spectra
strength is weak, which makes NMR an from dense heterogeneous tissue, such as
inherently insensitive technique. Only seeds, and from tissues containing air-
those nuclei that have a non-zero spin spaces (leaves and roots).
quantum number resulting in non-zero The T1 (spin–lattice or longitudinal) and
magnetic moment can be used. The split- T2 (spin–spin or transverse) relaxation
ting between energy levels depends on the times characterize the magnetization decay
strength of the magnetic field and the mag- because of the interaction of the nuclear
netogyric ratio of the nucleus. The highest magnetic moments with the environment
magnetogyric ratio and the almost 100% (T1) and with each other (T2). Relaxation
natural abundance make proton (1H) NMR times are mostly determined by the
the most sensitive. The reasonably high motional properties of the nucleus.
magnetogyric ratio and 100% natural abun- Measurements of relaxation are particu-
dance of 31P nuclei give moderately good larly important in NMR studies of tissue
receptivity for in vivo phosphorus NMR. In water when information about the exis-
contrast, the 13C nucleus has very low tence of different water fractions in the tis-
receptivity because of its low natural abun- sue is required. In practice, the
dance, but, as a label, this isotope could be measurements are easier to conduct than to
useful (Schneider, 1997; Roberts, 2000). interpret (Ratcliffe, 1994).
In basic NMR experiments, the sample is cal systems because of the generally high
placed in a magnetic field, and the NMR sig- water content, the high natural abundance
nal is generated by irradiation of the sample and the high magnetogyric ratio of 1H. This
with a radio-frequency field, given as pulses allows the use of low-field NMR instead of
of different sequences. A single pulse cre- expensive high-field NMR magnets.
ates a net magnetization, which is regis-
tered. The magnetization decays to zero, MEASUREMENTS OF WATER CONTENT. 1H low-field
and the time-dependence of the decay (free NMR allows the non-destructive measure-
induction decay) is recorded. In low-field ment of the water content in biological sys-
studies, this decay is analysed directly. In tems with high precision. There are two
high-field NMR, the decay is converted into types of analytical NMR commonly used in
spectra by Fourier transformation. The NMR this respect – continuous wave (CW) NMR
spectrum is the plot of intensity against fre- (wide-line) (Pohle and Gregory, 1968) and
quency of the radio-frequency field. All pulsed NMR (Martin et al., 1980), the latter
NMR applications developed for studying now being generally adopted. In CW-NMR
living systems can be divided into four the amount of liquid water is estimated from
groups: (i) detection of water signal; (ii) the area under the absorption peak. The sig-
NMR imaging; (iii) high-resolution multinu- nal from water strongly ‘bound’ to biopoly-
clear NMR spectroscopy; and (iv) solid-state mers is not visible because of broadening.
NMR spectroscopy (Ratcliffe, 1994). The signals from liquid water and oil are not
To exploit the advantage of a non-inva- resolved, but the contribution of oil to the
sive technique, NMR experiments need to signal can be estimated by drying.
minimize the physiological perturbation Pulsed NMR can be used to analyse the
and maintain the tissue in a physiologi- different water fractions. In pulsed NMR
cally controllable state. In this respect, the all protons are excited by a short intense
whole plant, cell suspensions and intact radio frequency (RF) pulse resulting in a
seeds are the easiest tissues, and excised free induction signal, which decays when
tissues the most demanding (Ratcliffe, the pulse is switched off. The initial
1994). Often, it is necessary to submerge a amplitude of free induction decay (FID) is
sample in water to avoid differences in proportional to the total number of pro-
magnetic susceptibility between air and tons in a sample. The signals due to nuclei
cellular material, a practice that is incom- in different physical states decay at differ-
patible with drying organisms. Proper O2 ent rates: signals due to protons in solid
supply and illumination have to be main- state decay faster (microseconds) than
tained, especially in densely packed sam- those in liquid phase (from milliseconds
ples. In solid-state NMR, when magic angle to seconds). This signal decay can be
spinning is applied, it is impossible to con- analysed to reveal the contribution of dif-
trol the physiological state because of the ferent proton fractions. To avoid the influ-
extreme conditions (more than 1000 rota- ence of inhomogeneity of magnetic field
tions per minute) imposed on the sample. and water diffusion on the rate of decay,
special sequences of pulses such as spin-
echo (SE) or Carr–Purcell–Meiboom–Gill
4.4.2.2. The NMR study of water in living (CPMG) are used (Farrar and Becker,
systems 1971). In air-dry samples, the signal decay
from water associated with polymers
GENERAL REMARKS. The study of water in (mainly starch) can be distinguished easily
anhydrobiotes is of particular interest from that of oil protons on the basis of the
because with drying and rehydration both considerable differences in spin–spin
water content and water properties change. relaxation time T2. Such an approach is
NMR is a powerful tool to study water in widely used for rapid and non-destructive
vivo. There is no problem with the sensitiv- determination of moisture and oil content
ity of detecting the water signal in biologi- in air-dry seeds (e.g. Tiwari et al., 1974;
Gambhir and Agarwala, 1985; Brusewitz Compartmentation is the reason why

and Stone, 1987; Gambhir, 1992; Rubel, more than one fraction of water is generally
1994; Warmsley, 1998). In hydrated seeds, observed in hydrated living systems. A the-
drying or D2O exchange can be used to ory of transverse relaxation in compart-
separate the NMR signal of free water from mented systems has been developed, based
that of oil (Ratkovic et al., 1982a). on the chemical exchange and diffusion
Because the different water fractions properties of the water (Belton and
have the same chemical shift, only pulsed Ratcliffe, 1985). Two to three water frac-
NMR can be used to characterize them in tions have been shown in hydrated tissue
living tissues. The changes in water frac- originating from different plant cell com-
tions with different relaxation characteris- partments (Bacic and Ratkovic, 1984;
tics can be followed during the Belton and Ratcliffe, 1985; Snaar and van
dehydration or rehydration of anhydrobi- As, 1992). However, it appears that there is
otic systems. This gives insight into the no simple relationship between the multi-
role of the different water fractions in bio- exponential character of T2 and the com-
logical systems (Seewaldt et al., 1981; partmentation of the water (Ratcliffe, 1994).
Ratkovic et al., 1982a; Aksyonov and The heterogeneity in cellular size and com-
Golovina, 1986a,b; Ishida et al., 1987, position, subcellular compartmentation,
1988b; Bacic et al., 1992; Golovina and and plasmalemma and tonoplast permeabil-
Aksyonov, 1993; Marconi et al., 1993). ity could have influenced the multi-expo-
However, data on different water fractions nential decay curves (Snaar and van As,
must be interpreted with extreme caution 1992). The detection of the simultaneous
(Ratcliffe, 1994). Different water fractions presence of water of different relaxation
with specific relaxation times can be dis- behaviour in anhydrobiotes with reduced
criminated only if there is no fast exchange MC may have been caused by the inhomo-
of protons between the fractions in the geneous water distribution within the
NMR time window. In the case of fast organisms. Thus, the water with long T2 (or
exchange between protons, only one relax- slow-relaxing water) observed in wheat ker-
ation time is observed. The number of pro- nels during the first hours of imbibition is
tons of different mobility and their thought to be localized around the embryo
relaxation times will determine the and in the vascular bundle, whereas the
observed effective relaxation time. When fast-relaxing water is thought to be associ-
associated with macromolecules, water ated with starchy endosperm (Golovina and
protons have shorter relaxation times, Aksyonov, 1993).
which will influence the overall relaxation
time. This is the reason why T1 (spin–lattice WATER SELF-DIFFUSION COEFFICIENT. The behav-
relaxation time) and T2 (spin–spin relax- iour of water in living systems can also be
ation time) values are lower in cellular characterized by the water self-diffusion
water than in bulk water and decrease fur- coefficient. The diffusion coefficient is
ther with water loss. Thus, T2 values can measured by the pulsed (spin-echo) NMR
also be used to measure moisture content technique in the presence of a (pulsed)
(MC) (Ratkovic, 1987). Below 0.2 g H2O g1 field gradient (Fukushima and Roeder,
dry weight, the relationship between relax- 1981). In addition to nuclear magnetic
ation times (T1 and T2) and moisture con- relaxation, the spin-echo amplitude
tent is reversed (Clegg et al., 1982; Ratkovic decreases in the presence of a field gradient
et al., 1982b; Wolk et al., 1989). Because if water changes its position during the
the increase in T1 and T2 at low water con- measurement. Diffusion coefficients as a
tents has also been observed in measure of water mobility can be calcu-
starch/water systems besides anhydrobiotic lated from the signal decay in the presence
organisms, the increase might be attributed of a field gradient. As in the case of relax-
to water molecules jumping from one sorp- ation times, self-diffusion coefficients of
tion site to another. cellular water are lower than those of bulk
water and decrease with drying (Clegg et 4.4.2.3. NMR imaging

al., 1982). This can be caused by the pres-
ence of diffusion barriers (membranes or GENERAL REMARKS. NMRI is mainly based on
cell walls) or macromolecules. These the detection of the water signal. 1H reso-
macromolecules can cause either obstruc- nance frequency is independent of the
tion of the diffusion or water binding (Seitz location of the water in a tissue, so that tis-
et al., 1981; Back et al., 1991). As a result, sue water signal is averaged across the
the diffusion coefficient in hydrated anhy- whole sample. The spatial distribution of
drobiotes has been shown to be 2–5 times the water signal can be obtained if a mag-
smaller than that of bulk water (Clegg et netic field gradient is applied, which arises
al., 1982; Fleischer and Werner, 1992). In from the dependence of the resonance fre-
Artemia cysts the diffusion coefficient has quency of NMR signals on the magnetic
been measured from 0.02 to 1.49 g water field strength. In spite of the simplicity of
g1 dry weight, the minimum value being the principle of NMRI, its practical appli-
almost 50 times lower in the dry cysts than cation is rather complicated. Information
in the hydrated cysts (Seitz et al., 1981). on the spatial distribution of water or water
properties (relaxation times or diffusion
MEMBRANE PERMEABILITY. Paramagnetic ions coefficients) can be obtained. Dynamic
(Mn2+) cause a decrease in relaxation times information can be obtained from time-
due to their interaction with nuclei. Conlon dependent properties of the image. There
and Outhred (1972) proposed a method of are two different experimental approaches
measuring membrane permeability to water, in NMRI: imaging large objects (roots,
based on the change in relaxation time of stems or whole plants) with low spatial
intracellular water that is in diffusional resolution, and imaging small samples
exchange with an extracellular MnCl2 solu- (seeds, excised tissues) with high spatial
tion. From the estimated water-exchange resolution (NMR microscopy) (Ratcliffe,
time and the cell dimension, the diffusion 1994; Ishida et al., 2000).
permeability coefficient Pd can be calcu- Spatial resolution is mainly determined
lated (Stout et al., 1977, 1978; Bacic and by the signal/noise ratio, but other factors
Ratkovic, 1984). Unfortunately, this such as short relaxation times and the pres-
approach cannot be applied to the systems ence of air space cause intensity loss and a
that are subjected to drying, because the tis- decrease in spatial resolution. The develop-
sue has to be in Mn2+ solution. ment of NMRI has led to a resolution that
The pulsed-gradient spin-echo method approaches the dimension of single cells in
proposed by Stejskal and Tanner (1965) plant tissues (Connelly et al., 1987). The
can be used to study the in situ membrane theoretical limit is considered as 10 10
permeability for water during drying. The 10 µm (Ratcliffe, 1994). While NMR is not
method allows the water diffusion to be yet able to compete with optical microscopy
measured over the time between two in its resolution of cellular structures, it has
pulses of field gradient. The presence of the great advantage of being non-invasive
partly permeable barriers causes the and, thus, can be used to monitor function-
decrease in the apparent diffusion coeffi- ing plant tissue. The ability to resolve struc-
cient for water, so that the permeability of tures depends not only on resolution but
membranes for water and the size of water also on the image contrast, which is deter-
compartments can be calculated (Tanner, mined by the differences in signal intensity
1978; von Meerwall and Ferguson, 1981). between different regions of the sample.
This approach has been applied to follow Knowledge of relaxation properties of the
the changes in membrane properties in tissue water is central to the understanding
developing barley seeds (Ishida et al., of image contrast. Nitroxide radicals
1995) and to calculate the size of oil bodies (Magin et al., 1986; Swartz et al., 1986) and
in rape seeds (Fleischer et al., 1990; paramagnetic ions (Ishida et al., 2000) can
Fleischer and Werner, 1992). be used as contrasting agents.
WATER DISTRIBUTION IN SEEDS DURING MATURATION signal of the externally supplied water
AND GERMINATION. It is possible to map sta- (Connelly et al., 1987). The changes in relax-
tionary, diffusing and flowing water in ation times of tissue water during seed matu-
plant tissue (Ratcliffe, 1994). NMRI enables ration or germination cause changes in the
the water distribution inside seeds to be image contrast. Relaxation times of water
determined. The brightness of the image is depend on the interaction of water with
proportional to the proton density. macromolecules. The synthesis of storage
Experiments with seeds of different species substances during maturation and their
have shown that the signal/noise ratio in hydrolysis during germination result in an
the image is sometimes limited by the short apparent decrease or increase in brightness
relaxation time for tissue water (T2 < 10 of the NMR image (Ishida et al., 1990, 1995;
ms) (Connelly et al., 1987). The sensitivity McIntyre et al., 1995), so that solubilized
problem can be overcome to some extent parts of the storage tissue can become visi-
by signal averaging, since the time scale for ble. The changes in image contrast during
detectable structural changes in germinat- precocious germination of Phaseolus vul-
ing seeds is long in comparison with the garis seeds after ethylene treatment have
time required to obtain an image. In NMR been attributed to changes in the water sta-
images of seeds, a clear distinction tus and water redistribution from the cotyle-
between axis and storage tissue can be don to the axis (Fountain et al., 1998).
obtained (Connelly et al., 1987; Kano et al.,
1990; Hou et al., 1996; Fountain et al., THE DISTRIBUTION OF OIL AND SUCROSE IN SEEDS.
1998; Carrier et al., 1999). The changes in The spatial image of other compounds,
water distribution during drying and rehy- mainly lipids and carbohydrates that accu-
dration have shown the transfer routes for mulate in storage tissue, can be mapped in
water (Ruan and Litchfield, 1992; Ruan et vivo using the chemical-shift imaging (CSI)
al., 1992; Song et al., 1992; Kovacs and technique (Bottomley et al., 1984). The 1H
Nemenyi, 1999). NMR spectra of water, oil and sugars have
The water content may be more uni- different chemical shifts, but the peaks are
formly distributed in seeds than proton not resolved unless the water peak is sup-
NMRI indicates. This discrepancy arises pressed. The CSI technique applied to 1
from the inhomogeneity of the susceptibil- day germinating mung bean seeds has
ity of the sample associated with the pres- shown uniformly distributed oil, which
ence of cell walls and storage substances allowed the changes in the image with ger-
(Back et al., 1991). Eccles et al. (1988) mination to be attributed to the bulk water
applied pulsed gradient spin-echo and fraction (Connelly et al., 1987). Oil and
steady gradient NMRI to maturing wheat sucrose have been mapped in fresh maize
kernels and found the spatial distribution kernels (Koizumi et al., 1995), germinating
of the self-diffusion coefficient of water. barley seeds (Ishida et al., 1990) and in
The diffusion was slowest in endosperm developing pea seeds (Tse et al., 1996).
and highest in the vascular bundle. Back et
al. (1991) used the dependence between
the self-diffusion coefficient of water and 4.4.2.4. High-resolution multinuclear NMR
the relative water content obtained by spectroscopy
Callaghan et al. (1979) to correct the proton
map for wheat grain and showed the more GENERAL REMARKS. High-resolution multi-
uniform distribution of water in the cor- nuclear NMR is used to detect ions and
rected image. metabolites of low molecular weight, the
For experiments in which germination of intracellular pH, the subcellular compart-
seeds has to be followed over many hours in mentation of compounds and the flux
the magnet, it is necessary to maintain a con- through metabolic pathways (Ratcliffe,
tinuous water supply to the seeds, while at 1994; Schneider, 1997; Roberts, 2000). Low
the same time minimizing the spectroscopic concentration of the molecules of interest
and low receptivity of nuclei other than 1H than that of the 1H nucleus, which reduces
make this approach rather insensitive. The overlapping in the spectra. Secondly, the
sensitivity increases with increasing field low natural abundance of 13C opens possi-
strength. High-resolution NMR spectrome- bilities for labelling the tissue and monitor-
ters are usually equipped with high-field ing metabolic pathways. The biological use
superconducting magnets in the range of NMR to study metabolism is described in
4.7–14.1 T, corresponding to a 1H fre- Section 4.3.2. 13C NMR has also been used
quency of 200–600 MHz. The sensitivity to establish changes in soybean seeds dur-
can be increased by multiple scanning and ing maturation and germination. The mois-
usually permits the detection of millimolar ture content-dependent disappearance or
concentrations of metabolites (Ratcliffe, appearance of narrow peaks associated with
1994). To increase the sensitivity further, sugars in in vivo NMR spectra is indicative
the tissue volume within the detector has of the presence of free water in these seeds
to be maximized. Cell suspensions and (Ishida et al., 1987, 1988a). The sensitivity
excised tissues are more suitable for such of natural abundance 13C NMR can be
experiments than whole plants or seeds. enhanced, by applying low-speed magic-
angle spinning (Ni and Eads, 1992) or by
1H NMR. Different nuclei can be used for dif- the detection of 13C by protons coupled to
ferent purposes. The high sensitivity makes the 13C nucleus (Heidenreich et al., 1998).
1H attractive for metabolite detection. 13C labelling gives opportunities for probing
Nevertheless, the need to suppress the water different metabolic pathways, such as lipid
signal and the complexity of spectra limit synthesis in soybean ovules (Schaeffer et
the possibilities for in vivo 1H NMR. The al., 1975) and the metabolism of dormancy-
small differences in chemical shift and con- breaking chemicals in red rice (Footitt et
siderable overlapping of broad signals in tis- al., 1995).
sues make 1H spectra poorly resolved. For
example, in germinating seeds only peaks 31P NMR. In vivo 31P NMR has many applica-
from sugars and oil under conditions of par- tions because of the convenient magnetic
tial water signal suppression can be properties of the 31P nucleus and the physi-
resolved (Koizumi et al., 1995; Ishida et al., ological importance of the information that
1996). 1H NMR spectra of oil in dry seeds can be deduced from the spectra. The mea-
can be obtained because the signals from surement of cytoplasmic and vacuolar pH is
other nuclei are broadened due to immobi- one of the most important applications of in
lization. However, the resolution of lines is vivo 31P NMR, which is based on the depen-
not good because of differences in magnetic dence on pH of the chemical shift of Pi.
susceptibility. The magic-angle sample This, together with the slow exchange of Pi
spinning (MASS) technique eliminates line across the tonoplast, allows the origin of the
broadening arising from differences in mag- Pi signal – either cytoplasmic or vacuolar –
netic susceptibility due to fast mechanical to be determined and, consequently, the
rotation about an axis, making a magic angle cytoplasmic and vacuolar pH. A number of
(54°55), and resulting in 1H spectra from important phosphorylated metabolites can
dry seeds with a good resolution (Rutar, be resolved in 31P spectra. For some of them
1989). 1H NMR is widely used to analyse (Pi, polyphosphates), information on the
tissue extracts for the presence of specific subcellular distribution can also be obtained
compounds such as, for example, betaine in because of the pH-dependent chemical shift.
wild-type and transformed Arabidopsis 31P NMR has been applied to study the pH
thaliana seeds (Alia et al., 1998). of intracellular compartments in germinat-

ing seeds of Phacelia tanacetifolia (Espen et
13C NMR. 13C NMR is more attractive for al., 1995). Changes in chemical shifts of the
application in vivo for two reasons. First, pH-dependent 31P signal from cytoplasmic
the chemical shift scale of the 13C nucleus and vacuolar inorganic phosphate correlate
is more than an order of magnitude greater with seed germination. 31P can also be used
to monitor phosphorus compounds and choline (DPPC). It was shown that the head
their changes during maturation and germi- groups are in a rigid state above and below
nation of seeds, both in extracts and in vivo. the phase transition for both dry DPPC and
Because of line broadening in in vivo exper- a mixture of dry DPPC and trehalose.
iments, a lower number of phosphorus com- Tsvetkova et al. (1998) used 31P NMR in a
pounds can be resolved (Ishida et al., 1987, comparative study of the interaction of glu-
1988a) in comparison with extracts (Ricardo cose, trehalose and hydroxyethyl starch
and Santos, 1990). 31P spectra can be used with dry DPPC. The differential effect of car-
for the identification of the appearance or bohydrates on the behaviour of head groups
disappearance of vacuoles in seeds during has been related to the role of trehalose in
germination and maturation (Ishida et al., membrane protection upon drying.
1990). Phospholipids arranged in bilayers or in
an inverted hexagonal phase have different
line shapes (31P pattern) (Cullis and de
4.4.2.5. Structure and dynamics of cellular Kruijff, 1979). These differences between
membranes bilayer and hexagonal phase spectra arise
from the fact that the lipids are restricted in
GENERAL REMARKS. NMR provides a rapid, motion to the plane of the membrane in the
non-invasive method for investigating the lamellar state. In the case of the hexagonal
state of membranes in isolated cellular phase, a rapid motion about the cylinder
fractions and in living tissues. The axis averages the chemical shift anisotropy.
approach in the study of membrane struc- These differences in 31P pattern can be used
ture and dynamics is solid-state NMR, to detect the presence of either phase.
because of the anisotropic nature of the For many years researchers have been
membranes. The main nuclei used for this interested in the membrane transition upon
study are 31P and 2H. Sometimes, labelling drying from the bilayer into the hexagonal
with 13C has been used, although the line phase (Simon, 1974). In an attempt to
shape is difficult to analyse. detect this membrane transition, Priestley
and de Kruijff (1982) applied 31P NMR to
31P NMR The chemical shift of the phospho- several dry biological systems. The in vivo
lipids depends on the orientation of the spectra were complicated by the superposi-
phosphate groups with respect to the mag- tion of the signals from phospholipids and
netic field. In the case of unrestricted phosphorus-containing compounds. Pollen
motion, all directions are averaged and the of Typha latifolia was the most suitable for
spectrum is isotropic and contains the nar- spectra analysis. At 5.2% MC, the line
row symmetrical 31P NMR line (Cullis and shape of the spectrum was broad and not
de Kruijff, 1979). In some cases, peaks from suitable for analysis. At MC 8.8%, only
different phospholipids can be resolved isotropic signals from phosphorus low-
(Smith, 1985). In the case of restricted weight molecules could be identified, but,
mobility of phospholipids in membranes, at 10.9% MC, a clear peak from phospho-
the spectrum is anisotropic. The shape of lipids organized in bilayers became evi-
the anisotropic 31P NMR spectrum depends dent. Thus, no evidence was obtained for
on the type and rate of motion of the phos- the presence of a hexagonal phase in the
pholipids. Thus, 31P NMR spectra are sensi- pollen on drying to 10.9% MC.
tive to the physical state of the
phospholipids. From the spectra, the order 2H NMR. The relatively small quadrupole
parameter can be calculated (Smith, 1985). moment of deuterium makes it an ideal

There are a few examples of the successful probe of membrane lipids (Smith, 1985).
application of 31P NMR in the field of desic- Fatty acids labelled with 2H at different
cation tolerance. Lee et al. (1986, 1989) positions must be synthesized. The 2H
studied the interaction of trehalose with the NMR spectrum of membranes contains
phospholipid, dipalmitoylphosphatidyl- three clearly separated lines (‘rabbit ears’),
and the separation relates to the ordering of netic field. IR spectroscopy is sensitive to
the 2H-labelled segment. Quadrupole split- vibrations that modulate a molecule’s
ting, overall pattern and relaxation times dipole moment. The range of frequencies
are usually used to characterize 2H spectra. is around 1012–1014 Hz or 400–4000 cm1.
Spin–lattice relaxation is sensitive to rela- The main problem of IR spectroscopy is
tively rapid motions, whereas spin–spin high water absorption in the IR region.
relaxation is sensitive to slow motions D2O substitution or dry films are often
(Smith, 1985). This technique can be used used. In plotting IR spectra, the intensity
to study membrane phase transitions, the of absorption (A) against wave number
influence of acyl chain saturation on mem- (1/
) is used. The main characteristics of
brane fluidity and changes in membrane the absorption band are wave number of
fluidity. the maximum absorption (Amax), the width
2H NMR was applied by Lee et al. (1986, of the band determined at half of the
1989) in a study on the effect of interaction height of Amax, the optical density at Amax
of trehalose with dry DPPC on the behav- and the shape of the band. Every band can
iour of acyl chains. 2H quadrupole spectra be assigned to a certain chemical group
of dry DPPC labelled at the 7th position and a certain type of vibration. In the case
showed that the disorder of lipid acyl of simple molecules, IR spectra consist of
chains is much greater in the case of inter- narrow lines. In the case of macromole-
action of DPPC with trehalose above the cules, the spectrum is characterized by rel-
phase transition than in hydrated or dry atively broad bands because of the
DPPC without trehalose. The new type of overlapping of a great number of individ-
liquid-crystalline phase observed in the dry ual lines corresponding to different types
mixture of trehalose and DPPC is believed of bonds and different conformations.
to play a main role in maintaining mem-
brane stability in dehydrating organisms.
4.4.3.2. Biological applications
13C NMR. 13C-labelledphospholipids can be With the introduction of FTIR spectrome-
used to study the particular dynamics of ters in the 1970s, in vivo studies became
membranes in the interfacial region. Lee et possible, which was not the case with the
al. (1989) used 13C-labelled sn-2-carbonyl grating IR spectrometers because of their
of DPPC to study the influence of the inter- low energy throughput. FTIR spectroscopy
action of dry DPPC with trehalose on inter- can be used for the analysis of certain com-
facial behaviour. No changes in 13C NMR pounds, or to study the interaction between
powder spectra were observed during the molecules. In dry organisms, the technique
phase transition of a dry mixture of is particularly useful because of the
DPPC/trehalose, whereas hydrated DPPC ‘absence’ of water. The absorption of water
exhibited pronounced changes during the usually obscures other absorption bands
phase transition. and thus complicates the interpretation of
spectra. A considerable advantage of in vivo
FTIR spectroscopy is that it permits the
4.4.3. Fourier transform infrared (FTIR)
analysis of macromolecules in their natural
spectroscopy
environment as opposed to in a solvent. A
disadvantage is that information is obtained
4.4.3.1. General description of infrared
on the average vibrational absorption of all
spectroscopy
molecular groups contributing to the IR-
Infrared (IR) spectroscopy deals with the absorption band under study.
transition between vibrational energy lev- For analysis of small samples or the loca-
els that permanently exist in a system, in tion of certain compounds in specific tis-
contrast to NMR and EPR where the transi- sues, an IR microscope fixed to the optical
tion occurs between energy levels that bench can be used. Improvement in sensi-
arise in a system only in an external mag- tivity has been reached by the application of
liquid nitrogen-cooled MCT (mercury/cad- tein secondary structure with dehydration

mium/telluride) detectors, which allow (Wolkers and Hoekstra, 1995, 1997;
pollen, microorganisms or slices of seeds to Golovina et al., 1997c; Wolkers et al.,
be studied. Peak positions or presence of 1998a,b). Conformational changes of pro-
shoulders in the spectra can be analysed by teins can be derived from peak positions
computer-assisted derivative analysis (Susi in the amide I (1600–1700 cm1) and II
and Byler, 1983) and deconvolution (Byler (around 1550 cm1) regions (Byler and
and Susi, 1986) procedures, respectively. Susi, 1986; Surewicz and Mantsch, 1988).
Depending on transmittance and scattering The amide I band mainly arises from the
characteristics of a sample, a transmission, CO stretching vibration of the peptide
reflection or attenuated total reflection groups, and the amide II band from the
(ATR) mode can be used. N–H bending vibration of the protein
An example of the in vivo analysis of cer- backbone (Susi et al., 1967). The CO
tain compounds in seeds is scanning in the stretching frequency is very sensitive to
transmission mode along a slice of tissue. changes in the nature of the hydrogen
Thus, it has been confirmed that the aleu- bonds arising from the different types of
rone layer is enriched in proteins and the secondary structure. This causes a charac-
endosperm in starch. In the case of dehy- teristic set of IR-absorption bands for each
drating organisms, the change in molecular type of secondary structure (Susi et al.,
interactions or conformation is of interest. 1967). Curve fitting of the different bands
The occurrence of an absorption band allows, to a certain extent, the amounts of
around 2850 cm1 originating from the sym- -helix, random coil, turn and -sheet
metric stretching vibration of CH2 can safely structures to be established (Surewicz et
be attributed to acyl chains, either from oil al., 1993). In some model enzyme sys-
or from membranes. If the organism is low tems, a highly characteristic low wave
in oil, it is possible to follow, in vivo, the number band (around 1625 cm1 in the
decrease in C–H vibrational freedom in the amide I region (the intermolecular
acyl chains of membranes with dehydration extended -sheets) is indicative of the for-
(Cameron et al., 1983; Crowe et al., 1989; mation of large protein aggregates with
Hoekstra et al., 1992). Restriction of vibra- drying (Prestrelski et al., 1993). These
tional freedom by molecular interaction aggregates have also been found in vivo on
(van der Waals interactions in the case of gel heat denaturation. The stability of pro-
phase formation) leads to shifts of the teins against heat denaturation can be fol-
absorption peaks to lower wave number and lowed by scanning over a range of
sharpening of these peaks. If the sample temperatures (Wolkers and Hoekstra,
holder is temperature-controlled, it is possi- 1997; Wolkers et al., 1998a). In the situa-
ble to determine the gel-to-liquid crystal tion where the absorption band of water
transition temperature of these membranes (HOH scissoring vibration band at
from shifts in the absorption maxima with 1650 cm1) interferes with the proper esti-
temperature. The same information can be mation of the different protein secondary
obtained from shifts in other absorption structures, H2O can be replaced by D2O,
bands, e.g. the asymmetric CH2 stretch which causes a downward shift in wave
around 2920 cm1 and the CO stretch of number. The accessibility of the proteins
the ester bond of the acyl chains around for D2O can help identify the protein sec-
1740 cm1 (Sowa et al., 1991). Although the ondary structure.
general melting behaviour of oil in seeds Recently, it was established that the
can also be analysed by other techniques glassy state can be studied in vivo by
(e.g. differential scanning calorimetry), that inspection of the OH-stretch at around
of membranes is difficult with other meth- 3300 cm1 (Wolkers et al., 1998c, 1999).
ods because of the small amount involved. The interaction of sugars with proteins or
In vivo FTIR spectroscopy has been with polar head groups has been verified
successfully applied in the study of pro- in dry model systems in the 3300 cm1
(Wolkers et al., 1998d) and 1240 cm1 thereby limiting the range of moisture
regions (Crowe et al., 1996), respectively. content that can be studied (Buitink et al.,
Such interaction upon desiccation has not 1996; Sacandé et al., 2000). However, the
been established with certainty in vivo due future of DSC in studying anhydrobiosis
to the possible absorption of other molecu- is questionable since no major difference
lar groups in these regions. Although in in the calorimetric properties of water was
vivo FTIR spectroscopy has disadvantages found between desiccation-tolerant and
in that it is an averaging technique and sensitive organisms (Sun et al., 1994;
that it is difficult to establish with cer- Buitink et al., 1996; Fig. 10.2, Chapter 10).
tainty from which molecules the spectra
originate, it has the considerable advan-
tage that molecules are studied in their 4.5.2. Electron microscopy
native environment. The disadvantages
can be partly alleviated by parallel in vitro Owing to technical difficulties in studying
experiments, also employing other meth- ultrastructural characteristics of organ-
ods of analysis. elles in the dry state and upon rehydration,
two promising microscopic techniques are
worth mentioning because they can be con-
4.5. Additional Techniques to Study sidered as non-invasive techniques: atomic
Biochemical and Biophysical Aspects of force microscopy (AFM) and low-tempera-
Desiccation Tolerance ture scanning electron microscopy
(LTSEM). AFM is particularly suitable for
4.5.1. Differential scanning calorimetry imaging, non-invasively, the surface topog-
(DSC) raphy of membranes at a nanometer scale.
Furthermore, AFM can be used to obtain
DSC is applied to the study of thermal information on the mechanical properties
events associated with lipid and water of surfaces (Heinz and Hoh, 1999;
phase/state transition. In plant anhydro- Claessens et al., 2000). LTSEM overcomes
biotes, it is used for two main purposes: problems linked to aqueous fixation. It
(i) to determine the calorimetric proper- allows a fast and direct observation of
ties of water present in the system; and freeze–fractured specimens with great reso-
(ii) to construct a state–phase diagram in lution without altering the sample water
which the glass transition temperature content. Application of LTSEM was found
(Tg) and the ice formation/melting temper- to be powerful for studying ultrastructural
ature are plotted as a function of moisture damage resulting from imbibitional injury
content (Vertucci, 1990; Leprince and in seeds (Leprince et al., 1998; Nijsse et
Vertucci, 1995; Buitink et al., 1996). The al., 1998; Sacandé et al., 2001) and cellu-
calorimetric behaviour of the glass transi- lar collapse in lichens (Scheidegger et al.,
tion can be characterized although DSC 1995). In the near future, new technologi-
does not give direct access to the physical cal developments (so-called semi-in-lens)
and biological properties of glasses. will improve the resolution, which is cur-
Sometimes, the heat released during the rently limited to 100 nm in most commer-
glass transition is below the sensitivity of cially available equipment. Non-invasive
the equipment. For example, in seed fixation (freeze-substitution) and a new
species such as rice and tobacco, Tg can- non-aqueous fixative for immunocyto-
not be detected by DSC (O. Leprince and J. chemistry (acrolein) are becoming avail-
Buitink, unpublished data). Furthermore, able for transmission electron microscopy
in oily seeds such as neem and Impatiens, studies (Grote et al., 1999), allowing
the lipid melting transitions often mask microscope observation without disturb-
the thermal events associated with water, ing the sample water content.
4.6. Acknowledgements l’Agriculture et de la Pêche, the Contrat

de Plan Etat-Région and INRA; E.A.G.
The authors thank Dr F.A. Hoekstra for gratefully acknowledges the financial
his contribution to the section on spec- support by a grant from the Wageningen
troscopy methods and for critically read- Centre for Food Sciences and by NATO
ing the manuscript. O.L. acknowledges collaborative linkage grant # LST.CLG
the financial support of the Ministère de 975082.
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Part III
Biology of Dehydration
5 Desiccation Sensitivity in Orthodox and

Recalcitrant Seeds in Relation to Development
Allison R. Kermode1 and Bill E. Finch-Savage2

1Department of Biological Sciences, Simon Fraser University, Burnaby, BC,
V5A 1S6, Canada; 2Horticulture Research International,Wellesbourne,
Warwick CV35 9EF, UK

5.2. Development and Acquisition of Desiccation Tolerance 151
5.2.1. Changes in water status during development of
orthodox seeds 151
5.2.2. Acquisition of desiccation tolerance during development
of orthodox seeds 152
5.2.3. Loss of desiccation tolerance following germination of
orthodox seeds 153
5.2.4. Effects of the rate and extent of desiccation on the
acquisition of tolerance of orthodox seeds 153
5.2.5. Variation in desiccation tolerance across species 155
5.2.5.1. Seed development in recalcitrant species 157
5.2.5.2. Time-dependent effects of storage and drying rate 159
5.2.5.3. Desiccation tolerance differs between seed tissues 160
5.2.6. Mechanisms underlying the acquisition of desiccation
tolerance: recent findings and speculations 161
5.2.6.1. The effects of premature desiccation during the
tolerant and intolerant stages of orthodox seed
development 161
5.2.6.2. Cellular and metabolic changes during the
transition to a desiccation-tolerant state 161
5.2.6.3. Desiccation-tolerance mechanisms in sensitive
seeds 170
5.3. Conclusions 174
5.4. References 175
150 A.R. Kermode and B.E. Finch-Savage
5.1. Introduction the embryo passes into a metabolically

inactive or quiescent state.
The development of most seeds can be The majority of seeds are referred to as
divided conveniently into three confluent ‘orthodox’, in which desiccation occurs as
stages (Fig. 5.1). During histodifferentiation, a pre-programmed and final stage in their
the single-celled zygote undergoes exten- development (Fig. 5.1). Seeds of the ortho-
sive mitotic division, and the resultant cells dox type and other desiccation-tolerant
differentiate to form the basic body plan of structures such as spores and pollen are
the embryo (the axis and cotyledons); con- unique in the degree of water loss toler-
currently, there is the formation of the ated; as much as 90–95% of the original
triploid endosperm or haploid megagameto- water is removed during their develop-
phyte. Thereafter, cell division ceases dur- ment. In this dehydrated state, the seed can
ing the seed expansion stage and there is survive the vagaries of the environment
cell expansion and the deposition of and, unless dormant, will resume full meta-
reserves (normally proteins along with bolic activity, growth and development
lipids or carbohydrates), primarily in the when conditions conducive to germination
storage tissues (i.e. cotyledons, endosperm are provided (Fig. 5.1). This chapter dis-
or megagametophyte). Finally, the develop- cusses some of the mechanisms underlying
ment of most seeds is terminated by some desiccation tolerance of seeds and recent
degree of drying (maturation drying), which approaches to elucidate the precise roles of
results in a gradual reduction in metabo- protective molecules and repair processes
lism as water is lost from seed tissues and at the cellular and subcellular levels.
Development Germination growth
Histodifferentiation Maturation Desiccation Dry

(Expansion)
Cell division Reduced Quiescence Renewed Reserve

Cell expansion metabolism (Mature dry metabolism breakdown
seed) (respiration,
Reserve deposition nucleic acid and
protein synthesis)
Dormancy Cell elongation

(sometimes) Cell division
Desiccation-intolerant Desiccation-tolerant Desiccation-intolerant
Histodifferentiation Cell expansion Maturation drying Germination and growth
Fig. 5.1. Some events associated with seed development, germination and growth. (From Kermode, 1995.)
Desiccation Sensitivity in Relation to Seed Development 151
An important approach to elucidating age upon subsequent rehydration and an

the basis of desiccation tolerance in seeds inappropriate proportion or distribution of
is comparative analyses between seeds that freezable and non-freezable (bound) water
differ in their capacity to withstand water within the seed (Berjak et al., 1992;
loss, i.e. seeds of orthodox and recalcitrant reviewed in Bewley and Oliver, 1992;
plant species. ‘Orthodox’ seeds can be Vertucci and Farrant, 1995). Recent research
stored for long periods under conventional in this area will be discussed briefly but see
conditions, i.e. in the dry state and at low also Chapters 6–8 of this volume.
temperature. Recalcitrant seeds, on the
other hand, do not undergo maturation
drying, nor are they capable of withstand- 5.2. Development and Acquisition of
ing water loss of the magnitude of that Desiccation Tolerance
experienced by orthodox seeds. The seeds
are shed at relatively high moisture con- 5.2.1. Changes in water status during
tents and are highly susceptible to desicca- development of orthodox seeds
tion injury; in order to remain viable, they
must not undergo any substantial change The three major phases of seed develop-
in moisture. They are not storable under ment characteristic of orthodox seeds
conditions suitable for orthodox seeds and, (namely histodifferentiation, expansion
even when stored under moist conditions, and maturation drying; Fig. 5.1) are marked
their viability is frequently brief and only by distinctive changes in fresh weight, dry
rarely exceeds a few months (reviewed in weight and water content (Fig. 5.2). During
Chin and Roberts, 1980; Bewley and Black, histodifferentiation and early cell expan-
1994; Smith and Berjak, 1995; Vertucci and sion, there is a rapid increase in whole
Farrant, 1995; Berjak and Pammenter, seed fresh weight and water content.
1997; Pammenter and Berjak, 1999). Thus Generally, a period of rapid dry-weight
the terms ‘orthodox’ and ‘recalcitrant’ have gain follows (when whole seed fresh
been used to describe the storage behaviour weight is relatively stable); this takes place
of seeds. A category intermediate between during the later part of the seed expansion
orthodox and recalcitrant is now recog- phase of development. Most seeds lose
nized (e.g. coffee) in which seeds survive water during this phase as reserves are
desiccation but become damaged during deposited primarily within storage tissues,
dry storage at low temperatures (0°C and displacing water from the cells. This
20°C) (Ellis et al., 1990, 1991a). It is decline in water content slows as the seed
important to note, however, that the situa- approaches its maximum dry weight. Then,
tion is more complex and there is a gradual as the seed undergoes maturation drying
continuum of desiccation tolerance across and approaches quiescence, there is a
orthodox and recalcitrant species. period of fresh weight loss accompanied by
The question arises as to whether the a rapid decline in whole seed water con-
desiccation sensitivity of recalcitrant seeds tent (Kermode, 1990; Fig. 5.2).
is at least partially the result of an insuffi- Little is known about the mechanism and
cient accumulation of protective proteins, route of water loss from seeds. Some studies
or whether other factors (including a lack suggest the existence of a passive mecha-
of protective sugars) are more important. nism whereby water is lost primarily by
Since desiccation tolerance is arguably a evaporation from the surface of surrounding
quantitative feature (Vertucci and Farrant, seed structures (Nechiporenko and
1995), the amount of protective proteins, or Rybalova, 1983; Lee and Atkey, 1984;
the rate at which the proteins accumulate, Goncharova et al., 1985). Another suggestion
may determine the level of tolerance. is that water moves from the seed to the par-
Other features that may be part of the ent plant by a metabolically active process,
basis of desiccation sensitivity include an i.e. the plant actually ‘pumps’ the water from
inability to repair desiccation-induced dam- the seed (Meredith and Jenkins, 1975).
Expansion Maturation
Histodifferentiation (reserve deposition) drying
fw
dw
Grams
WC
I II III
Days of development
Fig. 5.2. A general scheme of changes in whole seed fresh weight (fw), dry weight (dw) and water content
(WC) during the histodifferentiation, expansion and maturation drying phases of development of orthodox
seeds. Three major periods are noted: I, rapid fresh-weight gain; II, rapid dry-weight gain; III, fresh-weight
loss. (From Kermode and Bewley, 1986.)
In soybean and castor bean, the desicca- drying of seeds at a desiccation-tolerant

tion period is most probably initiated by stage of their development promotes germi-
the severing of the vascular supply to the nation upon subsequent rehydration. Air-
seed (funiculus detachment) and senes- dried grains of wheat not only germinate at
cence of the pod or capsule (Greenwood an earlier stage of development than non-
and Bewley, 1982; Murray and Nooden, dried grains, but at later stages may also
1986). This would suggest that relocation germinate at a faster rate than their non-
of water from the seed to the parent plant dried counterparts (Mitchell et al., 1980;
is not the means by which water loss Symons et al., 1983). Seeds of Phaseolus
occurs. Similarly, pectic substances in the vulgaris (French bean) undergo a transition
lumina of xylem elements of the rachis of to a desiccation-tolerant state around 26
wheat and barley (laid down during the DAP (days after pollination) approximately
final stages of grain maturation) may lead halfway through development (Dasgupta et
to the progressive dehydration of the ear by al., 1982). Seeds at 26–32 DAP can be
cutting off its water supply (Cochrane, induced to germinate to increasing extents
1985). if first dried over silica gel, whereas those
dried at 22 DAP fail to germinate when
subsequently rehydrated and they eventu-
5.2.2. Acquisition of desiccation tolerance ally deteriorate. The 22 DAP seeds do not
during development of orthodox seeds recover their full cellular and metabolic
integrity following the premature drying
Seeds of orthodox plant species cannot tol- treatment. At later times of development,
erate drying at all stages of their develop- however, the seeds acquire a tolerance of
ment. During very early development, seeds desiccation and also germinability is
are generally intolerant of drying, but they induced.
later undergo a transition to a desiccation- A similar situation exists for the castor
tolerant state at a particular time (reviewed bean seed (Ricinus communis) (Kermode
in Kermode, 1990, 1995). In many cases, and Bewley, 1985). Here, germinability is
not achieved until 50–55 DAP, whereas pre- (brought about by air-drying to 10% water
mature drying will promote the germina- content) during the course of germination,
tion ability of seeds as young as 25 DAP. At while the cotyledons remain tolerant for a
earlier stages of development, drying not considerably longer period (Senaratna and
only fails to induce germination ability, but McKersie, 1983).
also kills the seed. For both P. vulgaris and As will be discussed below, changes that
R. communis, the seeds acquire a tolerance occur on dehydration of the most
of desiccation and an ability to be potenti- desiccation-sensitive seeds (e.g. those of the
ated to germinate by this treatment around mangrove, Avicennia marina) can be very
25 days after development commences. similar to changes brought about by desic-
The transition to a desiccation-tolerant cation of orthodox seeds during the intoler-
state approximately midway through ant stage following germination (Farrant et
development is also characteristic of other al., 1986). Some recalcitrant seeds initiate
seeds, e.g. soybean, maize, barley, germination-related metabolism shortly
Agrostemma githago (Adams and Rinne, after shedding (reviewed in Vertucci and
1981; deKlerk, 1984; Bartels et al., 1988; Farrant, 1995) and, in A. marina, 10–15
Bochicchio et al., 1988). Tolerance of desic- days before shedding (Farrant et al., 1993b).
cation is gained over only a few days of As germination events progress, the seeds
development (e.g. between 20 and 25 DAP become increasingly sensitive to drying and
in R. communis); it is achieved well before attempting to store these seeds is akin to
the completion of major developmental storage of germinated, orthodox seeds
events such as reserve deposition and the (Farrant et al., 1986, 1988). There is no
commencement of normal maturation dry- clear-cut event delineating the end of seed
ing (Kermode and Bewley, 1985; Kermode development and the start of germination;
et al., 1986) (Figs 5.1 and 5.3). during both phases, recalcitrant seeds
appear to remain metabolically active,
although the axes may undergo a very brief
5.2.3. Loss of desiccation tolerance following period of relative quiescence.
germination of orthodox seeds
During germination, seeds initially remain 5.2.4. Effects of the rate and extent of
tolerant of reimposed desiccation, but at desiccation on the acquisition of tolerance of
some stage after axis elongation this ability orthodox seeds
is lost (Fig. 5.1). Germinating soybean
seeds are tolerant of drying during the The rate at which drying is imposed during
early stages, up to 6 h after commencing early development is critical for the subse-
imbibition, but they become increasingly quent expression of germinability, and
intolerant after this time. Thus, desiccation thus, when it is stated that a seed acquires
at 36 h after the start of imbibition kills the a tolerance of desiccation at a particular
seed (Senaratna and McKersie, 1983). The stage during its development, it is neces-
plasma membrane appears to be a major sary to define the rate of water loss to
site of damage in seeds during the desicca- which it is subjected (see Chapters 2 and
tion intolerant stage of germination, as 3). Whole seeds of several legumes (Adams
indicated by ultrastructural studies et al., 1983; Ellis et al., 1987) and R. com-
(Crevecoeur et al., 1976) and by the munis (Kermode and Bewley, 1985) are
increased solute and electrolyte leakage unable to withstand rapidly imposed dry-
upon subsequent rehydration (Senaratna ing (over silica gel or under regimes similar
and McKersie, 1983). There appears to be a to ambient laboratory conditions) at early
differential sensitivity of different seed tis- stages (i.e. during most of development,
sues with respect to the loss of desiccation prior to maturation drying) and exhibit no
tolerance. For example, axes of soybean germinability upon subsequent rehydra-
rapidly lose their tolerance to desiccation tion. This contrasts with seeds at the same
Histodifferentiation Cell expansion Maturation drying Germination/growth
154
(Water-stress
conditions)
Initiation of High rate of Cessation of

storage protein storage protein storage protein
synthesis synthesis synthesis Germination Induction of
and post- LEA protein
Inhibition of Initiation of Termination of germinative synthesis
18/3/02
germination LEA protein development growth

synthesis Inhibition of
Adjustment to reserve
Inhibition of drying breakdown
germination +
2:07 pm
Preparation for
+ germination
+ +
+ +
Page 154
Desiccation ABA Water

‘Vascular stress
ABA High
factors’ osmolarity
from
mother
A.R. Kermode and B.E. Finch-Savage
plant Water
stress –
– Precocious
germination
Vivipary
Fig. 5.3. Events during the development and germination/growth of seeds that are affected by desiccation, high osmolarity or abscisic acid (ABA). Desiccation
tolerance is generally acquired by seeds around mid-maturation, when late embryogenesis abundant (LEA) proteins and other protective substances are synthesized.
Induction of a subset of LEA proteins also occurs following the transition to germination and growth, i.e. in seedlings and plant vegetative tissues when they are
subjected to water-deficit-related stresses. (From Kermode, 1995.)
stage of development dried slowly over sat- occurs during the first 55 days of develop-
urated salt solutions or air-dried while ment of R. communis seeds, although these
enclosed in the pod, where full germinabil- seeds can tolerate slow drying at stages as
ity is evident. Tolerance of rapid drying early as 25 DAP (Kermode and Bewley,
generally occurs only at or near the com- 1985). Seeds (and isolated embryos) of some
pletion of reserve deposition (as indicated members of the Gramineae, on the other
by the attainment of maximum dry weight) hand, can survive and germinate following a
just after the onset of natural drying drastic drying treatment (which reduces
(Rogerson and Matthews, 1977; Kermode their water content to around 5%) at rela-
and Bewley, 1985; Ellis et al., 1987), tively early stages of development (Bartels et
although there are exceptions (see subse- al., 1988; Bochicchio et al., 1988). The rea-
quent discussion). sons for the variation between species in the
Gradual water loss may allow protective rate of water loss tolerated during their
changes to occur and hence increase the development are not known.
seed’s resistance to disruption by dehydra-
tion. Rapid drying presumably would not
allow such protective changes to take place 5.2.5. Variation in desiccation tolerance
and may cause considerable disruption to across species
cellular membranes and internal structures (see also Chapter 8)
(see Section 5.2.6). As a result, the seed
requires time for metabolic readjustment A wide range of species growing in differ-
(i.e. repair) following rapid drying. This ent habitats produce seeds that cannot sur-
cannot take place during drying itself vive drying to the low moisture content
because the seed reaches a critical dry (and that enables prolonged storage of orthodox
quiescent) state before the repair processes seeds. These species are spread widely
can be initiated. Such repair is also through the plant kingdom and updated
impeded upon imbibition because of a too- lists continue to be produced (Chin and
rapid influx of water, which cannot be Roberts, 1980; Hofmann and Steiner, 1989;
accommodated by the weakened or dam- Hong et al., 1996). Since Roberts (1973)
aged structural components of the cell. In introduced the terms orthodox and recalci-
fact, rapid rates of drying may predispose trant, it has become clear that the degree to
seeds to imbibitional injury, as indicated which seeds can survive desiccation varies
by increased rates of solute leakage, a greatly both within and between non-
symptom of cellular membrane disruption. orthodox species. The moisture contents to
However, slowing the rate of hydration which seeds of different species can sur-
may prevent a loss of germinability of vive dehydration ranges from just less than
rapidly dried seeds by allowing time for that of vegetative tissues to almost com-
repair to occur. Germinability of soybean plete tolerance. A recent study has shown a
seeds (following an accelerated ageing continuous scale of desiccation tolerance
treatment) is increased from 10% to 90% across 64 orthodox and recalcitrant
by controlling the rate of imbibition species, with critical water contents for
(Tilden and West, 1985). Since seeds survival ranging from 0.1 to > 1.2 g g1 dry
become capable of surviving rapid water weight (Sun, 1999). Even in genetically
loss during later development, they must related species there is wide variation in
acquire a greater cellular and metabolic desiccation tolerance at shedding, for
resistance to this event or have an example, within the genera Shorea, Hopea
enhanced ability to effect repair during the and Dipterocarpus (Tompsett, 1987), Citrus
early stages of imbibition. and Quercus (Sun, 1999) and Coffea
The capacity to withstand rapid or slow (Dussert et al., 1999). Species within the
desiccation during the tolerant phase of genus Acer (Hong and Ellis, 1990; Dickie et
orthodox seed development varies between al., 1991; Fig. 5.4) produce orthodox and
species. An intolerance of rapid desiccation recalcitrant seeds.
The restrictive categorization of seeds Consideration of variation in the desicca-

introduced by Roberts (1973), particularly tion tolerance of seeds across species as a
with the introduction of an intermediate continuum of behaviour in response to dry-
category (Ellis et al., 1990, 1991a,b), ing is arguably more realistic (Farrant et
remains useful in so far as it fulfils its origi- al., 1988; Berjak and Pammenter, 1997;
nal purpose to describe storage behaviour. Pammenter and Berjak, 1999). Such varia-
However, it does not accurately reflect tion, while tedious to categorize, provides
knowledge of seed response to desiccation. an opportunity to increase our understand-
Seed dry weight
Norway maple
Sycamore
Shedding
100
Seed moisture content (%)
80
60
40
20
Seed development
100
80
Germination (%)
60
40 Germination Germination
before drying after drying
20
August September October November
Seed development
Fig. 5.4. Seed development on adjacent trees of orthodox Acer platanoides (Norway maple) and recalcitrant
Acer pseudoplatanus (sycamore). Adapted from data in Hong and Ellis, 1990 and Dickie et al., 1991.
ing of the basis of desiccation tolerance, known what proportion of this variation is
which has only just begun to be exploited. genetic in origin or due to the environ-
A number of studies have shown that ments in which seed development, storage
desiccation-sensitive seeds do not pass or drying occurred. Temperature of drying
through a fully desiccation-tolerant phase can alter tolerance to desiccation (Ellis et
during their development, but tolerance al., 1990, 1991a; Berjak et al., 1994). Rate
tends to increase to a maximum near the of drying and the extent of storage before
time of shedding as moisture content drying are also important time-dependent
declines (Figs 5.4 and 5.5; reviewed by factors that could alter the extent of metab-
Finch-Savage, 1996; Berjak and olism-induced damage accumulated dur-
Pammenter, 1997). Thus, the extent of ing desiccation (Pammenter and Berjak,
seed maturity at harvest is one of a num- 1999). Such damage would alter the seed’s
ber of factors, discussed below, that deter- inherent capacity for desiccation tolerance
mines the degree of desiccation tolerance and may obscure discrete critical water
observed in sensitive seeds (e.g. as deter- potentials for survival. A further compli-
mined by critical water content experi- cating factor is that the onset of metabo-
ments). The increase in desiccation lism leading to the completion of
tolerance as water content declines during germination is often observed in recalci-
development (Fig. 5.5) and the apparent trant seeds following harvest or natural
continuous range of critical moisture con- shedding (Farrant et al., 1988; Pammenter
tents observed across species suggests that and Berjak, 1999), and this is known to
desiccation tolerance is a quantitative fea- progressively increase sensitivity in toler-
ture. However, it is more accurate to ant species (Section 5.2.3).
express the degree of desiccation tolerance
in terms of water potential as this reflects
5.2.5.1. Seed development in recalcitrant
the amount of water available to the cyto-
species
plasm (see Chapter 2). When data are pre-
sented in this way, there is convincing With the exception of A. marina, the phys-
evidence that tolerance appears to be iology of seed development following ini-
acquired in discrete water potential steps tial histodifferentiation has strong
during development (Farrant and Walters, similarities across the recalcitrant species
1998), and the tolerance of species may be so far studied in detail (reviewed by Finch-
grouped according to these steps (Walters, Savage, 1996; Berjak and Pammenter,
1999; see Chapter 9), though in other stud- 1997). This general pattern of recalcitrant
ies this was thought unlikely (Sun, 1999). seed development is also similar to that of
It is argued that these critical water poten- orthodox seeds before they reach maxi-
tials, which have different water activities mum dry weight (mass maturity) and
and associated metabolic processes rapidly lose water following vascular sepa-
(reviewed by Vertucci and Farrant, 1995; ration (Finch-Savage, 1996; Farrant et al.,
see also Chapters 2 and 9), may be related 1997). For example, when adjacent trees of
to specific desiccation stresses and dis- the sympatric species Acer pseudoplatanus
crete patterns of gene expression (Walters, (recalcitrant) and Acer platanoides (ortho-
1999). Thus, critical water potentials may dox) are compared, there is a strong tempo-
be determined by specific tolerance mech- ral correlation in developmental events
anisms or by sufficient accumulation of such as the accumulation of seed reserves
desiccation protectants. and the development of germinability (Fig.
Within a species, seed tolerance of des- 5.4). However, there are a number of com-
iccation can vary according to provenance mon characteristics of recalcitrant seed
even to the extent that one species, neem development that contrast with those of
(Azadirachta indica), has been described orthodox seeds and which result in seeds
as both orthodox and recalcitrant (Berjak adapted for rapid germination and estab-
and Pammenter, 1997). However, it is not lishment. In general, recalcitrant seeds at
Seed development
70 Reserve accumulation
(a)
60
50
40
30
Moisture content at 50% viability (%)
20
40 45 50 55 60 65 70 75 80 85
35
(b)
30
25
20
45 46 47 48 49 50 51 52 53 54 55
Moisture content at harvest (%)
Fig. 5.5. The relationship between moisture content at harvest and desicccation tolerance (moisture content
at 50% viability) (a) during seed development in Quercus robur in 1989, and (b) following shedding in 1989
( ), 1990 ( ), 1991 (), early in 1993 ( ) and late in 1993 (∆ ). The linear regression in (a) and (b) is
fitted to 1989 data (r 2 = 0.937, d.f. 5). (From Finch-Savage and Blake, 1994.)
shedding have had almost no net loss of 1993; A. pseudoplatanus, Hong and Ellis,
water; they can still be accumulating dry 1990). In contrast, viviparous germination
weight, they retain active metabolism, is a common event in other tropical recal-
remain desiccation-sensitive and have no citrant species such as Telfairia occiden-
requirement for desiccation to stimulate talis (Akoroda, 1986) and A. marina
subsequent germination. Despite these (Farrant et al., 1993b).
apparent adaptations for rapid germina- During development, tolerance to desic-
tion, a few temperate recalcitrant species cation increases throughout reserve accu-
are dormant at shedding (Aesculus hip- mulation as percentage moisture content
pocastanum, Tompsett and Pritchard, decreases in most recalcitrant seeds as it
does in orthodox species (Hong and Ellis, opment in recalcitrant species have not sig-
1990; Finch-Savage, 1996; Berjak and nificantly improved their desiccation toler-
Pammenter, 1997; Farrant et al., 1997). ance, suggesting inherent limitations to the
These changes are concomitant with a development of full tolerance.
reduction in vacuolar volume, the appear- A. marina will tolerate little drying and
ance of a semi-viscous state and other can be considered to be at the extreme sen-
changes associated with desiccation toler- sitive end of the tolerance continuum
ance (Farrant and Walters, 1998). However, across species; developmental age has lit-
in recalcitrant seeds there appears to be no tle influence on the desiccation sensitivity
clear end point to development. For exam- of seeds (Farrant et al., 1993b). In some
ple, seeds of Quercus robur are shed at dif- other species, maximum desiccation toler-
ferent moisture contents in different years ance is reached at a point before shedding
on the same tree and those shed with the (e.g. A. pseudoplatanus, Hong and Ellis,
lowest moisture content are most tolerant 1990) and tolerance may then subse-
to desiccation (Fig. 5.5; Finch-Savage and quently decline (e.g. Litchi chinensis,
Blake, 1994). There is a linear relationship Clausena lansium and Coffea arabica)
between moisture content at harvest (pre- (Ellis et al., 1991a; Fu et al., 1994). This
mature and at shedding) and the moisture decrease in tolerance may be due to the
content at which 50% of seeds remain initiation of germination (Farrant et al.,
viable during drying. This contrasts with 1988; Hong and Ellis, 1992). A gradual
orthodox species, where desiccation toler- decrease in tolerance is then shown as ger-
ance continues to increase after the acqui- mination proceeds in desiccation-sensitive
sition of maximum seed dry weight, during seeds (Farrant et al., 1988) as it does in
maturation drying, which results in a qui- orthodox seeds (Hong and Ellis, 1992).
escent seed (Sanhewe et al., 1996). In Q.
robur, development is indeterminate, but
5.2.5.2. Time-dependent effects of storage
consistent in several respects with that of
and drying rate
orthodox seeds shed early before mass
maturity and therefore before full desicca- Many recalcitrant seeds are characteristi-
tion tolerance is acquired (Finch-Savage cally large and consequently dry slowly.
and Blake, 1994). It is therefore tempting to But even when the seeds are of similar size
suggest that the level of desiccation toler- to comparable orthodox seeds, such as in
ance may depend upon how far seeds of a A. pseudoplatanus (recalcitrant) and A.
species progress through development, platanoides (orthodox), the recalcitrant
possibly an evolutionary consequence of Acer takes 12 times as long to reach 20%
the environmental and selection pressures moisture content under the same drying
that were exerted on them in the past. conditions (Greggains et al., 2000a). A fur-
These ideas are taken further in a compari- ther characteristic of recalcitrant seeds is
son of development in orthodox P. vulgaris that even in the absence of desiccation they
seeds, and the recalcitrant seeds of A. hip- deteriorate rapidly and are therefore short-
pocastanum and A. marina (Farrant et al., lived. Differences in the reported critical
1997). One possibility in temperate cli- water contents for recalcitrant seeds can be
mates is that the onset of winter truncates greatly influenced by these factors, and the
seed development so that full tolerance threshold water potentials for a number of
does not develop. However, seeds of Q. other physiological processes can be highly
robur produced on trees grown outside the dependent on the postharvest history of the
temperate climate zone in the apparently seed (Tompsett and Pritchard, 1998). A
non-limiting environment and season common observation is that if seeds are
length of South Africa also failed to pro- stored before drying they become more
duce desiccation-tolerant seeds (Finch- desiccation-sensitive (Finch-Savage et al.,
Savage and Blake, 1994). In addition, 1996). This may be because of damage
experimental manipulations of seed devel- accumulated during storage (Greggains et
al., 2000b) or because storage has allowed (Vertucci et al., 1991). It appears from this
the initiation of germination (Farrant et al., and similar published work that desicca-
1985; Berjak et al., 1989). However, in tion sensitivity in recalcitrant seeds and
some other species, particularly in seeds excised embryonic axes can occur at a min-
shed early, development may continue so imum of two levels, which are influenced
there is a considerable delay before the by the rate of drying:
initiation of germination (Berjak and
1. The removal of freezable water is toler-
Pammenter, 1997).
ated and minimum survivable moisture
Drying rate can affect the apparent toler-
content coincides with the quantity of non-
ance of whole seeds (Farrant et al., 1985;
freezable (matrix-bound) water in the tis-
Pritchard, 1991; Pammenter et al., 1998,
sue. Farrant et al. (1988) suggested that
1999; Chapter 3), although this is not
recalcitrant seeds, unlike orthodox seeds,
always the case (Finch-Savage, 1992). In
require this bound water for the mainte-
embryonic axes, as discussed below, rapid
nance of membrane integrity.
drying consistently improves their survival
2. Seed viability is lost as freezable (free)
to lower moisture contents, perhaps
water is removed.
because there is less time for damage to
accumulate during drying. However, The former situation usually occurs in
Pammenter and Berjak (1999) pointed out rapidly dried embryonic axes (Berjak et
that rapid drying does not confer improved al., 1992), but has also been reported in
desiccation tolerance because axes that relatively desiccation-tolerant recalcitrant
have been rapidly dried to lower moisture seeds (Finch-Savage, 1992; Finch-Savage
contents do not survive long under ambi- et al., 1993). The second situation occurs
ent conditions. in more sensitive recalcitrant species. A
third level of sensitivity, which is unaf-
fected by drying rate, is thought to occur
5.2.5.3. Desiccation tolerance differs
in the most sensitive seed: mechanical
between seed tissues
damage resulting from a reduction in cell
In general, when isolated, the embryonic volume in the early stages of drying
axis of desiccation-sensitive species is (Pammenter and Berjak, 1999). In all
more tolerant than when it is dried in the cases, solute leakage precedes viability
whole seed (Berjak et al., 1990; Pammenter loss during desiccation, suggesting that
et al., 1991; Leprince et al., 1999), which significant membrane damage has
may result from its more rapid drying occurred. These differences in critical
when excised than when in situ (Berjak et moisture levels are consistent with the
al., 1990). It may also relate to a signifi- concept of discrete water potential steps
cantly smaller proportion of ‘matrix-bound’ associated with desiccation tolerance
(Finch-Savage, 1992; Finch-Savage et al., (Farrant and Walters, 1998; Walters, 1999)
1993) or non-freezable water (water that is and may be related to the different desic-
bound or structure-associated; Berjak et al., cation stresses encountered. In species
1992) in the axis compared to storage tis- where loss of viability appears to coincide
sues. Desiccation tolerance of Landolphia with removal of non-freezable water
kirkii axes is also affected by developmen- (Berjak et al., 1992; Finch-Savage, 1992),
tal status and, in contrast to the whole it may be that these seeds lack mecha-
seed, immature axes are more tolerant than nisms required to stabilize membranes as
mature ones (Berjak et al., 1992) as a result water is removed. Where viability is lost
of a lower content of non-freezable water in as freezable water is removed, other
the immature axes. Despite their greater mechanisms may be limiting, such as the
desiccation tolerance, the immature axes, provision of adequate protection against
unlike mature axes, do not survive expo- free-radical attack. The provision of puta-
sure to very low temperatures and are tive protective mechanisms in seeds is
therefore unsuitable for cryopreservation reviewed in the following section.
5.2.6. Mechanisms underlying the ment), but the physical nature of such
acquisition of desiccation tolerance: recent changes is enigmatic. While drastic
findings and speculations changes to membranes have been observed
upon their drying in vitro (Crowe et al.,
5.2.6.1. The effects of premature desiccation 1992), the evidence suggests that mem-
during the tolerant and intolerant stages of branes (in the desiccation-tolerant state)
orthodox seed development appear to be protected against certain
major alterations (e.g. transformation of the
Premature desiccation during the early
bilayer arrangement to hexagonal-type
developmental stages of P. vulgaris (up to
arrangements). The importance of a preser-
22 DAP, i.e. during the desiccation-sensi-
vation of basic membrane composition dur-
tive stage) drastically reduces the metabolic
ing drying is obvious, for cells would
and cellular integrity of the axis upon sub- surely perish without the prompt re-estab-
sequent rehydration. Particularly evident is lishment of functioning membranes upon
a loss in the capacity to recover polyribo- rehydration when they are challenged by a
some levels and to resume protein synthe- swiftly changing hydration environment.
sis (Dasgupta et al., 1982). Considerable Fourier transform infrared microspec-
damage is inflicted upon cellular organelles troscopy has been useful for elucidating
(including protein bodies and mitochon- some of the changes to membranes and
dria) and upon the nuclear membrane. In other cellular constituents (e.g. proteins) fol-
contrast, such severe perturbations do not lowing desiccation at the tolerant and sensi-
occur following desiccation at a tolerant tive stages of seed development (Wolkers et
stage, e.g. at 32 DAP. Moreover, the limited al., 1998b, 1999; see Chapter 4). Isolated
damage that is sustained during drying at immature maize embryos acquire a toler-
this stage is rapidly reversed following ance to rapid drying between 22 and 25
rehydration; cells regain their normal DAP, but can tolerate slow drying from 18
appearance within a very short time. DAP onwards. Rapid drying at the tolerant
Studies on the effects of desiccation stages is associated with lower membrane
during the sensitive stages of seed develop- permeability upon rehydration in contrast
ment (or germination) suffer the limitation to embryos rapidly dried at a sensitive stage,
of not distinguishing between the causes of in which there is an almost complete loss of
desiccation intolerance and changes during membrane integrity. In addition, there is a
the death of cells as a consequence of greater proportion of -helical protein struc-
undergoing desiccation. Nevertheless, a tures in embryos rapidly dried at a tolerant
few of these studies (particularly those that versus an intolerant stage (Wolkers et al.,
have compared the effects of drying at the 1998b). The proportion of -helical protein
sensitive and tolerant stages) have pro- structures increases in the axes of embryos
vided some useful information on the cel- during slow drying of 20 and 25 DAP seeds
lular sites and/or metabolic processes that (as compared with that within fresh devel-
are most susceptible to damage during des- oping seeds at these stages), and this factor
iccation/rehydration, and hence require coincides with the acquisition of additional
protection for retention of viability (see tolerance of desiccation.
Chapters 9 and 12). As implied earlier, the
integrity of membranes in seeds is of cru-
cial importance to the maintenance of via- 5.2.6.2. Cellular and metabolic changes
bility; any undue disruption of the during the transition to a desiccation-tolerant
membrane systems during drying is likely state
to be of immediate consequence once the
seed imbibes. It is probable that some DEHYDRINS AND LATE EMBRYOGENESIS ABUNDANT
changes in membrane structure are pro- PROTEINS ARE PRODUCED AS PART OF THE DEVELOP-
voked as a consequence of desicccation MENTAL PROGRAMME. As noted earlier,
(even during the tolerant stages of develop- orthodox seeds are not capable of with-
standing desiccation at all stages during glycine content and a high hydrophilicity
their development, but their potential index) also accumulate in Escherichia coli
acquisition of tolerance is usually sub- and in the yeast Saccharomyces cerevisiae
stantially earlier than the onset of the nat- as an adaptive response to hyperosmotic
ural drying event itself. A highly conditions; the authors suggest that most
abundant set of hydrophilic proteins LEA proteins are part of a more wide-
exhibiting temporal regulation during spread group that they term ‘hydrophilins’
seed development (i.e. the late embryoge- (Garay-Arroyo et al., 2000).
nesis abundant (LEA) proteins first A subset of the LEA proteins (including
described in cotton) has been implicated the LEA D-11 family and some denoted
in desiccation tolerance (Dure, 1993; see RAB (responsive to abscisic acid (ABA)) in
also Chapters 1, 10 and 11). The genes rice) have been termed dehydrins; they
encoding these proteins arise as highly exhibit some common features in their
coordinately regulated sets, which on this structure that may be important for their
basis comprise two distinct classes in cot- putative protective function (Close, 1996).
ton (Hughes and Galau, 1989); the mRNAs Dehydrin genes exhibit a flexible expres-
that correspond to the two classes peak sion repertoire, being responsive to both
just prior to, or during, desiccation developmental and environmental cues
(Hughes and Galau, 1989). LEA protein (reviewed by Thomas et al., 1991).
synthesis constitutes a large proportion of Transcription of these genes is also
the translational activity of the cotton induced in virtually all seedling tissues
embryo during late maturation (up to subjected to water stress (i.e. non-lethal
25%), regulated at the level of transcrip- desiccation). Thus, the protective role of
tion, i.e. by the abundance of lea mRNAs dehydrins in the survival of water loss is
(Hughes and Galau, 1987). In mature cot- purported to be dual: during maturation
ton embryos they comprise about 2% of drying of the developing seed and follow-
the total soluble protein (Dure, 1993) or ing germination/growth of the mature seed
about 30% of the non-storage protein moi- (i.e. in seedlings or plant vegetative tissues
ety (Hughes and Galau, 1987). undergoing mild water stress) (Fig. 5.3).
Since their description in cotton, mes- Precocious appearance of the proteins and
sages homologous to the lea cDNAs of cot- their mRNAs can be induced in cultured
ton (representing at least five conserved immature embryos by exogenous ABA
families of corresponding proteins) have treatment. It has been hypothesized that,
been found in abundance in mature dry during normal development, high levels of
embryos and storage organs of many ABA induce the accumulation of these
diverse plant species including polypeptides and hence prepare the
Arabidopsis thaliana, several crop species embryo for desiccation or possible cellular
and gymnosperms. Protein families related disruption upon subsequent rehydration
(reviewed by Kermode, 1990, 1995; Bray,
to some of the LEA proteins are induced
1991, 1993; Bewley and Oliver, 1992;
during drying of xerophytic species, e.g.
Chandler and Robertson, 1994; Ingram and
the desiccation-tolerant resurrection plant
Bartels, 1996).
(Craterostigma plantagineum), which is
capable of surviving in the desiccated state REGULATION OF DEHYDRIN AND LEA GENE EXPRESSION
for long periods and resumes full physio- BY ABA. Since ABA has been implicated as a
logical activity within several hours of mediator of stress responses, especially
rehydration (Bartels et al., 1990; where water stress is concerned, its poten-
Piatkowski et al., 1990; reviewed in tial role as the primary regulator of lea
Ingram and Bartels, 1996; see Chapter 11). genes in vegetative tissues has been inves-
The desiccation-related proteins accumu- tigated (reviewed by Bray, 1991; Chandler
late in leaves; some are also present within and Robertson, 1994; Kermode, 1995;
roots and in seeds. Proteins sharing fea- Ingram and Bartels, 1996; Plant and Bray,
tures with plant LEA proteins (e.g. a high 1999). In some cases (but not all) ABA
application stimulates the accumulation of of maize has been undertaken in order to

the mRNAs in the absence of water stress, elucidate the possible regulatory role of
and there is some evidence that endoge- ABA and to address whether discrete paral-
nous ABA plays a regulatory role in their lel ABA and stress response pathways exist
expression in seedling tissues (Fig. 5.3). in developing maize embryos (Finkelstein,
For example, there is excellent correlation 1993). However, substantially different
(e.g. in barley and maize) between the results have been obtained depending on
amounts of mRNA and ABA in shoots, the type of LEA protein under study and
roots and aleurone layers from either well- more systematic and detailed investigation
watered, dehydrated or dehydrated/rehy- is needed. Several 23- to 25-kDa proteins
drated seedlings (Chandler et al., 1988; corresponding to RAB17 are expressed nor-
Gomez et al., 1988). Other stresses that mally in the ABA-deficient mutants of
lead to increased endogenous ABA (e.g. maize (vp-2 and vp-5) in contrast to the
salt, cold and wounding) are often also dependency on applied ABA for their
capable of eliciting expression of these expression in vegetative tissues of the
genes. The most convincing evidence for mutant seedlings (Pla et al., 1989). Likewise,
the role of ABA in dehydrin gene expres- the regulation of the Rab28 gene (a homo-
sion comes from studies of ABA-deficient logue of the cotton lea D34 gene) in excised
mutants of maize (Pla et al., 1989, 1991). young embryos of the ABA-deficient vp-2
When exposed to dehydration stress, mutant closely resembles that found in non-
seedlings homozygous for mutations lead- mutant excised young embryos (Pla et al.,
ing to vivipary (e.g. vp2 and vp5) fail to 1991). In contrast, embryos of the ABA-
elevate ABA levels and show a correspond- insensitive mutant of maize (vp-1) do not
ing inability to produce dehydrins. Similar accumulate Rab28 transcripts to significant
results have been found in an ABA-defi- amounts during development; surprisingly,
cient mutant of tomato (Cohen and Bray, induction of Rab28 mRNA can be achieved
1990; reviewed by Bray, 1991). in these young vp-1 embryos by ABA treat-
What is the evidence that ABA plays a ment (Pla et al., 1991). Expression of the
central regulatory role in the expression of maize Em gene (a group 1 lea gene) may be
lea genes within the developing seed? dependent on both the presence of ABA
Generally, the mRNAs encoding LEA pro- within embryos and its perception via a
teins are detected in embryos around mid- functional Vp-1 gene product; it is barely
development; highest levels of expression detectable in the ABA-deficient mutant
occur either at incipient desiccation or embryos and is undetectable in the ABA-
during maturation drying itself (Gomez et insensitive vp-1 embryos. Nevertheless, vp-1
al., 1988; Mundy and Chua, 1988; Close et embryos do exhibit a response to both ABA
al., 1989). The mRNAs are preserved in and osmotica at the molecular level, since
the mature dry seed but are rapidly they accumulate specific gene products (22-
degraded upon imbibition, although, in and 30-kDa polypeptides) differentially
some cases, certain proteins persist follow- upon imposition of osmotic stress or exoge-
ing imbibition (Han et al., 1996). nous ABA (Butler and Cumming, 1993).
Precocious appearance of the proteins and The Vp-1 gene is thought to encode a
their mRNAs can be induced in cultured novel type of transcription activator, which
immature embryos by exogenous ABA. plays a role in the expression of ABA-
Thus, high levels of ABA during mid- responsive genes during seed development
development are thought to induce the (e.g. maize globulin and Em genes), similar
accumulation of these polypeptides and to the Abi-3 gene product in Arabidopsis
hence prepare the embryo for desiccation and other species (reviewed by Giraudat et
or possible cellular disruption upon subse- al., 1994; Hattori et al., 1995; McCarty,
quent rehydration (Fig. 5.3). 1995). The promoter elements of the rice
A comparative analysis of wild-type, Osem gene (an Em-type gene) required for
ABA-deficient and ABA-insensitive mutants regulation by VP-1 have been identified
(Hattori et al., 1995). These include 1 plantlets subjected to drought stress.

TACGTGTC (an ABA-responsive element or Thus, there may be two regulation path-
ABRE), a small sequence located just down- ways that mediate dehydrin transcript
stream of the ABRE and a quantitiative ele- accumulation in seeds and stressed vegeta-
ment (the sph box/RY repeat), conserved in tive tissues – an ABA-dependent pathway
many seed-specific gene promoters. and an ABA-independent pathway;
Accumulation of group 3 LEA proteins together, these pathways may have cumula-
in maturing maize embryos may be depen- tive effects (Giordani et al., 1999).
dent upon ABA but appears to have no Ectopic expression of the Abi-3 gene
specific requirement for the Vp-1 gene product (Giraudat et al., 1992) allows the
product (Thomann et al., 1992). ABA-mediated activation of lea genes in
Interestingly, when an ABA-deficient, vegetative tissues of A. thaliana (Parcy et
viviparous mutant of maize (vp-5) is al., 1994). Seed viability is not altered in
manipulated either genetically or via ABA-deficient (aba) and ABA-insensitive
biosynthesis inhibitors to induce gib- (abi-3) mutants of A. thaliana, yet seeds of
berellin (GA) deficiency during early seed double mutants exhibiting these two traits
development, vivipary is suppressed in do not undergo desiccation on the parent
developing kernels and the seeds acquire plant, are intolerant of artificial desiccation
desiccation tolerance and storage longevity and fail to produce some of the late abun-
(White et al., 2000). Major accumulation of dant proteins (Koornneef et al., 1989;
GA1 and GA3 occurs in wild-type maize Meurs et al., 1992). These double-mutant
kernels, just prior to a peak in ABA content seeds accumulate only low amounts of the
during development. It is speculated that major storage proteins and are deficient in
these GAs induce a developmental pro- several low-molecular-weight polypep-
gramme that leads to vivipary in the tides, both soluble and bound, some of
absence of normal amounts of ABA, and which are heat-soluble. During develop-
that a reduction of GAs re-establishes an ment (14–20 DAP), the low amounts of var-
ABA/GA ratio appropriate for suppression ious maturation-specific proteins are
of germination and induction of matura- degraded and proteins characteristic of ger-
tion. Induction of GA deficiency does not mination are induced, in the absence of
suppress vivipary in vp-1 mutant kernels, germination. Here, the seed developmental
suggesting that VP-1 acts downstream of programme is not completed, and there is a
both GA and ABA in programming seed premature (yet incomplete) switching to a
development (White et al., 2000). germination programme in the absence of
Two ABA-deficient mutants of sun- substances presumed to be protective
flower have been isolated – nd-1, an albino, against desiccation. Seeds become desicca-
non-dormant and lethal mutant exhibiting tion-tolerant when the plants are watered
a very low ABA content and no accumula- with an ABA analogue (LAB 173711) or by
tion of ABA in response to stress, and w-1, incubating isolated immature seeds (11–15
a wilty mutant, with reduced ABA accu- DAP) with ABA and sucrose. Whereas
mulation during embryo and plantlet sucrose may protect desiccation-sensitive
development and drought stress (Giordani structures from damage, ABA inhibits pre-
et al., 1999). The w-1 mutant exhibits a cocious germination and may be required
reduction of dehydrin transcripts in the for, or is accompanied by, completion of
early stages of embryo development as the seed developmental programme and
compared with wild-type embryos, indicat- associated acquisition of desiccation toler-
ing that ABA affects dehydrin accumula- ance (Meurs et al., 1992). In another study,
tion; however, the amount of dehydrin expression of a specific lea gene (a group 1,
transcripts appears to be independent of D19/Em homologue) was found to be
ABA content during late embryogenesis. slightly reduced in seeds of the
Accumulation of dehydrin transcripts Arabidopsis aba mutant, but was reduced
occurs in the leaflets and cotyledons of nd- by approximately tenfold in the abi-3
mutant; expression in the double mutant OTHER PROTECTIVE PROTEINS IMPLICATED IN DESICCA-
was not studied (Finkelstein, 1993). A dif- TION TOLERANCE. Specific small heat-shock
ferent Arabidopsis abi-3 mutant (abi-3-3, proteins (HSPs) of the cytosolic classes (I
isolated by screening for mutants that ger- and II) accumulate in seeds of several plant
minate in the presence of the GA biosyn- species. These proteins appear to be homo-
thetic inhibitor, Uniconazol) showed geneously distributed in all tissues of the
abnormal seed development, remaining seed and a role in the acquisition of desic-
green until maturity, had dramatically cation tolerance has been suggested (Coca
reduced amounts of storage proteins, was et al., 1994; Wehmeyer et al., 1996; see
desiccation-sensitive, and lacked dor- Chapters 1 and 10). In Arabidopsis and
mancy, indicative of a possible role for the other species, class I small HSPs are first
Abi-3-3 gene in the control of the synthesis detected during mid-maturation and
of seed storage proteins and desiccation become most abundant in dry seeds
protectants (Nambara et al., 1992). ABI5, a (Wehmeyer et al., 1996; Carranco et al.,
member of the family of basic leucine zip- 1999). In some seeds (e.g. Arabidopsis), the
per transcription factors, regulates a subset proteins decline rapidly during germination
of lea genes during seed development and (Wehmeyer et al., 1996); in others (e.g. sun-
in vegetative tissues in the presence of flower), they persist (Coca et al., 1994). The
ABA (Finkelstein and Lynch, 2000). Abi-3 gene product may activate expression
Further studies to clarify the role of of genes encoding specific small HSPs dur-
ABA and other components of the signal ing seed development. Transcriptional acti-
transduction pathway leading to lea gene vator mutants of Arabidopsis (abi-3-6,
expression and other late maturation fus3-3 and lec1-2) that are desiccation-sen-
events in developing seeds will be awaited sitive have undetectable amounts of HSP17.4
with interest. Recessive mutants of (abi-3-6) or highly reduced amounts of the
Arabidopis with lesions at the Fusca3 protein (fus3-3 and lec1-2), i.e. less than 2%
(fus3) and Leafy Cotyledon (lec1) gene loci of that in wild-type seeds (Wehmeyer and
lead to various abnomalities during mid- Vierling, 2000). Interestingly, a chimeric
embryogenesis and late embryogenesis, gene consisting of the small HSP gene pro-
including loss of dormancy and failure to moter linked to -glucuronidase (GUS)
acquire desiccation tolerance (Kirik et al., shows strong expression in mutant seeds
1998). FUS3 and LEC1 modulate the abun- that are heat-stressed, indicating that the
dance of ABI3 protein in seeds and syner- genes are under distinct developmental
gistic interactions between the three and stress regulation.
proteins (ABI3, FUS3 and LEC1) are Polypeptides produced in sunflower
thought to control various key events, seeds (e.g. HSP17.6 and HSP17.9, belong-
including accumulation of chlorophyll and ing to different families of cytoplasmic
anthocyanins, sensitivity to ABA and small HSPs) are indistinguishable from
expression of individual members of the low-molecular-weight HSPs expressed in
12S storage protein gene family (Parcy et vegetative tissues in response to water
al., 1997). Interestingly, part of FUS3 (a deficit, but they are different from homolo-
continuous stretch of 100 amino acids) gous proteins expressed in response to
shows significant similarity to the B3 thermal stress (Coca et al., 1994; Carranco
domain of the ABI3 and VP-1 proteins et al., 1997, 1999). Proteins immunologi-
(Luerssen et al., 1998), a domain which cally related to two sunflower small HSPs
interacts with the RY cis promoter motif of are detected in unstressed vegetative tis-
several seed proteins. Thus, both FUS3 and sues of the desiccation-tolerant resurrec-
ABI3 may be essential components of a reg- tion plant C. plantagineum and are
ulatory network acting in concert through induced to higher levels in these tissues
the RY-promoter element to control gene by water stress and heat shock. In desicca-
expression during late embryogenesis and tion-sensitive Craterostigma callus tissue,
seed development (Reidt et al., 2000). there are no detectable small HSP-related
polypeptides, but their expression, and the However, it is possible that the attainment
concurrent acquisition of desiccation tol- of a critical level of reserves is required
erance is induced by exogenous ABA before the seed can withstand desiccation
(Alamillo et al., 1995). (Kermode, 1997). Highly vacuolated cells
Small HSPs, immunologically related to (hence containing little reserve material)
a 20-kDa HSP from desiccation-sensitive may undergo severe mechanical disrup-
chestnut (Castanea sativa) seeds have been tion during water loss, and tearing or
detected in orthodox and recalcitrant seeds shearing of membranes (or other cellular
of 13 woody species; hence additional pro- components) could lead to irreversible
teins or mechanisms are likely to be changes in their internal morphology. The
involved in desiccation tolerance (Collada presence of a critical level of cellular
et al., 1997) (see later discussion). reserves would limit such changes (Table
Major intrinsic proteins (MIPs) are a 5.1). The quantity of reserves may also
family of channel proteins that are mainly merit consideration in relation to the loss
represented by aquaporins in plants. They of tolerance during seed germination. As
are generally divided into TIPs (tonoplast noted earlier, while soybean seed axes
intrinsic proteins) and PIPs (plasma mem- rapidly lose their tolerance to desiccation
brane intrinsic proteins) according to their during the course of germination, the
subcellular localization (reviewed by cotyledons remain tolerant for a consider-
Maurel et al., 1997). The vacuolar mem- ably longer period (Senaratna and
brane protein, -TIP (a water-channel pro- McKersie, 1983). The major breakdown of
tein), accumulates during seed maturation reserves within the cotyledons is a post-
in the parenchyma cells of seed storage germinative event; however, catabolism of
organs. Synthesis of this integral membrane reserves within the axes occurs relatively
protein does not appear to be related (in a early (i.e. during germination) to provide a
quantitative manner) to storage protein source of nutrients. The decline of
deposition and a role in seed desiccation, reserves below a critical level within the
cytoplasmic osmoregulation and/or seed axes may contribute to a loss of desicca-
rehydration has been suggested (Johnson et tion tolerance within this germinating tis-
al., 1989). The water-channel activity of the sue. Interestingly, certain seed storage
protein can be regulated by phosphoryla- proteins are suggested to play a more
tion and the protein assembles as a 60 Å direct role in desiccation tolerance. One
60 Å square in which each subunit is member of the vicilin superfamily in pea
formed by a heart-shaped ring comprised of (psp54) is expressed during seed desicca-
-helices. This structure is remarkably sim- tion and is not detected prior to this stage;
ilar to that of mammalian PIPs, suggesting the mRNA encoding the protein declines
that the molecular design of functionally soon after imbibition, but can be detected
analogous and genetically homologous in vegetative tissues in response to water-
aquaporins is maintained between the plant deficit-related stresses and ABA (Castillo
and animal kingdoms (Daniels et al., 1999). et al., 2000). A lower-molecular-weight
In the desiccation-tolerant resurrection protein (p1), which corresponds to the C-
plant C. plantagineum, homologues to PIPs terminal third of p54, shares some proper-
and TIPs are regulated by dehydration and ties with dehydrins and is suggested to
ABA, with members of a subset of PIPs protect chromatin structure during desic-
(PIPa) being regulated by ABA-dependent cation (Castillo et al., 2000). Seed storage
and ABA-independent pathways (Mariaux globulins of spermatophytes are thought to
et al., 1998). have evolved from a group of ancient
In many seeds, the acquisition of desic- single-domain proteins of prokaryotes
cation tolerance during the seed expansion and fungi functional in cellular desicca-
stage of development occurs well before tion/hydration processes (Baumlein et al.,
the completion of reserve deposition. 1995; Shutov et al., 1998).
Table 5.1. Possible components of desication tolerance in seeds and their protective action.a (Based on Kermode (1990). With permission from
CRC Press, Inc.)
Site or process
affected Protective component Protective action Possible mode of protection
Membranes Carbohydrates: sucrose Prevent changes in selective permeability Hydroxyl groups of sucrose replace water on
plus raffinose and/or due to lateral phase separation of hydrophilic (polar) end groups of membrane
stachyose phospholipids in the bilayer and the phase phospholipids; oligosaccharide (raffinose and/or
transition from liquid crystalline to gel stachyose) inhibits sucrose crystallization during
18/3/02
drying, preventing loss of its protective potential

Lipid-soluble antioxidants As above; prevent de-esterification of Scavenging activity increases resistance to
(e.g. tocopherols) membrane phospholipid and free fatty free-radical-mediated desiccation injury
acid accumulation
2:07 pm
Structure/metabolism Reserves: carbohydrates, Prevent ‘whole scale’ mechanical Critical level of reserves in vacuoles/storage bodies
lipids, proteins disruption of cellular components confers mechanical strength to whole cell
Prevent loss of tightly bound (‘vital’) water Water-binding capacity of cells enhanced with
necessary for structural and functional increased number of sorption sites
integrity of biomolecules
Page 167
Hydrophilic, denaturation- As above Water-binding capacity of cells enhanced with

resistant proteins (e.g. LEAs, increased number and strength of sorption sites;
other desiccation-inducible native conformation of protective molecules maintained
polypeptides) throughout drying; bind ions and thereby counteract
damaging effects of increasing ionic strengths of
cytosol during drying
‘Repair’ proteins, proteases, Rapid re-establishment of structural Efficient repair of membranes and other cellular
ubiquitin and extension and metabolic integrity following components restores normal functioning; aid
protein, HSP/molecular imbibition proteins in recovering their native conformation;
chaperones, some LEAs degradation of damaged or denatured proteins
Desiccation Sensitivity in Relation to Seed Development
aRefer to review by Kermode (1990) and Ingram and Bartels (1996) and references therein.
167
ROLE OF SUGARS (see also Chapters 1, 10 and humidity (Blackman et al., 1992). However,
11). It is likely that the underlying basis an increase in the amount of raffinose is
of desiccation tolerance is diverse and is not correlated with the acquisition of des-
not simply restricted to the synthesis of iccation tolerance of wheat embryos (Black
specific proteins. The ability to withstand et al., 1999). Accumulation of fagopyritol
desiccation may also depend upon B1 (a galactopinitol) in buckwheat seeds is
increased amounts (or a heightened capac- temporally associated with the acquisition
ity to synthesize) molecules which stabi- of desiccation tolerance during develop-
lize membranes. Carbohydrates such as ment (Horbowicz et al., 1998). This major
trehalose (a non-reducing disaccharide of soluble carbohydrate, which comprises
glucose) are effective in preserving the 40% of the carbohydrate of the mature
structural and functional integrity of mem- buckwheat embryo, declines with the loss
branes in vitro at low water contents of desiccation tolerance following germina-
(reviewed by Crowe et al., 1992). Drying tion. Temperature conditions during seed
and rehydration of the model membrane development that have a favourable effect on
sarcoplasmic reticulum usually results in vigour and storability of buckwheat seeds
the fusion of vesicles and loss of the ability result in seeds having a lower sucrose-to-
to transport calcium. However, when disac- fagopyritol ratio as compared with those
charides such as trehalose are present in that develop under non-optimal tempera-
concentrations equivalent to those in desic- ture conditions (Horbowicz et al., 1998).
cation-tolerant organisms, functional vesi- Although trehalose is not abundant in
cles are preserved. Membrane fusion vascular plants, it has been identified as a
during desiccation is thought to be pre- major carbohydrate in more than 70
vented as a result of the sugars’ hydroxyl species of desiccation-tolerant lower plants
groups interacting (i.e. forming hydrogen (reviewed by Muller et al., 1995). There are
bonds) with the polar head groups of phos- recent reports of trehalose in relatively
pholipids and functional groups of pro- high amount in two desiccation-tolerant
teins (Crowe et al., 1992). Thus, the sugars angiosperms (reviewed by Muller et al.,
are thought to alter physical properties of 1995; see references therein). One is
dry membranes so that they resemble those Myrothammus flabellifolia, a dicotyledo-
of fully hydrated biomolecules (Crowe et nous plant living in arid, rocky regions in
al., 1992) (Table 5.1). southern Africa, the leaves of which con-
The occurrence of trehalose in high contain about 3% trehalose on a dry weight
centrations in anhydrobiotic organisms basis. The other is the grass Sporobolus
such as yeast and nematodes (up to 20% of stapfianus, in which trehalose comprises
their dry weight) suggests that this sub- 2–5% of the total soluble carbohydrates.
stance may be involved in their desiccation Interestingly, even though most higher
tolerance (Crowe et al., 1992). A role for plants contain low amounts of trehalose,
sucrose and raffinose (carbohydrates found high activities of trehalase, an enzyme
in much greater abundance in seed tissues which degrades trehalose, have been found
than trehalose) in the preservation of mem- (reviewed by Muller et al., 1995). A.
branes during drying has been suggested by thaliana possesses genes for at least one of
Leopold and Vertucci (1986) and several the enzymes required for trehalose synthe-
others. Orthodox seeds accumulate consid- sis, trehalose-6-phosphate phosphatase
erable amounts of soluble proteins and sug- (Vogel et al., 1998).
ars throughout maturation, and these As noted above, the protective effect of
collectively may be important in the acqui- sugars probably extends to preventing irre-
sition of a desiccation-tolerant state (Amuti versible changes to proteins. For example,
and Pollard, 1977). Stachyose accumulates phosphofructokinase is a tetrameric
in immature soybean seeds subjected to enzyme, which is irreversibly denatured
slow drying, but does not increase signifi- during desiccation, dissociating into inac-
cantly when seeds are maintained at high tive dimers. However, the disaccharides
sucrose, maltose and trehalose stabilize the dried embryos cannot account for their
activity of the enzyme (in vitro) during dry- enhanced viability as compared with
ing (Carpenter et al., 1987). Although the rapidly dried embryos; however, enhanced
evidence from these experiments carried synthesis of LEA proteins embedded in the
out in vitro is convincing, the role of sugars glassy matrix may be a contributing factor
(in vivo) in protecting cells during water (Wolkers et al., 1999).
deficit and during desiccation of orthodox The proteins of maturation-defective
seeds remains to be elucidated. mutants of Arabidopsis (e.g. abi-3 and lec
One way sugars may protect the cell mutants) appear to be more susceptible to
during severe desiccation is by glass forma- denaturation during heating (Wolkers et
tion (see Chapter 10); in the presence of al., 1998a). Proteins in dry wild-type seeds
sugars a supersaturated liquid is produced do not denature at temperatures up to
with the mechanical properties of a solid 150°C; those of dry desiccation-sensitive
(Koster, 1991). Only sugar mixtures equiva- seeds (lec1-1, lec1-3 and abi3-5) denature
lent in concentration and composition to at 68, 89 and 87°C, respectively. In con-
those of desiccation-tolerant embryos are trast, in desiccation-tolerant seeds (abi3-7
able to form glass at ambient temperature and abi3-1), denaturation commenced
(Koster, 1991) and this ability has been above 120 and 135°C, respectively. The dif-
associated with retention of viability of ferential sensitivity of the seed proteins of
maize embryos (Williams and Leopold, the mutants to denaturation has been
1989). Glass formation has been suggested attributed in part to differences in molecu-
to prevent cellular collapse during desicca- lar packing density, which is higher in dry
tion and to promote a state of metabolic desiccation-tolerant seeds than in dry des-
quiescence by restricting diffusion of sub- iccation-sensitive seeds (Wolkers et al.,
strates and products within cells (Koster, 1998a).
1991). Carrot somatic embryos, when pre-
treated with ABA, are able to tolerate slow OTHER MECHANISMS UNDERLYING DESICCATION TOLER-
drying, but are still intolerant of rapid dry- ANCE. The loss of desiccation tolerance dur-
ing. This appears to be due in part to the ing germination of soybean seeds is not
extent of protein denaturation, which is associated with any compositional changes
greater after rapid drying (Wolkers et al., in fatty acids, but is correlated with a
1999). In contrast to slowly dried embryos, decline in the quantity of lipid-soluble
which form a glassy state at room tempera- antioxidants in the membrane (Senaratna et
ture, no clearly defined glassy matrix is al., 1985a,b). These antioxidants may con-
formed in rapidly dried embryos. The aver- tribute to the desiccation tolerance of axes
age strength of hydrogen bonding is less in during the early stages of germination by
rapidly dried versus slowly dried embryos, preventing changes in membrane fluidity
which may be indicative of less extensive caused by free-radical attack on phospho-
‘molecular packing’ in the former. Sucrose lipids in response to drying (Senaratna et
accumulates following rapid drying of al., 1985a,b). The transition to desiccation
embryos; following slow drying, the trisac- sensitivity following germination of pea and
charide umbelliferose is accumulated at cucumber seeds is accompanied by a desic-
the expense of sucrose. In phospholipid cation-induced imbalance of metabolism
model systems, both carbohydrates are able (i.e. increased emission of CO2 and fermen-
to form a stable glass with drying; they tation products such as acetaldehyde) in the
depress the transition temperature of dry radicle, which precedes loss of membrane
liposomal membranes to an equal extent as integrity (Leprince et al., 2000). Imbalanced
well as preventing leakage from dry lipo- metabolism is significantly reduced when
somes upon subsequent rehydration. sensitive axes are dried in 50% O2 instead
Likewise, both exhibit an equal capacity to of air and it is suggested that a balance
protect a desiccation-sensitive protein. between down-regulated metabolism and O2
Thus, increased umbelliferose in slowly availability is associated with desiccation
tolerance. Products resulting from imbal- by facilitating the conversion of abnormal

anced metabolism (e.g. acetaldehyde) dis- L-isoaspartyl residues to normal L-aspartyl
turb the phase behaviour of phospholipid residues (Mudgett and Clarke, 1994). A
vesicles and thus may aggravate membrane summary of some of the possible compo-
damage induced by dehydration (Leprince nents of desiccation tolerance in seeds is
et al., 2000). presented in Table 5.1.
Within the developing seed, the thiol-
requiring (1-cysteine) peroxiredoxin family DIFFICULTIES IN ASSESSING THE ROLE OF PROTECTANTS
of antioxidants may protect tissues (e.g. the IN DESICCATION TOLERANCE. As indicated
embryo and aleurone layer of cereals) from above, a wealth of information has been
reactive oxygen species during desiccation derived from the study of desiccation-toler-
and early imbibition (Haslekas et al., 1998; ant systems, such as seeds and resurrection
Stacy et al., 1999). PER1, a protein belong- plants, and from the various molecular and
ing to this family, is maintained in imbibed biochemical analyses that have contributed
dormant barley seeds, but declines in the to our understanding of gene and protein
non-dormant seeds. In immature embryos function. In orthodox seeds, the metabolic
and aleurone layers, the protein resides in changes that occur either prior to or during
the nucleus and is most abundant within maturation drying (including the accumu-
the nucleolus (Stacy et al., 1999). In con- lation of oligosaccharides, sugars and LEA
trast, in mature imbibed dormant seeds, an proteins) may have functional significance
equivalent amount of protein is present in in protecting them against the rigours of
the cytosol. In Arabidopsis, the expression desiccation and/or subsequent rehydration.
of AtPer1, a gene encoding a protein with However, some of the changes in metabo-
similarity to barley PER1, is reduced in lism of orthodox seeds during the time of
seeds of the ABA-insensitive mutant, acquisition of desiccation tolerance may
abi3-1, but is unaltered in an ABA-defi- not directly contribute to the ability to
cient mutant of Arabidopis (aba-1) withstand water loss, but rather may be
(Haslekas et al., 1998). pre-programmed changes that are part of
Serotonin accumulation in walnut other seed developmental programmes ulti-
cotyledons is thought to protect seeds from mately important for seedling survival.
toxic ammonia concentrations following What are other research strategies that may
seed desiccation (Schroder et al., 1999). contribute to our understanding of the
In conclusion, the basis of desiccation underlying basis of desiccation tolerance?
tolerance of developing seeds is still a One strategy is the transfer of genes encod-
poorly understood phenomenon. What ing putative desiccation protectants into
emerges from the evidence available at pre- transgenic host plants with the ultimate
sent is a complex process involving various goal of testing protein function and
metabolic and/or structural adjustments, enhancing stress tolerance (see Chapter
which allow cells to undergo extensive 11). Another approach is the comparative
water loss with a minimum of damage analysis of LEA- and dehydrin-related pro-
(Table 5.1). However, while maturation teins and other putative desiccation protec-
drying may inflict limited damage on cells tants in recalcitrant versus orthodox seeds
of orthodox seeds, the capacity to reverse (discussed below). Both approaches have
such changes (i.e. to effect repair) upon limitations.
subsequent rehydration is probably an inte-
gral feature of desiccation tolerance
5.2.6.3. Desiccation-tolerance mechanisms
(Bewley and Oliver, 1992; Ingram and
in sensitive seeds
Bartels, 1996; O’Mahony and Oliver,
1999a,b). An L-isoaspartyl protein methyl- In the previous sections we have shown
transferase that accumulates in wheat that development of orthodox seeds fol-
seeds during the late stages of caryopsis lows a largely predetermined sequence of
development may repair damaged proteins events that leads to desiccation and then
shedding of the seed in a quiescent (and sidered to be particularly sensitive to des-

sometimes dormant) state. In evolutionary iccation. The pattern of ABA concentration
terms, it is not known whether the ability in seeds of the tropical wetland species, A.
to develop full desiccation tolerance has marina, during seed expansion differs from
been lost in species with recalcitrant seeds, that of other recalcitrant species; low and
was never gained, or is just not fully decreasing concentrations of ABA are pre-
expressed. The progress of evolution is also sent in the axis during reserve accumula-
likely to have differed among taxa (von tion (Farrant et al., 1993a). Moreover, ABA
Teichman and van Wyk, 1994; see Chapter concentration does not increase with dry-
8). Despite increasing interest in recalci- ing and dehydrin proteins are not detected
trant seeds, it is clear from recent reviews (Farrant et al., 1996). In general, the pres-
(Berjak and Pammenter, 1997; Pammenter ence of dehydrins in recalcitrant seeds is
and Berjak, 1999) that the cause of their associated with those species that have
desiccation sensitivity is still far from high ABA concentrations and are most
understood. However, comparison of likely to be exposed to moisture stress
desiccation-sensitive seeds with tolerant (Farrant et al., 1996). In addition, an earlier
orthodox seeds can clarify our understand- peak in ABA concentration of recalcitrant
ing of desiccation-tolerance mechanisms. Q. robur seeds is associated with greater
In the following section, the occurrence of desiccation tolerance at shedding (Finch-
these putative mechanisms in desiccation- Savage and Farrant, 1997). However, in T.
sensitive seeds is reported. cacao embryos in vitro, ABA is associated
with maturation events as it is in orthodox
ABSCISIC ACID AND PROTEINS. As noted in the seeds, but does not influence desiccation
previous sections, ABA may play a role in tolerance (Pence, 1992).
the regulation of dehydrin and lea gene In orthodox cotton, lea mRNAs that
expression in orthodox seeds. In the recal- have protein homology with dehydrins
citrant seeds of Theobroma cacao (Pence, accumulate relatively slowly during the
1991) and Q. robur (Finch-Savage et al., period of cotyledon expansion, but then
1992; Finch-Savage and Blake, 1994), there increase rapidly at the point of vascular
is a clear pattern of ABA accumulation separation (Galau et al., 1991). Recalcitrant
during seed expansion, similar to that seeds are often shed at a time when dry
reported in orthodox species, such as P. weight is still increasing and may therefore
vulgaris (Prevost and Le Page-Degivry, lack the phase of rapid increase in lea
1985a,b). In both the axis and cotyledons, mRNAs that occurs at the end of orthodox
ABA increases to a maximum and then seed development. Desiccation sensitivity
decreases before shedding. However, the may therefore be due in part to an inability
decline in ABA concentration prior to to accumulate a sufficient quantity of dehy-
shedding in recalcitrant seeds is limited drins or other LEA proteins.
and consistent with a continuing role for Small HSPs have also been associated
ABA in preventing precocious germination with desiccation tolerance in orthodox
(Finch-Savage and Farrant, 1997). seeds (see earlier discussion; DeRocher and
Dehydrin proteins accumulate during seed Vierling, 1994; Wehmeyer et al., 1996), and
development, and in response to seed dry- have been shown, like dehydrins, to accu-
ing, in a number of recalcitrant species mulate to significant levels in recalcitrant
(Finch-Savage et al., 1994; Gee et al., 1994; C. sativa seeds during development and to
Farrant et al., 1996; Han et al., 1997; be present in seeds of other recalcitrant
Greggains et al., 2000a). However, dehy- species (Collada et al., 1997). As discussed
drin proteins are not detected in mature earlier, the presence of dehydrin and small
undried axes of a range of recalcitrant trop- HSPs in recalcitrant seeds supports the
ical wetland species (Farrant et al., 1996). view that they alone are not sufficient to
These species would not normally be confer desiccation tolerance. However,
exposed to significant drying and are con- Farrant et al. (1996) suggested that the con-
verse might be true, i.e. their absence may sue in recalcitrant seeds tends to have a
imply an inability to tolerate desiccation, much lower oligosaccharide:sucrose ratio
and the absence of a specific protective than that generally present in orthodox
protein cannot be ruled out as the cause of seeds (Steadman et al., 1996). This ratio is
desiccation sensitivity. For example, the therefore a potential indicator of seed
membranes surrounding oil bodies of seeds behaviour; however, seeds of cocoa and A.
contain integral proteins, called oleosins, marina are exceptions (Steadman et al.,
which may maintain the integrity of these 1996). Pammenter and Berjak (1999)
organelles during desiccation and subse- pointed out that the proposed mechanisms
quent imbibition (Murphy et al., 1995; for the involvement of sugars in desicca-
Leprince et al., 1998). Oleosins are pre- tion tolerance operate at moisture contents
sent in the membranes of oil bodies in below those at which most recalcitrant
desiccation-tolerant seeds, but are absent, or seeds can survive. It is therefore perhaps
their amount is diminished, in desiccation- not surprising that there is no clear rela-
sensitive seeds (Leprince et al., 1998). tionship between the degree of desiccation
Thus, a lack of oleosins may be an impor- tolerance in recalcitrant seeds and sugar
tant factor in the desiccation sensitivity of accumulation.
oil-storing recalcitrant seeds. High monosaccharide levels have been
linked with desiccation sensitivity and the
SUGARS. Studies with recalcitrant seeds potential for damage resulting from the
show that there is no clear link between Maillard reaction (Koster and Leopold,
the presence of sugars and the level of des- 1988). In the later stages of development in
iccation tolerance in seeds. For example, orthodox seeds, monosaccharide levels are
large amounts of sugars including sucrose reduced and this also occurs in some
and stachyose accumulate during develop- (Farrant and Walters, 1998), but not all,
ment in the highly desiccation-sensitive species with recalcitrant seeds (Farrant et
seeds of A. marina (Farrant et al., 1993b). al., 1992, 1993b; Finch-Savage et al., 1993).
In the more tolerant Q. robur, sucrose and Monosaccharide levels were generally low
raffinose accumulate in the cotyledons and in most of the 18 species studied by
axes during the later stages of reserve accu- Steadman et al. (1996), including the recal-
mulation (Finch-Savage et al., 1993; Finch- citrant ones.
Savage and Blake, 1994) and, in mature
axes of Quercus rubra, desiccation sensitiv- IS DESICCATION SENSITIVITY DUE TO RETENTION OF
ity is not caused simply by the absence of METABOLIC ACTIVITY AT SHEDDING? Recalcitrant
non-reducing sugars (Sun et al., 1994). In a seeds, perhaps because they remain moist,
more comprehensive study, Steadman et maintain active metabolism throughout
al. (1996) determined the sugar composi- development to the time of shedding. For
tion of a range of recalcitrant, intermediate example, respiration of A. marina seeds,
and orthodox species and combined this after a small decline at the start of reserve
with published data for additional species. accumulation, remains relatively constant
They found no simple relationship between until abscission (Farrant et al., 1992). High
seed type and total sugar content or sucrose respiration rates have also been recorded in
level; however, the content of raffinose and the seeds of other recalcitrant species at
stachyose was generally lower in recalci- shedding (Farrant et al., 1992, 1997;
trant than in orthodox seeds. These Poulsen and Eriksen, 1992; Finch-Savage
oligosaccharides were also found to be and Blake, 1994; Salmen Espindola et al.,
lower in seeds of recalcitrant A. pseudopla- 1994; Leprince et al., 1999). The absence of
tanus than in seeds of the orthodox A. pla- substantial developmental arrest as seeds
tanoides (Greggains et al., 2000a). In approach shedding is confirmed by ultra-
general, there are large variations in the structural and biochemical studies (Dodd
content of sugars between tissues of desic- et al., 1989; Berjak et al. 1992; Farrant et
cation-sensitive seeds, but at least one tis- al., 1992; Farrant and Walters, 1998).
Indeed, in A. marina the limited de-differ- like that thought to occur in orthodox
entiation of subcellular components species.
towards the end of development allows In most cases, the viability of recalci-
changes indicative of germination to begin trant seeds is lost during drying in region
immediately upon shedding (Farrant et al., three of the five hydration levels summa-
1992). Interestingly, in contrast to this and rized in Vertucci and Farrant (1995). In this
other recalcitrant species studied, respira- region (c. 3 to 11 MPa), seeds are meta-
tion in the dormant seeds of A. pseudopla- bolically active, respiration is measurable
tanus declines to a rate similar to that of and presumably membranes are still
the orthodox A. platanoides at shedding hydrated. However, at this level of hydra-
(Greggains et al., 2000a). tion, it is thought that metabolism becomes
There are differences in the activities of ‘unregulated’, repair processes become
respiratory enzymes between the recalci- inoperative and catabolic activities con-
trant Guilfoylia monostylis and the ortho- tinue unabated, but the processes utilizing
dox Erythrina caffra (Nkang and Chandler, the high-energy intermediates are impaired
1986). These differences may be indicative (Vertucci and Farrant, 1995). As Q. robur
of the seeds’ different germination strate- seeds are dried, increasing quantities of
gies; the recalcitrant seeds maintained a several harmful volatiles are produced,
balance of enzymes suitable for immediate including ethanol and acetaldehyde
germination, whereas, in the orthodox (Finch-Savage et al., 1993). These volatiles
seed, biosynthetic processes were drasti- are indicative of ‘unregulated’ respiration
cally reduced (Nkang and Chandler, 1986). and are a potential source of the free radi-
In contrast to recalcitrant seeds, the meta- cals that accumulate around the time of
bolic activity of orthodox seeds is thought viability loss in Q. robur (Hendry et al.,
to decline in a programmed way before or 1992). These free radicals could alter the
during the early stages of maturation dry- physical/chemical properties of mem-
ing, so that seeds are shed in a quiescent branes, causing them to lose liquid/crys-
state (Rogerson and Matthews, 1977; Miller talline structure (McKersie and Leshem,
et al., 1983; Farrant et al., 1997). This orga- 1994). Thus, it is reasonable to speculate
nized decline in metabolic activity, which that membrane damage and viability loss
presumably has a role in protection against during drying of recalcitrant seeds may
desiccation damage (reviewed by Vertucci result from unregulated metabolism
and Farrant 1995), does not occur in enhanced by inadequate protection by free-
species with the most sensitive seeds and radical scavengers.
is not completed in those species with
more tolerant recalcitrant seeds (Berjak and ANTIOXIDANT SYSTEMS. Lipid peroxidation and
Pammenter, 1997; Farrant et al., 1997). free-radical activity have been associated
Recent studies on seeds of a number of with seed viability loss in several recalci-
more tolerant temperate recalcitrant trant species during desiccation (Hendry et
species suggest that, although high at shed- al., 1992; Chaitanya and Naithani, 1994;
ding, respiration rates decline like those of Finch-Savage et al., 1996; Li and Sun,
orthodox seeds during desiccation (V. 1999). So far, it is difficult to tell whether
Greggains and W.E. Finch-Savage, unpub- the reported accumulation of free radicals
lished data). However, in the more sensi- is a cause or a consequence of viability
tive Araucaria angustifolia, respiration is loss. In either case, adequate protective
only reduced by levels of desiccation that systems to limit free-radical damage are
cause viability loss (Côme and Corbineau, likely to be essential for the maintenance of
1996). In temperate Castanea sativa seeds, viability (Côme and Corbineau, 1996) and a
disruption of the electron transport chain range of antioxidant systems is present in
occurs during drying (Leprince et al., recalcitrant seeds (Hendry et al., 1992;
1999), suggesting that the decline in respi- Chaitanya and Naithani, 1994; Li and Sun,
ration due to drying is not programmed, 1999; Tommasi et al., 1999). In Q. robur,
there appear to be different protective sys- differentiation that occurs in orthodox seeds
tems in the embryonic axis and cotyledons during maturation drying (reviewed by
(Hendry et al., 1992). In the axis, protec- Vertucci and Farrant, 1995).
tion occurs predominantly through the It is essential that there is effective
antioxidants ascorbic acid and -toco- maintenance of the integrity of DNA during
pherol, whereas in the cotyledons protec- desiccation and that any damage is
tion is largely enzymatic, with relatively repaired on rehydration for seed viability
high and increasing activities of superox- to be maintained (see Chapter 12). In A.
ide dismutase and glutathione reductase. marina, DNA repair processes are
Decreased levels of protection from lipid markedly compromised after limited dry-
peroxidation during desiccation may con- ing and DNA replication does not fully
tribute to the loss of seed viability (Hendry recover after only 8% water loss (Boubriak
et al., 1992). Increased lipid peroxidation et al., 2000). The arrest of cell cycle activ-
during desiccation, which precedes viability at the stage where DNA per nucleus is
ity loss in Shorea robusta (Chaitanya and lowest may render embryos more resistant
Naithani, 1994) and T. cacao (Li and to stress conditions (Deltour, 1985).
Sun, 1999) seeds, was associated with Desiccation tolerance may therefore be
decreased activities of free-radical-scaveng- related to the stage of cell cycle activity at
ing enzymes. In contrast to these findings, which desiccation occurs (Bino et al.,
in a comparison of orthodox and recalci- 1992). However, Sacandé et al. (1997) have
trant Acer species, it was concluded that presented data suggesting this is not true,
the limitation to desiccation tolerance does and further convincing evidence against
not result from inadequate free-radical the possibility comes from a comparison of
scavenging (Greggains et al., 2000a). the orthodox species A. platanoides and
However, A. pseudoplatanus used in the the recalcitrant species A. pseudoplatanus
study can be placed at the most tolerant (Finch-Savage et al., 1998). Both species
end of the continuum of desiccation sensi- produce seeds with stable high levels of 4C
tivity among recalcitrant species. Its respi- DNA during the later stages of develop-
ration rate at shedding is similar to that of ment, and both contain nuclei arrested at
the orthodox Acer species, and there is no the 2C and 4C levels at maturity.
evidence of increased lipid peroxidation as So far there has been little emphasis on
viability is lost. the study of post-desiccation repair mecha-
nisms in seeds and few studies have been
OTHER FACTORS. Differences in cellular struc- published on this topic in recalcitrant
ture could influence desiccation tolerance seeds; this may well be a limiting factor in
(reviewed by Ruhl, 1996) such that the ini- desiccation-sensitive seed tissues.
tial loss of water and consequent reduction
in cell volume in very sensitive seeds
causes mechanical damage (reviewed by 5.3. Conclusions
Berjak and Pammenter, 1997; Pammenter
and Berjak, 1999). For example, the seeds of Essential components of desiccation toler-
A. marina are highly vacuolated and this ance of seeds include the accumulation of
has been connected to their level of desicca- protective substances, which limit the
tion sensitivity compared with other species amount of damage that otherwise would be
(Farrant et al., 1997). However, Vertucci and induced by water loss, and the ability to
Farrant (1995) showed that the evidence for repair cellular components upon subse-
this is conflicting. Considerable disruption quent rehydration. Sugars (disaccharides,
of the cytoskeleton has also been observed such as sucrose, and oligosaccharides, such
during drying of Q. robur axes, and it is not as raffinose and stachyose) have been sug-
reassembled during rehydration (Mycock et gested to play a key protective role by
al., 1999), which may contrast with the accumulating under water deficit condi-
apparently organized intracellular de- tions and functioning to replace water, thus
stabilizing membranes and other sensitive 1998) for examining protein–protein inter-
systems. Another protective mechanism actions and the use of differential display
may involve dehydrins (Table 5.1). reverse-transcription PCR (Rodriguez-Uribe
Desiccation tolerance is acquired during et al., 2000), gene and enhancer trap tag-
development of orthodox seeds; tolerance to ging (Rojas-Pierce et al., 2000) and micro-
full desiccation is generally lost after germi- arrays (Nevarez et al., 2000) to identify
nation. Recalcitrant seeds, unlike orthodox water-deficit-regulated genes. Proteomics
seeds, are sensitive to desiccation when approaches could yield invaluable infor-
shed from the parent plant, and thus pro- mation concerning post-translational con-
vide a system to study temporal and stress- trols over desiccation-induced gene
induced changes in dehydrins and other expression. These and similar research
putative desiccation protectants. Although avenues may yield more decisive results
studies on recalcitrant seeds provide indi- than the traditional approaches.
rect evidence in relation to elucidating the Finally, it is noteworthy that desicca-
roles of putative desiccation protectants, tion tolerance of seeds is a complex and
more work needs to be done in this area. multifaceted property involving a multi-
It is currently an exciting time to under- tude of genes whose expression ultimately
take the challenges of understanding the leads to mechanisms of both cellular pro-
biochemical and genetic components of tection, to sustain limited damage during
desiccation tolerance of seeds. Several drying itself, and cellular repair, to
novel approaches are now available, includ- reverse any desiccation-induced changes
ing the yeast one- and two-hybrid systems when the appropriate hydrated conditions
(Ingram and Bartels, 1996; Frank et al., are re-established.
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Desiccation - Chap 06 18/3/02 1:56 pm Page 185
6 Pollen and Spores: Desiccation Tolerance

in Pollen and the Spores of Lower Plants and
Fungi
Folkert A. Hoekstra
Laboratory of Plant Physiology, Department of Plant Sciences, University of
Wageningen, Arboretumlaan 4, 6703 BD Wageningen, The Netherlands

6.2. Pollen 187
6.2.1. Desiccation tolerance 187
6.2.2. Characteristics 188
6.2.3. Longevity 190
6.2.4. Imbibitional stress 191
6.2.5. (Cryo)preservation 191
6.3. Fern Spores 192
6.3.4. Cryopreservation 193
6.4. Moss Spores 193
6.4.1. Desiccation tolerance, longevity and imbibitional stress 194
6.5. Spores of Horsetails, Lycopodia and Selaginella 194
6.6. Fungal Spores 195
6.6.4. Imbibitional stress 197
6.7. Conclusion 197
6.8. Acknowledgement 197
6.9. References 199
186 F.A. Hoekstra
6.1 Introduction other propagules is linked with consider-

able differences in physiology.
Pollen grains of seed plants and spores of Pollen is dispersed mostly through the
lower plants and fungi have diverse devel- air by wind, insects or vertebrates to meet a
opmental histories. However, they are simi- female receptive structure, where it is pro-
lar in size, usually below 200 µm, which voked to germinate immediately after rehy-
promotes their effective dispersal via the air. dration, sometimes depending on
The propagules are exposed to often hostile recognition and/or the presence of self-
environmental conditions, without the abil- incompatibility. The rapid start of pollen
ity to actively control their own hydration tube growth might be linked with the com-
status. Under similar environmental condi- petition of the grains for the available
tions, microscopic propagules dry out much ovules (Mulcahy, 1979), as discussed in
faster than, for example, the much larger Section 6.2. Those pollen grains that fail to
seeds. It is, therefore, not surprising that land on the proper site are lost, because
pollen and spores are endowed with mecha- they have very little subsequent chance to
nisms that allow them to withstand a cer- meet the appropriate vehicle for transport.
tain degree of water loss and to survive the After dehiscence, rainwater is generally
period from their release from the produc- detrimental to pollen, as it causes loss of
tion site to the target site and, often, beyond viability, bursting or germination at inap-
that. The seeds of about 15% of all plant propriate sites (Lidforss, 1896). This
species are supposed to have problems as a extreme sensitivity to rainwater is associ-
result of water loss (Hong et al., 1996). ated with the absence of dormancy in
Drought tolerance is the term used to pollen. Only in some gymnosperms has it
indicate tolerance of moderate dehydration, been found that the pollen remains viable
for example, not below the moisture con- after several hours of soaking of dry pollen
tent (MC) at which bulk water has disap- in rainwater followed by redrying
peared (approx. 20–25%, on a fresh weight (Hoekstra, 1983).
basis, or 0.25–0.33 g H2O g1 dry weight). Fungal, fern and moss spores are also
Desiccation tolerance refers to further dehy- often transported via the air after the
dration and is understood to include not release from the mother plant upon slight
only the ability of cells to become air-dry drying of the spore-bearing structures.
without loss of viability, but also to success- They will dehydrate to some extent,
fully rehydrate. The period of anhydrobio- depending on the niche in which the par-
sis in between these two events is referred ent organism grows. In these propagules,
to as longevity or life span in the dried dormancy mechanisms occur, which sup-
state. This chapter focuses on the possible press germ tube emergence in the presence
tolerance of spores of eukaryotic plants to of sufficient water, when the environmen-
reduced levels of hydration. tal conditions are unfavourable for sup-
Pollen, the male gametophyte of porting growth. This also implies the
higher plants, is designed to deliver its activity of dormancy-breaking mechanisms
haploid sperm cells to the ovules in at some time when the conditions improve.
order to bring about fertilization. There As a consequence of the difference in tar-
is a specific and highly specialized target get, pollen does not survive in the hydrated
for the pollen to land on – the stigma in state for a long time, whereas the other
the case of angiosperms, or a pollination propagules usually do.
droplet or pollen-collecting apparatus in A considerable amount of physiological,
the case of gymnosperms. In contrast, the biochemical and biophysical research in
propagules of the spore-forming plants relation to desiccation tolerance has been
and fungi are less restricted as to their performed on pollen and yeast, but com-
initial target site, which is usually the paratively little on the other propagules.
soil or a host organism. This difference For each particular propagule described in
in specialization between pollen and the this chapter, special emphasis is placed on
Desiccation Tolerance in Pollen and Spores 187
how widely spread the phenomenon of Bruinsma, 1975; Linskens et al., 1989), or
desiccation tolerance is (as far as is pollen may form germ tubes inside the
known), including possible problems with anthers (Pacini and Franchi, 1982). When
the rehydration of the dried specimens. In viable pollen rehydrates on the appropriate
tests for desiccation tolerance, improper female receptive structure, it germinates,
rehydration may kill otherwise viable dry producing a filiform structure (pollen
specimens, leading to false negatives. tube), which grows through the style
Longevity in the dried state is surveyed, towards the ovules by a process much like
because the span may be indicative of the a parasitic invasive action.
depth of desiccation tolerance. The occur-
rence of compounds generally associated
with anhydrobiosis is listed, particularly 6.2.1. Desiccation tolerance
sugars, compatible solutes and dehydration
proteins. The feasibility of (cryo)preserva- The pollens of a large number of plant
tion is highlighted as a practical guideline species can withstand air-drying (Stanley
for those interested in preserving the and Linskens, 1974; Towill, 1985; Hoekstra,
propagules. 1986, 1995). Studies by the author of the
pollen literature have revealed that the pol-
lens of plants belonging to some genera of
6.2. Pollen about half of all plant families have been
tested for their tolerance to various levels of
The male gametophytes of higher plants dehydration. While desiccation tolerance
develop in the anthers (Stanley and appears to be widespread among pollen of
Linskens, 1974). When the flower opens, most plant families studied, there are a few
the anthers become exposed to the environ- exceptions. It is expected that more such
ment and dehydration then triggers a recalcitrant pollen types will be found in
mechanism in the anthers for the release of very humid climates and niches not yet
the pollen. In the process, pollen also investigated, particularly in the hot, humid
dehydrates. Continuous rain may prevent tropics.
anthers from opening. Once pollen has From the results of in vitro and in vivo
passed physiological maturity, ageing may germination experiments, it is clear that the
start within the anthers under such condi- pollen of genera in Araceae, Cucurbitaceae,
tions. Ultimately, pollen with reduced via- Gramineae and Zingiberaceae experience
bility and vigour is released (Hoekstra and problems during drying (see Table 6.1 for
Table 6.1. List of plant families and their genera in which desiccation-sensitive pollens have been
reported.
Family Genus Reference
Araceae Dieffenbachia Henny, 1980a,b
Aglaonema Henny, 1985
Cucurbitaceae Cucurbita Wang and Robinson, 1983; Gay et al., 1987
Momordica Dubey and Gaur, 1989
Gramineae Avena Wallace and Karbassi, 1968
Hordeum Anthony and Harlan, 1920; Firbas, 1922
Saccharum Sartoris, 1942; Moore, 1976
Secale Chaudhury and Shivanna, 1987; Shi and Tian, 1989a
Sorghum Sanchez and Smeltzer, 1965
Triticum Firbas, 1922; Goss, 1968
Zea Barnabas, 1985; Digonnet-Kerhoas and Gay, 1990
Pennisetum Aken’ova and Chheda, 1970
Zingiberaceae Zingiber Adaniya and Higa, 1988; Adaniya, 2002
188 F.A. Hoekstra
details). They can be kept alive only under more subtle rehydration requirements and
100% relative humidity (RH) are often difficult to germinate on such
(Zingiberaceae) or at slightly lower RH, media (Bar-Shalom and Mattsson, 1977).
albeit for short periods of time. Although During the development of the male
Gramineae pollens are generally sensitive angiosperm gametophyte, two nuclear divi-
to air-drying, they usually have a consider- sions take place. The first one leads to one
able tolerance to drought. Thus, pollen of vegetative and one generative cell that
Secale cereale is tolerant to MCs as low as gives rise to two sperm cells in the subse-
11.5% (fresh weight basis) (Shi and Tian, quent division. Depending on whether
1989a), and that of Zea mays to 13% MC dehiscence occurs after the first or the sec-
(Digonnet-Kerhoas and Gay, 1990). In the ond mitosis, mature pollen is bicellular or
genus Pennisetum, tolerance (Pennisetum tricellular. Bicellular pollen grains have to
typhoides (Pennisetum americanum), perform the second mitosis during tube
Chaudhury and Shivanna, 1986, 1987; growth. Thus, tricellular pollen is ontoge-
Hoekstra et al., 1989) as well as intoler- netically advanced. This characteristic
ance (Pennisetum purpureum, Aken’ova occurs in about 30% of angiosperm families
and Chheda, 1970) to air-drying have been that are considered as being evolutionarily
observed. Some orchid pollens rapidly advanced (Brewbaker, 1959, 1967; Sporne,
lose viability on storage above silica gel, 1969). Typical families with tricellular
which cannot be restored by a day of pre- pollen are the Asteraceae, Caryophyllaceae,
hydration in humid air prior to the in vitro Chenopodiaceae, Cruciferae, Gramineae,
test (Pritchard and Prendergast, 1989). Juncaceae and Umbelliferae. Tricellular
This may point to reduced desiccation tol- pollen tends to be associated with dry stig-
erance, but may also indicate that the mas and is not readily germinated in vitro
dried pollen is sensitive to the humid air (Heslop-Harrison and Shivanna, 1977).
treatment (see Section 6.2.4). The time from contact with the germi-
nation medium or appropriate stigmatic
surface to emergence of a pollen tube is
6.2.2. Characteristics generally short. It ranges from a few min-
utes for Gramineae and Compositae pol-
On dehydration, pollen changes in shape lens to a few hours for some other pollens
in different ways, dependent on the (Hoekstra and Bruinsma, 1978; Hoekstra,
species. While indentation is observed in 1986). Only in the case of gymnosperm
some pollens, e.g. those of Gramineae, in pollens does it take a few days before
others the major to minor axis ratio is emergence occurs (e.g. Pettitt, 1985). A
increased as the shape changes from rapid start of tube emergence is generally
round into ellipsoid along special fur- followed by extremely fast tube growth of
rows, as in the pollens of Solanaceae and up to 1–2 cm h1 (Hoekstra and Bruinsma,
Papaveraceae (for low-temperature scan- 1978). It has been argued that recurrent
ning electron micrographs, see Fig. 6.1). competition for the fertilization of avail-
The pattern of size reduction with dehy- able ovules has led to such short lag peri-
dration also depends on the distribution ods and fast growth, which may have
and number of germ pores. made mature pollen extremely sensitive to
Germination ability of pollen is assayed stress. This problem may have been suc-
in vivo by the analysis of tube growth in cessfully evaded by the regular production
compatible stigmas or styles, or in vitro on of fresh pollen during the flowering
artificial germination media (see Hoekstra, period. Some characteristics of fast-grow-
1995, for a review). Those pollens that are ing pollens that are often tricellular are the
adapted to plants having wet stigmatic sur- high proportion of polyunsaturated fatty
faces easily germinate in liquid or solidified acids (linolenic acid) in the phospholipids
germination media. In contrast, pollen grains and neutral lipids, the high rates of metab-
from plants with dry stigmatic surfaces have olism associated with well-developed
mitochondria and, in the case of short tional and desiccation-tolerant at approxi-

styles, even the absence of protein synthe- mately 1–2 days prior to anthesis, coinci-
sis during tube growth (see Hoekstra, dent with the degradation of starch and a
1986, for a review). The high level of doubling of the amount of sucrose.
polyunsaturated acyl chains that are par- Precocious drying leads to loss of viability
ticularly sensitive to peroxidation might and damaged plasma membranes. When
contribute to the intrinsically short life immature pollen is liberated mechanically
span. from the anthers and allowed to mature in
Mature pollen is generally endowed humid air, starch degrades and sucrose
with high contents of sucrose, ranging from content nearly doubles, and the grains
7 to 23% of the pollen dry weight, but become largely functional and dehydration-
oligosaccharides have not been encoun- tolerant. Apparently, maturation during the
tered (Hoekstra et al., 1992). Sucrose may last 3 days of development is independent
play an essential role in the acquired toler- of the parent plant, and sucrose may play
ance of severe dehydration, as illustrated an essential role in the acquired desicca-
by the following examples. Developing tion tolerance (Hoekstra and van Roekel,
pollen of Papaver dubium becomes func- 1988). During the slow dehydration of
Fig. 6.1. Low-temperature scanning electron micrographs of partly dehydrated (A,C) and hydrated pollens
(B,D). Maize pollen, (A) partly dehydrated and (B) hydrated (fresh); poppy (Papaver rhoeas) pollen, which
was liberated from an anther in a partly dehydrated state (C) and upon rehydration in water (D). Upon
dehydration, maize pollen becomes indented, whereas poppy pollen becomes elongated. Bars are 10 µm.
(Micrograph, courtesy of Adriaan van Aelst, Laboratory of Experimental Plant Morphology and Cell Biology,
Wageningen University.)
190 F.A. Hoekstra
fresh maize pollen, the sucrose content to biopolymers under conditions when
increases from 5 to 12% of the dry weight. there is still bulk water present during
When fresh pollen is dried in the cold dehydration, is discussed in Chapter 10.
(2°C) or at a high rate, the increase in As in seeds, heat-stable proteins with
sucrose content is curtailed, and the toler- late embryogenesis abundant (LEA)
ance of dehydration is affected, from which (Wolkers et al., 2001) and dehydrin (Wang
it has been concluded that survival of et al., 1996) characteristics have been iso-
dehydration is correlated with the presence lated from pollen. There are further indica-
of sucrose (Hoekstra et al., 1989). The tions that, as a result of osmotic stress,
mode of action of sugars in dry plant mate- dehydration and ABA, a number of pro-
rial is discussed in detail in Chapter 10. teins are produced in pollen with homolo-
Free amino acids are abundant in gies to stress proteins in seeds. The
mature pollen grains. On the basis of an possible mode of action of these proteins is
analysis of the pollen of a large number of discussed in Chapter 10.
plants (about 200 species from 63 families),
it has been shown that pollen differs from
other organs in having an unusually high 6.2.3. Longevity
content of free proline, exceeding 1.5% per
crude weight in many species (Britikov and Longevity of pollen varies considerably
Musatova, 1964). A high content of free among species and is dependent on water
proline has repeatedly been confirmed for content and temperature (see Hoekstra,
other pollens, even up to 3% of the dry 1986, for a review). Tricellular pollen tends
weight. Instead of proline, free arginine to be shorter-lived than bicellular pollen.
and glutamate may also occur. There are However, short storage life and extremely
indications that the free proline content of rapid tube emergence and growth have also
pollen is positively related to viability been found in bicellular pollen, e.g.
(Palfi and Köves, 1984; Palfi and Mihalik, in Balsaminaceae, Cucurbitaceae and
1985; Lansac et al., 1996). It is remarkable Commelinaceae. The average survival peri-
to note that the desiccation-sensitive ods at 20–25°C and equilibrium RHs of
pollen of Cucurbita has low proline con- approximately 40% range from a few hours
tents (Gulyas and Palfi, 1986). On the other in desiccation-sensitive pollens to several
hand, dehydration-sensitive maize pollen, months in the most stable, desiccation-tol-
which can withstand dehydration to erant pollens (Hoekstra, 1995). Only for
approximately 13% MC, does contain con- gymnosperm pollens have longevities of
siderable amounts of proline (Linskens and over 1 year under these conditions been
Pfahler, 1973; Palfi and Köves, 1984). The reported. The survival of pollen in storage
accumulation of proline is preceded by a conforms to a cumulative negative normal
peak in free abscisic acid (ABA) (Lipp, distribution (van Bilsen and Hoekstra,
1991; Chibi et al., 1995). Whereas the ABA 1993; Buitink et al., 1998b). Based on the
content decreases towards maturity (added data of Buitink et al. (1998b), an empirical
ABA inhibits germination), the proline model for the storage behaviour of pollen
content further increases to a maximum, has been constructed, which can predict
coinciding with the stage of anther desicca- the viability of a pollen lot over time at a
tion (Zhang et al., 1985). Proline added to broad range of different water contents and
the germination medium makes pollen storage temperatures (Hong et al., 1999a).
more resistant to heat (Zhang and Croes, There is a negative logarithmic relation
1983), which suggests that the high between longevity and pollen MC and a
endogenous amounts in pollen may have a curvilinear semi-logarithmic relation
role in conferring resistance to between longevity and temperature.
unfavourable temperatures. The role of free It has long been known that there is a
proline and some amino acids as compati- lower MC limit, below which further reduc-
ble solutes, providing structural protection tion in MC does not increase pollen
longevity, but, instead, decreases it (Pfundt, is predicted to be considerably above 10%

1910; Hoekstra, 1986). At high MC, pollen MC at cryogenic temperatures. This may be
longevity is not extended as in dormant the reason for the successful cryogenic
spores and seeds, but the pollen germi- storage of desiccation-sensitive Gramineae
nates, or engages in inappropriate synthe- pollen, as mentioned below.
ses, such as callose deposition (Hoekstra,
1986). As mentioned earlier, dormancy
mechanisms are absent in pollen. An 6.2.4. Imbibitional stress
exception to the rule that fully hydrated
pollen does not survive for a long time is Sensitivity of pollen to imbibitional stress
orchid pollen that is enclosed in pollinia in has been known for many years (see
long-lasting flowers. On agar, survival times Hoekstra, 1986, for a review). The imbibi-
of a few weeks at 2°C have been reported tion of dry pollen in germination medium,
(Pritchard and Prendergast, 1989). particularly at chilling temperatures, can
Recently, it has been established that lead to loss of endogenous solutes and
pollen is in a glassy state (see Chapter 10) delay of tube emergence, or even a com-
at below approximately 10% MC at room plete failure of the grains to become turges-
temperature. In a glass, molecular mobility cent and form a pollen tube. The problem
is considerably reduced. It has been found is widespread among pollens and can be
that the molecular mobility of a small guest circumvented by prehydration in humid
(spin-probe) molecule in the cytoplasm is air or by warm imbibition, or a combina-
inversely correlated with storage longevity tion of both (Hoekstra, 1984; Hoekstra and
(Buitink et al., 1998a). Thus, the slower the van der Wal, 1988). Some caution has to be
molecular mobility, the greater is the life exercised with prehydration in humid air,
span. Elevated water contents and tempera- as this treatment may lead to a rapid loss of
tures increase molecular mobility and, viability in some cases. The extensive leak-
thus, decrease the life span. For long-term age as a result of imbibitional stress is
survival it is therefore important that the indicative of problems at the level of the
pollen cytoplasm is in the glassy state. plasma membrane. Few pollens have been
From the linear relationship between the found that are resistant to this stress. They
logarithms of molecular mobility and are characterized by highly polyunsatu-
longevity, Buitink et al. (2000a) have been rated acyl chains in their membranes
able to predict storage longevities under (reviewed by Hoekstra and Golovina,
varying conditions of temperature and MC. 1999), which may increase membrane flu-
Thus, survival times at low temperatures, idity and thus more easily accommodate
for which experimental determination is the expanding protoplast on rehydration.
practically impossible, can be estimated.
This approach on the basis of molecular
mobility probably gives more accurate esti- 6.2.5. (Cryo)preservation
mates of survival times for sub-zero tem-
peratures than does the empirical model Desiccation-tolerant pollen that is air-
proposed by Hong et al. (1999a), which is dried to approximately 7% MC can be
more conservative. stored for extended periods of time at
A possible cause of the critical lower sub-zero temperatures. The experience of
MC limit may be encompassed in the phe- the author with air-dried cattail (Typha
nomenon that, during the removal of water, latifolia) pollen is that a half-life of about
molecular mobility reaches a minimum, 25 years is possible at 18°C in a deep-
but increases again on further drying to freezer. However, for long-term storage of
very low water contents (Buitink et al., many decades or even longer, cryogenic
2000b). In addition, the MC at which this storage is essential. Some excellent
minimum molecular mobility occurs reviews of this subject have been pub-
increases with decreasing temperature, and lished (Binder et al., 1974; Stanley and
192 F.A. Hoekstra
Linskens, 1974; Franklin, 1981; Shivanna 6.3.1. Desiccation tolerance

and Johri, 1985; Towill, 1985).
The storage of desiccation-sensitive Because the release of spores from the spo-
pollen is limited to several days or at most rangia requires a certain degree of dehydra-
a few weeks under conditions of high RH tion, some tolerance to dehydration in the
at 0–2°C (see Hoekstra, 1995). Desiccation- spores is likely. This has, indeed, been
sensitive Gramineae pollen can usually established in the spores of a number of
withstand drying to 20–25% MC. This is ferns, e.g. Adiantum capillus-veneris
the MC at which bulk water has disap- (Uchida et al., 1998), Dryopteris filix-mas
peared and ice crystals do not rapidly form (Haas and Scheuerlein, 1991), Dryopteris
at sub-zero temperatures. When pollen, paleacea (Scheuerlein et al., 1988; Haupt
carefully dried to just below this water and Psaras, 1989), Anemia phyllitidis
content, is quenched in liquid nitrogen, a (Grill, 1988), Lygodium japonicum
glassy state is rapidly formed, and viability (Manabe et al., 1987) and Onoclea sensi-
is maintained because detrimental ice crys- bilis (Raghavan, 1992), but precise informa-
tals cannot grow. In this way, successful tion on longevity is scarce.
storage at 196°C is possible with maize
pollen (Barnabas and Rajki, 1976; Barnabas
et al., 1988; Shi and Tian, 1989b; Barnabas, 6.3.2. Characteristics
1994 (even for more than 10 years)) and rye
pollen (Shi and Tian, 1989a). After pollina- To detect spore survival by germination
tion with the thawed pollen, excellent seed tests is laborious and slow. Fern spores ger-
set can be obtained. minate in soil or on aseptic agar-containing
It has been reported that storage in vac- or liquid nutrient media after a dormancy-
uum ampoules at 5°C after vacuum-drying releasing treatment. Criteria of viability are
allows maize pollen to survive for more swelling of the cell, coat splitting, greening
than 1 year (Jensen, 1964). Also freeze-dry- and rhizoid formation, which usually
ing followed by storage at 0–5°C gives require weeks after induction for their
some survival up to 5 months (Nath and expression. A chlorophyll fluorescence
Anderson, 1975). Short lyophilization, fol- technique has been applied, which has the
lowed by vacuum storage at 40°C pro- advantage that it can be used to quantify
longs the life span of pollen of the grass the germination capacity of non-green
Dactylis glomerata for more than 2 years spores (D. paleacea) just 2 days after
(Cauneau-Pigot, 1991). The beneficial photoinduction (Scheuerlein et al., 1988).
effect of the vacuum during dehydration There is ample evidence for the occur-
and storage might be associated with the rence of dormancy in fern spores. Spore
exclusion of oxygen: oxidative stress germination in hybrid Azolla increases
might be an important factor in the desic- progressively to attain a maximum after 5
cation sensitivity and storability of months of storage and declines after 7
Gramineae pollen. months to reach a minimum after 11
months (Bhattacharyya and Kushari,
1999). It is clear that fern spores do not
6.3. Fern Spores simply germinate when they come into
contact with water. They are stimulated by
Fern spores are formed by meiosis in spo- red light and reversibly inhibited by far
rangia, often underneath the leaves and red light. The red-light-induced germina-
above ground. They are released when tion is irreversibly inhibited by ultraviolet
dehydration causes rupture of the sporan- (275 nm) and blue (440 nm) light (Sugai
gia. They are dispersed and can germinate, and Furuya, 1985). The induction of spore
giving rise to prothalli, where eventually germination is mediated by phytochrome,
fertilization takes place followed by out- and the effect of a red pulse irradiation
growth of the sporophytic plant. can be enhanced by nitrate added to the
culture medium (Haas and Scheuerlein, collected in the field (Janes, 1998). From
1991). The application of gibberellic acid the scanty data in the literature, one cannot
(GA3) also allows spores to germinate, but get an idea of how widespread the occur-
this hormone strongly inhibits further rence of desiccation tolerance is among
gametophyte development (Fernandez et fern spores.
al., 1997). The red-light-induced spore ger- Some fern species (e.g. A. filix-femina,
mination is inhibited by AMO 1618, an Gymnocarpium dryopteris, Phegopteris
inhibitor of gibberellin biosynthesis, from connectilis) form spore banks, which are
which it has been suggested that red light soil reservoirs of viable spores that remain
induces the biosynthesis of gibberellin via dormant while buried, but germinate in
the phytochrome system and that gib- light if brought to the surface (Dyer, 1994).
berellin induces spore germination Fern spores remain stable in the soil for
(Kagawa and Sugai, 1991). Added ABA more than a year (Dyer and Lindsay, 1992).
significantly inhibits spore germination
(Singh et al., 1996).
Spores accumulate reserve lipids 6.3.4. Cryopreservation
(Gemmrich, 1977) and storage proteins that
are genetically similar to seed storage pro- It has been found that spores of Cyathea
teins (Templeman et al., 1988). The spores of spinulosa survive storage in liquid nitro-
Osmunda japonica contain large amounts of gen (196°C) followed by slow thawing
free proline and arginine (Wada et al., 1998). (Agrawal et al., 1993). In this case, the
water content of the spores was uncon-
trolled. Prior dehydration to at least below
6.3.3. Longevity the level of the non-frozen water content
(approximately 0.25–0.33 g H2O g1 dry
Storage of spores at 20°C in the air-dried weight) is expected to provide possibilities
state and under hydrated conditions has for long-term survival under conditions of
been compared for four species (Athyrium cryogenic storage, if the spores survive
filix-femina, Blechnum spicant, Polystichum dehydration to this low water content.
setiferum and Phyllitis scolopendrium)
(Lindsay et al., 1992). The viability of their
hydrated, non-green spores remained un- 6.4. Moss Spores
changed after 2 years of storage, while that
of the air-dried spores decreased with time. Although the release of moss spores from
In addition, the time required for germina- the sporophyte capsules upon drying
tion of the air-dried spores increased dur- would suggest that desiccation tolerance in
ing storage. The chlorophyllous spores of these spores could be common, little is
Todea barbara, which cannot be stored for known about how widely distributed this
long periods by conventional methods, also phenomenon really is among species. In
lost viability during storage much less the soil, the unicellular haploid spore ger-
quickly when hydrated than when air- minates to form a small green protonema.
dried. From this, Lindsay et al. (1992) have Later on, plants grow from these protone-
concluded that wet storage of fern spores is mata. Beside the spores that arise from the
far more effective than dry storage. inner tissue of the capsule by meiotic divi-
However, prior dehydration allows spores sion, a variety of gametophytic cells and
(Cyathea delgadii) to be stored at sub-zero fragments, called ‘diaspores’, can be pro-
temperatures, which extends survival duced from the gametophytic plant and
times considerably in comparison with protonemata by different cellular separa-
other storage methods (Simabukuro et al., tion mechanisms (Duckett and Ligrone,
1998). Sporocarps of Azolla filiculoides 1992). Here, diaspores will also be consid-
are able to survive storage in water for 3 ered in relation to their possible desicca-
years and to germinate from mud samples tion tolerance.
194 F.A. Hoekstra
6.4.1. Desiccation tolerance, longevity and present. These authors have found that on
imbibitional stress agar the germination of Archidium alterni-
folium spores continues to increase over a
When comparing the survival of spores period of several months. Percentage ger-
from five species (Schistidium rivulare, mination was consistently less than 65% in
Racomitrium aciculare, Dicranoweisia freshly collected material, and increased
crispula, Oligotrichum hercynicum and with the age of the spores up to 4 years of
Ceratodon purpureus) in water and after storage. There is no information about the
desiccation, Dalen and Soderstrom (1999) major constituents of spores. For compari-
found that the spores generally survived son, sucrose is the main sugar in the dried
better in the dried state than in water. gametophyte (Smirnoff, 1992).
However, the survival times were limited,
i.e. not exceeding 6 months. In these five
species, fragments (diaspores) have been 6.4.3. Cryopreservation
found to survive equally well in water as in
the dried state. The (dia)spore bank of Spores can be expected to survive drying,
bryophytes that are soil reservoirs of viable and also to survive well in the dried state
spores appears to play a role similar to that at sub-zero temperatures. However, cryo-
of the soil seed bank in seed plants. preservation of (hydrated) cultures solves
Redifferentiation of moss protonemata problems associated with long-term cul-
into spherical, thick-walled brood cells ture, particularly when spores are short-
(brachycytes) is a widespread phenome- lived or not produced. Improvements in
non, which occurs when protonemal survival after cryopreservation have been
colonies are cultured for long periods of made by preconditioning cultures in ABA
time, allowed to dry out or are treated with and proline (Christianson, 1998). There is
ABA. Brood cells in some species retain information that suggests that moss spores
viability for long periods even in a desic- buried in the Siberian permafrost have sur-
cated state and germinate rapidly in new vived 40,000 years, as they could still form
medium lacking ABA (Duckett et al., 1993; protonemata upon culture (www.science.
Schnepf and Reinhard, 1997). It is likely nasa.gov/newhome/headlines/ast27jul99_
that ABA is the natural compound that 1.htm).
triggers brood cell development and
induces tolerance to desiccation (Goode et
al., 1993), including the synthesis of 6.5. Spores of Horsetails, Lycopodia and
extremely heat-resistant soluble proteins Selaginella
(Werner and Bopp, 1992). It has been con-
cluded that ABA has the same function in The chlorophyllous spores of the sole
bryophytes as in higher plants, acting as a horsetail genus, Equisetum, survive desic-
mediator in stress conditions (Bopp and cation, yet do not live for more than
Werner, 1993). Dry intact moss gameto- approximately 2 weeks when desiccated at
phytes are sensitive to imbibitional stress 2% RH and 25°C (Lebkuecher, 1997).
(Schonbeck and Bewley, 1981). Whether Under these conditions, disseminated
dry spores and diaspores are similarly sen- spores of Equisetum hyemale have an
sitive has not been studied. extremely short life span, possibly due to
the inability to recover losses of water oxi-
dation and photosystem II core function.
6.4.2. Characteristics Storing ripe cones of E. hyemale at 70°C
extends the viability of the spores for more
Although no special studies have been con- than a year (Whittier, 1996).
ducted as to possible dormancy mecha- Spores of Equisetum arvense, when cul-
nisms in spores, the study of Miles and tured in Murashige and Skoog liquid
Longton (1992) suggests that dormancy is medium, germinate 2–3 days after sowing
(Kuriyama and Maeda, 1999). Large amounts duce a powdered formulation of skimmed
of free proline and arginine have been milk/P. bilaji spore particles that could be
detected in these spores (Wada et al., 1998). kept alive under refrigeration for at least 3
The spores of the Lycopodiaceae, some months. Drying techniques for spores and
of them chlorophyllous, germinate rela- yeast cells that have been applied include
tively slowly (months/years). There air-drying, freeze-drying, spray-drying and
appears to be no information on desicca- fluid bed-drying by contact-sorption.
tion tolerance of the spores, including
those of Selaginella.
6.6.2. Characteristics
6.6. Fungal Spores Spores change their shape as a result of

dehydration, with size reductions to
Spores of terrestrial fungi will be consid- 70–80% of the hydrated size. The critical
ered here and not those of aquatic fungi. At RH, above which spores are expanded and
spore maturity, ejection mechanisms occur below which they are collapsed, ranges
that ensure the dispersal of spores into the between 80 and 90% RH, as established for
air, but sometimes spores develop in an a number of urediniospores (Littlefield and
aqueous environment. Schimming, 1989).
Desiccation tolerance of the spores is
enhanced when the fungi are grown in spe-
6.6.1. Desiccation tolerance cial media, e.g. enriched with glycerol, glu-
cose and casamino acids, or when spores
Desiccation tolerance has been established are harvested at mature rather than early
in the spores of a wide variety of fungi (see stages of development. It has been found
Table 6.2). Particularly well studied are the that the life span of Metarhizium
desiccation-tolerant spores of economically flavoviride conidia is greater after slow
important fungi, including yeast. Among rather than rapid drying (Hong et al.,
them are the entomopathogenic fungi of the 2000). This suggests that there is a certain
genera Beauveria, Metarhizium and time required for protective mechanisms to
Paecilomyces, the phytopathogenic fungi of be expressed during the stress, which is
the genera Alternaria, Helminthosporium, also observed in other anhydrobiotic
Pseudopezicula, Puccinia, Sclerotinia, propagules, e.g. seeds and pollen.
Venturia, Uromyces and Ustilago, and the A compound that typically accumulates
bioherbicidal fungus, Stagonospora. The in fungal spores is trehalose (see Feofilova,
main issues in the research were the explo- 1992, for a review). Additional stresses,
ration of how long spores can survive under such as heat shock or stationary phase
natural conditions, which is important in (stress because of crowding) (Pedreschi and
relation to plant pathogens, and the estab- Aguilera, 1997; Pedreschi et al., 1997) and
lishment of culture conditions and drying high osmolality (Hallsworth and Magan,
protocols that give adequate shelf stability 1994; Eleutherio et al., 1997), are particu-
of spores in the case of commercial biologi- larly effective at increasing the content of
cal products for agricultural application. trehalose, and enhancing viability and des-
Desiccation-sensitive spores have been iccation tolerance, such as in yeast cells.
found as a spin-off in the search for toler- Apart from its effect as an osmoprotectant,
ance. For example, the conidia of trehalose causes a depression of the gel-to-
Penicillium bilaji appear to be desiccation- liquid crystalline transition temperature of
sensitive, because they could be kept alive membranes in dried yeast (Leslie et al.,
only under conditions of 100% RH 1994; see Chapter 10), alleviates the effects
(Cunningham et al., 1990). However, using a of ethanol stress (Mansure et al., 1994) and
fluidized bed drier and drying system, can be involved in the formation of a glassy
Tadayyon et al. (1997) have managed to pro- state in the dried organism (Wolkers et al.,
196 F.A. Hoekstra
Table 6.2. List of spore types of different fungal species in which desiccation tolerance has been
established.
Species Type of spore Reference
Alternaria porri Conidia Hong et al., 1997
Aspergillus japonicus Conidia Gornova et al., 1992
Beauveria bassiana Conidia Hallsworth and Magan, 1994; Pfirter et al., 1999
Beauveria brongniarti Conidia Hong et al., 1997
Colletotrichum gloeosporioides Conidia Cunningham et al., 1990
Helminthosporium oryzae Conidia Hong et al., 1997
Metarhizium anisopliae Conidia Hong et al., 1997
Metarhizium flavoviride Conidia Moore et al., 1997; Hong et al., 2000
Neosartorya fischeri Ascospores Beuchat, 1992
Paecilomyces farinosus Conidia Hallsworth and Magan, 1994
Paecilomyces fumosoroseus Blastospores Cliquet and Jackson, 1997; Jackson et al., 1997
Conidia Stephan and Zimmermann, 1998
Phycomyces blakesleeanus Zygospores Rivero and Cerda Olmedo, 1994
Pseudopezicula tracheiphila Ascospores Pearson et al., 1991
Puccinia graminis Urediniospores Eversmeyer and Kramer, 1994
Puccinia recondita Urediniospores Eversmeyer and Kramer, 1994
Saccharomyces cerevisiae Cells van Steveninck and Ledeboer, 1974
Saccharomyces uvarum Cells Eleutherio et al., 1997
Sclerotinia sclerotiorum Ascospores Hong et al., 1997
Sordaria macrospora Ascospores Read and Lord, 1991
Stagonospora convolvuli Conidia Pfirter et al., 1999
Talaromyces flavus Ascospores Beuchat, 1992
Trichoderma harzianum Conidia Jin et al., 1996; Pedreschi and Aguilera, 1997
Uromyces appendiculatus Urediniospores Hong et al., 1997
Ustilago scitaminea Teliospores Hoy et al., 1993
Venturia inaequalis Conidia Becker and Burr, 1994
1998). Saccharomyces cerevisiae mutants, the percentage germination increases with

in which proline and the charged amino spore age (Perry and Fleming, 1989; Ben
acids such as glutamate, arginine and lysine Ze’ev et al., 1990; Gazey et al., 1993) or after
are increased, have a marked cryoprotective chemical treatment (Rivero and Cerda
activity, nearly equivalent to that of glycerol Olmedo, 1994). Usually, exposure of the
or trehalose, both known as major cryopro- spores to low temperatures for some time, or
tectants in this yeast (Takagi et al., 1997). to heat, certain chemicals or light, increases
Other typical products are heat-shock pro- the germination percentage. The presence of
teins (HSPs), which, for example, in yeast a thick wall may play an important role in
are produced in the stationary phase of fer- the constitutive dormancy of some spores
mentation (Praekelt and Meacock, 1990; (Ulanowski and Ludlow, 1989). In the
Sanchez et al., 1992). They are thought to be spores of ectomycorrhizal and several sapro-
crucial for the naturally high thermo-toler- trophic fungi, fluorescein diacetate (FDA)
ance of these cell types and for their long- staining has been found to be unreliable as
term viability at low temperatures. A small an indicator of viability, but a good predic-
HSP in yeast may function in the protection tor of dormancy (Miller et al., 1993). As the
of the plasma membrane against desiccation spores age, the percentage of fluorescent
and ethanol-induced stress (Sales et al., spores increases. It is not known whether
2000). Further, high superoxide dismutase this increase is the result of a better penetra-
(SOD) activity is essential for stationary tion of FDA through the spore wall, or of the
phase survival in yeast (Longo et al., 1996). synthesis or activation of esterase capable of
Evidence for the existence of dormancy cleaving the fluorogenic ester to form the
in spores comes from the observation that fluorescent dye, fluorescein.
6.6.3. Longevity dry intervals (Becker and Burr, 1994). Spores

can also survive for considerable periods of
Longevity of spores varies considerably time when stored fully hydrated. Septoria
among species. Thus, the viability of nodorum spores have been shown still to be
teliospores of Ustilago scitaminea in air-dried infectious after 24 months of storage in water
soils does not begin to decrease until after 18 (Wilkinson and Murphy, 1990).
weeks, but is lost after maintenance, free of
soil, for 23 weeks at ambient RH (Hoy et al.,
1993). Air-dried spores of Stagonospora con- 6.6.4. Imbibitional stress
volvuli remain viable at 3°C for 180 days
(Pfirter et al., 1999). Particularly long-lived As in pollen, there is evidence in fungi and
are the heat-resistant ascospores of yeast that dried cells suffer from being
Neosartorya fischeri and Talaromyces flavus, plunged into liquid medium, particularly at
which have been shown to survive dry stor- low temperatures. For example, dry conidia
age in fruit powders (RH = 23%; 25°C) for up of M. flavoviride (4–5% MC) display
to 30 months (Beuchat, 1992). It has been reduced viability when rapidly rehydrated
reported that some Talaromyces spores sur- in free water. Prehydration in an atmosphere
vive in coating material of dry seeds for as of high humidity allows dry conidia to
long as 17 years at room temperature absorb sufficient moisture to avoid imbibi-
(Nagtzaam and Bollen, 1994). tional damage (Moore et al., 1997). A similar
The survival of conidia conforms to a avoidance of damage has been demonstrated
cumulative negative normal distribution. As when dried yeast was allowed to imbibe at
in seeds and pollen, longevity of conidia is elevated temperatures of 39–42°C (van
determined by MC and temperature, beside Steveninck and Ledeboer, 1974). The mecha-
endogenous factors (Hong et al., 1997, 1998). nism of imbibitional damage is dealt with in
These authors have constructed an empirical Chapters 10 and 12.
model for the storage behaviour of conidia,
which can predict the viability of a spore lot
over time at a broad range of different water 6.6.5. Cryopreservation
contents and storage temperatures. A nega-
tive logarithmic relation has been observed Spores of vesicular–arbuscular (VA) myocor-
between longevity and conidia MC. The MC rhizal fungi are usually stored in soil just
of the conidia is dependent on the sorption above 0°C, but some of them may be stored
properties of the endogenous compounds frozen. When cryoprotectants such as
and the RH in which the conidia are equili- dimethylsulphoxide (DMSO), glycerol, man-
brated. The relation between longevity and nitol or sucrose are ineffective, incubation of
equilibrium RH can be described by a nega- freeze-sensitive spores for 2 days in
tive semi-logarithmic relation. There is a 0.75–1.0 M trehalose confers a measure of
lower MC limit, below which value further freeze-damage protection. However, it has
reduction in MC does not increase conidia been found that slow drying of spores in situ
longevity, and an upper MC limit, above (in soil) prior to freezing gives satisfactory
which longevity no longer decreases. survival of the VA spores (Douds and
Analysis of the effect of different tempera- Schenck, 1990). In the case of full desiccation
tures has shown that the Q10 for the loss in tolerance, sub-zero temperature storage of the
conidia viability increases, the warmer the dried spores will generally be successful.
temperature (Hong et al., 1999b). With fluc-
tuating day/night temperatures, the warmer
temperature mainly determines conidia 6.7. Conclusion
longevity. Viability of ungerminated desicca-
tion-tolerant conidia is not affected by expo- In the introduction, it was suggested that
sure to wet and dry intervals, but germinated pollen and spores are likely to have at
conidia (germlings) are generally sensitive to least a certain level of tolerance to dehy-
198 F.A. Hoekstra
dration. This expectation appears to be which makes them more attractive to

generally correct. study. The economic importance of pollen
Most pollens are tolerant to MCs and fungal spores has also encouraged
below 10%. However, pollens of some considerable research, particularly into the
plant families have an elevated lower MC fundamental aspects of stress tolerance.
limit (>10% MC), below which viability The occurrence of imbibitional damage in
is lost. Such a reduced tolerance is com- dry pollen and fungal spores/yeast, and
parable with that of the so-called ‘inter- probably also in the other propagules, may
mediate’ seeds. It seems that real have reduced the enthusiasm for the appli-
desiccation sensitivity (recalcitrance), cation of dry storage: the probably still
i.e. sensitivity already below 40% MC, as viable propagules may have been killed on
is frequently observed in seeds, is rehydration. Considering the generally
uncommon among pollens. This might be limited longevity of the pollen/spores at
explained by the usually larger size of room temperature, sub-zero temperature
the seeds: under the same humid condi- storage has to be applied for successful
tions, the comparatively large seeds do long-term storage.
not dry out as fast as the microscopic The life span in the dry state at room
pollens. Thus, desiccation tolerance is temperature generally ranges from a few
less imperative for seeds that are dis- days to several months for both desicca-
persed in humid climates. tion-tolerant pollen and spores. This is in
The phenomenon of desiccation toler- sharp contrast to plant seeds, which often
ance has been extensively studied in survive a number of years (see Priestley,
pollen. Some of the compatible solutes, 1986, for a review). Pollen, for example,
such as sucrose and proline, that are typi- is not typically designed to overcome
cally associated with drought and desicca- long periods of adverse conditions, as is
tion tolerance are abundantly present. The common for seeds. Although the cause of
heat-stable dehydration proteins, known the difference in longevity between
from seeds, also occur in pollen. pollen and spores, on the one hand, and
A similar picture emerges for fungal seeds, on the other hand, is a matter of
spores and yeasts. However, the situation speculation, it may be envisaged that the
is less clear for fern and moss spores, comparatively large size of seeds gives
mainly because considerably less work has them an extra protection against molecu-
been done on this material. Desiccation- lar oxygen and peroxidative degradation.
tolerant fern and moss spores have been The exceptionally long life span, as found
found, nevertheless, and drought tolerance in some thick-walled fungal spores, might
might at least be common. Only for the depend on a possible impermeability of
spores of the fern Osmunda has analysis these walls to oxygen.
shown that free proline and arginine The major difference between pollen
abound. Little is known about horsetail, and the other propagules is the lack of dor-
Lycopodium and Selaginella spores, mancy in the former. In hydrated pollen,
except that dehydrating horsetail spores metabolism seems unrestricted, which leads
have a very short life span and contain to a rapid loss of viability. Thus, while sur-
free amino acids. The problem with vival in the hydrated state is a common
screening for desiccation tolerance in strategy in spores, it is not in pollen.
these propagules (not pollen and fungal
spores) is that germ tube emergence is
comparatively slow, taking at least a week 6.8. Acknowledgement
and often much longer. Until rapid assay
methods for viability are developed, it will The author is indebted to Dr Elena A.
not be attractive to study anhydrobiosis in Golovina from the Timiryazev Institute of
these spores. In contrast, germ tube emer- Plant Physiology, Russian Academy of
gence is generally a matter of hours in fun- Sciences, Moscow, for critically reading
gal spores and even minutes in pollen, the manuscript.
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7
Vegetative Tissues: Bryophytes, Vascular
Resurrection Plants and Vegetative Propagules
Michael C.F. Proctor1 and Valerie C. Pence2

1School of Biological Sciences, University of Exeter, Washington Singer Laboratories,
Perry Road, Exeter EX4 4QG, UK; 2CREW, Cincinnati Zoo and Botanical Garden,
3400 Vine Street, Cincinnati, OH 45220, USA

7.2. Bryophytes 208
7.2.1. Desiccation time and recovery time: the typical pattern of
desiccation response 210
7.2.2. The effect of intensity of desiccation 211
7.2.3. Effects of temperature 213
7.2.4. Events on rehydration 213
7.2.5. Drying rate and drought hardening 215
7.2.6. Constitutive and induced tolerance 216
7.2.7. How long is needed for complete recovery? Processes and
criteria of recovery; long-term survival 216
7.3. Vascular Plants 217
7.3.1. Ecological and morphological adaptations 224
7.3.2. The effect of the intensity of desiccation 225
7.3.3. Effect of the rate of desiccation 226
7.3.4. Morphological and cytological changes that occur
with drying 226
7.3.5. Rehydration and recovery 227
7.3.6. Vegetative propagules: bulbils, corms, tubers and plant
fragments 228
7.4. Concluding Comments 228
7.5. References 230
7.1. Introduction iccation tolerance in vegetative parts of

plants. However, desiccation tolerance is a
We take for granted the desiccation toler- widespread phenomenon (Bewley and
ance of seeds and pollen, but see it as a Krochko, 1982; Stewart, 1989; Crowe et al.,
matter for remark when we encounter des- 1992; Oliver and Bewley, 1997). It is found
208 M.C.F. Proctor and V.C. Pence
in many microorganisms, in animals such There are probably some features and
as tardigrades, rotifers and nematodes and mechanisms common to all desiccation-
in larval cysts of crustacea of seasonal tolerant cells, but there are also some major
water bodies, and in plants it is a frequent differences. In general, the more tolerant
and characteristic feature of the vegetative bryophytes are what have been character-
cells of terrestrial algae, lichens and ized as ‘fully desiccation-tolerant’ (Bewley
bryophytes. In vascular plants it is the and Oliver, 1992; Oliver, 1996; Oliver and
norm in spores, pollen and seeds, but Bewley, 1997; Oliver et al., 2000); their tol-
uncommon in vegetative tissues. erance is essentially constitutive and little
Desiccation tolerance implies the ability affected by the rate of drying. By contrast,
of an organism to dry to equilibrium with many desiccation-tolerant vascular plants
the ambient air, and to recover and return to show little tolerance if they are dried fast,
normal metabolism on remoistening. It is a but tolerance is induced in the course of
qualitatively different phenomenon from slow drying, which, because of their vascu-
drought tolerance. Drought-tolerant plants lar system and relatively large size, is gen-
can maintain more or less normal metabo- erally what happens in nature. Some of the
lism in the presence of soil water potentials less tolerant bryophytes probably behave
that may often be low enough to wilt the similarly. These have been referred to as
leaves, but are killed if the relative water ‘modified desiccation-tolerant’ plants
content (RWC) of the tissues falls below (Oliver and Bewley, 1997). Desiccation-tol-
~ 0.2–0.3, i.e. 20–30% of full turgor, corre- erant plants vary greatly in the length and
sponding to a tissue water potential of frequency of the wet and dry periods to
perhaps 5 to 10 MPa. Many desiccation- which they are adapted. Small bryophytes
tolerant plants withstand drying to water of sun-exposed dry habitats may achieve a
potentials of 100 MPa or lower, at which positive carbon balance from moist periods
no liquid phase remains in the cells and of an hour or less and can benefit from
metabolism is at a standstill. Desiccation tol- brief showers, or even dewfall following
erance must have evolved early in the colo- clear nights. Vascular plants that retain
nization of land by microorganisms their chlorophyll when desiccated may
including cyanobacteria and simple algae. typically require around a day for recovery.
Amongst more complex photosynthetic land In poikilochlorophyllous species, which
organisms, the bryophytes are a numerous lose their chlorophyll on drying, regreen-
group, growing in diverse habitats from the ing and return to normal metabolism takes
tropics to the polar regions, in which a several days and these plants generally
degree of desiccation tolerance seems to be a occupy situations with relatively long alter-
fundamental part of their life strategy, and is nating wet and dry periods.
presumably primitive. Much the same may
be said of the symbiotic associations
between fungi and the photosynthetic organ- 7.2. Bryophytes (see also Chapter 1)
isms that we call lichens (Kershaw, 1985;
Nash, 1996). On the other hand, the vascular Bryophytes are the second largest group of
plants that dominate the world’s flora gener- photosynthetic land plants, with some
ally depend on maintenance of a high inter- 25,000–30,000 species occupying diverse
nal water content by conduction of water habitats from the tropics (Pócs, 1982;
from the soil, with varying degrees of control Richards, 1984) to polar regions (Longton,
of water loss by the stomata (Raven, 1977). 1988). By contrast with the vascular plants,
Desiccation-tolerant vascular plants are the they may be seen as having followed an
rare exception. They are relatively few in alternative strategy of adaptation to the
number and taxonomically scattered and irregular and intermittent availability of
isolated – products of independent selection water on land. Conduction of water is typi-
for intermittently or seasonally desiccated cally external and water is freely gained
habitats, mostly warm-temperate to tropical. and lost over much or all of the surface of
Desiccation Tolerance of Vegetative Tissues 209
the plant, and dry periods are passed in a early investigators were well aware
state of suspended metabolism until wet (Irmscher, 1912; Höfler, 1946; Clausen,
conditions return. Clearly, bryophytes are 1952; Abel, 1956; Hosokawa and Kubota,
in some respects limited by this mode of 1957), though appreciation of their com-
life; they cannot compete with vascular plexity was constrained by limited tech-
plants in size or in productivity on fertile niques. The earlier studies generally used
water-retentive soils. On the other hand, plasmolysis as an indicator of cell survival.
they are freed from some constraints: they This necessarily gave a somewhat static
can occupy hard substrates that are impene- picture, but it allowed exploration of the
trable to roots and so untenable to vascular effect of different times and intensities of
plants, and they are well adapted to nutri- desiccation, and demonstration of drought-
ent capture in N- and P-deficient situations. hardening (Höfler, 1946; Abel, 1956);
The majority of bryophytes will with- Höfler, Clausen, Abel, Hosokawa and
stand at least modest levels of desiccation Kubota and others laid a foundation of
(to equilibrium with, for example 20 to knowledge which is still valuable as back-
40 MPa) for at least a few days, but some ground for more analytical research. Ried
are much more tolerant than that. Breuil- (1960a,b), in his work on lichens, measured
Sée (1993) observed regrowth following photosynthesis and respiration by classical
remoistening of gametophytes of the liver- gas-analysis techniques, which, though
wort Riccia macrocarpa kept as dried laborious, gave newly dynamic insights
herbarium specimens for over 23 years. into the process and timing of recovery.
Maheu (1922) observed regeneration of pro- Various authors in the 1970s and early
tonema from leaves of ‘Barbula’ (= Tortula, 1980s (Hinshiri and Proctor, 1971; Dilks
Syntrichia) ruralis that had been dry for 14 and Proctor, 1974; Schonbeck and Bewley,
years. Keever (1957) found that most speci- 1981a,b) measured gas exchange manomet-
mens of Grimmia laevigata were still viable rically, but by the mid-1980s that had been
after 3 years’ dry storage, but only 20% largely superseded by infrared gas analysis
remained viable after 10 years. Of taxo- (IRGA). This allowed continuous measure-
nomic orders within the bryophytes, those ment and, as IRGA systems developed, pro-
that stand out as particularly desiccation- gressively better stability, sensitivity and
tolerant include the Andreaeales (Andreaea: time resolution (Šesták et al., 1971; Dilks
‘granite mosses’), Encalyptales (Encalypta), and Proctor, 1976; Kershaw, 1985; Lange,
Pottiales (Tortula, Syntrichia, Tortella, etc.), 1988; Long and Hällgren, 1993; Tuba et al.,
Orthotrichales (Orthotrichum, Ulota, 1996). Since the 1990s, chlorophyll fluores-
Zygodon), Hedwigiales (Hedwigia) and cence has provided a powerful new
Grimmiales (Grimmia, Racomitrium, etc.), method for rapid in vivo assessment of
but a number of other orders include at some aspects of photosynthetic function to
least some highly tolerant species, e.g. complement gas-exchange measurements
Polytrichales (Polytrichum piliferum, etc.), (Seel et al., 1992b; Deltoro et al., 1998a,b;
Dicranales (Dicranoweisia cirrata, Csintalan et al., 1999; Marschall and
Cheilothela chloropus, etc.), Hypnales (e.g. Proctor, 1999).
Eurhynchium pulchellum, Scleropodium Alongside this strand of research centred
tourretii, Hypnum spp., Leucodon sci- on gas exchange and carbon balance,
uroides, Anomodon viticulosus). Some Bewley, Oliver and their co-workers have
other groups, such as the Hookeriales concentrated particularly on effects of desic-
among the mosses and many genera of leafy cation and rehydration on protein synthesis.
and thalloid liverworts, are relatively sensi- Their very extensive work, reviewed by
tive and characteristically confined to shel- Bewley (1979), Bewley and Krochko (1982),
tered humid places. Most bryophytes fall Bewley and Oliver (1992), Oliver (1996) and
somewhere between these two extremes. Oliver and Bewley (1997), leads naturally
The responses of bryophytes to desicca- into modern molecular biological tech-
tion are multifaceted, a fact of which the niques (Wood et al., 1999) and to the possi-
bility of genetic engineering for increased viticulosus (measured as net O2 evolution),

drought tolerance in arid-zone crops. as a function of desiccation time (at c. 94
MPa) on one axis and rehydration time on
the other (Hinshiri and Proctor, 1971). For
7.2.1. Desiccation time and recovery time: periods of up to about 10–15 days desicca-
the typical pattern of desiccation response tion, recovery is quick and essentially com-
plete within 3–4 h. This may be seen as a
Figure 7.1 shows the photosynthetic per- period of ‘pure’ desiccation tolerance.
formance of a rehydrated moss, Anomodon From this point to about 40–45 days’ desic-
20
Hours
rehydration
10
12
22
28
35
42
+500 Oxygen output

49
l g–1 h–1
56
0
67
–500 Days
desiccation
Fig. 7.1. The course of the net photosynthesis rate of Anomodon viticulosus following moistening after various
periods of desiccation at 50% RH, 20°C. Curves are interpolated for net photosynthesis rates attained 2.5, 5,
10, 15 and 20 h after moistening. The shaded area indicates the part of the graph over which net C assimilation
is negative. Warburg manometer measurements of oxygen evolution or uptake; CO2 concentration maintained
by carbonate–bicarbonate buffer in centre wells; with a few exceptions points are means of two readings.
Measurements up to 28 days were made at 20°C, the remainder at 25°C. Somewhat simplified and in part
redrawn from Hinshiri and Proctor (1971). (Reproduced with permission from Proctor, 2001.)
cation, recovery becomes progressively Hookeria lucens

1
slower and less complete. Evidently, one or
more progressive damaging processes take –3 MPa
0.8
effect, which can at least to some extent be –9 MPa
repaired given sufficient recovery time. 0.6
After prolonged desiccation, remoistening –37 MPa
leads to long-persistent oxygen uptake, 0.4 –154 MPa
implying corresponding carbon loss. This
probably reflects major metabolic disrup- 0.2
tion, from which ultimate recovery, if it
takes place at all, can only be slow. 0
0 20 40 60 80
7.2.2. The effect of intensity of desiccation Rhytidiadelphus loreus
1
In addition to the factors considered in
Fig. 7.1, recovery and long-term survival 0.8
depend on the equilibrium water potential
to which the bryophyte has been desiccated. 0.6
Fv/Fm
Dilks and Proctor (1974) gave recovery data

for several species following desiccation at 0.4
different water potentials, which showed
that some, like Plagiothecium undulatum, 0.2
are more severely damaged the lower the
water potential to which they have equili- 0
brated, while others, such as Racomitrium 0 20 40 60 80
lanuginosum, show better long-term sur- Tortula ruralis
vival at low than at high water potentials. 1
Experiments using chlorophyll fluorescence
as a measure of plant performance show the 0.8
same pattern (Proctor, 2001; Fig. 7.2).
Species of more-or-less shaded habitats, 0.6
never exposed in the field to extreme desic-
cation stress, were most quickly and
0.4
severely damaged at the lowest water poten-
tial, at which species of dry, open, sun-
0.2
exposed situations, such as R. lanuginosum
and Tortula ruralis, showed best survival of
prolonged drying. Given a modest level of 0
daylight illumination (comparable with a 0 20 40 60 80
shady woodland floor) all the investigated Desiccation time (days)
species survived well for several weeks at Fig. 7.2. The chlorophyll-fluorescence parameter
3 MPa, corresponding to a cell RWC of Fv /Fm (estimating the maximum quantum efficiency
about 50%. Over this period, the highly des- of photosystem II), measured 20 min after
iccation-tolerant species, R. lanuginosum remoistening, following desiccation at a range of
and T. ruralis, performed best at either this water potentials (3 to 154 MPa). The curves are
smoothed from the original data (n = 3) by a single
highest water potential or at the lowest, and
pass through a binomial-average smoothing routine.
it was only after about 6 weeks that material Collecting sites: Hookeria lucens, Stoke Woods,
stored dry outperformed material kept lightly Exeter, Devon, UK, August 1999; Rhytidiadelphus
wilted. It was noteworthy that the more des- loreus, White Wood, Holne, Devon, UK, August
iccation-tolerant species were damaged most 1999; Tortula ruralis, Fülöpházá, Kiskunság
rapidly in the range 9 to 22 MPa. In fact, National Park, Hungary, July 1998.
1
212
Dessication 07
Ulota crispa
0.8
0.6
18/3/02
0.4
0.2
1:57 pm
Tortula ruralis Rac. lanuginosum

0
1
Fv /Fm
Pleurochaete squarrosa Anomodon viticulosus
Page 212
0.8
0.6 24 h
20 min
0.4
0.2
M.C.F. Proctor and V.C. Pence
Grimmia pulvinata
0
–400 –300 –200 –100 0 –400 –300 –200 –100 0 –400 –300 –200 –100 0
Fig. 7.3. The chlorophyll-fluorescence parameter Fv /Fm measured 20 min () and 24 h () after remoistening, following desiccation for 60 days at different water
potentials. Data points are means of three replicate readings; error bars (in some cases hidden within the data symbols) show standard deviations. Collecting sites:
Racomitrium lanuginosum, O Brook, Dartmoor; Tortula ruralis, Felsötárkány, near Eger, Hungary, October 1999; Grimmia pulvinata, Clyst St Mary; Pleurochaete
squarrosa, Chudleigh Rocks; Ulota crispa, New Bridge, Dartmoor; Anomodon viticulosus, Chudleigh Rocks (all except T. ruralis, Devon, UK, November 1999).
even for the most desiccation-tolerant to less than 0.05 K in liquid helium, and
species, there is an optimum range of water Bewley (1973) reported recovery of protein
potential for longest survival and most rapid synthesis in T. ruralis shoots that had been
recovery, generally around 100 to 300 cooled in the dry state to –196°C in liquid
MPa. After 24 h recovery from desiccation nitrogen. This is perhaps no matter for sur-
periods of up to 60 days, the most tolerant prise. However, by analogy with seeds in
species showed very little difference storage, increased temperature would be
between 41, 113, 218 and 412 MPa, expected to shorten survival time, and this
the chlorophyll-fluorescence parameter is indeed so. Hearnshaw and Proctor (1982)
Fv/Fm (a measure of the maximum quantum measured mean survival of seven species
efficiency of photosystem II, widely used as (in terms of chlorophyll content after a
a general indicator of plant stress) recover- period of moist recovery) at temperatures
ing within 24 h to normal unstressed levels from 20 to 100°C. Five of the bryophytes
after all of them (Fig. 7.3). The water content gave good straight-line relationships on an
of a bryophyte (as % dry weight (dw)) is ‘Arrhenius plot’ relating the logarithm of
closely determined by the water potential half-survival time to the reciprocal of
with which it is in equilibrium (Dilks and absolute temperature, as did data for viabil-
Proctor, 1979; Schonbeck and Bewley, ity of rice in storage (Roberts, 1975;
1981a). Water contents of 5% dw or less Proctor, 1982). This is the pattern that
may be reached by bryophytes in exposed would be expected for the temperature
places in the heat of the sun, corresponding response of chemical reactions in general
to water potentials below 300 MPa. (Morris, 1974). Two Racomitrium species
gave non-linear plots, but for all the
bryophytes survival times ranged continu-
7.2.3. Effects of temperature ously from minutes at 100°C to weeks or
months at normal ambient temperatures.
When fully hydrated, bryophytes show Ambient ‘shade’ temperatures are com-
generally similar temperature responses to monly in the range 0–30°C and rarely
C3 vascular plants. The lower limit for exceed 40°C, but dry bryophytes in the sun
most species is probably set by loss of on hot days can reach 40–60°C, and it is
water as the cell contents equilibrate with probably these high temperature episodes
the water potential of extracellular ice that drive selection for the apparently
(Kallio and Heinonen (1975) found positive extravagant levels of desiccation tolerance
net photosynthesis in R. lanuginosum and seen in, e.g. Tortula ruralis, Grimmia
Dicranum elongatum down to 8°C); pos- laevigata, R. lanuginosum and Andreaea
sible chilling effects in warm-climate rothii.
bryophytes at non-freezing temperatures
seem not to have been explored. The upper
lethal limit for metabolically active 7.2.4. Events on rehydration
bryophytes is usually in the range
40–50°C. Bryophytes become much more The preceding sections have principally con-
tolerant of extremes of temperature as they sidered responses along the desiccation-
dry out (Meyer and Santarius, 1998). time axis of Fig. 7.1. There are also impor-
Although there has been a general aware- tant questions to answer on the rewetting
ness that temperature must influence the (recovery) axis. As the time-resolution of
survival of dry bryophytes, there has been measurement techniques has improved, it
rather little systematic work on the subject has become increasingly apparent that
(Irmscher, 1912; Lange, 1955; Nörr, 1974). recovery of normal photosynthetic function
Desiccation-tolerant bryophytes, when dry, on remoistening can be very rapid.
can survive extremely low temperatures. Manometric and IRGA measurements have
Becquerel (1951) reported survival of demonstrated recovery to near-normal rates
Barbula and Grimmia leaves after cooling of net photosynthesis within an hour or
Tortula ruraliformis: two of remoistening in a number of species

8 days desiccation (Hinshiri and Proctor, 1971; Dilks and
8 (a) Proctor, 1974, 1976; Fig. 7.4). IRGA data
show net photosynthesis in T. ruralis to be
6 negative 15 min after remoistening, but
4 almost completely recovered after 30 min
(Tuba et al., 1996). Measurements from
2 modulated chlorophyll fluorometers show
0
that photosystem II (PSII) can return to
near-normal quantum efficiency within a
–2 few minutes. Initial recovery of the ratio
Fv/Fm on remoistening shoots of T. ruralis
–4
in the dark after 3 days air dry (c. 100
–6 MPa; water content c. 10% dw) approxi-
mated closely to an exponential curve with
–8
a half-recovery time of c. 20 s. The corre-
0 60 120 180 sponding initial half-recovery time for the
pendulous African forest moss Pilotrichella
Grimmia pulvinata:
14 days desiccation ampullacea (after 20 h at 37 MPa) was c.
20 40 s (M.C.F. Proctor, unpublished data).
(b)
This rapid initial phase of recovery is fol-
10 lowed by a much longer phase of slow
recovery, which may last an hour or two in
CO2 uptake (vpm)
0 the most tolerant species and many hours

in the more sensitive. Recovery of
–10 (FmFm)/Fm (PSII, a measure of effective
quantum yield of PSII) in light is slower
–20 than recovery of Fv/Fm. Even so, in tolerant
Net photosynthesis species recovery of photosynthetic electron
–30 flow (which of course may include a pho-
Dark respiration torespiratory component as well as carbon
–40 fixation) can be largely complete within
15–20 min (Csintalan et al., 1999).
0 60 120 180 240
It is clear that full recovery must involve a
Andreaea rothii: diversity of processes. First, membrane
4 days desiccation integrity must be re-established. Probably all
6 bryophytes (indeed, all desiccation-
(c)
tolerant cells (Crowe et al., 1992)) show
4
detectable leakage of solutes immediately on
remoistening (Brown and Buck, 1979;
2
Bewley and Krochko, 1982) but in tolerant
species this in only transient and most leaked
0
Fig. 7.4. Rate of net CO2 uptake () and dark
–2 respiration () of some desiccation-tolerant mosses
following remoistening, after various periods air dry
–4 (at ~ 100 MPa), measured by infrared gas analysis
(LCA-2, ADC, Hoddesdon, UK); PPFD 500 mol
–6 m2 s1; ~ 23°C. (a) Tortula ruralis ssp. ruraliformis,
8 days; Braunton Burrows, Devon, UK; (b) Grimmia
0 30 60 90 120 pulvinata, c. 14 days; stone parapet of bridge, Clyst
St Mary, Devon, UK; (c) Andreaea rothii, 4 days;
Recovery time (min)
granite outcrop, Haytor, Devon, UK.
solutes are rapidly reabsorbed; little or no net water stress, but recommences within a
leakage of inorganic ions is detectable after a minute or two of remoistening. In T. ruralis
few minutes (Deltoro et al., 1998a). Of the the pattern of protein synthesis in the first
major metabolic systems, photosynthesis, hours of rehydration is distinctly different
dark respiration and protein synthesis from that of hydrated controls, but no novel
(Gwózdz et al., 1973; Oliver, 1991) are reiniti- transcripts are made in response to desicca-
ated very rapidly (within a minute or two) in tion; the changes appear to be due to alter-
desiccation-tolerant species such as R. lanug- ations in translational controls. Oliver
inosum, T. ruralis and A. viticulosus. Much (1991) showed that in T. ruralis the synthe-
or all of the process of the recovery of all sis of 25 proteins ceased (or substantially
three systems seems likely to be essentially decreased) and the synthesis of 74 proteins
physical, involving reinstatement of water was initiated (or substantially increased)
into macromolecules and re-establishment of during the first 2 h of rehydration. He
spatial and conformational relationships. The named these protein groups ‘hydrins’ and
evidence indicates that recovery of the photo- ‘rehydrins’, respectively. The synthesis of
systems is essentially independent of protein all the hydrins returned to control levels
synthesis, but that some protein synthesis is within 2–4 h, but, while some rehydrins
needed for full return to predesiccation rates were synthesized only transiently within
of photosynthetic carbon fixation (Proctor the first hour or two of rehydration, others
and Smirnoff, 2000; Proctor, 2001). were still being synthesized at elevated lev-
Respiration recommences immediately els 10–12 h later. Synthesis of all proteins
on remoistening; indeed, in lichens there had returned to normal levels within 24 h.
are indications of low levels of respiratory The exact nature and function of most of
activity even at water potentials as low as these hydrins and rehydrins is not yet
100 MPa (Cowan et al., 1979). The initial known, but some homologies with known
rate of respiration on remoistening is proteins are beginning to emerge from mol-
always higher, and sometimes very much ecular biological investigations (Wood et
higher, than the normal steady rate of dark al., 1999; Chapters 1 and 11).
respiration, perhaps reflecting some (proba- Growth of the bryophyte plant requires
bly variable) combination of uncoupling of not only a positive carbon balance and the
respiration from other metabolic systems ability to synthesize proteins, but also the
and the demands of repair processes. In the re-establishment of the cell cycle, and
IRGA measurements of Dilks and Proctor translocation of metabolites, mineral nutri-
(1976), dark respiration of A. viticulosus ents and growth regulators to the meristem-
and Rhytidiadelphus loreus took 5–10 h to atic regions. Cell division and probably also
return to steady levels following a few days’ translocation depend on processes mediated
desiccation. The data of Tuba et al. (1996) by microtubules, which are broken down on
showed dark respiration in T. ruralis and desiccation and must be reconstituted as a
the dry-grassland lichens Cladonia convo- part of the recovery process. A degree of
luta and Cladonia furcata, following a few repair of DNA will always be necessary, and
hours’ desiccation, falling rapidly within 2 the requirement for this is likely to increase
h to a rather steady level still greater than with increasing desiccation time.
that measured before or during drying.
The recovery of protein synthesis has
been the subject of extensive research by 7.2.5. Drying rate and drought hardening
J.D. Bewley, M.J. Oliver and their co-
workers, reviewed by Bewley (1979), Höfler (1946) and Abel (1956) found that the
Bewley and Krochko (1982), Bewley and desiccation tolerance of bryophytes was
Oliver (1992), Oliver (1996), Oliver and enhanced following a period of less severe
Bewley (1997), Oliver et al. (1998) and in drying. Abel surveyed the recovery of a wide
Chapters 1 and 11 of this book. Protein syn- range of moss species after 24–48 h desicca-
thesis declines and ceases quickly under tion at a range of humidities from 0.08 to
96% relative humidity (RH), with and with- 1997; Oliver et al., 1998), R. lanuginosum
out a previous light drying at 96% RH for 24 and A. viticulosus. However, in many species
h. The predrying treatment led to clearly of more mesic habitats, tolerance is induced
greater desiccation tolerance in the majority to varying degrees. The physiology of the
of species, sometimes very strikingly so, as in induction of enhanced tolerance in such
Bryum capillare, Bryum caespiticium, important groups as the Bryaceae, Mniaceae
Mnium marginatum, Plagiomnium rostra- and the common pleurocarpous families in
tum, Ceratodon purpureus, Fissidens the Hypnobryales is almost entirely unex-
adiantoides, Pohlia elongata, Timmia austri- plored. Abscisic acid (ABA)-induced toler-
aca and P. undulatum. In some species (gen- ance (involving synthesis of specific proteins
erally the most sensitive and the most during drying) has been demonstrated in pro-
tolerant) predrying made little difference, as tonema of Funaria hygrometrica (Werner et
in Bryum pseudotriquetrum (bimum), al., 1991; Bopp and Werner, 1993; Schnepf
Mielichhoferia elongata, Philonotis seriata and Reinhard, 1997), and short, thick-walled
and Rhynchostegium riparioides (Platy- desiccation-tolerant protonemal cells form
hypnidium rusciforme) (sensitive), and T. under the influence of desiccation or ABA in
ruralis, Encalypta streptocarpa (contorta), Aloina aloides (Goode et al., 1994) and
Grimmia pulvinata, Hedwigia ciliata (albi- Diphyscium foliosum (Duckett, 1994).
cans), Pleurozium schreiberi and Beckett (1999) showed that partial dehydra-
Rhytidiadelphus spp. (tolerant). Schonbeck tion of the moss Atrichum androgynum for 3
and Bewley (1981a) explored the effects of days increased resistance to desiccation-
slow and fast drying and rehydration on pho- induced cation leakage, and that treatment
tosynthesis by T. ruralis following 2 days’ with ABA produced the same effect. Induced
and 7 days’ desiccation at 21 or 208 MPa. or seasonally switched desiccation tolerance
Rate of drying made little difference to recov- is probably common in marchantialean liver-
ery after desiccation at 21 MPa, but recov- worts of seasonally dry habitats. In Lunularia
ery of photosynthesis was severely impaired cruciata, the switch from the desiccation-sen-
after rapid drying to 208 MPa. Damaging sitive winter state to the tolerant summer
effects of rapid drying to –600 MPa were state appears to be mediated by lunularic
largely eliminated if the moss samples were acid (Schwabe and Nachmony-Bascomb,
first dried slowly to 21 MPa, and were 1963). Hellewege et al. (1994) showed that
greatly reduced if the samples were equili- ABA induces desiccation tolerance in the liv-
brated for 5 h in saturated air before remoist- erwort Exormotheca holstii, and Hellewege et
ening. Oliver and Bewley (1997) suggested al. (1996) have shown that ABA can bring
that the limited ‘hardening’ effect seen in T. about the transition from the aquatic to the
ruralis may reflect sequestration of ‘recovery’ land form of Riccia fluitans. The ability to
mRNAs during slow drying. Krochko et al. respond to ABA with enhanced desiccation
(1978) found very large differences between tolerance thus seems likely to be widespread
fast- and slow-dried material of the relatively among bryophytes, but how widely endoge-
desiccation-sensitive moss Cratoneuron fil- nous ABA occurs within the bryophytes is
icinum. There is clearly much of interest in unknown. ABA is undetectable in T. ruralis,
the effects of different rates of drying and in which a high level of desiccation tolerance
rehydration, which invites further investiga- is constitutive (Oliver and Bewley, 1997).
tion and study of a wider range of species.
7.2.7. How long is needed for complete

7.2.6. Constitutive and induced tolerance recovery? Processes and criteria of recovery;
long-term survival
Desiccation tolerance apears to be largely
constitutive in the highly desiccation-tolerant The recovery processes outlined in Section
bryophytes on which most work has been 7.2.4 proceed on different time scales.
done, such as T. ruralis (Oliver and Bewley, What should we take as the criteria of over-
all ‘recovery’? There may be no hard and total, have been documented as being
fast answer to this question. Very short desiccation-tolerant in their vegetative parts
moist periods will lead to net carbon loss. (Table 7.1; Porembski and Barthlott, 2000).
Moist periods long enough for a positive Because the rehydration of some of these
net carbon balance may be insufficient for plants gives the appearance of a revitaliza-
cell division and growth, but might per- tion of apparently dead tissues, they are
haps allow significant DNA repair. This is often referred to as ‘resurrection plants’.
conjectural, but indicates the kind of ques- The first scientific report of a resurrection
tions on which research is needed. The species was made by Hooker (1837) in a
limited available measurements indicate description of Selaginella lepidophylla from
that the moist periods experienced by des- the southwestern USA and Mexico.
iccation-tolerant bryophytes in the field Subsequent reports were made in the next
vary greatly in length (Proctor, 1990; century, the earliest from field observations
Proctor and Smith, 1995). Maintenance of a made in dry areas of central Asia and sub-
positive net carbon balance must be impor- Saharan Africa. The remarkable ability of
tant, even on a rather short time scale, these plants to exhibit the effects of extreme
whereas a bryophyte may have an entirely desiccation and yet to remain alive and
viable annual life cycle in which growth is revive when rewetted was reported with
largely confined to particular seasons, interest and often careful notes (Thoday,
while for the rest of the year the plant is 1921; Vassiljev, 1931; Hambler, 1961, 1964;
doing no more than maintaining its Hornby and Hornby, 1964). Carex physodes,
foothold in the habitat. Whether ‘recovery’ Myrothamnus flabellifolia and Craterostigma
is seen as return to a normal rate of carbon plantagineum were among the first desicca-
fixation, or as requiring full restoration of tion-tolerant angiosperms described, while
all metabolic systems to their optimum early reports confirmed the phenomenon in
moist-period activity, is a question of other pteridophytes, such as Polypodium
context – indeed, there may be no single polypodioides, Notochlaena marantae,
‘optimum’. Further, bryophyte shoots com- Selaginella njam-njamensis and Platycerium
monly show progressive formation of new stemaria (Pessin, 1924; Iljin, 1931; Hambler,
leaves and death of old ones; recovery from 1961, 1964).
desiccation of the apical parts of the shoot Resurrection species have been docu-
may be accompanied by accelerated senes- mented thus far in nine families of pterido-
cence and death of the older parts. phytes and ten families of angiosperms
(Table 7.1). They are conspicuously lacking
in gymnosperms, though foliage of
7.3. Vascular Plants (see also Chapter 1) Welwitschia mirabilis shows some degree
of tolerance (Gaff, 1972). In angiosperms,
Although often observed in seeds, spores they occur among both monocotyledons
and pollen, desiccation tolerance is the and dicotyledons. Although a higher pro-
exception in vegetative tissues of vascular portion of pteridophyte taxa than seed
plants. The combination of vascular tissue plants are tolerant of desiccation,
and intercellular spaces with cuticle and angiosperms often exhibit a higher degree
stomata allows these species to maintain a of tolerance than ferns (Gaff, 1977).
water potential higher than that of their Desiccation tolerance shows a wide tax-
above-ground environment (homoiohydry), onomic scatter, and appears to have
avoiding the need to tolerate large fluctua- evolved independently a number of times
tions in moisture availability. Nevertheless, as an adaptation to extremes in water avail-
in intermittently arid habitats some species ability. Some genera, such as Cheilanthes,
have adapted to survive desiccation rather Pellaea, Selaginella and Xerophyta, have
than avoid it. However, of the quarter of a large numbers of tolerant species while
million or so species of vascular plants, others, such as Boea, have only a single
only some 330 species, or < 0.15% of the species that is known to be tolerant. A
Table 7.1. Desiccation-tolerant (DT) vascular plants. With a few exceptions, names and authorities are
those used by the cited authors. Species of a genus recorded in a single publication from the same
geographical area are listed together; otherwise the arrangement is alphabetical within major taxonomic
categories.
Species Country Reference
PTERIDOPHYTES
LYCOPSIDA (Clubmosses)
Isoetaceae
Isoetes australis Williams Australia Gaff and Latz (1978) (Only the corms
are DT; other terrestrial Isoetes spp. of
seasonally desiccated habitats, e.g. in
southern Europe, are likely to behave
similarly)
Selaginellaceae
Selaginella caffrorum (Milde) Hieron., South Africa Gaff (1977)
S. digitata Spring, S. dregei (C. Presl)
Hieron., S. echinata Baker,
S. imbricata (Forsk.) Spring ex
Decaisne, S. nivea Alston
Selaginella convoluta Spring, South America Gaff (1987)
S. peruviana (Milde) Hieron.,
S. sellowii Hieron.
Selaginella lepidophylla (Hook. and Southwestern USA, Hooker (1837), Gaff (1971), Eickmeier
Grev.) Spring Mexico (1979), Iturriaga et al. (2000)
Selaginella njam-njamensis Hieron. West Africa Hambler (1961)
Selaginella pilifera A. Br. Southwestern US Eickmeier (1980)
Selaginella sartorii Hieron. Mexico Iturriaga et al. (2000)
PTEROPSIDA (Ferns)
Adiantaceae (sensu lato)
Actiniopteris dimorpha Pic. Serm. South Africa Gaff (1977)
A. radiata (Sw.) Link South Africa, India Gaff (1977), Sharma and Purohit (1986)
Adiantum incisum Forsk. South Africa, India Gaff (1977), Sharma and Purohit (1986)
Cheilanthes albomarginata India Sharma and Purohit (1986)
Cheilanthes bonariensis (Wild) Proctor, Mexico Iturriaga et al. (2000)
C. integerrima (Hook.) Mickel.,
C. myriophylla Desv.
Cheilanthes buchtienii (Rosenst.) South America Gaff (1987)
Capurro, C. glauca (Cav.) Mett.,
C. marginata H.B.K.
Cheilanthes farinosa (Forsk.) Klf. South Africa Gaff (1977), Sharma and Purohit (1986)
Cheilanthes capensis (Thunb.) Desv., South Africa Gaff (1977)
C. depauperata Bak., C. dinteri Brause,
C. eckloniana (Kunze) Mett., C. hirta
Sw., C. inaequalis (Kunze) Mett.,
C. marlothii (Hieron) Mett., C. multifida
(Sw.) Sw., C. parviloba Sw.
Table 7.1. Continued
Cheilanthes distans (R. Br.), Australia Gaff and Latz (1978)

C. fragillima F. Muell., C. lasiophylla
Pic.-Ser., C. paucijuga Benth.,
C. tenuifolia (Burm.f.) Sw., C. vellea
(R. Br.) F. Muell.
Cheilanthes lendigera Swartz Wittrock (1891)
Cheilanthes pringlei Daven., Southwestern USA, Helvy (1963)
C. wrightii Hooker Mexico
Cheilanthes sieberi Kunze Australia, Gaff and Latz (1978), Gaff and
South Africa McGregor (1979)
Doryopteris concolor (Lag. and Risch.) South Africa Gaff (1971, 1977)
Kahn
Doryopteris kitchingii (Bak.) Bonap. South Africa Gaff (1977)
Doryopteris pedata (L.) Fée, D. triphylla South America Gaff (1987)
(Lam.) Christ
Notholaena marantae R.Br. Europe Iljin (1931)
Notholaena parryi D. C. Eat. North America Witham (1972), Nobel (1978)
Notholaena R. Br. sp. South America Porembski and Barthlott (2000)
Paraceterach muelleri (Hook.) Copel. Northeastern Gaff and Latz (1978)
Australia
Pellaea atropurpurea (L.) Link Mexico Pickett and Manuel (1926)
Pellaea boivinii Hook., P. calomelanos South Africa Gaff (1977)
(Sw.) Link, P. hastata (L.f.) Link,
P. quadripinnata (Forsk.) Prantl.,
P. viridis (Forsk.) Prantl.
Pellaea falcata (R. Br.) Fée Australia Gaff and Latz (1978)
Pellaea glabella Mett. USA Pickett and Manuel (1926)
Pellaea longimucronata Hooker Southwestern USA, Helvy (1963)
Mexico
Pellaea ovata (Desv.) Weatherby (commercial) Iturriaga et al. (2000)
Pellaea rotundifolia (Forsk.) Hk. (RBG Kew: Gaff and Latz (1978)
origin unknown)
Pellaea sagittata (Cav.) Link f. var. Mexico Iturriaga et al. (2000)
cordata (Cav.) Tyron.
Pellaea ternifolia (Cav.) Link South America Gaff (1987)
Aspleniaceae
Asplenium aethiopicum (Burm. f.) South Africa Gaff (1977)
Bech., A. rutifolium (Berg.) Kunze var.
bipinnatum (Forsk.) Schelpe,
A. sandersoni H. K.
Asplenium bourgaei Mediterranean Greuter et al. (1983)
Asplenium pringlei Davenp. Wittrock (1891)
Continued
Asplenium ruta-muraria L. Western Kappen (1964)

Europe
Asplenium septentrionale (L.) Hoffm. Europe Kappen (1964)
Asplenium trichomanes L. Europe Wittrock (1891), Kappen (1964)
Ceterach cordatum (Thunb.) Desv. South Africa Gaff (1977)
Ceterach officinarum Lam. et DC Southern and Oppenheimer and Halevy (1962),
Western Europe, Schwab et al. (1989), M.C.F. Proctor
Mediterranean (unpublished data)
Pleurosorus rutifolius (R. Br.) Fée Western Australia Gaff and Latz (1978)
Woodsia ilvensis (L.) R.Br. Europe Wittrock (1891)
Davalliaceae
Arthropteris orientalis (Gmel.) Porth. South Africa Gaff (1977)
Grammitidaceae
Ctenopteris heterophylla (Labill.) Tindale New Zealand Gaff and Latz (1978)
Hymenophyllaceae
Hymenophyllum tunbridgense (L.) Southwestern
Smith, H. wilsonii Hook. England M.C.F. Proctor (unpublished data)
Hymenophyllum sanguinolentum New Zealand J.G. Duckett and M.C. Proctor
(Forst. F.) Swartz (unpublished data)
Polypodiaceae
Platycerium stemaria (P. Beauv.) Desv. West Africa Hambler (1961)
Polypodium cambricum L. Southwestern M.C.F. Proctor (unpublished data)
England
Polypodium polypodioides (L.) North America Pessin (1924), Stuart (1968), Gaff
Hitchcock (1977), Iturriaga et al. (2000)
Polypodium virginianum L. North America Reynolds and Bewley (1993)
Polypodium vulgare L. Wittrock (1891), Kappen (1964)
Schizaeaceae
Anemia tomentosa (Sav.) Swartz South America Gaff (1987)
Mohria caffrorum (L.) Desv. South Africa Gaff (1971, 1977)
Schizaea Sm. sp. East Africa, Porembski and Barthlott (2000)
Seychelles
ANGIOSPERMS
MONOCOTYLEDONS
Cyperaceae
Afrotrilepis pilosa (Boeck.) J. Raynal West Africa Hambler (1961), Owoseye and Sanford
(1972)
Carex physodes M. Bieb Central Asia Vassiljev (1931)
Coleochloa pallidior Nelmes South Africa Gaff and Ellis (1974)

Coleochloa setifera (Ridley) Gilly South Africa Gaff (1971), Gaff and Ellis (1974)
Cyperus bellis Kunth South Africa Gaff and Ellis (1974)
Fimbristylis dichotoma (L.) Vahl Australia Gaff and Latz (1978)
Fimbristylis Vahl Tropical Africa Porembski and Barthlott (2000)
Kyllinga alba Nees South Africa Gaff and Ellis (1974)
Mariscus capensis Schrad. South Africa Gaff and Ellis (1974)
Microdracoides squamosa Hua West Africa Porembski and Barthlott (2000)
(monotypic)
Trilepis Nees South America Porembski and Barthlott (2000)
Liliaceae (Anthericaceae)
Borya inopinata Australia Forster and Thompson (1997)
Borya nitida Labill. Australia Gaff and Churchill (1976)
Borya septentrionalis F. Muell. Australia Gaff and Latz (1978)
Poaceae
Brachyachne patentiflora (Stent and South Africa Gaff and Ellis (1974)
Rattray) C.E. Hubb.
Eragrostiella bifaria (Vahl) Bor. Australia Gaff and Latz (1978)
Eragrostiella brachyphylla (Stapf) Bor., India Gaff and Bole (1986)
E. nardioides (Trin.) Bor.
Eragrostis hispida K. Schum., South Africa Gaff and Ellis (1974)
E. nindensis Fic. and Hiern,
E. paradoxa Launert
Eragrostis invalida Pilger West, East and Gaff (1986), Nugent and Gaff (1989)
South Africa
Micraira adamsii Australia Gaff (1989)
Micraira spinifera Lazar, M. tenuis Lazar Australia Gaff and Sutaryono (1991)
Micraira subulifolia F. Muell. Australia Gaff and Latz (1978)
Microchloa caffra Nees, M. kunthii Desv. South Africa Gaff and Ellis (1974)
Microchloa indica (L.f.) O. Kunze South America, Gaff (1987), Iturriaga et al. (2000)
Mexico
Oropetium capense Stapf South Africa Gaff (1971), Gaff and Ellis (1974)
Oropetium roxburghianum (Steudel) India Gaff and Bole (1986)
S. Phillips, O. thomaeum Trin.
Poa bulbosa L. Europe Gaff and Latz (1978)
Sporobolus atrovirens (Kunth) Kunth Mexico Iturriaga et al. (2000)
Sporobolus elongatus R. Br. Australia Gaff and Sutaryono (1991)
Sporobolus festivus Hochst. South Africa Gaff and Ellis (1974), Kaiser et al.
(1985)
Continued
Sporobolus fimbriatus Australia Gaff and Ellis (1974)

Sporobolus lampranthus Prig. South Africa Gaff and Ellis (1974)
Sporobolus pellucidus Hochst. East Africa Gaff (1986), Nugent and Gaff (1989)
Sporobolus stapfianus Gandoger South Africa Gaff and Ellis (1974), Kaiser et al.
(1985), Sgherri et al. (1994)
Tripogon capillatus Jaub. et Spach, India Gaff and Bole (1986)
T. filiformis (Stapf) Nees ex Steud.,
T. jacquemontii Stapf, T. lisboae Stapf,
T. polyanthus Naik. et Patunkar
Tripogon curvatus Phillips and Launert Africa Gaff and Sutaryono (1991)
Tripogon lolioformis (F. Muell.) Australia Gaff and Latz (1978)
C. E. Hubbard
Tripogon minimus (A. Rich.) Hochst. South Africa Gaff and Ellis (1974)
ex Steud.
Tripogon spicatus (Nees) Ekman South America, Gaff (1987), Iturriaga et al. (2000)
Mexico
Velloziaceae
Aylthonia blackii (L.B.Smith) Menezes South America Gaff (1987)
Barbacenia flava Martius ex Schultes f., South America Gaff (1987)
B. longiflora Martius, B. riedeliana
Goethart and Henrare, B. sellovii
Goethart and Henrard
Barbaceniopsis boliviensis (Baker) South America Gaff (1987)
L.B. Smith, B. humahuaguensis Noher
Nanuza plicata (Speng) L.B. Smith South America Rosetto and Dolder (1996)
and Ayensu
Pleurostima Raff. South America Porembski and Barthlott (2000)
Vellozia Vand. (~ 124 spp., probably all South America Gaff (1987), Porembski and Barthlott
desiccation-tolerant) (2000)
Xerophyta Juss. (~ 28 spp., probably all Sub-Saharan Gaff (1971, 1977), Owoseye and
desiccation-tolerant) Africa, Madagascar Sanford (1972); Gaff and Hallam (1974),
Hallam and Gaff (1978), Tuba et al.
(1993), Porembski and Barthlott (2000)
DICOTYLEDONS
Acanthaceae
Talbotia elegans Balfour South Africa Gaff and Hallam (1974), Hallam and
Gaff (1978)
Cactaceae
Blossfeldia liliputana South America Barthlott and Porembski (1996)
Gesneriaceae
Boea hygroscopica F. Muell. Australia Gaff and Latz (1978), Kaiser et al.
(1985)

Haberlea rhodopensis Friv. Southeastern Bewley and Krochko (1982), Müller et
Europe al. (1997)
Ramonda myconi Reichb. Southwestern Gaff and McGregor (1979), Schwab et
(= R. pyrenaica Rich.) Europe al. (1989)
Ramonda nathaliae Panc. and Petrov. Southeastern Bewley and Krochko (1982), Müller et
Europe al. (1997)
Ramonda serbica Panc. Southeastern Markovska et al. (1994)
Europe
Streptocarpus Lindley spp. Africa Porembski and Barthlott (2000)
Labiatae
Satureja gilliesii (Benth.) Briq. South America Montenegro et al. (1979)
Myrothamnaceae
Myrothamnus flabellifolia Welw. South Africa Thoday (1921), Child (1960), Gaff
(1971, 1977)
Myrothamnus moschata (Baillon) Madagascar Gaff (1977)
Niedenzu
Scrophulariaceae
Chamaegigas intrepidus Dinter ex Heil South Africa Gaff (1971, 1977)
Craterostigma monroi S. Moore, South Africa Gaff (1977)
C. nanum Engl.
Craterostigma plantagineum Hochst. South Africa Gaff (1971, 1977), Schwab et al. (1989)
Craterostigma wilmsii Engl. South Africa Gaff (1971, 1977)
Ilysanthes purpurascens Hutch., South Africa Gaff (1977)
I. wilmsii Engl. and Diels
Limosella L. South Africa Porembski and Barthlott (2000)
Lindernia All. spp. Tropical Africa Porembski et al. (1997)
large number of desiccation-tolerant plants, and early studies centred on mea-

species have been documented from cen- suring the rates of drying and the loss of
tral and southern Africa, Australia and moisture in leaves of plants observed in the
South America (Gaff, 1971, 1977, 1986, field or laboratory (Child, 1960; Hambler,
1987; Gaff and Ellis, 1974; Gaff and 1961; Hornby and Hornby, 1964).
Hallam, 1974; Gaff and Churchill, 1976; Regreening of species that lose chlorophyll
Gaff and Latz, 1978; Gaff and Giess, 1986). during desiccation, as well as the use of
Although fewer have been described from vital stains, was soon added to confirm tis-
Europe, Asia and North America, new des- sue viability after rehydrating (Gaff and
iccation-tolerant species continue to be Okon’O-Ogola, 1971; Gaff and Ellis, 1974;
found as dry habitats are further explored Gaff, 1977; Hallam and Gaff, 1978), and
(Gaff and Bole, 1986; Iturriaga et al., 2000). early measurements of respiration and pho-
Because these plants exhibit such a dra- tosynthesis progressed from simple gas
matic change in morphology during desic- exchange to IRGA (Stuart, 1968; Vieweg
cation and rehydration, revival of normal and Ziegler, 1969; Eickmeier, 1979). Solute
appearance after apparent death was, at leakage from desiccated tissue was also
first, sufficient to indicate survival of used as a measure of damage (Leopold et
al., 1982). By the 1980s, research exploring promoting soil development and the estab-
the relationship of metabolism to the desic- lishment of other species (Child, 1960).
cation phenomenon was well under way, In addition to physiological desiccation
with a few species being notable models tolerance, some species share water-
around which much of this research cen- absorbing or retaining characteristics with
tred (e.g. S. lepidophylla, C. plantagineum, desiccation-intolerant species. Xeromorphic
M. flabellifolia, Chamaegigas intrepidus, characteristics in the liliaceous desiccation-
Polypodium virginianum, Sporobolus stap- tolerant plant Borya nitida and the grass S.
fianus, Boea hygroscopica). This has natu- stapfianus help reduce water loss, with the
rally led to molecular studies, particularly latter retaining almost 80% RWC after a
with Craterostigma plantagineum (see week of desiccation (Gaff and Churchill,
reviews by Hartung et al., 1998; Scott, 1976; Vecchia et al., 1998). In other
2000), and these lines of research are exam- species, specialized structures aid in
ined in detail in Chapter 12. absorbing or dispersing water. Scales on
the underside of the leaf of P. polypodi-
oides appear to function in distributing
7.3.1. Ecological and morphological water over the surface, thereby aiding in
adaptations absorption (Pessin, 1924; Stuart, 1968),
while dead leaf bases around the stem of A.
Resurrection species are often small, low- pilosa help retain water and slow the rate
growing plants with short internodes and of drying (Hambler, 1961). Velamen on the
compact growth that are found as pioneers roots of some species (Afrotrilepis pilosa,
in shallow soils or on rocky outcroppings, Coleochloa setifera, Xerophyta pinnifolia)
areas that can experience extreme variations allows for rapid absorption when water is
in moisture availability. When the rains do available (Porembski and Barthlott, 1995).
come, these plants must be able to rehy- Other resurrection plants, such as
drate, photosynthesize and grow before dry- Craterostigma plantagineum, C. wilmsii
ing occurs again. At some point, they must and C. nanum, possess few morphological
also remain active for a period long enough characteristics which help to avoid or slow
to reproduce. Some species are adapted for desiccation, and it is not surprising that
seasonal changes in water availability. these show a greater physiological toler-
Others dry and are rewetted at more irregu- ance for rapid desiccation than do more
lar intervals. Chamaegigas intrepidus grows xeromorphic species (Sherwin, 1995;
in southern Africa in situations where water Farrant et al., 1999). It should also be noted
collects into ephemeral pools. This aquatic that some desiccation-tolerant species are
plant may be subjected to as many as 20 able to grow under conditions of extreme
desiccation/rehydration cycles annually in water deficits as well as in areas where
addition to surviving through at least 8 soils are richer and water more plentiful,
months of dry dormancy (Schiller et al., and that morphologies, such as leaf shape,
1998, 1999). The ability to survive rapid and may differ in the different environments
frequent desiccation and to quickly re-estab- (Hornby and Hornby, 1964).
lish normal function is a necessity under Although they are adapted to extreme
such conditions. desiccation, often in hot, dry environ-
As pioneers, resurrection species may ments, not all resurrection plants are toler-
be restricted to areas that are uninhabitable ant of either high light levels or high
by other vascular species. The sedge temperatures. Many resurrection species
Afrotrilepis pilosa forms monospecific do tolerate full sun, but others, especially
stands, which persist without encroach- pteridophytes, typically require a shaded
ment by less well-adapted species habitat and may be damaged by excessive
(Porembski et al., 1996). Plants such as sunlight (Gaff, 1977; Lebkuecher and
Myrothamnus flabellifolia, however, pro- Eickmeier, 1991). Similarly, some resurrec-
vide shelter and trap organic materials, tion species, again often pteridophytes, fre-
quently occur in thermally buffered micro- Child, 1960; Oppenheimer and Halevy,
habitats, such as in rocky crevices or out- 1962; Kappen, 1964; Stuart, 1968; Gaff and
croppings. This environment provides Churchill, 1976; Reynolds and Bewley,
some shelter from direct sunlight, reducing 1993). While some research on the limits of
daytime temperatures while holding heat desiccation tolerance has been done
during the cooler nights and winter days. directly on field-dried tissues, in the labo-
The optimal temperature for photosynthe- ratory tissues have been air-dried, equili-
sis in the desert fern, Notholaena parryi, brated over solutions producing known
has been measured as several degrees RHs or dried over desiccating agents, such
cooler than the mean air temperature of its as sulphuric acid or silica gel, for more
general environment, suggesting a modify- rapid drying. The humidity over silica gel
ing effect of its microhabitat (Nobel, 1978). and other strong drying agents is often
Moderating the extremes of temperature reported as ‘0% RH’, but this limit (corre-
and light can also be important for surviv- sponding to a water potential of ∞) is
ing desiccation, since desiccation damage unattainable in practice.
can be enhanced by photodamage and high Levels of desiccation tolerance differ
temperatures (Eickmeier, 1986; Muslin and among species. Gaff (1977) examined 37
Homann, 1992; Chapter 9). desiccation-tolerant ferns and angiosperms
Although research on desiccation toler- for their RH tolerance levels and found
ance in vascular species has focused pri- that only 30% of the pteridophytes could
marily on the sporophyte, gametophytes of survive equilibration with an RH close to
some pteridophytes also display desicca- 0%, while 76% of the angiosperm taxa did
tion tolerance. The extent of this phenome- so, reflecting the tendency of pterido-
non has not been well documented, but phytes to inhabit somewhat protected
several reports indicate that it may be a areas. The least tolerant resurrection fern,
survival mechanism in at least some pteri- however, was still able to survive equili-
dophyte species (Mottier, 1914; Kappen, bration with 30% RH, significantly lower
1965; Page, 1979; Quirk and Chambers, than the RH tolerated by non-resurrection
1981). Some success has been achieved in plants. The RWCs of most dry resurrection
recovering in vitro-grown fern gameto- plants are in the range of 5–10% or less
phytes of Adiantum tenerum, Adiantum (Scott, 2000).
trapeziforme, Cibotum glaucum, Davallia As with dry seeds, vegetative tissues
fejeensis and Drymaria quercifolia after that are sufficiently dry may remain viable
exposure to liquid nitrogen when the for relatively long periods of time,
gametophytes were first air-dried, indicat- although moisture levels, temperature and
ing that the tissues were dry enough to species differences will affect longevity.
avoid freezing injury (Pence, 2000). Dried Afrotrilepis pilosa remained viable
for a year at room temperature, and also
survived storage overnight at 10°C,
7.3.2. The effect of intensity of desiccation whereas undried tissues did not survive
freezing (Hambler, 1961). Dry tissues of
The vegetative tissues of most vascular seven vascular species survived for at least
plants can survive equilibration with RHs 3.5 years as field-dried leaves when they
only in the range of 85–98%; the Namibian were sealed in plastic or air-tight glass
Welwitschia mirabilis will stand drying (Gaff, 1977), and two of these species,
only to a RWC of 56% (Gaff, 1972). Truly Xerophyta squarrosa and C. setifera,
desiccation-tolerant species, however, can showed 100% survival even after 5 years.
equilibrate with much lower RHs, often As with seeds, lower temperatures might
< 10%, and still recover on remoistening. be expected to prolong viability of dry tis-
Drying tissues may lose from 70 to 95% of sues, and such tissues should be good can-
their original water content, generally over didates for low-temperature storage or
a period of several days (Pessin, 1924; cryopreservation.
7.3.3. Effect of rate of desiccation 7.3.4. Morphological and cytological

changes that occur with drying
In contrast to bryophytes, vascular plants
are buffered to varying degrees from ambi- Drying of resurrection plants brings about
ent moisture levels by their larger size, the various morphological changes. These
presence of a cuticle and stomata, and result from the loss of water at the cellular
their ability to access underground mois- level, but provide protection at the whole
ture. In addition, desiccation-tolerant plant level. The curling of leaves to form
species with xeromorphic characteristics long, threadlike structures and the curling
can slow water loss even further. As a of older leaves and stems over younger
result, the desiccation-tolerance mecha- leaves and buds slow the rate of drying in
nisms in desiccation-tolerant vascular taxa younger, growing tissues, as well as pro-
appear to be, in large part, inducible rather tecting inner dried tissues from photodam-
than constitutive, as in many bryophytes, age (Pessin, 1924; Child, 1960; Gaff and
with tolerance developing over the course Churchill, 1976; Gaff, 1977). Younger leaf
of 12–24 h. tissue, in general, appears more tolerant of
Natural drying of resurrection species in desiccation (Gaff and Ellis, 1974; Norwood
the field generally occurs over a period of et al., 1999).
days or even weeks. In Craterostigma plan- Changes in shape at the whole plant
tagineum signs of desiccation were visible level are accompanied by a dramatic reduc-
within 1 week of the last rain (Hornby and tion in size, loss of turgor and a decrease in
Hornby, 1964). Gaff (1977) observed 11 cell volume. Some resurrection species may
species in the field in southern Africa for shrink to less than 20% of their original leaf
the first signs of water stress in the leaves area upon drying. These species are able to
and noted that, for ten of the species, dry- maintain connections between the plasma
ing times ranged from 40 to 96 h. The membrane and the cell wall and to undergo
exception was Chamaegigas intrepidus, a controlled and extensive folding of the cell
small, aquatic plant of ephemeral rock wall during drying, allowing the collapse of
pools, which was air-dry within an hour. the tissue without the fatal results seen in
Critical evaluation of the maximum tol- non-tolerant species (Hartung et al. 1998;
erated rates of drying support the observa- Vicre et al., 1999).
tion that natural drying of resurrection Damage from light has been observed in
species is generally slow. A number of S. lepidophylla and P. polypodioides when
studies have shown that these species do curling of the leaves was manually inhib-
not survive rapid desiccation, although ited during illuminated drying (Muslin and
slower rates of drying will allow survival Homann, 1992; Lebkuecher and Eickmeier,
to very low levels of moisture. For exam- 1993), and it is thought that a number of
ple, leaves of Borya nitida which were morphological and physiological processes
air-dried could not survive below equili- associated with desiccation are adaptations
bration with 85% RH or less. If, however, to minimize damage from light in the dry
the leaves were first exposed to 96–98% tissues. In several species, hairs or other
RH for 2 days, they were then capable of substances help reflect light from the abax-
surviving close to 0% RH (Gaff and ial leaf surfaces, which are exposed during
Churchill, 1976). Studies with Selaginella desiccation-induced curling (Nobel, 1978;
lepidophylla, Boea hygroscopica and other Sherwin and Farrant, 1998), as well as
species have demonstrated similar from adaxial surfaces in species with
responses (Eickmeier, 1983; Sgherri et al., leaves that do not curl (Vecchia et al.,
1994). Slow drying can be replaced by 1998). Pigments in many desiccation-
ABA in several systems, suggesting that it tolerant plants also help to reduce photo-
is closely involved with the induction of damage, and colour changes in dried leaves
protective mechanisms in resurrection have been reported for a number of species.
species (Reynolds and Bewley, 1993). In C. wilmsii, Xerophyta viscosa and
Myrothamnus flabellifolia, anthocyanin remain intact, although the vacuole may

levels increased significantly in dried tis- fragment into many small vacuoles. These
sues, helping to mask chlorophyll and species are generally adapted to areas that
reduce damage by free radicals (Sherwin experience fewer fluctuations in water
and Farrant, 1998; Koonjul et al., 2000). availability and thus do not require a rapid
Photoprotection by mechanisms associated recovery response.
with the production of zeaxanthin has also An inherent problem in cytological stud-
been demonstrated (Casper et al., 1993; ies of these desiccated tissues has been that
Eickmeier et al., 1993). aqueous fixatives can initiate changes asso-
Changes in colour are accentuated in ciated with rehydration in desiccated tis-
some species by the loss of chlorophyll sues. Fixatives with high osmolality have
during desiccation. Resurrection plants can been effective in some studies (Platt et al.,
be classified as either poikilochlorophyl- 1998), but the use of freeze-substitution
lous desiccation tolerants (PDTs), which techniques has proved most efficient in
lose chlorophyll during drying, or maintaining the structure of desiccated
homoiochlorophyllous desiccation toler- cells for electron microscopy (Thomson and
ants (HDTs), which retain chlorophyll Platt, 1997; Platt et al., 1998).
throughout desiccation. Gaff and Hallam
(1974) reported that most resurrection
pteridophytes and dicots were HDTs, while 7.3.5. Rehydration and recovery
about half of the monocots surveyed were
PDTs. PDTs avoid photodamage to their The ‘resurrection’ of desiccation-tolerant
photosynthetic apparatus by dismantling vascular species centres on the events of
it, a trade-off against the greater time rehydration and recovery from apparent
needed for the re-establishment of photo- lifelessness. Very little rehydration occurs
synthetic activity. Both HDT and PDT from dew, but rains of 10 mm or greater
species can withstand severe desiccation generally stimulate rehydration (Gaff,
(Gaff, 1989), but HDTs are fully functional 1977). Rapid water uptake may occur
within about 24 h of exposure to water, through the leaves, and in experimental
while PDTs may take 48–72 h or longer to systems this is often the method used for
regreen and re-establish photosynthetic rehydrating desiccated tissues. C. plan-
apparatus and function (Gaff and Ellis, tagineum can recover 85% RWC within
1974; Tuba et al., 1993; and see below). 5–6 h when submerged or substantially
Although the cells shrink during dehydra- indundated, and desiccation-tolerant
tion and the cell walls and membranes grasses regain most of their shape within
become convoluted, most of the organiza- 4–5 h of rehydration (Gaff and Ellis, 1974),
tion of the chloroplasts and other In nature, however, water uptake will also
organelles is maintained in HDT species occur through the roots, and larger species,
(Platt et al., 1994; Thomson and Platt, such as M. flabellifolia, which rely primarily
1997). on rehydration from the roots, will take
In contrast, desiccation in PDT species longer to rehydrate and recover than species
leads to the degeneration of chloroplast taking up water primarily through the
membranes and the loss of the grana, leaves. During desiccation, the flow of
stroma and thylakoid structure, and the water through the vascular system is dis-
unprotected chlorophyll suffers photode- rupted, leaving gaps of air. Upon rehydra-
struction (Owoseye and Sanford, 1972; Gaff tion, the xylem must be refilled by
et al., 1976; Hallam and Gaff, 1978; Bartley capillary action and/or root pressure in
and Hallam, 1980; Bergstrom et al., 1982; order to resume functioning (Sherwin et
Tuba et al., 1993; Vecchia et al., 1998). In al., 1998).
these species, there is also loss of inner Several factors can influence the rate
mitochondrial membranes and cristae. and type of recovery. Species of Selaginella
Nuclear, plastid and tonoplast membranes from drier habitats resume photosynthesis
more quickly than those from moister habi- flowers (as in Ranunculus ficaria,
tats (Eickmeier, 1980), suggesting a natural Cardamine spp., Saxifraga spp. and Allium
adaptation to the less frequent availability spp. (Richens, 1947)). There are also vari-
of water in the drier areas. There may also ous kinds of stem and root tubers, and
be developmental differences in desicca- viable shoot fragments, which establish as
tion tolerance. Recovery is limited to independent plants. Desiccation tolerance
young growing tissues in a number of has been noted in tubers of Anemone coro-
species, suggesting a greater desiccation naria and Ranunculus asiaticus (Antipov
tolerance than in older tissues (Gaff and and Romanyak, 1983). Some other tubers
Churchill, 1976; Sherwin and Farrant, and at least some bulbs, bulbils and similar
1996). When C. plantagineum was rehy- structures are likely to be desiccation-toler-
drated through the roots, rather than ant too, but there seems to have been little
leaves, water uptake was slower and many systematic work to determine which of the
of the older leaves did not recover com- more persistent of these are truly desicca-
pletely (Bernacchia et al., 1996). tion-tolerant and which are simply highly
The time needed for the recovery of resistant to water loss.
function will depend on how much of the
photosynthetic apparatus was dismantled
during the drying process. Photosynthesis 7.4. Concluding Comments
is re-established in HDT plants within a
few hours to a day after rewatering Various facets of the desiccation responses
(Eickmeier, 1979; Bernacchia et al., 1996). of vegetative tissues have parallels in seed
HDT species generally retain a portion of biology, though these should be seen as
the thylakoid system, as well as chloro- illustrative and a prompt to thought rather
phyll, during desiccation, and, within a than as necessarily implying close physio-
day of rehydration, functioning grana and logical correspondence. The shutting-down
lamellae are re-formed. In contrast, PDT of metabolism on drying and recovery on
species lose membrane structure as well as remoistening after moderate periods of des-
chlorophyll when dried, and these must all iccation suggest parallels in the maturation
be re-formed when water is available and then in the imbibition and germination
(Hallam and Gaff, 1978; Markovska et al., of seeds. The gradual loss of viability on
1995; Sherwin and Farrant, 1996). prolonged desiccation and the relation of
Chlorophyll synthesis begins within a few this to intensity of desiccation and temper-
hours of water availability, but it can take ature have obvious (even if not complete)
up to several days to re-establish function parallels in the well-researched field of
(Tuba et al., 1993; Drazic et al., 1999). PDT seed storage. Fundamental prerequisites for
species are generally adapted to seasonal desiccation tolerance are maintenance (or
changes in water availability, while HDT rapid recovery) of membrane integrity,
species may experience more frequent fluc- preservation of macromolecules in a func-
tuations in moisture. tional state and maintenance of spatial
relationships between functional compo-
nents of the cell. Protective substances
7.3.6. Vegetative propagules: bulbils, corms, such as sucrose and dehydration proteins
tubers and plant fragments probably combine to allow vitrification of
the cell contents on drying (Crowe et al.,
Many normal homoiohydric vascular 1998; Buitink, 2000; Chapter 10) to provide
plants produce vegetative propagules, a living (and reversible) equivalent of good
which often serve to carry the plant fine-structural fixation for electron
through unfavourable dry periods. These microscopy. Some other factors, such as
include bulbils formed either in the soil (as enhanced activity of anti-oxidant systems
in many Oxalis species (Robb, 1963)) or in (Dhindsa and Matowe, 1981; Seel et al.,
leaf axils above ground, often replacing 1991, 1992a; Smirnoff, 1993), may be seen
as extensions of processes common to all potentials (Gaff, 1980), and there are indica-
living cells (Foyer et al., 1994; Alscher et tions that molecular mobility and rates of
al., 1997). ageing generally decrease with falling water
This underlines the distinction between content, but increase again at water contents
effects related to ‘pure’ desiccation toler- in equilibrium with air at less than c. 10%
ance, characteristic of (and inseparable RH (Buitink et al., 2000). The results in Fig.
from) a drying and rewetting event, and the 7.3 are broadly consistent with the conclu-
cumulative damaging effects of longer-term sion that ‘desiccation-tolerant tissues are
desiccation. The effects of cumulative desic- often least damaged at values of of about
cation damage are certainly diverse and 20 to 40% RH or 1300 to 2200 bars’
complex (see, for example, Chapter 9). Gaff (130 to 220 MPa) (Gaff, 1980).
(1980) distinguished a number of possible Bewley (1979) introduced the concept of
sources of injury operating at different rates, ‘repair’ in the recovery of bryophytes from
of which the slower types may be either desiccation, primarily in relation to the
stress-parallel or stress-inverse. Injury at restoration of membrane integrity, and he
high water potentials may arise from contin- and Oliver (Oliver and Bewley, 1984, 1997;
ued activity of metabolic processes. In Bewley and Oliver, 1992; Oliver et al.,
osmotic stress experiments, measurable res- 2000) have extended the concept and asso-
piration and photosynthesis were found ciated it particularly with the recovery
down to c. 10 MPa in Anomodan viticulo- processes in bryophytes. However, as they
sus, and between 10 and 20 MPa in point out, ‘repair’ must be an element in
Homalothecium lutescens (Dilks and the recovery of all desiccation-tolerant
Proctor, 1979); and photosynthetic activity plants, most of all in the poikilochloro-
has been detected in the lichen phyllous vascular species. The broad con-
Dendrographa minor down to almost 40 cept of ‘repair’ needs to be considered
MPa (Lange, 1988). In various lichens, it analytically and quantitatively in terms of
was found that incorporation of tritium into the wide range of processes that it must
sugar alcohols, amino acids and some TCA- embrace. Some systems, such as protein-
cycle intermediates was taking place rather synthesis mechanisms and the photosys-
freely at 40 MPa, and still detectable at tems, survive a drying–rewetting event
100 MPa (Cowan et al., 1979). Many essentially intact and are functional within
bryophytes, especially those of woodland seconds or minutes of remoistening, but
habitats, are remarkably tolerant of being experimental evidence shows that recovery
kept at or near full turgor in the dark for of some other systems must be slower, and
periods of weeks, but under these condi- full return of cell function to a steady state
tions Tortula ruralis dies within a few days. may typically take hours or days.
As Fig. 7.2 shows, given a low level of light, Underlying processes of recovery span a
it survives well at 3 MPa, but much less similar range of time scales. Reinstatement
well at water potentials between 9 and of water into macromolecules and re-estab-
37 MPa. As has been noted for resurrec- lishment of normal membrane integrity are
tion vascular plants, slow growth of likely to be primarily physical, and fast,
pathogens, especially fungi, may be a com- taking place within seconds or minutes.
mon cause of deterioration in this range Other processes are slower, such as re-
(Gaff, 1997). At lower water potentials, other establishment of normal water relations in
factors are likely to be important. High-light vascular ‘resurrection plants’ and synthesis
damage to dry thalli of the forest lichen of the rehydrins (which themselves evi-
Lobaria pulmonaria has been demonstrated dently represent a diversity of processes
(Gauslaa and Solhaug, 1999), and photon proceeding at varied rates (see Chapter
damage is likely to be significant under field 12)). There are important aspects of recov-
conditions for other desiccation-tolerant ery of cell function after desiccation, such
organisms too. Very slow stress-parallel as reinitiation of the cell cycle, of which
effects may be envisaged at very low water we know very little (Paolillo, 1984).
What determines the success of desicca- carbon fixation, though still fast, is measur-
tion-tolerant plants in some habitats and ably slower. For species of shady habitats,
their exclusion from others? In broad such as R. loreus, Mnium hornum or
terms, desiccation-tolerant plants are char- Polytrichum formosum, recovery is slower
acteristic of situations where availability of again. Homoiochlorophyllous desiccation-
water is strongly intermittent, and either tolerant vascular plants are especially char-
the physical nature of the site or climate acteristic of situations where a thin soil
makes a continuous closed cover of vascu- cover allows normal vascular-plant water
lar plants impossible. However, there is a relations to function for much of the year,
wide range of possibilities within these but the soil is desiccated for more or less
constraints. Lichens on exposed rock faces extended periods. The ‘high-inertia’
such as Rhizocarpon geographicum (Ried, extreme is represented by poikilochloro-
1960a,b) or the crustose and endolithic phyllous desiccation-tolerant plants such
lichens in the Negev Desert studied by as the Xerophyta species of central and
Lange et al. (1970) represent a ‘low-inertia’ southern Africa, which, essentially, are
extreme (Tuba et al., 1998). Species with adapted to the switch between a wet and a
this pattern of adaptation extend from the dry season. Tropical inselbergs provide the
wettest to quite arid climates. Their water habitats for a large proportion of all desic-
content tracks closely the incidence of pre- cation-tolerant vascular plants (Porembski
cipitation – rain, dew or impacted mist and Barthlott, 2000).
droplets – from the atmosphere, and they By contrast with all desiccation-tolerant
dry quickly when precipitation ceases. plants, drought-tolerant plants require con-
Survival in such situations demands rapid tinued access to soil water at a physiologi-
return to a positive carbon balance on cally tolerable water potential. Vascular
remoistening after desiccation. Some therophytes, such as desert ephemerals and
mosses such as Tortula ruralis respond to temperate winter annuals, are drought
changing hydration almost equally quickly. evaders, and succulents are drought
For larger lichens and many bryophytes the avoiders. Both groups commonly grow in
time scale is longer. Cushions of the com- the same habitats as desiccation-tolerant
mon wall-top moss Grimmia pulvinata species, which arguably (Proctor, 2000)
store substantial amounts of water follow- may be seen as drought evaders no less
ing rain and may take a number of hours to than the therophytes, substituting desicca-
dry out (Proctor, 1990; Proctor and Smith, tion-tolerant vegetative tissues for desicca-
1995; Zotz et al., 2000), and recovery of tion-tolerant seeds.
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8 Systematic and Evolutionary Aspects of

Desiccation Tolerance in Seeds
John B. Dickie and Hugh W. Pritchard

Seed Conservation Department, Royal Botanic Gardens Kew, Wakehurst Place,
Ardingly, West Sussex RH17 6TN, UK

8.1.1. Reviews and compilations of seed desiccation tolerance 240
8.1.2. Classification of seed storage responses 241
8.1.3. The phylogenetic classification of plants 242
8.1.4. Evolution of desiccation tolerance in land plants 243
8.2. Systematics and Evolution of Seed Desiccation Tolerance 244
8.2.1. The complete dataset 244
8.2.2. Gymnosperms 244
8.2.2.1. Araucariaceae 246
8.2.3. Angiosperms 247
8.2.3.1. Fagaceae 249
8.3. Seed Desiccation Tolerance and Ecology sensu lato 250
8.3.1. Are recalcitrant seeds bigger than orthodox seeds? 250
8.3.2. Are desiccation-sensitive seeds morphologically or anatomically
distinct from tolerant ones? 251
8.3.3. Are desiccation-sensitive seeds associated with particular
habitats? 252
8.4. Future Directions 253
8.5. Conclusion 254
8.6. References 254
8.1. Introduction comparative biology of seed desiccation

tolerance, looking at broad patterns in the
The ability of seeds to tolerate desiccation occurrence of this functional trait through-
is a trait of major adaptive importance to out the spermatophytes and examining sys-
their survival and dispersal role. That tematic, ecological and other potentially
seeds of some species do not withstand informative correlations. We intend to
drying presents a challenge for ex situ con- complement the in-depth mechanistic
servation. In this review, we explore the studies on a number of species described
240 J.B. Dickie and H.W. Pritchard
elsewhere in this volume by discussing (Hong et al., 1998b), hereafter referred to as

seed desiccation tolerance in an evolution- the Compendium.
ary context. In so doing, we aim to high-
light gaps in knowledge and hint at
possible new research directions. At a 8.1.1. Reviews and compilations of seed
more practical level, a better understanding desiccation tolerance
of the evolution of seed desiccation toler-
ance is likely to suggest approaches to ex Significant compilations of recalcitrant seed
situ conservation for those species with data are listed in Table 8.1. The earliest list
desiccation-sensitive seeds. of recalcitrant (desiccation-sensitive) seeded
Much of the consideration of desicca- species included 73 species from 37 genera
tion tolerance in seeds focuses on the (Salix and Swietenia at genus level only) and
‘other side of the coin’, sensitivity to desic- 29 families (King and Roberts, 1979). Within
cation, as this appears to be the exception a year, the list had been shortened, firstly to
in seeds and leads to a number of practical 68 species/42 genera/29 families (King and
problems. Our account relies on a survey of Roberts, 1980) and then to 49 species/36
the literature, as well as some new analysis genera (Juglans and Swietenia at genus level
of existing data. In relation to the former, only) and 27 families (Roberts and King,
two other recent reviews (Farnsworth, 1980). Major divergences between the lists
2000; Pammenter and Berjak, 2000) have related to, for example, the inclusion/exclu-
also dealt with the subject from a compara- sion of Citrus spp., of which at least some
tive and evolutionary aspect. For the latter, were now considered to be orthodox, and
heavy reliance is placed here on the data Coffea spp., about which there was some
accumulated in the Compendium of doubt. A later review identified 186 recalci-
Information on Seed Storage Behaviour trant-seeded species across 124 genera, after
Table 8.1. Compilations of recalcitrant-seeded species.
Number of species Number of genera Number of families Reference
73 37 29 King and Roberts (1979)

(Salix and Swietenia at genus level only)
68 42 29 King and Roberts (1980)

(Malus and Swietenia at genus level only)
49 36 27 Roberts and King (1980)

(Juglans and Swietenia at genus level only)
186 124 64 Hofmann and Steiner (1989)

(Anthurium, Malus, Mauritia, Oxalis,
Roystonea, Sabal, Swietenia and
Thrinax at genus level only)
NGa NG 45 von Teichman and

van Wyk (1994)
195 143 75 Farnsworth (2000)

514b 192c 65d Hong et al. (1998b)
a NG, full details not given.
b Out of a total of 6919 species considered, i.e. 7.4%.
c Out of 2146 listed in electronic version, i.e. 9%.
d Out of 251 families for which there are seed storage data, i.e. 25.9%.
NB: there are c. 462 Angiosperm Phylogeny Group (APG) families.

Systematic and Evolutionary Aspects of Desiccation Tolerance 241
the exclusion of 19 reclassified species intermediate and recalcitrant. Orthodox

(Hofmann and Steiner, 1989). Research since seeds can be dried without damage, to low
then indicates that the seeds of numerous moisture contents, usually much lower
other species can now be considered not to than those they would normally achieve in
be recalcitrant as long as they are treated nature. They can be conserved ex situ for
carefully, e.g. Corylus avellana (hazelnut), relatively long periods (at least decades) in
Zizania aquatica (Indian wild rice), Elaeis seed banks, and many of them, but not all,
guineensis (oilpalm) and Azadirachta indica form persistent seed banks in the soil. Over
(neem). The difficulties associated with allo- a wide range of storage environments, their
cating seeds to the recalcitrant grouping can longevity increases with reductions in both
be gauged by the fact that 15–28% of the moisture content and temperature, in a
species in the early lists were reported as quantifiable and predictable way (Ellis and
having recalcitrant behaviour that had not Roberts, 1980; Dickie et al., 1990).
yet been fully confirmed. Recalcitrant, or desiccation-sensitive, seeds
Similarly, there is discrepancy between do not survive drying to any large degree,
the more recent lists of species. For exam- although the critical moisture level for sur-
ple, 17 species listed by Farnsworth (2000) vival varies among species, from about
as recalcitrant or viviparous are shown in 25% to 40% seed/embryo moisture content
the Compendium (Hong et al., 1998b) to (fresh mass basis) for cacao and red oak,
have seeds which are probably orthodox. respectively (e.g. Leprince et al., 1998).
The species are: Amomyrtus luma, Agathis Thus, they are not amenable to long-term
robusta, Caltha palustris, Chenopodium storage for conservation, nor are they likely
quinoa, Cordia alliodora, Cupressus macro- to form persistent soil seed banks. For this
carpa, Dovyalis hebecarpa, Fagopyrum review, this category includes those seeds,
esculentum, Fagraea fragrans, Flacourtia of some aquatic species in particular,
indica, Hedera helix, Michelia champaca, described as viviparous (Farnsworth,
Muntingia calabura, Nyssa aquatica, Piper 2000). Intermediate seeds are more tolerant
hispidum, Santalum album and Vochysia of desiccation than recalcitrants, though
honurensis. that tolerance is apparently more limited
The latest hard-copy version of the than is the case with orthodox seeds, and
Compendium (Hong et al., 1998b) draws on when dry they generally lose viability more
data for 6919 species, of which 514 (7.4%) rapidly at 0°C and 20°C than at warmer
from 65 families are recorded as being temperatures around 15°C (Ellis et al.,
recalcitrant or likely to be recalcitrant. That 1990, 1991).
version is an update of an earlier version Assignment of species to these classes of
with very limited circulation, sponsored by response is not always clear-cut and sev-
the International Plant Genetic Resources eral ‘likely’ and ‘probable’ epithets are
Institute (IPGRI). Readers should note that used in the Compendium. One of the main
the analysis described below was actually difficulties relates to a lack of a unified
carried out on an electronic version of the approach to measuring the level of desicca-
dataset (see IPGRI website for download – tion (in)tolerance. Whilst most studies use
www.ipgri.cgiar.org/), consisting of records moisture content on a fresh mass and
for 7146 species, and which forms the basis sometimes dry mass basis (e.g. Tompsett,
of part of the Royal Botanic Gardens Kew’s 1984a,b; Pritchard and Prendergast, 1986;
Seed Information Database see Tweddle et Farrant et al., 1988), estimates of critical
al., 2002 for information on Release 2.0. water potentials for desiccation stress have
been used more recently (e.g. Roberts and
Ellis, 1989; Pritchard, 1991; Poulsen and
8.1.2. Classification of seed storage responses Eriksen, 1992; Pritchard and Manger, 1998;
Sun, 1999; Walters, 1999). Although such
The Compendium recognizes three main an approach removes the potentially con-
categories of storage response: orthodox, founding effects of differing chemical com-
position on the assessment of critical mois- the Seed Information Database at

ture contents for desiccation stress, espe- www.rbgkew.org.uk/data/sid).
cially the effect of oil content on the
sorption properties of seeds (Cromarty et
al., 1984), critical water potentials for 8.1.3. The phylogenetic classification of
stress have been suggested for the onset, plants
mid-point and end of the viability loss
response in seed populations (Pritchard, Since Darwin’s Origin of Species, the aim of
1991; Tompsett and Pritchard, 1998; successive generations of plant systematists
Dussert et al., 1999; Sun, 1999; Walters, has been to produce a classification that is
1999). In addition, the physiological state ever more ‘natural’, reflecting as closely as
of the seeds prior to dehydration is influ- possible the phylogeny or evolutionary
enced by developmental age and post-har- descent of species and higher groupings
vest storage conditions and this can have a (see, for example, Woodland (2000) or Judd
profound effect on the level of desiccation et al. (1999) for an account of the history of
tolerance (e.g. Probert and Longley, 1989; plant classification systems and especially
Finch-Savage, 1992; Tompsett and the more modern ones, such as those of
Pritchard, 1993, 1998; Pammenter et al., Cronquist (1981) or Dahlgren (1983)). The
1998; reviewed by Pammenter and Berjak, development of cladistic methods based on
1999). Finally, the methods of desiccation, parsimony, together with the recent rapid
rehydration and germination testing also increase in the availability of DNA sequence
impact on our perception of desiccation data, has led to the emergence of robust
stress (in)tolerance (Grout et al., 1983; phylogenetic classifications of seed plants.
Kovach and Bradford, 1992; Leprince et al., A radically new ordinal classification of the
1998; Sacandé, 2000). flowering plants has arisen from the cooper-
The consequent variability in data, and ative work of the Angiosperm Phylogeny
interpretation, has led to divided opinion Group (APG, 1998). This classification
on whether there is a continuum of seed recognizes only monophyletic families and
desiccation tolerances between species orders, most, but not all, of which fit into
(Berjak and Pammenter, 1994; Dussert et informal higher-level groups or clades.
al., 1999; Sun, 1999) or approximately five Concordance with other, more formal, clas-
discrete levels of desiccation tolerance sification systems may be rather forced at
(Walters, 1999). In addition, variability in ordinal and higher levels; and family cir-
response has tended to a proliferation of cumscriptions are not exactly the same in
‘classes’, for example ‘sub-orthodox’ all cases, e.g. the sinking of Aceraceae into
(Bonner, 1990; Dickie and Smith, 1995) Sapindaceae, and Chenopodiaceae into
and ‘minimally recalcitrant’ (Berjak et al., Amaranthaceae. Like all classification sys-
1989; Dickie et al., 1991). However, and tems, the APG system is a working hypothe-
despite the caveats about assigning species sis, not fact. Nevertheless, we believe that
to three classes of storage, the practical sys- its powerful representation of likely evolu-
tem of classification used in the tionary pathways and relationships pro-
Compendium is retained here. Also, for vides the best background for comparative
reasons of simplicity, our analyses of the investigations of physiological or ecological
Compendium data rely totally on the com- traits such as seed desiccation tolerance,
pilers’ assignment of species to a particular and we use it in the analysis of the flower-
seed storage category, and we include those ing plants in the Compendium dataset
cases where a classification is likely or below. The classification we use for the
probable, as well as those where the gymnosperms is basically the arrangement
observed responses are more definite. used by Judd et al. (1999), referring also to
Readers are referred to the extensive bibli- two more recent molecular treatments of the
ography in the Compendium (and in group (Bowe et al., 2000; Chaw et al., 2000).
8.1.4. Evolution of desiccation tolerance in greatest opportunity for spatial and tempo-
land plants ral movement, and having desiccation-tol-
erant propagules would offer a distinct
Vegetative desiccation tolerance is a wide- ecological advantage in environments not
spread but uncommon occurrence in plants constantly moist, e.g. seasonally dry tem-
(Oliver et al., 2000; Chapters 1, 7). Although perate and tropical habitats. Oliver et al.
the algae, lichens and bryophytes contain (2000) speculated that such propagules
the most desiccation-tolerant plants, about may have evolved as a modification of veg-
120 spp. of ferns, fern-allies and etative desiccation tolerance, i.e. using
angiosperms exhibit vegetative desiccation existing genes.
tolerance. The facts of its early evolution As noted by Pammenter and Berjak
are not certain, but it probably coincided (2000), little is actually known about the
with the colonization of the land by primi- evolution of seed desiccation tolerance
tive plants during the Ordovician period itself, though rather more has been
(from about 510 million years ago (mya)), inferred. Inference has relied on the associ-
although it is frequent in the spores and ation of seed desiccation tolerance with
cysts of extant representatives of more various aspects of systematics, ecology
primitive organisms. For instance, the (particularly habitat and plant habit) and
conidia of Metarhizium flavoviride with- seed and fruit structure, among others (e.g.
stand drying, and once dry they respond Tompsett, 1994; Hong and Ellis, 1998;
quantitatively to moisture content and tem- Hong et al., 1998b).
perature in the same way as orthodox seeds Systematic analyses, mainly based on
(Hong et al., 1998a). Oliver et al. (2000) seed structural features that are regarded as
argued that desiccation tolerance was the primitive (see later), have led to the pro-
ancestral state for early land plants (liver- posal that seed desiccation sensitivity is
worts, hornworts and mosses), but that this the ancestral state, with tolerance evolving
trait was lost early in the evolution of tra- early, and several times independently (e.g.
chaeophytes, possibly beginning in the von Teichman and van Wyk, 1991, 1994;
Silurian (from 439 mya). It has been postu- Pammenter and Berjak, 2000). Meanwhile,
lated that desiccation tolerance was inde- based on a review of 195 sensitive species,
pendently evolved (or possibly re-evolved) Farnsworth (2000) has suggested that the
in both Selaginella and ferns, and at least most parsimonious explanation of the cur-
eight times in the angiosperms. In the last rent distribution of species’ seed desicca-
group, vegetative desiccation tolerance is tion sensitivity is by convergent loss of
found in the order Hamamelidales and the tolerance from tolerant ancestors. Oliver et
families Poaceae (grasses), Cyperaceae al. (2000) inferred that this may have been
(sedges), Velloziaceae, Liliaceae, Labiatae, the case in seeds. We are aware of the
Gesneriaceae and Scrophulariaceae (Oliver inherent risks of inferring evolutionary pat-
et al., 2000). (Note that Hamamelidales is terns by extrapolating from extant species
not recognized as monophyletic by the on purely phylogenetic grounds, and thus
APG, and the species concerned, our interpretation of the evolution of seed
Myrothamnus flabellifolius, is not assigned desiccation tolerance is placed in a wider
to an order, being placed in the Core ecological and functional biology context.
Eudicots clade.) As plants adapted further The factors or features concerned are
to an existence on land, structural and closely interrelated and it is often difficult
morphological modifications permitted to disentangle the relative importance of
greater control of plant water status one from another in explaining seed
(homoiohydry), together with an increase responses to drying. However, from a prac-
in size and growth rate, and vegetative des- tical standpoint the ability to diagnose or
iccation tolerance was lost. A stationary predict the response from such diverse
existence was countered by the evolution information would be a valuable tool for
of dispersal structures that facilitated the seed conservation planning.
For convenience we have chosen to con- The evidence so far available indicates
sider systematic aspects separately first, and that desiccation tolerance is the rule for
then to cover ecology in the broad sense, mature seeds of the overwhelming majority
including both habitat and parent plant fea- of species. Overall, of the 7146 species
tures, as well as seed and fruit structure, listed in the Compendium (electronic ver-
including size. We believe that analyses of sion) the great majority (c. 90%) are ortho-
such associations will ultimately illuminate dox, with c. 7% recalcitrant and 2%
the evolution of the extremely valuable trait intermediate. This is in stark contrast to
of seed desiccation tolerance. the tolerance of vegetative desiccation by
adult plants: the overwhelming majority of
spermatophytes are sensitive (Oliver et al.,
8.2. Systematics and Evolution of Seed 2000), and resurrection plants are very
Desiccation Tolerance rare.
8.2.1. The complete dataset

8.2.2. Gymnosperms
Species covered in the Compendium prob-
ably represent less than 2.5% of all seed Among the gymnosperms, seed desiccation
plant species, and the issues of coverage sensitivity is just as uncommon as it is
and potential bias (e.g. under-representa- overall and as it is in angiosperms (see
tion of tropical moist forest species) in the Section 8.2.3), with the same relative pro-
dataset are real. The low level of sampling portions found among the species repre-
of terminal taxa is of particular concern in sented (87% tolerant, 6% intolerant and
light of the incidence of variation of behav- 4% limited tolerance). Of the gym-
iour within genera; for example, the fol- nosperms represented in the Compendium
lowing genera contain species with both the majority are conifers (200 species), and
orthodox and recalcitrant seeds: Acer of these 174 are recorded as orthodox.
(see also Hong and Ellis, 1990, 1992a; Though their extant members are few and
Dickie et al., 1991), Agathis, Araucaria, sampling is limited, all the cycads, ginkgos
Calophyllum, Castanopsis, Citrus, and gnetophytes so far examined have des-
Coprosma, Diospyros, Garcinia, Magnolia, iccation-tolerant (or at least not desicca-
Pittosporum, Spondias, Vitex. However, tion-sensitive) seeds. With the demise of
the Compendium is substantially larger the ‘anthophyte hypothesis’ (Bowe et al.,
than any of the earlier compilations used 2000; Chaw et al., 2000; Qiu et al., 2000), it
by other authors as the basis for review is not now so easy to suggest that the puta-
(e.g. Hofmann and Steiner, 1989; von tive sister group to the angiosperms (gneto-
Teichman and van Wyk, 1994), and is more phytes), and hence a common ancestor,
rigorous in its assignment of species to possessed only orthodox seeds. However,
storage category. For example, apparently the ancestral status of seed desiccation toler-
mainly on account of its preference for ance within the gymnosperms still has sig-
moist habitats, Caltha palustris was classi- nificant support, from the fact that
fied as having recalcitrant seeds by recalcitrant behaviour has not been observed
Hofmann and Steiner (1989); and this has in the cycads or ginkgos. These are regarded
been perpetuated in subsequent reviews by as basal in the group (e.g. Bowe et al., 2000;
von Teichman and van Wyk (1994) and by Chaw et al., 2000). Indeed, so far, desiccation
Farnsworth (2000). Yet seeds of this sensitivity appears to be restricted to two
species have been successfully stored air- derived conifer families, Podocarpaceae and
dry and frozen for a number of years in the Araucariaceae. In the Podocarpaceae only
Royal Botanic Gardens Kew (now Podocarpus usambarensis is recorded as
Millennium) Seed Bank at Wakehurst Place possibly orthodox, of ten species covered,
– its seeds are clearly orthodox and the remainder having no or limited toler-
recorded as such in the Compendium. ance of desiccation (seven recalcitrant, two
intermediate). Within the Araucariaceae, bution (South America, Australia, New

seed storage behaviour is polymorphic, Zealand, New Caledonia, New Guinea and
with orthodox, intermediate and recalci- other South Pacific Islands) (Setoguchi et
trant behaviour shown by congeners in al., 1998). In a comprehensive survey of
both Agathis and Araucaria (Tompsett and seed storage responses in this family,
Kemp, 1996). Tompsett and Kemp (1996) showed that
In the gymnosperm fossil record, while four of 14 species investigated had recalci-
well-preserved gametophytic tissue is trant seeds. All the recalcitrant species
sometimes contained in Late Palaeozoic belong to the genus Araucaria, which con-
sediments, embryos are rarely found. Post- stitutes about two-thirds of the species in
zygotic development in the earliest gym- the family. The genus can be split into
nosperms is thought to have been rapid Sections, with Eutacta being the largest. All
and continuous, making it less likely that investigated species of this Section (eight in
embryos would be fossilized (Mapes et al., total) produce seeds that are tolerant of des-
1989). Thus, the appearance in the Permo- iccation to low levels, from 15 to 2%. In
Carboniferous (c. 370–240 mya) strata of contrast, species investigated in the Sections
well-developed cotyledonary embryos in Araucaria (Araucaria araucana, Araucaria
relatively small seeds (6–7 mm long) has angustifolia), Intermedia (Araucaria hun-
been used to suggest the development of a steinii) and Bunya (Araucaria bidwillii) pro-
quiescent phase in embryo growth, i.e. the duce recalcitrant seeds. Based on the
embryos may have been dormant (Mapes et consensus tree of the 20 equally parsimo-
al., 1989). Thus, by Permo-Carboniferous nious ones for Araucariaceae, the cpDNA
times, conifer seeds may have already rbcL sequence information shows a major
evolved two functionally important traits – branching of Agathis from Araucaria after a
dormancy and, possibly, desiccation toler- split from Wollemia (Setoguchi et al., 1998).
ance. Readers should note that Mapes et al. However, there is no agreement as to
(1989) used the word ‘dormancy’, as does whether Wollemia is closer to either
Farnsworth (2000), in its broad sense, to Araucaria or Agathis (Gilmore and Hill,
denote the capability for embryonic devel- 1997; Setoguchi et al., 1998). Interestingly,
opmental arrest, no matter how long sus- Wollemia, Agathis and the non-Eutacta
tained or controlled, in contrast to Araucaria have two cotyledons, whilst the
‘vivipary’. Most seed biologists use the Eutacta Araucaria have four. All araucar-
word ‘quiescence’ to describe this capabil- ian fossil species from the Mesozoic (c.
ity, reserving ‘dormancy’ for specific physi- 245–65 mya) have dicotyledonous embryos,
cal or physiological mechanisms that delay suggesting that the common ancester of the
seed germination, even when hydration Section Eutacta had two cotyledons. As
and the typical temperature requirements Agathis, the Eutacta Araucaria and
are satisfied (e.g. Baskin and Baskin, 1998). Wollemia have desiccation-tolerant seeds,
As we discuss below, there is a general we can postulate that the increase from two
inverse relationship between dormancy to four cotyledons was not associated with
and desiccation sensitivity (an exception is the loss of desiccation tolerance.
Aesculus hippocastanum). Desiccation tolerant seeds ( 15%
moisture content) of the Section Eutacta
vary in size or mass from about 300 mg for
8.2.2.1. Araucariaceae
Araucaria cunninghamii to 1870 mg for
This family includes 41 species across Araucaria heterophylla (Tompsett and
three genera, Agathis, Araucaria and Kemp, 1996). Similarly, desiccation-tolerant
Wollemia (Farjon, 1998). Wollemia is repre- seeds of Agathis sp. are around 200 mg and
sented by one species, the Wollemi pine those of W. nobilis measure about 1.1
(Wollemia nobilis) discovered in the last 10 0.9 cm (Offord et al., 1999). In contrast,
years (see Offord et al., 1999). The family the desiccation-sensitive seeds of the
primarily has a southern hemisphere distri- Sections Araucaria, Intermedia and Bunya
are generally bigger, varying from 580 mg for seeds (Collinson, 1999; Eriksson et al.,
A. hunsteinii to c. 10,000 mg in A. bidwillii 2000). At least for angiosperms, the behav-
(Dooley, 1990). There are abundant palaeo- iour of modern species would predict that
botanical data for this family, which have such ancient seeds were orthodox, i.e. tol-
allowed an assessment to be made between erant of desiccation.
the evolution of seed size and current trends In the angiosperms, all but two of the
in the level of seed desiccation tolerance. monophyletic orders recognized by the APG
The fossil record for Section Bunya are covered in the Compendium dataset (see
seeds indicates a relatively small seed size Fig. 8.1), and all of those contain some
for Araucaria mirabilis (c. 1 0.4 cm), species with orthodox seeds. Seed desicca-
Araucaria brownii (0.8 0.3 cm) and tion sensitivity, or recalcitrance, is spread
Araucaria sphaerocarpa (1.6 0.7 cm) across all major clades, but, with the excep-
(see Setoguchi et al., 1998, and references tion of Ericales, is quite rare in the asterids.
therein). This is in stark contrast to the Examples of ‘hot spots’ for this state are
seeds of the extant species A. bidwillii at c. Malvales, Arecales and Laurales in the
5 3 cm, and it has been suggested, on the eudicots, monocots and basal angiosperms,
basis of other morphological features, that respectively, and others are indicated in
this species should be treated separately Fig. 8.1. Of the most basal ‘ANITA’ group
from Mesozoic (c. 245–65 mya) araucarians (Amborellaceae, Nymphaeales, Illicales,
assigned to the Section Bunya. Trimeniaceae and Austrobalyaceae; see
The current molecular data suggesting a Barkman et al., 2000; Qiu et al., 2000), rep-
monophyletic origin for the Sections resentation is restricted to Nymphaea spp.
Araucaria, Intermedia and Bunya agree (see below), and the other families and
with the fossil record, which suggests their orders should be targeted for study. Moving
evolution into Sections was possibly com- to the other basal orders, less than half the
plete before South America separated from species listed from the Magnoliales have
Antarctica during the Eocene at the latest. recalcitrant seeds and, of the few Piperales
Thus, it is possible that desiccation-sensi- (see also Vázquez-Yanes and Orozco-
tive seeds in these Sections of the family Segovia, 1982) examined, none has them.
may have been present at least 40–60 mya. Seeds of Ceratophyllum demersum
Molecular data also suggest that Section (Ceratophyllales), while difficult to germi-
Eutacta (containing extant species with nate, are almost certainly desiccation-
desiccation-tolerant seeds) is older than the tolerant (Hong et al., 1998b; Hay et al.,
other Sections. Although there is the need 2000; F. Hay, Ardingly, 2001, personal com-
to review Mesozoic fossils of Eutacta, our munication). Thus, the extant members of
working hypothesis is that desiccation the basal groups do not show anything like
tolerance may have been associated with exclusively, or even predominantly, recalci-
an ancestral small-seeded state in trant behaviour.
Araucariaceae. At the family level, Table 8.2 shows the
percentages of the three types of seed stor-
age behaviour occurring in a selection (44)
8.2.3. Angiosperms of the largest angiosperm families. These
are ordered by % species coverage in the
From comparative studies of DNA amounts Compendium, to give some idea of the cur-
(Leitch et al., 1998), it seems likely that rent sampling levels. In all, about half of all
angiosperms at least originated as rapid- angiosperm families are represented, and
cycling species of ephemeral habitats of these only c. 25% have members with
(Midgley and Bond, 1991), where seed des- desiccation-sensitive seeds. Mean coverage
iccation tolerance would have been a dis- of genera in families is around 18%, rang-
tinct advantage. It is also likely that the ing from 88% in the Fagaceae to less than
earliest angiosperms were early succes- 2% in the Melastomataceae. Species cover-
sional herbs or shrubs with relatively small age within family goes from a low of
0/1
Ceratophyllales
28/42
Laurales
12/42
Magnoliales
0/7
Piperales
(Acorales)
4/31
Alismatales
3/199
Asparagales
Monocots
0/18
Dioscoreales
(Pandanales)
0/22
Liliales
Angiosperms
25/97
Arecales
4/584
Poales
0/10 Commelinoids
Commelinales
1/15
Zingiberales
0/158
Ranunculales
2/81
Proteales
2/404
Caryophyllales
2/7
Santalales
0/80
Eudicots
Saxifragales
0/17
Geraniales
31/200
Malpighiales
4/10
Oxalidales
15/1133
Fabales
25/338 Eurosid I
Rosales
4/69
Cucurbitales
Rosids
69/161
Fagales
Core eudicots
22/314
Myrtales
2/569
Brassicales
97/296 Eurosid II
Malvales
75/285
Sapindales
0/30
Cornales
32/202
Ericales
0/1
Garryales
6/137
Gentianales
Asterids
5/430 Euasterid I
Lamiales
0/181
Solanales
0/8
Aquifoliales
4/107
Apiales
0/489 Euasterid II
Asterales
0/62
Dipsacales
Fig. 8.1. Seed storage behaviour at ordinal level. No data for the two orders in parentheses. All other orders
contain some species with desiccation-tolerant seeds. Numbers of desiccation-sensitive species are shown as
fractions of total no. examined – orders having at least one sensitive sp. are shown in bold. Tree derived from
several cladistic analyses by the Angiosperm Phylogeny Group – see www.rrz.uni-hamburg.de/biologie/
b_online/apg/APG.html
0.04%, again in the Melastomataceae, to a family. Families with a high incidence

high of 17% in the Brassicaceae, and a (> 10% of species examined) of recalcitrant
mean value between 2 and 3%. The overall species are comparatively rare (ten, or less
percentage occurrence of species with des- than a quarter of those listed) and widely
iccation-sensitive seeds is around 10% per scattered taxonomically (examples in Table
Table 8.2. Representation of seed storage ‘groups’ in 44 plant families in relation to the number of
genera and species for which there are data. Bold signifies families with the highest percentage
(10.9–80.2%) of recalcitrant species out of the total species investigated.
Representation Species’ seed storage behaviour

Genera Species Orthodox Intermediate Recalcitrant
Family (%) (%) (%) (%) (%)
Brassicaceae (incl. Capparaceae) 37.5 16.9 99.6 0.2 0.2

Fagaceae 87.5 8.2 17.4 1.2 80.2
Amaranthaceae
(incl. Chenopodiaceae) 20.7 7.1 100.0 0.0 0.0
Caryophyllaceae 27.6 7.1 100.0 0.0 0.0
Rosaceae 46.3 7.0 96.5 0.0 1.5
Fabaceae 34.1 6.3 98.6 0.2 1.2
Sapindaceae (incl. Aceraceae,
Hippocastanaceae) 16.8 5.8 63.1 1.2 31.0
Cucurbitaceae 18.2 5.4 95.1 0.0 4.9
Poaceae 22.6 5.3 98.6 0.6 0.8
Solanaceae 23.2 4.9 100.0 0.0 0.0
Proteaceae 23.4 4.7 90.7 0.0 2.7
Malvaceae (incl. Sterculiaceae,
Bombaceae, Tiliaceae) 24.5 4.2 91.1 1.2 5.9
Polygonaceae 26.5 4.0 93.2 0.0 2.3
Arecaceae 30.5 3.6 27.8 12.4 25.8
Ranunculaceae 29.0 3.6 100.0 0.0 0.0
Moraceae 28.9 3.6 51.2 0.0 48.8
Rutaceae 16.7 3.6 50.0 28.1 10.9
Myrtaceae 25.6 3.1 83.9 0.0 14.7
Clusiaceae 20.0 2.8 57.9 0.0 42.1
Gentianaceae 12.0 2.5 100.0 0.0 0.0
Sapotaceae 18.9 2.4 3.8 23.1 65.4
Scrophulariaceae 12.8 2.3 100.0 0.0 0.0
Apiaceae 11.2 2.2 98.7 0.0 0.0
Ericaceae 15.9 2.1 100.0 0.0 0.0
Lamiaceae 17.5 2.0 100.0 0.0 0.0
Convolvulaceae 10.9 1.8 100.0 0.0 0.0
Boraginaceae 15.4 1.8 100.0 0.0 0.0
Asteraceae 12.6 1.8 99.6 0.0 0.0
Iridaceae 11.0 1.4 100.0 0.0 0.0
Lauraceae 28.8 1.2 5.7 0.0 77.1
Celastraceae 9.1 1.2 80.0 13.3 6.7
Bromeliaceae 22.0 1.0 100.0 0.0 0.0
Cyperaceae 10.6 0.9 100.0 0.0 0.0
Apocynaceae (incl. Asclepiadaceae) 6.3 0.9 88.9 0.0 4.4
Urticaceae 8.3 0.7 100.0 0.0 0.0
Annonaceae 7.8 0.6 62.5 0.0 12.5
Euphorbiaceae 8.2 0.5 85.4 0.0 6.3
Cactaceae 3.1 0.4 100.0 0.0 0.0
Rubiaceae 4.6 0.4 83.9 5.4 7.1
Orchidaceae 5.5 0.3 63.9 25.3 0.0
Araceae (incl. Lemnaceae) 10.2 0.3 83.3 0.0 8.3
Zingiberaceae 6.3 0.3 66.7 33.3 0.0
Piperaceae 12.5 0.2 83.3 16.7 0.0
Melastomataceae 1.1 0.0 100.0 0.0 0.0
8.2 indicated by bold type). This ‘top ten’ The Fagaceae consist of eight or nine
list is quite familiar to students of seed des- genera (depending on authority) and close
iccation sensitivity – Fagaceae, Lauraceae, to 1000 species. What is particularly inter-
Sapotaceae, Moraceae, Clusiaceae, Sapin- esting about this family is that desiccation
daceae (including Aceraceae), Arecaceae tolerance is mainly delimited by genera
(= Palmae), Myrtaceae, Annonaceae and (note that this is the case in a number of
Rutaceae; and all of these also have at least other families). Species in the genera Fagus
some, and usually many, orthodox species. and Nothofagus are desiccation-tolerant,
Other ‘hotspots’ for recalcitrant seeds in whilst those in Quercus and Castanea are
smaller families, not shown in the table, desiccation-sensitive – although one species
include Anacardiaceae, Dipterocarpaceae, of Quercus (Quercus emoryi) appears to be
Meliaceae and Rhizophoraceae. All the relatively desiccation-tolerant (Hong et al.,
extant Nymphaeaceae (a basal family – see 1998b).
above) are aquatic herbs, and intuitively The last complete descriptive treatment
might be expected to have desiccation-sen- of the family dates from about 150 years
sitive seeds (see remarks on ecology ago and various infrafamilial schemes have
below), yet Nymphaea gigantea is reported been proposed, with notable character par-
to have orthodox seeds (Ewart, 1908, cited allelisms and disagreement on the origin
in the Compendium). and evolution of the cupule. Thus, it is not
Below the family level, there are also possible to be categorical about the evolu-
some recognizable patterns in the distribution of the family. None the less, there is
tion of seed storage types. Intergeneric fossil evidence from North America
variation in seed desiccation tolerance is (Buchannan locality) to suggest that the
present in c. 25% of families for which two subfamilies – Castaneoideae and
there are data (i.e. c. 12% of all families). Fagoideae – may have split no later than
By overlaying the data from the the Palaeocene (c. 58 mya), and that the
Compendium on the APG cladogram, we Fagaceae probably originated in the late
have been able to identify a number of fam- Cretaceous (c. 65 mya) (Nixon and Crepet,
ilies of particular interest with respect to 1989). Fagus (desiccation-tolerant) appears
the level or proportion of species with des- to be basal for the subfamily Fagoideae,
iccation-sensitive seeds. These include the with Quercus arising later (Crepet and
monocotyledon family Arecaceae (c. 4% of Nixon, 1989). Thus, there is the possibility
species investigated and c. 26% recalci- that the ancestral seed state in this subfam-
trant) and the dicotyledon family Fagaceae ily may well have been desiccation-
(of the 8% of species investigated, 80% are tolerant. Moreover, the palaeoclimate at
recalcitrant). this locality is generally considered to have
been warm temperate to tropical with sea-
sonal drought. This type of environment is
8.2.3.1. Fagaceae
also consistent with the modern distribu-
The family Fagaceae belongs to the order tion of many species of Castaneoideae,
Fagales, which has 43% recalcitrant species including Castanopsis (variable desicca-
(i.e. 69 out of 161 species investigated) (Fig. tion tolerance!) in Asia. The mean seed
8.1). The vast majority of these recalcitrant weight for the > 50 species with recalci-
species (53) belong to this family. The trant seeds (principally in the genera
Fagaceae are distributed across temperate Quercus and Castanea) is close to 4 g. In
parts of the northern hemisphere and South contrast, seed weight for the handful of
America, and as far east as Malesia, orthodox species (in Fagus, Castanopsis
Australia and New Zealand, and adjacent and Nothofagus) is around 0.3 g. Thus,
areas. In most parts of the range, the family orthodox Fagaceae seeds tend to be smaller
is a very prominent part of broadleaved than the recalcitrants, as was found to be
forests (Govaerts and Frodin, 1998). the case in Araucariaceae.
8.3. Seed Desiccation Tolerance and 8.3.1. Are recalcitrant seeds bigger than
Ecology sensu lato orthodox seeds?
The ability of seeds of the vast majority of Seed size varies over ten orders of magni-
extant spermatophyte species (so far exam- tude (Harper et al., 1970), with a number of
ined) to tolerate desiccation is presumably potential ecological and evolutionary
a trait of major adaptive importance to their causes, as well as phylogenetic constraints.
survival and dispersal role. Assuming that The case has been made above, for two
it is an ancient trait (see above), what eco- families, that seeds of the recalcitrant
logical trade-offs have induced certain species are generally larger than those of
species to relinquish it from time to time the related orthodox species. But is this the
during evolution? Seed desiccation sensi- case across all species so far investigated?
tivity is not a problem for those species that The answer is ‘yes’, with intermediate
have it, only for people wishing to store the seeds somewhere in between. Figure 8.2
seeds. From a practical point of view, the shows the seed mass distributions for 1080
association between seed storage behaviour species for which ‘seed’ weights are given
and plant ecology and structural character- in the Compendium (1000-seed weights
istics has long been of interest (e.g. Roberts cited). The mean seed weight for recalci-
and King, 1980) in relation to the search for trant seeds (3958 mg) is greater than that
predictors of seed storage behaviour. This for intermediates (900 mg), which is greater
issue is bound up in the complex of inter- than for orthodox seeds (329 mg). However,
acting factors defining the ‘regeneration as in all large-scale comparisons of seed
niche’ (Grubb, 1977) of particular species. mass, the degree of overlap is considerable
As such, simple associations with single (see Leishmann et al., 2000), and except at
factors will not have broad applicability, the extremes it would be a relatively poor
and this has been recognized, for example predictor of seed storage behaviour. ‘Seed’
by Hong and Ellis (1997, 1998), in propos- mass is easy to measure. However, it is a
ing the use of multiple criteria. With partic- broad term, including true seeds as well as
ular reference to Meliaceae they suggested a variety of indehiscent fruits, confounded
four – seed size, shape and moisture con- by variation in the proportion of covering
tent at maturity, together with ‘plant ecol- structures removed in postharvest clean-
ogy’. The latter is not a single criterion, but ing, leading to the use by some authors of
presumably means ‘habitat’ in this context. terms such as ‘dispersule’ and ‘germinule’
Desiccation-sensitive seeds, so-called recal- (e.g. Grime et al., 1988). It is strongly asso-
citrants, are widely held to be generally ciated with a number of seedling character-
large and ‘fleshy’, more likely to occur in istics, habitat type and dispersal mode, and
forest-tree species and more frequently in the strength of the relation between growth
the moist tropics, or in aquatic species, and form depends on location, but woody
possibly in certain taxonomic groups. The plants and climbers generally have heavier
first part of this review has confirmed that seeds than graminoids and forbs (see
seed desiccation sensitivity is more fre- Leishmann et al., 2000). Seed size is also
quent in certain plant families, but that larger in tropical floras, independent of
those families are not exclusively recalci- growth form and dispersal mode (Lord et
trant, nor is there any systematic pattern to al., 1997). Thus, everything that might be
that distribution. The occurrence of recalci- positively associated with seed desiccation
trance is much more likely to result from sensitivity is associated with seed weight,
convergence in relation to ecological condi- which in turn is broadly associated with
tions. This section of the chapter examines seed desiccation sensitivity.
the validity of such broad statements by The very earliest seeds appeared in the
looking at the available evidence in the fol- late Devonian–Mississippian era (c. 400
lowing, sometimes obviously overlapping, mya) on seed ferns and similar plants and,
categories. while there may be doubts about the types
30
Recalcitrant
n = 205
20
10
30
Intermediate
% Frequency
n = 46
20
10
30
Orthodox
n = 839
20
10
0
0.01 0.1 1 10 100 1000 10,000 100,000
Seed mass (mg) (log scale)
Fig. 8.2. Seed size distributions for storage types in the Compendium. The number of species per storage
category is shown. NB: seed mass values shown are central between the ticks.
of environment in which they evolved, 8.3.2. Are desiccation-sensitive seeds

they were relatively quite small (volume morphologically or anatomically distinct
mostly less than 10 mm3) (Tiffney, 1986), from tolerant ones?
and thus, by reference to extant species,
more likely rather than less likely to be Are recalcitrant seeds generally ‘fleshy’?
desiccation-tolerant. This argument centres to some extent on
size and degree of hydration at dispersal, and have recalcitrant embryos (see Pritchard
as well as the proliferation of certain tis- et al., 1995).
sues. Many orthodox seeds are borne in The morphology of the dispersal unit
fleshy fruits, presumably animal-dispersed, coat (testa and/or pericarp, depending on
and are at high moisture content at matu- species) may offer some resistance to dehy-
rity/fruit shedding (e.g. tomato), though the dration in some recalcitrant seeds, e.g.
seeds themselves are not especially large Dipterocarpus tuberculatus (Tompsett,
and many of them are known to be ortho- 1992) and Acer pseudoplatanus (Dickie et
dox, e.g. apples (Dickie and Bowyer, 1985). al., 1991), compared with their more ortho-
Among gymnosperms there is also at least dox relatives, such as Dipterocarpus alatus
one example, Taxus brevifolia (Walters- and Acer platanoides, respectively. Such a
Vertucci et al., 1996), where seeds sur- property may have provoked the sugges-
rounded by a fleshy false fruit (aril) are tion that recalcitrant seeds are homoiohy-
nevertheless orthodox. Hong and Ellis drous (Berjak et al., 1989), but recalcitrant
(1998) regarded moisture content at the seeds do equilibrate to ambient relative
time of dispersal as a good marker of seed humidities, even if equilibration times are
storage type, along with other factors, but sometimes longer than expected. Short term
of itself it is unlikely to be particularly avoidance of desiccation is likely to be a
informative, and Dussert et al. (2000) viable alternative to tolerance, as a survival
found that it was not correlated with the mechanism under certain conditions.
level of seed desiccation tolerance in nine
Coffea spp.
Corner (1951, 1976) introduced the term 8.3.3. Are desiccation-sensitive seeds
‘overgrown seeds’ initially to describe seeds associated with particular habitats?
of a number of leguminous species that are
relatively large, non-endospermic and have There are general associations between habi-
a poorly developed testa. The list includes tat or macroclimate and seed storage
genera such as Castanospermum and responses, at least for some species.
Pentaclethra, known to have recalcitrant Tompsett (1994) noted that the more ortho-
seeds, along with others such as Bauhinia, dox-seeded members of the Aracauriaceae
Millettia and Pithecellobium, from which and Dipterocarpaceae were found in season-
desiccation-sensitive seeds have so far not ally dry tropical woodland. Dickie et al.
been recorded (Hong et al., 1998b). Von (1992) drew a similar conclusion from a lim-
Teichman and van Wyk (1991, 1994) ited survey of seed storage behaviour in the
attempted to relate recalcitrant seed behav- Arecaceae, and Dussert et al. (2000) have
iour to large size resulting from over- examined the relation between seed desicca-
developed chalazal tissue, along with several tion sensitivity and climate in the native
other seed structural characters presumed to environments of nine Coffea species. The
be primitive (e.g. bitegmic, crassinucellate desiccation-tolerant species of Meliaceae are
ovules, presence of substantial endosperm). often located in savannah regions of Africa
However, Corner (1992) doubted that pachy- (Tompsett, 1994; Hong and Ellis, 1998).
chalazy itself is primitive. However, recalcitrant-seeded species are
There is no evidence that the gross mor- known in the tropical drylands, e.g.
phological disposition of reserve materials, Vitellaria paradoxa (Pritchard and Daws,
i.e. either endospermic or cotyledonous, is 1997). It could be that such species are his-
associated with seed storage behaviour. In torical relics of an earlier more hydric envi-
Arecaceae (Palmae), Cocos nucifera is ronment and/or that the reproductive
endospermic and recalcitrant, Washingtonia strategy of the species is finely tuned to the
sp. are endospermic and orthodox (Dickie et dynamics of the local microenvironment,
al., 1992). In Fabaceae (Leguminosae) including the availability of seed dispersers.
Pisum spp. are non-endospermic and ortho- Vázquez-Yanes and Orozco-Segovia (1984)
dox, whilst Inga spp. are non-endospermic and Vázquez-Yanes et al. (2000) have com-
piled much of the available evidence show- tolerance, e.g. in Fagaceae, Arecaceae and
ing that perhaps a majority of species, espe- Rubicaceae (e.g. Coffea sp.), opens up the
cially the dominant trees, of humid moist prospect of creating a molecular cladogram
forests have seeds that germinate rapidly to for desiccation (in)tolerance in closely
produce a carpet of dormant or very slowly related species using existing methodolo-
growing seedlings. Many of these may be gies. Some of the families identified here
recalcitrant, but a number do persist as soil appear suitable for further investigation in
seed banks (e.g. Cao et al., 2000), especially this context, and in particular the basal
pioneer and gap species (Vázquez-Yanes et groups or species in those families.
al., 2000). The current Compendium dataset The existence of variation in seed desic-
almost certainly under-represents tropical cation response among species within a
moist forest species and more data are genus has been remarked upon above (see
needed. That such vegetation will have qual- also Hong and Ellis, 1995). Of particular
itatively more recalcitrant species than most interest recently has been information on
other vegetation types is perhaps beyond the inheritance of desiccation tolerance in
dispute, but by how much more is another interspecific crosses of Coffea (Dussert et al.,
question. There are a number of different 1999). Furthermore, there is growing evi-
types of tropical moist forests and many gra- dence of the potential for variation within
dations into drier vegetation types, where species. This follows the isolation of a grow-
the proportions of different functional types ing number of viviparous and seed desicca-
of trees (e.g. pioneer and gap versus canopy tion-sensitive mutants in Arabidopsis (e.g.
and emergent) may vary considerably. Ooms et al., 1993) and several crop species
Aquatic vegetation is the other main type (maize, rice, peppers), of which the wild-
assumed to contain a high proportion of types bear orthodox, desiccation-tolerant
species with desiccation-sensitive seeds. seeds. However, there are no reports of seed
This is largely on the grounds that there is desiccation-tolerant mutants in species with
plenty of water around, seeds would not recalcitrant seeds. The work on desiccation-
need to survive drying out and there may be sensitive mutants (e.g. Ooms et al., 1993)
selection pressure for very rapid germination suggests that relatively few genes seem to be
and establishment (Farnsworth, 2000). In associated with sensitivity. Moreover, it can
fact, this is frequently not the case, and the be assumed that desiccation tolerance in all
example of Caltha palustris has been cited orthodox seeds is limited to a ‘developmen-
above. Hay et al. (2000) have shown that, of tal window’ from maturation to germination
all the aquatic and marsh species investi- (see Hong and Ellis, 1992b), more or less
gated, over 90% are orthodox, with the per- coincident with embryo ‘dormancy’ (used in
centage recalcitrant at 7%, with the the broad sense and including quiescence
remainder having limited desiccation toler- (e.g. Mapes et al., 1989; Farnsworth, 2000)).
ance, e.g. Najas flexilis (Hay and Muir, 2000). In addition, it is known that many species
These proportions are no different from those have variable desiccation tolerance between
in seed plants as a whole (see earlier). tissues of the sporophyte (seed and vegeta-
Perhaps this is less surprising when it is tive tissues) and gametophyte (pollen), indi-
remembered that the seeds of many aquatic cating that transcriptional, translational or
species are dispersed over long distances on post-translational control of existing genes
the feet of birds, for instance, and others in is most probably involved in the expression
marshes may have to survive fluctuating of desiccation tolerance. It is probable that
moisture levels and seasonal desiccation. seeds of the few angiosperm resurrection
plants capable of surviving vegetative desic-
cation in the adult phase are desiccation-
8.4. Future Directions tolerant. Published data showing whether or
not the desiccation tolerance of the sporo-
The existence of congeneric species with phyte continues unbroken from mature
distinctly different levels of desiccation embryo to adult plant have been lacking.
However, J.M. Farrant (Cape Town, 2001, help with understanding as well as diagnos-
personal communication) has found that ing and predicting the condition. Apart from
seedlings of Craterostigma wilmsii, the highlighting by Hong and Ellis (1997) of
Xerophyta viscosa and Eragrostis nindensis the possible role of moisture content at
go through a desiccation-sensitive phase maturity, little has yet been done to compile
post-germination. Interestingly, very prelim- the large but widely scattered amount of
inary results suggest that in E. nindensis at information on seed and fruit dispersal (see,
least one gene expressed in the desiccation- for example, Jordano, 2000; Stiles, 2000;
tolerant seed and adult phases is not Willson and Traveset, 2000) in the context of
expressed during the desiccation-sensitive seed desiccation tolerance. This will be a
seedling stage (J.M. Farrant, Cape Town, significant component of the Royal Botanic
2001, personal communication). Moreover, Gardens Kew’s (Millennium Seed Bank)
it appears that species vary in the level of Seed Information Database (Tweddle et al.,
desiccation sensitivity displayed during the 2002). Another profitable area to pursue
seedling window, with relative tolerance in would be the ecological trade-offs involved
C. wilmsii possibly related to high levels of in seed size, provisioning and protection and
up-regulation of protection systems in that seedling establishment in relation to desicca-
species compared with others (Farrant et al., tion sensitivity (see, for example, Garwood,
1999). 1996; Mazer, 1998; Leishmann et al., 2000).
Clearly, the identification of a set of spe- Likewise, there is evidence of increasing
cific (homologous) genes or products asso- interest in the importance of ripening phe-
ciated with the loss or acquisition of nology in relation to environment at seed
desiccation tolerance, e.g. through gene dispersal (e.g. Dussert et al., 2000; Rodríguez
micro-array technology, will permit com- et al., 2000).
parative physiology and molecular screen-
ing of diverse species to give us greater
insight into the evolution of regulatory 8.5. Conclusion
pathways for stress tolerance. Some mole-
cules of potential value, e.g. late embryoge- Seed desiccation tolerance is a complex,
nesis abundant proteins, are already under labile trait, which is at least as likely to be
extensive investigation in plant and micro- ancestral as is sensitivity in the evolution
bial systems (see Chapters 1, 5 and 10). of seed plants. In view of its frequent and
Such comparative physiological studies wide occurrence in both gymnosperms and
need to be to a common standard to permit angiosperms, especially among extant
incorporation into a taxon-based database. members of basal groups, parsimony dic-
Such information will come from many tates that it is the likely ancestral state in
sources, including the Millennium Seed seed plants. The analysis here gives con-
Bank project (Royal Botanic Gardens Kew, siderable support to a similar suggestion by
UK), which may contribute to an increase Farnsworth (2000). Applying Dollo’s law
in seed storage information for species (see Judd et al., 1999, p. 21), parallel origin
from an existing level of around 70 families seems unlikely, and it is quite conceivable
to around > 200 families by the year 2010. that this trait has been lost repeatedly, pos-
Greater understanding of the ecology of sibly through single gene loss, in response
species with desiccation-sensitive seeds will to one or more ecological trade-offs.
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Part IV
Mechanisms of Damage and Tolerance

9 Desiccation Stress and Damage
Christina Walters,1 Jill M. Farrant,2 Norman W. Pammenter3 and

Patricia Berjak3
1USDA-ARS National Seed Storage Laboratory, 1111 South Mason Street, Fort Collins,
CO 80521, USA; 2Department of Molecular and Cellular Biology, University of Cape
Town, 7700, South Africa; 3School of Life and Environmental Sciences, University of
Natal, Durban 4041, South Africa

9.2. Water Stress 264
9.2.1 Drought vs. desiccation 264
9.2.2. Exacerbating stresses 265
9.2.3. Degrees of stress 265
9.3. Desiccation Damage 269
9.3.1. Mechanical strains and structural damage 269
9.3.1.1. Cellular and subcellular scales 269
9.3.1.2. Molecular scale 273
9.3.2. Metabolically derived damage 278
9.4. Perspectives on the Kinetics of Desiccation Damage 280
9.5. Conclusion 281
9.7. References 282
9.1. Introduction organism structures when water was not

available to provide physical support and
Terrestrial plants became established in the acquiring nutrients when the lack of water
Silurian Period (459–409 million years limited the movement of both organisms
ago), a few hundred million years after the and the necessary resources. The loss of
first appearance of multicellular organisms mobility also required plants to develop
on earth (Late Precambrian Period: mechanisms to tolerate a spectrum of other
900–545 million years ago) (Strickberger, stresses associated with life on land, partic-
2000). The time required for the necessary ularly temperature extremes and high lev-
adaptions to arise attests to the harshness els of radiation. The requirements for water
of a water-limited environment. The two are so fundamental (and obvious) that most
major challenges were maintaining cell and research has focused on the strategies used
264 C. Walters et al.
to address the challenges of life in non- what are generally considered to be

aquatic environments (i.e. protective mech- ‘drought-tolerant’ organisms, which usu-
anisms) rather than the physical evidence ally resist water loss by having imperme-
of failure – collapse and starvation. able outer coverings and reducing surface
Studies of cellular responses to water area-to-volume ratios. Drought-stressed
stress mostly focus on what cells need to organisms may grow relatively slowly, per-
tolerate or resist water loss. Direct evidence haps because of the reduced turgor pres-
concerning the damaging process is sparse, sure for cell expansion, but also because of
with the mechanisms of damage often the tremendous metabolic costs of main-
made by inference from the presence of taining structures that block water loss to
putative protectants. Often it is unclear the environment (e.g. Pimienta-Barrios and
whether a change in morphology, ultra- Nobel, 1998), supporting root structures
structure or metabolism is a simple conse- that seek water, and accumulating compati-
quence of drying, a protective strategy or a ble solutes that keep osmotic potentials
sign of damage. For example, cessation of low (Jones and Gorham, 1983). The degree
metabolism is considered a component of of drought tolerance can be based on how
all three possibilities (Vertucci and much the organism resists water loss (i.e.
Leopold, 1984; Leprince et al., 1999, 2000; the minimum water potential sustained),
Salmen Espindola et al., 1994, respec- the duration that the organism sustains low
tively). Damage by desiccation is often water potentials, or the productivity
measured by an irreversible change or a (growth) of the organism during the stress.
failure of the organism to revive once water When drought-tolerance mechanisms fail,
is plentiful again. These rather crude the organism either loses water essential
assays do not detect damage that is repara- for structure or compromises metabolism
ble, though the suite of repair enzymes pro- to an unsupportable level. Either of these
duced de novo upon rehydration attests to consequences is considered a subset of the
the turnover of cellular constituents (Oliver strains associated with desiccation damage.
et al., 1998). A better understanding of the The distinction between drought and des-
nature of desiccation stress and the result- iccation tolerance lies in the protection
ing strains is required if we are to under- mechanisms – mechanisms conferring tol-
stand fully the nature of protection and erance of drought avoid water removal
repair and, ultimately, exploit millions of while mechanisms conferring tolerance of
years of evolutionary adaptation to pro- desiccation enable the organism to survive
duce plants more capable of withstanding in spite of the water loss.
the basic challenges of life in a water- In the above context, drought tolerance
limited environment. is really desiccation avoidance. Because
the mechanisms required to scavenge and
sequester water may differ from those that
9.2. Water Stress enable the organism to exist without it, tol-
erance of drought does not necessarily
9.2.1. Drought vs. desiccation imply tolerance of desiccation. None the
less, both desiccation- and drought-tolerant
Most terrestrial organisms can grow (by organisms accommodate life at low water
mitotic divisions and cell expansion) at potentials ( 1 MPa). Mild drops in
water potentials greater than about 1 MPa water potential (from 1 to about 3 MPa)
(Levitt, 1980; Vertucci and Farrant, 1995, coincide with a series of metabolic changes
and references therein). Organisms that that make cells more tolerant of the water
successfully deal with lower water poten- stress (Ingram and Bartels, 1996; Bray,
tials can either cope with limited water 1997; Oliver et al., 1998; see Chapter 11).
availability while maintaining high inter- The products of these metabolic changes
nal water concentration, or cope with (antioxidants, low-molecular-weight carbo-
water loss. The former class constitutes hydrates, late embryogenesis abundant
Desiccation Stress and Damage 265
(LEA)-like proteins, heat-shock proteins) tective pigments such as anthocyanins

are putative protectants for both drought (Smirnoff, 1993; Foyer et al., 1994;
and desiccation, even though the mecha- Sherwin and Farrant, 1998; Farrant, 2000;
nism of protection is quite different for the Vander Willigen et al., 2001), increases in
two types of stress. Future research should xanthophyll pools and conversion to the
be directed towards resolving this apparent de-epoxide forms (Smirnoff, 1993; Foyer et
contradiction. al., 1994; Kranner and Grill, 1997) and pro-
duction of free-radical-scavenging enzymes
(Foyer et al., 1994; Wise, 1995; Pammenter
9.2.2. Exacerbating stresses and Berjak, 1999).
Low temperatures tend to intensify
Water-stressed plants (w 1 MPa) are water stress. The classic case describes
predisposed to damage by other stresses. temperatures at which water freezes extra-
Free-radical production appears to be a cellularly, thereby creating a water poten-
common effect of numerous stresses tial gradient and forcing intracellular water
including drought and desiccation, ageing, to migrate out of the cell (Meryman, 1974;
freezing, pollution, temperature extremes Steponkus, 1979). Lowering the tempera-
and radiation (Elstner et al., 1988; ture requires an increase in the water con-
McKersie et al., 1988; Puntarulo et al., tent of cells to maintain a constant w
1991; Hendry, 1993; Leprince et al., 1993; (w 10 MPa). This requirement for more
Foyer et al., 1994; Wise, 1995; Bowler and water at lower temperatures is related to
Fluhr, 2000), and so it is likely that these the exothermic nature of water condensa-
stresses may be cooperative or synergistic. tion on macromolecular surfaces at low
Stressed plants are particularly susceptible water potentials (Walters, 1998).
to photo-oxidative damage (Elstner et al., Consistently, critical water contents that
1988; Foyer et al., 1994; Wise, 1995). Light lead to desiccation damage are greater at
energy, which was efficiently harvested, lower temperatures (Kovach and Bradford,
transduced and assimilated in non-water- 1992; Vertucci et al., 1995; Eira et al.,
stressed cells, may be absorbed by the pho- 1999a). Indeed, the moisture content giving
tosynthetic apparatus and dissipated as rise to changes in membrane phase behav-
reactive oxygen molecules that damage cel- iour, the most often cited consequence of
lular constituents (Bewley and Krochko, desiccation stress in model systems,
1982; Vertucci et al., 1985; Kaiser, 1987; increases with decreasing temperature
Elstner et al., 1988; McKersie et al., 1988; (Crowe et al., 1989; Crowe and Crowe,
Smirnoff, 1993; Foyer et al., 1994; Tuba et 1992; Hoekstra and Golovina, 1999;
al., 1996, 1998; Sherwin and Farrant, 1998; Hoekstra et al., 1999; Bryant et al., 2001).
Csintalan et al., 1999; Farrant, 2000;
Vander Willigen et al., 2001). Desiccation-
tolerant plants initiate many processes that 9.2.3. Degrees of stress
are considered to be protective against pho-
tochemical damage. These processes There are many ways to measure water loss
include dismantling of the photosynthetic in cells. Water content (absolute or relative)
apparatus (Gaff and Hallam, 1974; (e.g. Berjak et al., 1992; Sun et al., 1994;
Hetherington et al., 1982a,b; Öquist and Farrant, 2000), water potential and related
Strand, 1986; Gaff, 1989; Demmig-Adams functions (Roberts and Ellis, 1989; Vertucci
and Adams, 1992; Vertucci and Farrant, and Roos, 1990; Tompsett and Pritchard,
1995; Tuba et al., 1996, 1998; Sherwin and 1993; Vertucci and Farrant, 1995; Vertucci
Farrant, 1998; Farrant et al., 1999; Farrant, et al., 1995; Farrant and Walters, 1998), cell
2000), chlorophyll shading by leaf folding volume (Meryman, 1974; Steponkus, 1979;
or rolling (Dalla Vecchia et al., 1998; Murai and Yoshida, 1998a,b), intracellular
Sherwin and Farrant, 1998; Farrant et al., viscosity (Vertucci and Roos, 1990; Koster,
1999; Farrant, 2000), accumulation of pro- 1991; Williams et al., 1993; Leopold et al.,
1994; Buitink et al., 1998b; Leprince and and Bradford, 1992) while other laborato-
Hoekstra, 1998; Bryant et al., 2001) inter- ries show detrimental effects of drying at
molecular proximity (Lis et al., 1982; much higher water contents (Probert and
Steponkus et al., 1995; Wolfe and Bryant, Longley, 1989; Vertucci et al., 1995).
1999; Bryant et al., 2001) and structural Similar discrepancies are reported for cof-
water (Ladbrooke and Chapman, 1969; fee (Coffea arabica), lemon (Citrus limon),
Vertucci and Leopold, 1984, 1987; Crowe neem (Azadirachta indica) and tea
et al., 1990; Pammenter et al., 1991) all (Camellia sinensis) (e.g. Ezumah, 1986;
change with desiccation (Fig. 9.1; see Ellis et al., 1990; Chaudhury et al., 1991;
Chapter 2). Each of these parameters has Berjak et al., 1993; Hong and Ellis, 1995;
been used to define the level of water Dussert et al., 1999; Eira et al., 1999a;
stress, but it is unclear which parameter(s) Sacandé et al., 2000). Even when the recal-
causes the stress and which is merely a citrant category is undisputed, there are
correlate of the stress. The distinction is differences among species in how much
important as it reveals the nature of the water can be removed and how long a
strain and the damage. A better under- water-stressed seed can survive (Berjak et
standing of the nature of desiccation stress al., 1989, 1990; Farrant et al., 1989, 1997;
and strain will also reveal whether damage Vertucci and Farrant, 1995; Pammenter and
accrues continuously, whether it occurs Berjak, 1999). To accommodate the vari-
when the stress or strain reaches a thresh- ability in desiccation tolerances observed
old, and whether numerous different among species, the categories of seed
strains result from the removal of water behaviour were further divided to distin-
from the cell. In other words, is damage by guish highly recalcitrant, recalcitrant,
desiccation a single event, a continuous intermediate, sub-orthodox and orthodox;
event or a series of insults to the cell or and Pammenter and Berjak (1999) have
organism? suggested that, in reality, a continuum
Desiccation tolerance/sensitivity has tra- exists among seed species, based on desic-
ditionally been regarded as a qualitative cation response.
feature: cells either do or do not survive Not only are there differences among
drying. The definition of ‘dry’ varies among species in response to desiccation, but
laboratories or experiments (i.e. 90% water there are also differences for seeds of the
loss; water contents 10% (0.10 g H2O g1 same species and among tissues as a func-
dry weight or fresh weight); water contents tion of developmental stage. Studies of the
in equilibrium with 75% or maybe even acquisition and loss of desiccation toler-
15% relative humidity (RH); water con- ance during embryogenesis and germina-
tents achieved after a material has been tion have demonstrated that tolerance to
freeze-dried or held in a laminar flow hood desiccation progressively increases with
for some period of time), and most studies seed maturation (Berjak et al., 1990, 1992,
use just one drying level. The binary 1993; Finch-Savage, 1992b; Farrant et al.,
approach suggested that damage was a sin- 1993; Sun and Leopold, 1993; Sun et al.,
gle event that either happened or did not 1994; Tompsett and Pritchard, 1993;
happen. Seeds were assigned to one of two Vertucci et al., 1995; Farrant and Walters,
categories, orthodox or recalcitrant, to dis- 1998; reviewed by Vertucci and Farrant,
tinguish between those that survived or did 1995; Pammenter and Berjak, 1999; see
not survive drying (Roberts, 1973; see Chapter 5) and decreases with germination
Chapter 5). Classification of seeds of cer- (Sargent et al., 1981; Senaratna and
tain species as recalcitrant has been dis- McKersie, 1983; Leprince et al., 1990;
puted as laboratories around the world Reisdorph and Koster, 1999). Correlative
have demonstrated variable success in dry- evidence suggests that there are similar
ing them. For example, some groups report stages of tolerance in vegetative tissues of
survival of Zizania palustris at water con- desiccation-tolerant angiosperms (Farrant,
tents as low as 0.07 g H2O g1 dw (Kovach 2000; Vander Willigen et al., 2001; J.M.
Stress
Dessication 09
Strain RH (%)
Viscosity 99.99 Hydration
level Binding State
(N s m–2) MPa sites
immature mature Volume (%) WC (g g–1) –0.1
0.06 immature mature immature mature Damage
18/3/02
99.9
V
none
? 0.1 1.0 1.0 17 3.0
solution
? 0.2 0.49 0.93 7.0 2.0 –1
1:58 pm
99
? 0.4 0.33 0.84 3.5 1.0
produced
IV
syrup
Osmotic excursions
Stress proteins
capillary
? 1.5 0.22 0.82 1.8 0.5
–10
deletions
90
Page 267
III
Metabolic imbalance
hydro-
phobic
rubber
Membranes
appressed
Membrane shearing and
Demixing
transitions
and fusion
? 63 0.13 0.76 0.15 0.1 Lipid phase
50
leather
II
–100
hydrophilic
glass
? 410 0.12 0.76 0.07 0.05

10
Desiccation Stress and Damage
Protein
1
deformations?
Mechanical
I
Unprotected oxidations
structures fail
glass?
porous
charged
<410 0.12 0.76 0.05 0.01 –1000
Fig. 9.1. Scales of water stress (water potential and relative humidity (RH)), water loss (water content) and strain (volume and viscosity changes) as they relate to
conceptual models of hydration (shaded rectangles) and projected damage to cells (white box). The figure is modified from a similar figure by Wolfe and Leopold
(Leopold, 1986). Water contents as a function of water potential are described for mature pea axes (Vertucci and Leopold, 1987; Walters et al., 2001) and immature
Aesculus hippocastanum axes (Farrant and Walters, 1998). Changes in volume are calculated for immature and mature bean axes from the amount of water lost
(assuming water density is 1 g ml1) and the proportion of space occupied by cell organelles (Farrant et al., 1997) at full hydration and an assumption that 8% of the
volume of the cytoplasmic matrix at full hydration is dry matter; the effect of positive turgor pressure on cell volume is not accounted for. Measures of cytoplasmic
viscosity are taken from Leprince and Hoekstra (1998) and Buitink et al. (1998b, 2000). Conceptual models of hydration are taken from Rupley et al. (1983), Vertucci
267
(1990) and Vertucci and Farrant (1995). Different mechanisms of damage are as described in the text.
Farrant, unpublished data). Vegetative tis- w) in most embryos (Avicennia marina,
sues from resurrection and cold-tolerant and perhaps other highly recalcitrant seeds,
angiosperms require time to adapt to water- excepted (Farrant et al., 1992, 1993)) was
stress situations (Steponkus et al., 1995; found to decline to about 10 to 15 MPa
Oliver et al., 1998; Farrant et al., 1999; see (Pritchard, 1991; Finch-Savage, 1992a;
Chapter 7), presumably to induce protec- Tompsett and Pritchard, 1993; Vertucci and
tive mechanisms. It is surmised that in vege- Farrant, 1995; Farrant and Walters, 1998).
tative tissues of angiosperms there is also a Embryos that are recalcitrant cannot be
developmental programme in response to dried below this level. During the final
stress that leads to progressively greater des- stages of maturation, species defined as
iccation tolerance. The variability in critical having intermediate postharvest physiology
water contents among different species, dur- (e.g. coffee, citrus and papaya) acquire the
ing maturation or germination of embryos ability to tolerate between about 60 and
and during adjustment of vegetative tissues, 80 MPa (Dussert et al., 1999; Eira et al.,
leads to the general conclusion that toler- 1999a; Sacandé et al., 2000) (neem is con-
ance/sensitivity is a quantitative feature. sidered to be in the intermediate category
The quantitative nature of desiccation as it has diminishing longevity at lower
tolerance/sensitivity suggests two broad water potentials (see Ellis et al., 1990)).
possibilities for the mechanism(s) of desic- Truly orthodox species survive the immedi-
cation damage in organisms. Desiccation ate effects of complete water loss, but suc-
damage may occur by a single mechanism cumb more rapidly if they are dried below
with species and tissue types differing in about 190 to 250 MPa (Vertucci and
how much damage they can accrue or how Leopold, 1987; Vertucci and Roos, 1990;
much stress they can endure. Alternatively, Walters, 1998). Vegetative tissues of resur-
damage by different mechanisms may rection plants acquire the same degree of
occur at multiple levels of stress and extreme tolerance (Bewley, 1979; Gaff,
species and tissue types differ in which 1989; Oliver et al., 1998). Cells that have
stresses, or combinations of stresses, they the genetic capacity to induce tolerance
can withstand. The former possibility sug- mechanisms progress towards tolerance
gests that a desiccation-tolerant organism and, with sufficient time, complete the
requires only a simple cadre of protectants, developmental programme leading to full
while the latter situation suggests that a tolerance of desiccation (e.g. Finch-Savage,
complex suite of protectants, each with a 1992b).
different function, is required for an organ- The differing levels of desiccation sensi-
ism to be truly tolerant of dehydration. tivity among developmental stages and seed
Evidence is accumulating to suggest that categories appear to correspond to levels of
desiccation damages cells and organisms by physiological activity documented in desic-
many mechanisms and that different types cation-tolerant organisms with studies of
of damage occur at different levels of stress. metabolic activity and properties of the
When expressed in terms of water poten- aqueous medium (Vertucci and Farrant,
tial, developing embryos acquire tolerance 1995). Five hydration levels designate the
stepwise (Vertucci and Farrant, 1995; cells’ ability to support growth (Level V, 0
Farrant and Walters, 1998). During histodif- to 1.5 MPa), to photosynthesize and effect
ferentiation, embryos are damaged by water stress-related metabolism (Level IV, 1.8 to
potentials less than about 1.2 to 2 MPa 4 MPa), to respire (Level III, 5 to about
(Vertucci and Farrant, 1995, and references 12 MPa), to carry out catabolic reactions
therein). During dry matter accumulation, (Level II, about 15 to 190 MPa), and to
embryos tolerate water potentials as low as be almost in stasis (Level I, 220 MPa)
about 4 to 5 MPa ( Farrant et al., 1992, (Fig. 9.1) (Clegg, 1986; Roberts and Ellis,
1993; Farrant and Walters, 1998). Following 1989; reviewed by Vertucci and Farrant,
vascular separation, the water potential at 1995; Farrant, 2000; Vander Willigen et al.,
which damage is first measured (i.e. critical 2001). These hydration levels correspond to
other parameters that measure the extent of by a reduced cell size or lack of integrated
desiccation (Fig 9.1) (Chapters 2 and 4). In metabolism, do not in themselves imply
Hydration Level V, turgor pressure is posi- damage. They may be purely consequences
tive and water behaves as it would in a of water removal and may be completely
dilute solution. At lower hydration levels, reversible once water is added back to the
cells shrink (Meryman, 1974; Steponkus, system. Therefore, damage from desicca-
1979; Steponkus et al., 1995), the properties tion is not indicated by differences
of water change (Rupley et al., 1983; between the hydrated and dry state, but
Vertucci, 1990) and the aqueous matrix rather by the resumption of normal activity
becomes more viscous having properties of upon rehydration.
syrups (Level IV), rubbers (Level III) and The number of different stresses that
leathers and glasses (Level II) (Vertucci and can be associated with removal of water
Roos, 1990; Slade and Levine, 1991; from cells can be attributed to the multiple
Williams et al., 1993; Leopold et al., 1994; roles that water plays in supporting life.
Buitink et al., 1998b; Leprince and Water plays a structural role: at the cellular
Hoekstra, 1998). Viscosity is minimized at scale, water, fills spaces and provides tur-
the transition from Level II to I (Vertucci gor, while, at the molecular scale, water
and Roos, 1990; Buitink et al., 1998b), a provides hydrophilic and hydrophobic
moisture level that also corresponds to a associations and controls intermolecular
discrete change in the heat capacity of distances that determine the conformation
water (Rupley et al., 1983; Vertucci, 1990; of proteins, polar lipids and the partition-
Buitink et al., 1996; M.T.S. Eira, unpub- ing of molecules within organelles. With
lished data) and poorly understood charac- water present, reactive surfaces of metals
teristics of water sorption (Vertucci and or molecules are not as exposed, and this
Leopold, 1987; Vertucci and Roos, 1990; limits reactivity among molecules. Water
Vertucci et al., 1994; Buitink et al., 1998a,b; also plays a role in controlling metabolism,
Walters, 1998; Eira et al., 1999b). With the as it is a reactant and product of many
exception of Hydration Level I (Vertucci reactions. As a dilutant, water affects the
and Leopold, 1987; Eira et al., 1999b; chemical potential of other molecules,
M.T.S. Eira, L.S. Caldas and C. Walters potentially shifting the likelihood of reac-
unpublished data), the relationships tions. Water also provides the fluid matrix
between physical properties of water and that allows diffusion of substances to reac-
water potential appear similar among tive sites. Changes in water concentration
diverse cells (Fig. 9.1), perhaps with only affect viscosity of the matrix and the over-
subtle differences to distinguish desiccation- all mobility of dissolved or suspended mol-
tolerant from less tolerant materials (Koster, ecules. The drier the medium becomes, the
1991; Berjak et al., 1993; Farrant and more viscous it becomes, until it is essen-
Walters, 1998; Leprince et al., 1999). If des- tially a solid matrix, trapping molecules
iccation stress occurs when cells traverse (Slade and Levine, 1991; Williams et al.,
critical water potentials or hydration levels, 1993; Leopold et al., 1994; Buitink et al.,
then the stresses experienced by desiccat- 1998b; Wolfe and Bryant, 1999). As one
ing cells are similar among organisms, but would expect from all the roles of water,
the responses to those stresses (i.e. damage there will be a number of strains that the
versus protection) differ with desiccation tissues undergo when water is removed.
tolerance (Pammenter et al., 1991).
9.3.1. Mechanical strains and structural
damage
9.3. Desiccation Damage
9.3.1.1. Cellular and subcellular scales
As water is removed from cells, the physi-
cal and physiological properties of the cells The first sign of desiccation/drought stress
change. These changes, often characterized is the loss of turgor pressure. This occurs at
water potentials of about 1 to 2 MPa, mature embryos (Fig. 9.1), this strain of cell
coinciding with the water potential range contraction can be avoided by accumulat-
designated as ‘permanent wilting point’ for ing dry matter.
non-transpiring vegetative tissue (Levitt, Differences in the degree to which cell
1980). At lower water potentials, cells lose walls contract compared with protoplasm
water and shrink (Meryman, 1974, may cause mechanical stress and damage
Steponkus, 1979; Levitt, 1980; Steponkus to the plasmalemma or plant cells during
and Lynch, 1989; Steponkus et al., 1995). dehydration. The tight attachment of the
Osmotic adjustments, which lessen the plasmalemma to the cell wall is believed to
water potential difference between cells create tension to the membrane in shrink-
and the environment and augment the ing cells (e.g. Murai and Yoshida, 1998b),
amount of dry matter in cells, can prevent which is most profound at the cell
water loss and cell contraction at water wall–plasmalemmma attachments near the
potentials between 1 and 2.5 MPa plasmodesmata (Iljin, 1957; Bewley and
(Levitt, 1980; Jones and Gorham, 1983). Krochko, 1982). Plasmolysis, where the
Osmotic adjustments are fairly ineffective plasma membrane separates from the cell
at reducing strains when cells are exposed wall, appears to mitigate damage to whole
to lower water potentials (Wolfe and cells during severe water stress (Murai and
Bryant, 1999). In slow-freezing experi- Yoshida, 1998b), and there is some evi-
ments, believed to mimic dehydration dence to suggest that cells in desiccation-
stress, protoplasts can undergo reversible tolerant seeds are slightly plasmolysed
contraction–expansion cycles, or ‘osmotic (Perner, 1965). Observations of plasmolysis
excursions’, when slowly cooled and may be an artefact of the aqueous fixatives
warmed from > 0°C (w ≈ 0.5 MPa) to used to study dry organisms (Öpik, 1985;
temperatures of 2 to 5°C (2.5 w Platt et al., 1997; Wesley-Smith, 2001). In
6 MPa) (Meryman, 1974; Steponkus, studies using anhydrous chemical fixation
1979; Steponkus and Lynch, 1989). A (Öpik, 1985) or freeze substitution (Wesley-
60–80% reduction in cell volume occurs Smith, 2001, Wesley-Smith et al., 2001),
when the water potential of cells decreases the plasma membrane remained closely
from about 0.5 MPa to about 4.5 to appressed to the cell walls, and both the
6 MPa (4 to 5°C) (Meryman, 1974; cell wall and the plasmalemma became
Steponkus, 1979; Steponkus and Lynch, highly convoluted during desiccation of
1989). Similar contraction was calculated tolerant cells. Öpik (1985) demonstrated
for immature embryo cells in which 88% that the plasmalemma separated from the
of the cell volume was occupied by water cell wall during rehydration as a result of
(Fig. 9.1). However, cells filled with dry differential swelling or weakening of the
matter reserves (mature embryos in Fig. cell wall–plasmalemma association caused
9.1) do not contract as much as highly vac- by detergents such as dimethylsulphoxide.
uolated cells (immature embryos in Fig. The mechanical properties of the cell wall,
9.1). For a similar reduction in water including its elasticity, ability to fold and
potential to 5 MPa, the cells of fully associations with plasmodesmata, influ-
mature bean axes contract only by about ence the degree of plasma membrane dis-
18%, and complete desiccation only causes ruption consequent upon contraction or
a 24% reduction in volume in these cells expansion (Webb and Arnott, 1982; Öpik,
(Fig. 9.1). When cells that have not been 1985; Murai and Yoshida, 1998b; Vicré et
acclimatized to the water stress shrink by al., 1999).
50–80%, they burst when returned to the Cell membranes must fold or vesiculate
original water potential. This observation to accommodate the volume changes dur-
led to the concept of ‘minimum critical ing cell contractions. Conservation of mem-
volume’ (Meryman, 1974), which describes brane surface area during contraction is
the limits to which a cell can contract in a critical for successful rehydration. If the
reversible osmotic excursion. As seen for surface area of the plasmalemma is
reduced too much, the cell bursts upon water stress, surviving to water potentials
rehydration, suggesting that there is a criti- of 12 MPa or less, compared with the
cal minimum surface area, rather than a benchmark of 5 MPa described above for
critical minimum volume, to which cells protoplasts from non-acclimatized cells.
can survive (Steponkus, 1979; Steponkus None the less, drying results in some
and Lynch, 1989; Steponkus et al., 1995). degree of cell contraction, which is mostly
Protoplasts from cells that are not acclima- completed when the water potential of the
tized to the cold contract through invagina- cell is reduced to 12 MPa (Fig. 9.1). In
tions of the plasma membrane, which cells that survive water potentials of about
eventually form endocytotic vesicles that 12 MPa but not lower, both endo- and
cannot be reincorporated into the plas- exocytotic vesicles have been observed (P.
malemma upon warming (Steponkus and Berjak and N.W. Pammenter, unpublished
Lynch, 1989; Steponkus et al., 1995). The date). These observations are not reported
plasma membrane of protoplasts from cells in extremely dried embryos, perhaps
more tolerant of water stress (i.e. acclima- because of technical problems of fixation.
tized by low temperatures) contracts In severely dried cells of fully desiccation-
through exocytotic extrusions which tolerant seeds, the plasmalemma stays
remain continuous with the plasma mem- intact and closely attached to the cell wall
brane and help to conserve the membrane as this folds, suggesting that the membrane
surface area (Steponkus and Lynch, 1989; surface area remains relatively constant
Steponkus et al., 1995). High phospho- during drying even though the cell volume
lipid:sterol ratios and high amounts of is diminished (Öpik, 1985). Some mem-
diunsaturated fatty acids in the plas- brane constituents may be removed during
malemma appear to facilitate exocytotic cell contraction as evidenced by whorls of
folding in shrinking protoplasts and greater membrane close to the plasmalemma in
elasticity of the expanding membranes seed cells (Webster and Leopold, 1977;
(Steponkus and Lynch, 1989; Steponkus et Öpik, 1985; Wesley-Smith et al., 2001) and
al., 1995). Protoplasts with these properties circular membrane structures and plas-
tend to survive to lower water potentials toglobuli within chloroplasts in sections of
(Steponkus et al., 1995). leaf tissue from desiccation-tolerant
The mechanism by which membrane angiosperms (Farrant et al., 1999; Farrant
surface area is conserved in intact cells is 2000; Mundree et al., 2000). These mem-
largely unknown. There are some studies brane bodies have been proposed to pro-
of the effect of dehydration on cell volume vide additional membrane reserves upon
and membrane configuration in cells from rehydration (Webster and Leopold, 1977;
plant embryos, but these are often con- Farrant et al., 1999; Mundree et al., 2000),
founded by problems associated with using although mechanisms by which they
aqueous fixatives (Platt et al., 1997; would be reinserted are not clear and their
Wesley-Smith et al., 2001). In addition, the very presence may be artefacts of aqueous
studies often use mature embryos (recalci- fixation. Alternatively, these membrane
trant or orthodox) where > 50% of the cell abnormalities may arise from other
volume is occupied by dry matter (e.g. organelles, such as endoplasmic reticula,
Farrant et al., 1997). These cells will not and may participate in autophagy or vac-
experience the same degree of shrinkage as uole formation (Wesley-Smith et al., 2001).
highly vacuolated cells (Fig. 9.1), and so The shapes of nuclei, mitochondria and
the need for conserving membrane surface plastids in dried cells of desiccation-toler-
area is not as critical. Circumventing the ant seeds are irregular and convoluted, sug-
problem of cell shrinkage may explain why gesting that the surface area of the
most orthodox and recalcitrant embryos membranes of these organelles are also
(except for A. marina and other recalcitrant conserved simply by folding (Öpik, 1985).
seeds with highly vacuolated cells (Farrant The membranes of cell vacuoles are
et al., 1992, 1993)) are fairly tolerant of likely to experience tensions similar to
those described for protoplast membranes mature orthodox seeds lack defined cristae
during osmotic excursions, and so are (Bergtrom et al., 1982; Thomson and Platt-
prone to rupture, with lethal consequences, Aloia, 1984; Farrant et al., 1997) and mito-
following exposure to water potentials of chondrial proteins are easily extractable
2.5 to 5 MPa (Murai and Yoshida, from dried pollen (Hoekstra and van
1998a). Highly vacuolated cells of imma- Roekel, 1983). Conversely, mitochondria
ture seeds (Berjak et al., 1984, 1994; from immature embryos and recalcitrant
Farrant et al., 1989, 1997) and desiccation- seeds are more defined, and the greater dif-
sensitive vegetative tissue (Farrant and ferentiation has been linked to greater sen-
Sherwin, 1997; Farrant, 2000) are particu- sitivity to desiccation (Farrant et al., 1997).
larly sensitive to tonoplast dissolution. Chloroplast structure also degrades during
Replacing the water in vacuoles with solid water stress. Dried leaves of the desicca-
material reduces the degree to which vac- tion-tolerant grasses Borya nitida and
uoles must contract, thereby lessening the Xerophyta humilis become yellow, concur-
tension on tonoplast membranes during rent with the loss of grana stacks in the
drying. Dry matter reserves naturally accu- chloroplasts (Gaff and Hallam, 1974;
mulate during embryogenesis in orthodox Farrant, 2000). Using fluorescence-induc-
and some recalcitrant seeds, and may tion kinetics to study partial processes of
explain the progressive tolerance to low photosynthesis, researchers found a
water contents in developing embryos decrease in the efficiency of photosystem II
(Vertucci and Farrant, 1995; Farrant et al., at water potentials between 3 and 4
1997; Farrant and Walters, 1998). There is MPa (Wiltens et al., 1978; Hetherington, et
also accumulation of dry matter in vac- al., 1982b; Vertucci et al., 1985; Sherwin
uoles of vegetative tissues in many of the and Farrant, 1998; Tuba et al., 1998;
desiccation-tolerant angiosperm species Csintalan et al., 1999) or during acclimati-
during acclimatization to water stress zation to winter (Öquist and Strand, 1986).
(Farrant, 2000). This decline could be a consequence of
Water loss results in a general contrac- photochemical damage, but is more likely
tion of cell volume. The plasmalemmae of to be a reflection of protective dismantling
plant cells can be damaged if they are of photosystem II (Demmig-Adams and
sheared from cell walls, which contract less Adams, 1992; Farrant, 2000). Indeed, the
than protoplasm, or if contraction results dismantling of the photosynthetic appara-
in an irreversible loss of membrane surface tus during drying of B. nitida and X.
area. In addition to protection by filling humilis is required for survival: plants
cells with dry matter (described above), the dried too rapidly stay green and do not
consequences of volume changes can also recover (Gaff and Hallam, 1974; Farrant et
be lessened by initial high surface area-to- al., 1999).
volume ratios of cells and vacuoles (Iljin, Slight water stress (1 w 3 MPa)
1957; Bewley, 1979) and may explain why enhances the protein synthesis that is
cells from non-vascular plants, which usu- believed to be important for conferring tol-
ally have small vacuoles and lack plasmo- erance (Ried and Walker-Simmons, 1993;
desmata, do not appear to suffer physical Vertucci and Farrant, 1995; Ingram and
damage upon contraction (reviewed by Bartels, 1996; Oliver et al., 1998; Mundree
Bewley and Krochko, 1982). Damaging et al., 2000; Whittaker et al., 2001). Further
effects of cell contraction are usually mani- drying reduces the rate of protein synthesis
fested during rehydration, suggesting that in both tolerant and sensitive cells (Bewley
the stress and damage are not direct effects and Krochko, 1982; Salmen Espindola et
of desiccation, but rather indications of al., 1994; Ingram and Bartels, 1996; Oliver
rehydration stress and mechanical failure. et al., 1998; Mundree et al., 2000;
A dismantling of mitochondria and Whittaker et al., 2001), perhaps because of
chloroplasts is associated with severe a dismantling of endoplasmic reticulum,
water stress. Mitochondria observed in dictyosomes and polysomes (Webster and
Leopold, 1977; Thomson and Platt-Aloia, occur. A consequence of these molecular

1984; Farrant et al., 1997; Wesley-Smith et aggregations is an increased ordering of
al., 2001). molecular structures, and it may seem
Indirect evidence from recalcitrant ironic that primary lesions during drying
seeds suggests that, during dehydration, are directly attributed to order rather than
the cytoskeleton is disrupted at fairly high to loss of it. Drying-induced compaction of
water potentials (3.8 MPa for Trichilia molecules requires greater packing effi-
dregeana and 3.5 MPa for Quercus robur) ciency, resulting in localized enrichments
leading to an abnormal distribution of of similar-type molecules in a process
organelles within cells (Berjak et al., 1999; known as demixing (Lis et al., 1982; Bryant
Mycock et al., 2000). Although it is tacitly and Wolfe, 1989; Rand and Parsegian,
assumed that cytoskeletal disassembly 1989; Bryant et al., 1992). Molecules remix
must occur during dehydration in desicca- upon rehydration, but the reactions that
tion-tolerant seeds (and vegetative tissues), occurred in the desiccated state may have
it is its failure to reconstitute that charac- irreversible consequences.
terizes this aspect of dehydration-related Intermolecular associations of polar
injury in recalcitrant material (Mycock et lipids are intrinsically linked to the water
al., 2000). content of the medium. Under aqueous
There is clearly a general trend towards conditions, polar lipids spontaneously
contraction or disassembly of cellular align to form micelles or bilayer structures
machinery during water stress to about 5 depending on the polar head group of the
MPa. In most desiccation experiments, lipid. Acyl chains within bilayers are
plant materials are stressed further and cell more-or-less mobile, giving considerable
survival is assayed by whether or not fluidity to the structure and allowing pro-
organelles reassemble upon rehydration. In teins and other constituents to be inserted.
desiccation-sensitive cells that do not rup- Drying brings membrane bilayers into close
ture, the protein-synthesizing machinery proximity and causes membrane con-
does not recover, nor do mitochondria and stituents to segregate laterally into different
chloroplasts resume normal function; domains enriched with particular lipid
organelles become irregularly shaped and classes or proteins (Lis et al., 1982; Bryant
disorganized (reviewed by Bewley and and Wolfe, 1989; Rand and Parsegian,
Krochko, 1982; Farrant et al., 1989; Berjak 1989; Bryant et al., 1992; Crowe and
et al., 1990; Mycock et al., 2000). The con- Crowe, 1992; Steponkus et al., 1995;
traction and dismantling of organelles Hoekstra and Golovina, 1999) (Fig. 9.2).
described above are clearly signs of water The closer packing between membranes
stress, but it is unclear whether these and among membrane constituents results
changes are symptoms of damage occurring in greater rigidity of the fatty acid domain
at 5 MPa, or means of protection when within the bilayer. There are two mecha-
water stress intensifies. It is also unclear nisms, based on either intra- or interlamel-
whether the failure to reconstitute lar events, used to explain why fatty acid
organelles indicates a primary site of dam- domains become more rigid. If water mole-
age or a general debilitation when cells die. cules are removed from between adjacent
These cause and effect arguments have led polar head groups, the associated fatty acids
researchers to study the primary effects of compress because of the increased strength
dehydration on the structure of macromol- of van der Waals attractions (Crowe et al.,
ecules. 1990; Crowe and Crowe, 1992; Hoekstra
and Golovina, 1999). Alternatively, as dif-
ferent bilayers come into close apposition,
9.3.1.2. Molecular scale
strong repulsive hydration forces keep
Removing water from cells pushes cellular them separate, but create isotropic tensions
constituents together, causing them to that lead to lateral compression within the
interact in ways that might not otherwise acyl domain (Lis et al., 1982; Wolfe, 1987;
Rand and Parsegian, 1989; Bryant and branes, but has been demonstrated in cells
Wolfe, 1992; Wolfe and Bryant, 1999). from non-acclimatized leaves that were
Increased rigidity eventually leads to phase lethally cooled to 5°C (oat, w ≈ 6 MPa)
transitions within the membrane from a or 10°C (rye, w ≈ 12 MPa) (Steponkus et
fluid to a gel state (Ladbrooke and al., 1995) and more frequently in animal
Chapman, 1969; Cullis and de Kruijff, cells (Cullis and de Kruijff, 1979; Crowe and
1979). While these phase transitions are Crowe, 1992). Evidence of cell fusion, but
completely reversible, they are believed to not via hexagonal-phase changes, is common
interfere with the semi-permeable proper- in desiccation-damaged cells, protoplasts
ties of membranes. Permanent damage and liposomes (e.g. Crowe et al., 1986;
comes from the exclusion of proteins from Steponkus et al., 1995). In oat and rye leaves
parts of the bilayer (Rand and Parsegian, acclimatized to cold (but clearly not fully
1989; Bryant and Wolfe, 1992; Crowe and desiccation-tolerant), fusion of plasmalemma
Crowe, 1992; Hoekstra and Golovina, 1999) and endomembrane systems is suggested at
(Fig. 9.2). Transient damage occurs upon temperatures between 10 and 40°C (12
rehydration: the rush of water on to an w 48 MPa), depending on the level of
inelastic membrane may cause it to rupture cold tolerance achieved (Steponkus et al.,
(Murphy and Noland, 1982; Steponkus et 1995). Upon rehydration, improperly fused
al., 1995; Hoekstra et al., 1999) or imper- membranes produce vesicles that exclude
fect packing among different domains may cell constituents or are combinations of dif-
cause leakage of cellular constituents ferent membrane systems (e.g. the plas-
(Crowe and Crowe, 1992; Hoekstra et al., malemma fuses with chloroplast outer
1999). membrane or with endoplasmic reticulum)
The close approach of membrane systems (Fig. 9.2). Because the osmotic balance
and the lateral demixing of membrane com- inside and outside the cells has been com-
ponents can lead to an even greater threat to pletely disrupted, vesicles produced from
membranes than lamellar fluid-to-gel transi- membrane fusions are identified by their
tions. Membranes can fuse together, causing inability to expand during rehydration
the complete loss of compartmentation (Steponkus et al., 1995).
within the cell (Crowe et al., 1986; Crowe Most of our understanding of how polar
and Crowe, 1992; Steponkus et al., 1995) lipids behave in water-stressed situations
(Fig. 9.2). Fusion is known to occur among comes from model studies of liposomes
liposomes and native membrane fractions, with known composition. In these systems,
although the mechanism that causes polar phase transitions are usually studied, even
lipids to cross over to a different bilayer is though they may only be harbingers of real
unclear. In principle, the hydration charac- damage. Phase transitions of prepared
teristics of individual lipids and lipids in a membrane systems occur at a range of
mixture, the intrinsic curvature of different water contents and temperatures depend-
head groups, the water content and the tem- ing on the saturation of the acyl chains and
perature allow the formation of inverted the presence of non-phospholipids (e.g.
micelles within closely appressed bilayers Ladbrooke and Chapman, 1969; Cullis and
(Cullis and de Kruijff, 1979; Crowe et al., de Kruijff, 1979; Crowe et al., 1989;
1986; Steponkus et al., 1995) (Fig. 9.2). In Steponkus et al., 1995). A water potential
domains enriched with non-bilayer-forming of about 12 MPa is often cited as critical.
lipids such as phosphatidylethanolamine- It has been suggested that structural water
diglycerides or monogalactosyl-diglycerides, needed for the proper spacing of polar
the polar head groups coalesce into rings and head groups is removed at w 12 MPa
the acyl chains extend radially outwards in (Ladbrooke and Chapman, 1969; Crowe et
what is known as a hexagonal phase (Cullis al., 1990). Also, at w ≈ 12 MPa, large,
and de Kruijff, 1979; Siegel et al., 1994; potentially deforming hydration forces
Steponkus et al., 1995). Fusion via hexago- result from the close approach of mole-
nal-phase changes is rare in native mem- cules (Wolfe, 1987).
Plasma membrane
Hydrated
membrane
systems Cytoplasm
Endomembrane
Exclusion of
–H2O intrinsic proteins
Demixing
Plasma
membrane
Dehydrated Membrane
membranes appression
Endomembrane
Plasma membrane
Non-bilayer phase formation

Dehydrated and membrane fusion
membranes
Endomembrane
+H2O
Plasma membrane
Cytoplasm
Rehydrated
membranes
Leakage of cell Endomembrane

contents
Fig. 9.2. Schematic drawing of the effect of dehydration on cellular membranes. Different membrane
systems may become closely appressed, leading to demixing of lipids and proteins and the loss of proteins
from parts of the bilayer. Closely appressed membranes may then form non-bilayer structures that lead to
fusion between different membrane systems. Upon rehydration, cellular contents leak out and fused
membrane particles do not swell (i.e. they are ‘osmotically unresponsive’). (Adapted from Steponkus et al.
(1993), with permission.)
There is little information for comparing loss between molecular surfaces, relieving
membrane phase behaviour among ortho- the size of hydration forces (Wolfe and
dox and recalcitrant embryos, maturing Bryant, 1999; Koster et al., 2000; Bryant et
embryos as they become more tolerant of al., 2001). As dehydration proceeds, the
desiccation, or leaves from desiccation-tol- concentration within the interfaces
erant angiosperms as they adjust to low increases, with a concomitant increase in
water potentials. Changes in bilayer spac- viscosity (Fig. 9.2). The high viscosity of
ings or lamellar fluid-to-gel transitions these interfacial solutions provides
have been detected in both desiccation- mechanical resistance to the further com-
tolerant and sensitive plant cells during pression of macromolecules (Wolfe and
dehydration, with little difference in Bryant, 1999; Koster et al., 2000; Bryant et
behaviour detected with degree of toler- al., 2001). In both protective models, the
ance (McKersie and Stinson, 1980; goal is to keep molecules separated so that
Seewaldt et al., 1981; Priestley and de harmful interactions are prevented. Sugars
Kruijff, 1982; Singh et al., 1984; Kerhoas et accomplish this capably in model mem-
al., 1987; Crowe et al., 1989; Hoekstra et brane systems (Crowe et al., 1986, 1989,
al., 1991, 1992; Sun et al., 1994; Hoekstra 1990; Crowe and Crowe, 1992; Wolfe and
and Golovina, 1999). In tolerant soybean Bryant, 1999; Koster et al., 2000; Bryant et
cotyledons, a gel-like transition occurred al., 2001). However, the presence of ade-
when seeds were dried to less than 0.2 g quate quantities of sugars in cells and the
H2O g1 dry mass (Seewaldt et al., 1981), a vitrification of cellular constituents do not
water content that corresponds to a water appear to prevent polar lipid phase
potential of about 12 MPa (e.g. Vertucci changes in desiccation-tolerant cells
and Roos, 1990) (Fig. 9.1). Water potentials (Seewaldt et al., 1981; Priestley and de
between 10 and 15 MPa also mark the Kruijff, 1982; Crowe et al., 1989; Hoekstra
survival limit for recalcitrant seeds et al., 1989, 1992, 1999; Leopold et al.,
(described above). A membrane-mediated 1994) or damage in desiccation-sensitive
mechanism is often invoked to explain cells (Berjak et al., 1992, 1993; Sun and
damage in desiccation-sensitive embryos Leopold, 1993; Still et al., 1994; Sun et al.,
and pollen because the membrane integrity 1994; Vertucci and Farrant, 1995; Vertucci
of these cells appears to be compromised et al., 1995; Farrant and Walters, 1998;
upon rehydration (McKersie and Stinson, Wolkers et al., 1998a; Hoekstra and
1980; Senaratna and McKersie, 1983; Golovina, 1999; see Chapter 10). Changing
Vertucci and Leopold, 1987; Berjak et al., the composition of membranes (reviewed
1992, 1993; Poulsen and Eriksen, 1992; by Steponkus et al., 1995) and reducing
Sun and Leopold, 1993; Sun et al., 1994; their surface area by dismantling
Wolkers et al., 1998a). endomembrane systems (described above)
The different views of dehydration may be the important tools for maintaining
stress (i.e. removal of structural water ver- compartmentation in drying cells.
sus enhancement of hydration forces) have Structural changes of proteins with
promoted different ideas for the mecha- hydration have received wide attention in
nisms of protection. According to the the literature. Early work using a variety of
‘Water Replacement Hypothesis’, if struc- proteins showed that protein structure was
tural water is removed, small hydrophilic conserved during drying to extremely low
molecules such as sugars must be inserted levels (Schneider and Schneider, 1972;
between polar lipid head groups to main- Kuntz and Kauzmann, 1974; Ruegg and
tain proper intermolecular spacings and Hani, 1975; Ruegg et al., 1975; Fujita and
membrane integrity (Clegg, 1986; Crowe et Noda, 1978; Careri et al., 1980; Takahashi
al., 1990; Crowe and Crowe, 1992). An et al., 1980; Jaenicke, 1981; Rupley et al.,
alternative, but not mutually exclusive, 1983). In parallel studies, it was demon-
model suggests that high concentrations of strated that some proteins even maintained
compatible solutes can help resist water functional activity (albeit at low levels)
when dry (Acker, 1969; Potthast, 1978; al., 1987, 1990; Franks et al., 1991;
Labuza, 1980; Rupley et al., 1983). Prestrelski et al., 1993). Enzymes such as
Secondary structure of cytoplasmic pro- lactate dehydrogenase and polypeptides
teins (extracted from desiccation-tolerant such as poly-L-lysine are particularly labile
pollen) was conserved upon drying in the (Prestrelski et al., 1993), and damage is
absence of protectant sugars, demonstrat- exacerbated if molecules are freeze-dried
ing innate stability perhaps because of the rather than air-dried (Franks et al., 1991).
high degree of -helical structures (Wolkers Rate of drying also has a large effect on the
and Hoekstra, 1995). The reversibility of conservation of protein structure, with
sorption–desorption isotherms of numer- greater preservation achieved by rapid dry-
ous proteins supported the idea that con- ing conditions (Wolkers et al., 1998a,b).
formational changes of proteins during Often, desiccation-labile proteins are used
hydration were slight and reversible, mak- to study the effects of protectants
ing proteins an ideal model for studying (Carpenter et al., 1987, 1990; Prestrelski et
hydration properties of biological materials al., 1993). Clearly, these studies are essen-
(Bull, 1944; D’Arcy and Watt, 1970). Slight, tial to the pharmaceutical industry, but
reversible changes in protein structure, similar mechanisms of protection must not
particularly secondary structure, have been be presumed to apply in vivo in dehydrat-
attributed to volumetric changes from the ing plants. A tremendous amount of work
loss of water rather than to changes in the has demonstrated that proteins are rather
native structure of proteins. These changes robust; thus, a need for protection must be
occur at fairly low moisture levels demonstrated before a protective mecha-
(between 0.2 and 0.1 g H2O g1 dry mass or nism is implied. Studies must show that
70 to 200 MPa) (Ruegg and Hani, 1975; desiccation-labile enzymes exist in vivo,
Griebenow and Klibanov, 1995). Drying, in that they are not produced de novo during
fact, stabilizes protein structures, making rehydration and that they are irreversibly
them particularly resistant to ageing damaged in desiccation-sensitive cells.
(Franks et al., 1991; Costantino et al., 1998) The structure and activity of proteins are
and heat denaturation (e.g. Echigo et al., compromised if they are stored under
1966; Ruegg et al., 1975; Fujita and Noda, extremely dry conditions of approximately
1978; Takahashi et al., 1980; Jaenicke, 0.1 g H2O g1 dry matter or about 200
1981; Leopold and Vertucci, 1986; Wolkers MPa or less (Kuntz and Kauzmann, 1974;
and Hoekstra, 1997). The extreme stability Luscher-Mattli and Ruegg, 1982; Sanches et
of protein structure with low hydration al., 1986; Labrude et al., 1987). Substantial
may be attributed to stronger intramolecu- deterioration of the lattice of protein crystals
lar associations compared with the situa- was attributed to the refolding of polypep-
tion of polar lipids. Such interactions tide chains to increase packing efficiency
would reduce the need for hydrogen bond- (Kuntz and Kauzmann, 1974; Luscher-Mattli
ing with water to maintain structural and Ruegg, 1982). Other studies have shown
integrity (obviating the need for water that severe drying exposes haem groups on
replacement by sugars as suggested by proteins, promoting free radical production
Crowe and co-workers (e.g. Crowe and (Sanches et al., 1986; Labrude et al., 1987).
Crowe, 1992)) and/or provide mechanical At such low water contents, proton
strength that resists deformation when exchanges among charged amino acids
molecules are compressed (obviating the could be measured, suggesting that these
need for mechanical barriers to compres- sites were exposed (Careri et al., 1980;
sion as suggested by Wolfe and co-workers Rupley et al., 1983). Deterioration at similar
(e.g. Wolfe and Bryant, 1999)). water potentials and in similar time frames
The conformations of some proteins and is observed in stored seeds and pollen (e.g.
polypeptides are irreversibly damaged by Vertucci and Leopold, 1987; Vertucci and
drying or freeze-drying in the absence of Roos, 1990; Buitink et al., 1996). Although
protectants (Hanafusa, 1969; Carpenter et these organisms survive the initial stress of
complete water removal, they age progres- material (Pammenter and Berjak, 1999) (see
sively more rapidly when stored at also Chapter 12).
w 220 MPa (< 20% RH). Perhaps mech- When unprotected cells are dried,
anisms suggested to cause damage in pro- organelles and macromolecules experience
teins at low water contents (e.g. exposure of mechanical or structural damage. This type
reactive sites on the proteins, increased of desiccation damage is termed sensu
relaxation of molecular structures as they stricto because the primary stress is water
fill voids left by water, or relaxation of the removal (Pammenter and Berjak, 1999;
glassy matrix that embeds the proteins) are Walters et al., 2001). Membrane structures
responsible for the deterioration of stored appear more prone to desiccation damage
seeds and pollen. Protein structure is stable sensu stricto than do proteins or DNA, per-
in seeds stored at about 30% RH (Golovina haps because of the intense hydrogen
et al. 1997), but stability of protein struc- bonding within proteins and nucleic acid
tures in seeds stored at lower humidities structures. Protection from damage often
has not been documented. Increased ageing lies in the ability of the structure or the
rates of seeds and pollen stored below a crit- surrounding medium to offer mechanical
ical water content have also been attributed resistance to the stress or accommodate the
to reduced viscosity of the aqueous medium stress through enhanced elasticity.
in cells that are almost completely dry
(Buitink et al., 1998b).
Upon dehydration, the same destabiliz- 9.3.2. Metabolically derived damage
ing forces that perturb lipid and same pro-
tein structures may also affect nucleic acid Loss of turgor precipitates a number of
structure (Rau et al., 1984). DNA is a partic- changes in metabolic pathways of plant
ularly stable molecule (Wayne et al., 1999) cells. Assimilation of CO2 (if the tissue is
which maintains its structure in the absence photosynthetic) and growth are impaired.
of water and reversibly unfolds at high tem- Often protein synthesis is temporarily
peratures (Bonner and Klibanov, 2000). The stimulated during mild water stress
intermolecular distances of dehydrating (reviewed by Farrant et al., 1989; Ingram
DNA strands are comparable to those of and Bartels, 1996; Oliver et al., 1998), with
condensed DNA in hydrated nuclei (Rau et a switch in metabolism believed to lead to
al., 1984), suggesting that DNA structures the production of putative protection
are resistant to perturbations resulting from mechanisms (reviewed by Vertucci and
dense packing. When DNA is replicated and Farrant, 1995; Ingram and Bartels, 1996;
so is decondensed during germination, the Oliver et al., 1998; Chapters 1, 5 and 11).
cells concomitantly become susceptible to Observations of increased polysomes and
desiccation injury (Deltour and Jacqmard, rough endoplasmic reticulum in slightly
1974; Crèvecoeur et al., 1988) and rapidly water-stressed recalcitrant embryos suggest
dividing cells during embryogenesis also that certain (possibly similar) metabolic
appear to be sensitive to desiccation (Myers pathways may also be induced in seeds
et al., 1992). Desiccation did not affect the that do not acquire full tolerance of desic-
structure of condensed or decondensed cation (Berjak et al., 1984; Farrant et al.,
chromatin in desiccation-tolerant or sensi- 1989; Pammenter et al., 1998). These
tive maize embryos, respectively (Leprince changes in metabolism do not indicate that
et al., 1995a). However, in those studies, the cells have already experienced damage;
chelation of Ca2+ (and other divalent when briefly stressed, most organisms
cations) by the ethylenediamine tetra-acetic resume normal metabolism once the water
acid (EDTA) present in the medium used for stress is relieved. However, prolonged mild
chromatin spreading, may have relaxed pre- stress (which could be considered akin to
viously condensed chromatin, possibly drought) is deleterious to both vegetative
accounting for the reportedly similar results and embryonic tissues. Many recalcitrant
from desiccation-tolerant and sensitive seeds lose viability if maintained for long
periods at constant high water contents A by-product of continued respiration

(e.g. Chin and Roberts, 1980; Berjak et al., and light harvesting when other metabolic
1989; Pammenter et al., 1994; Walters et processes are shut off is the accumulation
al., 2001), and similar damage is observed of high-energy intermediates that leak out
in orthodox seeds (Walters et al., 2001). of mitochondria and plastids and form
The loss of viability has been associated reactive oxygen species (ROS) and free rad-
with the continuation of metabolism icals (Puntarulo et al., 1991; Dean et al.,
(including cell division) (Farrant et al., 1993; Hendry, 1993; Leprince et al., 1993,
1989), which will ultimately lead to a 1994, 1995b; Smirnoff, 1993; Foyer et al.,
greater demand for water to maintain high 1994; Halliwell and Gutteridge, 1999).
water potentials (Berjak et al., 1989; Reactive oxygen species and free radicals
Pammenter et al., 1994). react with proteins, lipids and nucleic
Metabolism slows at water potentials acids, causing permanent damage to
less than about 2 MPa, but not all reac- enzymes (Wolff et al., 1986; Dean et al.,
tions are affected by dehydration in the 1993; Halliwell and Gutteridge, 1999),
same way. Protein synthesis slows down at membranes (Senaratna and McKersie,
relatively high water potentials (reviewed 1983, 1986; Chan, 1987; McKersie et al.,
by Bewley and Krochko, 1982; Clegg, 1986; 1988, 1989; Finch-Savage et al., 1996;
Salmen Espindola et al., 1994; Ingram and Halliwell and Gutteridge, 1999; Leprince et
Bartels, 1996; Mundree et al., 2000; al., 2000) and chromosomes (Dizdaroglu,
Whittaker et al., 2001), while respiration 1994). Peroxidation of lipids decreases the
continues to much lower levels (Vertucci fluidity within membranes (McKersie et
and Leopold, 1984; Vertucci and Roos, al., 1988, 1989), interfering with their
1990; Salmen Espindola et al., 1994; selective permeability upon rehydration (as
Leprince and Hoekstra, 1998; Leprince et described above). Upon dehydration, high
al., 1999; Farrant, 2000; Walters et al., levels of free radicals have been detected in
2001). Various reactions within photosyn- desiccation-sensitive embryos (Senaratna
thetic (Wiltens et al., 1978; Hetherington et and McKersie, 1983, 1986; McKersie et al.,
al., 1982b; Vertucci et al., 1985; Vertucci 1988; Hendry et al., 1992; Leprince et al.,
and Leopold, 1986; Farrant, 2000) and res- 1993, 1994, 1995b, 1999, 2000; reviewed
piratory (Vertucci and Leopold, 1986; by Vertucci and Farrant, 1995; Pammenter
Leprince and Hoekstra, 1998; Leprince et and Berjak, 1999). The origin and sequence
al., 2000) pathways respond differently to of events following the appearance of these
low water contents. The differing responses toxic compounds are still unclear. They
to water stress among and within metabolic may be produced by the water-stressed cell
pathways can lead to imbalances in metab- (Leprince et al., 1994, 1995b, 1999, 2000;
olism. Metabolic imbalances may be con- Leprince and Hoekstra, 1998) or as a result
founded by the respiration of fungi that of the associated fungi (Goodman, 1994;
occurs at water potentials as low as 20 Finch-Savage, 1999), and they may precede
MPa in orthodox and recalcitrant seed tis- (or precipitate) damage (Finch-Savage et
sues (Mycock and Berjak, 1990; Goodman, al., 1996; Leprince et al., 2000) or arise
1994; Calistru et al., 2000). Damage by after the cell has already died (Finch-
metabolic stress is most pronounced in Savage, 1999).
cells at water potentials between 2 and There are several ways that cells can
5 MPa with a diminishing effect as cells protect themselves from metabolic imbal-
are dried to 12 MPa (Leprince et al., 2000; ance and ROS-mediated damage. At higher
Walters et al., 2001). Both desiccation-sen- moisture levels, free-radical-scavenging
sitive and -tolerant organisms are damaged enzymes efficiently detoxify ROS (Bewley,
when stored at intermediate water poten- 1979; Dhindsa, 1987; Hendry, 1993;
tials, though the time-dependency of the Smirnoff, 1993; Foyer et al., 1994; Kranner
damage varies considerably among species and Grill, 1997; Sherwin and Farrant, 1998;
and tissues (Walters et al., 2001). Pammenter and Berjak, 1999; Farrant,
2000). These enzymes appear ineffective at changes of cellular constituents, may lead
low water contents, and tocopherol and to metabolically derived damage and vice
ascorbic acid may be more effective versa. Most model studies suggest that
(reviewed by McKersie et al., 1988; changes in molecular conformations with
Pammenter and Berjak, 1999). dehydration are reversible. Dehydration
Amphipathic molecules such as tocopherol slows chemical reactions and so organisms
can partition between aqueous and lipid that are dried sufficiently rapidly should
domains according to the water content of experience few, if any, changes in the
the cell and polarity of the molecule chemistry of their cells. Thus, it seems
(Golovina et al., 1998). A controlled shut- likely that the primary lesions resulting
down of metabolism upon drying may also from water stress, whether they are physi-
mitigate the consequences of unbalanced cal or chemical, are minor. It also appears
metabolism (as reviewed by Leprince et al., that the primary lesions are not exclusive
1993; Vertucci and Farrant, 1995; Hand to desiccation-sensitive cells. Gel-phase
and Hardewig, 1996; Pammenter and lipid transitions occur in both tolerant and
Berjak, 1999). Cells with more organelles sensitive pollens (e.g. Hoekstra and
and greater definition of organelle structure Golovina, 1999; Hoekstra et al., 1999);
appear to be more sensitive to desiccation metabolic imbalances occur in both desic-
(Bewley, 1979; Hetherington, 1982a; Gaff, cation-tolerant pea and desiccation-
1989; Berjak et al., 1990; Farrant et al., sensitive tea (Walters et al., 2001); changes
1997; Farrant and Walters, 1998; Farrant, in the secondary structures of proteins are
2000), either because there are more mem- comparable in both mature and immature
brane structures to protect (described maize embryos (Wolkers et al., 1998a).
above) or because the higher metabolism Irreparable desiccation damage must then
leads to greater ROS production. result from a cascade of reactions, initiated
Conditions that reduce metabolism such as by primary but subtle lesions, which per-
low temperature (Leprince et al., 1995b) or turb organization within the cell and ulti-
highly complex substrates (Leprince et al., mately lead to cell death. The extent of
1990) also tend to reduce sensitivity to des- desiccation damage can then be viewed as a
iccation. Desiccation-sensitive cells respire function of the rapidity at which the cas-
at comparatively greater rates than tolerant cade of deleterious reactions occurs. From
cells at the same water content (Leprince et this perspective, desiccation damage is a
al., 1999; Walters et al., 2001), which may time-dependent process – an ageing phe-
reflect properties of the mitochondria nomenon.
themselves or of the cellular matrix. It has Dehydration has contrasting effects on
been suggested that changes in viscosity the kinetics of both physical and chemical
with dehydration are not as marked in des- deteriorative reactions. Removing water
iccation-sensitive cells, and so metabolism from cells increases the concentrations of
is not as restricted (Leprince and Hoekstra, reactants but slows the molecular motions
1998). It has also been suggested that the necessary for reactions to occur. The degree
packing of macromolecules during dehy- to which cells are damaged by desiccation,
dration of desiccation-sensitive cells is not and by extension the critical moisture level
as dense (Wolkers et al., 1998a,c), and this to which they survive drying, is deter-
might facilitate the diffusion of oxygen mined by the treatment duration, the con-
through the cell matrix. centration of the reactants and the physical
barriers (e.g. viscosity and compartmenta-
tion) to thermodynamically favourable
9.4. Perspectives on the Kinetics of reactions. Given the same experimental
Desiccation Damage time, cells that tolerate more stress have
either fewer reacting substances or greater
This chapter has described how desicca- barriers to harmful reactions. For example,
tion damage, incurred from structural according to the Water Replacement
Hypothesis, the concentration of reacting ing to fusion occur in milliseconds (Siegel

phospholipids is reduced by inserting sug- et al., 1994). Drying within 2–3 min
ars between head groups (Clegg, 1986; reduces protein denaturation of purified
Hoekstra et al., 1989, 1991; Crowe and protein (Wolkers et al., 1998a,b), and dry-
Crowe, 1992; Hoekstra and Golovina, ing within a few hours allows recalcitrant
1999). Alternatively, vitrification between embryos to survive greater amounts of
bilayer interfaces provides a mechanical water loss (Pammenter et al., 1991, 1998;
resistance to the compression of bilayers Walters et al., 2001). Despite valiant
(Wolfe and Bryant, 1999; Koster et al., attempts, we have not been able to dry
2000; Bryant et al., 2001). According to recalcitrant embryos in less than 10 min
these two hypotheses, more tolerant organ- and have yet to observe a recalcitrant
isms have either proportionally more sug- embryo survive 15 MPa. This failure to
ars to insert (Hoekstra et al., 1989) or more extend the lower limits of survivable water
strength to repel (Wolfe and Bryant, 1999). contents in recalcitrant seeds suggests that
Continuing this line of thinking may help the damaging reaction that occurs between
to further distinguish between the two 10 and 15 MPa is extremely fast in des-
hypotheses. According to the Water iccation-sensitive cells and relatively slow
Replacement Hypothesis, once a finite (and perhaps non-existent) in tolerant
number of sites is filled, the membrane or cells. The low rate of damage at this water
cell should be able to tolerate complete potential in tolerant cells cannot be
drying and maintain structure through time explained by glass formation per se (e.g.
since reactive sites are protected. On the Leopold et al., 1994), as glasses form in
other hand, vitrification is likely to provide seeds and pollen at lower water potentials
imperfect protection, and macromolecular (w 70 MPa) (see Chapter 10). However,
structures will eventually be compromised the viscosity of leathery and rubbery states
given sufficient time or stress. This latter (Fig. 9.1) may effectively impede molecular
possibility is consistent with observations of movements leading to membrane damage at
naturally occurring desiccation-tolerant higher moisture levels. The greater viscos-
systems: desiccation-tolerant seeds (Vertucci ity measured in more desiccation-tolerant
and Roos, 1990), pollen (Buitink et al., cells (Leprince et al., 1999) may contribute
1998b) and other phylogenetically diverse to greater structural stability.
life forms such as Artemia cysts, leaves from
resurrection angiosferms, and yeast (C.
Walters, unpublished data) are progressively 9.5. Conclusion
damaged when dried to water potentials
less than about 200 MPa. Clearly desiccation sensitivity is not an ‘all
The effect of time on desiccation-related or nothing’ or qualitative feature as it was
damage has been explored at higher mois- once treated. Macromolecules, cells and
ture levels (12 w 3 MPa) where organisms succumb at a range of stress lev-
metabolism is believed to play a role els over a range of times. The primary
(Pammenter et al., 1998; Leprince et al., lesion may be slight and reversible by our
1999, 2000; Walters et al., 2001) and at standards of measurement and may occur
extremely low moisture levels (w 220 in both tolerant and sensitive cells. This
MPa) where mechanical properties are lesion, which arises from the contraction of
compromised and/or reacting substances the aqueous volume and the consolidation
are exposed (Vertucci and Roos, 1990; of cellular constituents, may lead to the
Buitink et al., 1996, 1998a,b; Walters, irreversible loss of plasmalemma surface
1998). In these systems, the time scale for area, disruption of normal metabolism,
measuring damage is days or years, respec- fusion of unrelated membrane systems and
tively. However, some damaging reactions protein aggregation. As deleterious reac-
may appear to be almost instantaneous. For tions cascade, it becomes difficult to deter-
example, membrane-phase transitions lead- mine the earliest sources of damage.
Protectants either mitigate unbalanced 9.6. Acknowledgements

metabolism or prevent the contraction and
consolidation of cellular constituents. In The authors gratefully acknowledge Drs
doing so, a level of quiescence and Peter L. Steponkus (Cornell University),
mechanical rigour is imposed. These Folkert Hoekstra (Wageningen University)
appear to be the most distinguishing and, especially, Karen L. Koster (The
characteristics of desiccation-sensitive and University of South Dakota) for helpful and
-tolerant angiosperms. thought-provoking discussions.
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10 Biochemistry and Biophysics of

Tolerance Systems
Julia Buitink,1 Folkert A. Hoekstra2 and Olivier Leprince1

1UMR Physiologie Moléculaire des Semences, Institut National d’Horticulture,
16 Bd Lavoisier, F49045 Angers, France; 2Laboratory of Plant Physiology, Department
of Plant Sciences, University of Wageningen, Arboretumlaan 4, 6703 BD Wageningen,
The Netherlands

10.2. Repression of Metabolism 294
10.3. Antioxidant Defence 295
10.4. Partitioning of Amphiphilic Compounds 296
10.5. Macromolecule Stabilization 298
10.5.1. Preferential hydration 298
10.5.2. DNA integrity and chromatin condensation 299
10.5.3. Water replacement hypothesis 299
10.5.4. Vitrification 302
10.5.4.1. Role of vitrification in desiccation tolerance
and longevity 303
10.5.4.2. Composition of glasses in desiccation-tolerant
organisms 305
10.6. Roles of Specific Compounds in Stability 306
10.6.1. Sucrose/oligosaccharides 306
10.6.2. Late embryogenesis abundant (LEA) proteins 307
10.6.3. Heat-shock proteins (HSPs) 309
10.7. Conclusion and Outlook 310
10.8. References 311
10.1. Introduction arrangements are lost when the water in

which they are formed is removed. For
Most living cells only survive dehydration instance, when a biological membrane is
to a limited extent. Water has a profound dehydrated, irreversible changes occur in its
influence on the association of amphiphilic structural and functional integrity (reviewed
phospholipids in bilayers and the folding of by Crowe et al., 1997a). Similarly, many
proteins (Tanford, 1978). These molecular labile proteins lose their functional and
294 J. Buitink et al.
probably structural integrity when they are lowed by partitioning of amphiphilic com-
desiccated (reviewed by Carpenter et al., pounds (Section 10.4). The mechanisms
1992). Also, removal of water from desicca- through which the macromolecules are sta-
tion-sensitive organisms leads to metabolic bilized at different hydration levels are dis-
imbalances, resulting in the production of cussed in Section 10.5, together with an
free radicals and oxidative damage. Whereas overview of the protective substances that
Chapter 9 discussed the damage linked to have been identified so far in relation to
desiccation, this chapter deals with the desiccation tolerance (Section 10.6).
biochemical and biophysical changes that
are part of the mechanisms by which
desiccation-tolerant organisms (anhydro- 10.2. Repression of Metabolism
biotes) cope with dehydration. The acquisi-
tion of desiccation tolerance is correlated The most reported degradative reactions
with the accumulation of considerable linked with desiccation sensitivity in seeds
quantities of non-reducing di- and are the extensive peroxidation and de-esteri-
oligosaccharides, compatible solutes and fication of phospholipids, leading to the loss
specific proteins such as the late embryoge- of membrane integrity (Senaratna et al., 1987;
nesis abundant (LEA) and heat-shock pro- Hendry et al., 1992; Leprince et al., 1994).
teins (HSPs) (see Chapters 1, 5 and 11). The cause of peroxidative damage is thought
Two strategies that confer desiccation to originate from an increased formation of
tolerance have been identified. The first reactive O2 species (ROS) as a result of the
strategy involves an initial avoidance of the impairment of the electron transport chains
accumulation of desiccation-induced dam- during drying (see Chapter 9).
age accompanied by the presence of pro- Characteristically, desiccation-tolerant
tecting factors such as sugars or LEA tissues suffer less from oxidative damage
proteins. The protecting factors can be pre- than do sensitive tissues (Leprince et al.,
sent before or synthesized during drying 1993, 1994; Vertucci and Farrant, 1995;
(Vertucci and Farrant, 1995). Desiccation Pammenter and Berjak, 1999). The produc-
tolerance probably depends on these tion rate of ROS is dependent on a number
responses acting in synergy during drying. of factors (Skulachev, 1996). It increases
For example, it has been shown that pro- with an increased lifetime of the electron
tective disaccharides do not exert their pro- carriers in the reduced state, with the
tective effects on dried membranes that depletion of ADP and with an increase in
contain more than 15% free fatty acids, a respiration rate. It has been surmised that
product that accumulates during desicca- desiccation-tolerant organisms reduce or
tion-induced oxidative stress (Senaratna adapt their metabolic activities to diminish
and McKersie, 1986; Crowe et al., 1989b). the chance of generating ROS (Leprince et
The second strategy is based on activa- al., 1994; Vertucci and Farrant, 1995;
tion of efficient repair mechanisms upon Pammenter and Berjak, 1999). Evidence
rehydration. This second strategy is dealt supporting this hypothesis is increasing,
with in Chapter 12. This chapter will give a but has been indirect so far. Rogerson and
critical assessment as to how desiccation- Matthews (1977) observed for garden pea
tolerant organisms have adapted their seeds that during maturation the ability to
strategies to cope with the stresses that withstand desiccation was preceded by a
occur during dehydration. The different fall in respiration rate. Furthermore, desic-
strategies will be discussed in sequence of cation-tolerant axes of pea and cucumber
hydration level at which they are thought were found to exhibit a much reduced CO2
to be active, from the hydrated to the dry production before dehydration compared
state, starting with the avoidance of oxida- with germinated, desiccation-sensitive
tive damage by means of regulation of axes, and this difference was maintained
metabolism (Section 10.2) and the presence during drying (Leprince et al., 2000; see
of antioxidant systems (Section 10.3), fol- Table 10.1). Respiration rates in the desicca-
Biochemistry and Biophysics of Tolerance Systems 295
Table 10.1. CO2 production rates during drying of desiccation-tolerant cucumber

axes (24 h imbibed at 20°C; DT), desiccation-sensitive axes (72 h imbibed at
20°C, radicle 3 mm protruded; DI) and germinated cucumber axes that were
incubated in polyethylene glycol (PEG) for 7 days to reinduce desiccation
tolerance (72 h imbibed at 20°C with a radicle 3 mm protruded, then 7 days in
PEG (1.5 MPa) at 10°C; DT PEG). (Adapted from Leprince et al., 2000.)
CO2 production rate (l h1 g1 dry weight)

g H2O g1 dry weight DT DI DT PEG
3.0 1.7 4.5 1.1

2.0 1.3 3.2 0.5
1.0 0.7 1.5 0.4
tion-sensitive cotyledons of Castanea sativa as anoxia, freezing and dehydration (Hand

Mill. increased at the onset of drying and and Hardewig, 1996; Hardie et al., 1998).
decreased only with the loss of membrane However, thus far there is only limited evi-
integrity. In contrast, dehydration of the dence for metabolic depression in develop-
more desiccation-tolerant axes resulted in a ing seeds (Kollöffel and Matthews, 1983)
rapid decline in O2 uptake rates at the onset and somatic embryos (Tetteroo et al., 1995).
of drying (Leprince et al., 1999). In general, Some indications that coordinated down-
mature recalcitrant seeds have relatively regulation might be essential to confer des-
high respiration rates (Salmen Espindola et iccation tolerance in seeds comes from
al., 1994; Leprince et al., 1999; Pammenter Leprince et al. (2000). Dehydration of desic-
and Berjak, 1999). Thus, the high respira- cation-sensitive axes resulted in an increase
tion rates in desiccation-sensitive seeds in the emission rates of acetaldehyde and
may promote free-radical-induced injury ethanol, which peaked well before the
during drying. The relationship between onset of membrane damage. Tolerant axes
the extent of metabolic activity and desicca- did not exhibit acetaldehyde or ethanol
tion sensitivity is strengthened by the production. The question remains as to
observation that treatments that limit rates whether controlled down-regulation is
of metabolism also reduce the incidence of exerted before drying (i.e. during seed mat-
desiccation injury (Leprince et al., 1995b). uration) or also during drying.
If metabolism must be down-regulated
for desiccation tolerance to be acquired, the
nature of this regulation is unknown. 10.3. Antioxidant Defence
Leprince et al. (1994, 1995b, 2000) sug-
gested that a coordinated down-regulation Since susceptibility to peroxidation may
of energy metabolism in seeds early during increase with drying (see Vertucci and
drying may play an important role in avoid- Farrant, 1995, for a review; Chapter 9), one
ing oxidative stress conditions and/or accu- may reason that free-radical-scavenging sys-
mulation of by-products of metabolism to tems are an important component among
toxic levels. During drying, the O2 availabil- the mechanisms of desiccation tolerance.
ity decreases with the increase in cellular ROS are natural by-products of the metabo-
viscosity. It is likely that to avoid metabolic lism, which are particularly present in
imbalances there will be a fine tuning chloroplasts and mitochondria. Thus,
between repression of metabolic activity plants are well endowed with antioxidant
and O2 availability (Leprince et al., 2000). molecules and scavenging systems (Larson,
Down-regulation of metabolism appears to 1988; Hendry, 1993). Enzymatic free-
be an ancient and widespread regulatory radical-processing systems include super-
mechanism that allows aerobes to with- oxide dismutase (SOD), which catalyses the
stand severe environmental stresses, such dismutation of superoxide (O2) into H2O2
and O2, and those that are involved in the radical-processing systems in seeds rely on
detoxification of H2O2 (i.e. catalase, glu- enzymatic activities, it is likely that they are
tathione reductase and peroxidases). mainly efficient early during drying and
Several studies have demonstrated the link may not function in reduced hydration con-
between tolerance of oxidative stress ditions. At the lower water contents, one
induced by water deficit and rise in antioxi- may expect molecular antioxidants (e.g. glu-
dant concentrations in photosynthetic tathione, ascorbate, tocopherol) to play a
plants (Winston, 1990; Price and Hendry, preponderant role in alleviating oxidative
1991). In vegetative tissues, removal of the stress. This is supported by a study on ger-
cytotoxic products resulting from oxidative minating seeds of soybean, which showed
events is considered to be of prime impor- that their desiccation tolerance was associ-
tance for survival of drought stress because ated with the presence of an unknown lipid-
genes encoding enzymatic antioxidants soluble antioxidant in extracted membranes
become up-regulated during drying (Ingram (Senaratna and McKersie, 1986). However,
and Bartels, 1996). In the resurrection plant in germinating maize, no correlation was
Craterostigma plantagineum, an inhibitor found between the presence of lipid-soluble
of lipoxygenase (the activity of which and hydrophilic antioxidants and desicca-
results in lipid hydroperoxide formation) tion tolerance (Leprince et al., 1990b). A
accumulates in the leaves during desicca- comparative study on ascorbate and dehy-
tion (Bianchi et al., 1992). In Craterostigma dro-ascorbate showed that the concentra-
wilmsii and Xerophyta viscosa, the activity tions of these antioxidants were higher in
of ascorbate peroxidase increases during recalcitrant seeds than in orthodox seeds
dehydration. During rehydration, the activ- (Tommasi et al., 1999). Future studies aimed
ity of SOD and glutathione reductase at establishing the importance of free-radi-
increases (Sherwin and Farrant, 1996). cal-processing systems in relation to desic-
Furthermore, C. wilmsii was also found to cation tolerance should take into account
accumulate large amounts of anthocyanins, both the hydration level during drying and
which have antioxidant capabilities. the fact that free-radical production will be
The protective role of antioxidants in localized within the mitochondria and/or
desiccation tolerance of seeds is far from microsomal membranes. Thus, efforts
resolved (reviewed by McKersie, 1991; should concentrate on antioxidant systems
Hendry, 1993; Leprince et al., 1993). within these organelles. Furthermore, coin-
Vertucci and Farrant (1995) have suggested cidental antioxidants such as quinones,
that it is particularly in the water content polyols, carbohydrates, amphipathic mole-
range corresponding to water potentials of cules (flavonoids, phenolics) and proteins
3 to 11 MPa that unregulated metabolic (e.g. peroxiredoxin) also deserve particular
events result in the first wave of free-radi- attention.
cal generation, although the upper limit
could be higher. Thus, it is assumed that
antioxidant systems should be maximally 10.4. Partitioning of Amphiphilic
effective during the initial stages of the Compounds
maturation drying of developing orthodox
seeds (Arrigoni et al., 1992). Cells may contain various cytoplasmic
Despite several studies that assessed in metabolites that have amphiphilic proper-
vitro (i.e. under hydrated conditions) the ties. Hoekstra et al. (1997) proposed that
activity of free-radical-processing systems desiccation may increase the transfer of
extracted from drying tissues, it remains to these amphiphiles from the polar cyto-
be ascertained whether these systems are plasm into the lipid phase, i.e. the mem-
also active at low water contents in vivo. branes and lipid bodies. On the one hand,
Considering that the metabolism is virtu- such partitioning into membranes could
ally nil in cells containing less than 0.3 g seriously perturb membrane structure, with
H2O g1 dry weight (dw), and that free- effects on permeability properties as the
result. On the other hand, partitioning into or liposomes with hydrated ones, the aver-
membranes might be extremely effective at age rigidity was similar, even in the pres-
automatically inserting amphiphilic ence of a protective sugar. This has been
antioxidants into membranes upon dehy- interpreted to mean that dry membranes
dration, which could promote desiccation inside anhydrobiotes are fluidized, presum-
tolerance and extend storage longevity. The ably by amphiphilic guest molecules
presumed transfer of amphiphiles has been (Hoekstra and Golovina, 1999). In isolated
experimentally tested in pollen and seeds membranes, such amphiphiles become lost
with electron paramagnetic resonance during the isolation procedure. Recent data
(EPR) spectroscopy, using amphiphilic based on the number of spin-probe mole-
spin probes inserted into the cytoplasm cules in either phase during dehydration
(Golovina et al., 1998; Buitink et al., (Golovina and Hoekstra, 2001) indicate that
2000d; Hoekstra and Golovina, 2000). The there is already a considerable transfer of
advantage of this method is that the EPR amphiphiles from the cytoplasm to mem-
spectra can be used to derive where the branes at the beginning of drying. This
spin probe resides – in the cytoplasm or in would mean that fluidization occurs early
the lipid phase. It has been demonstrated during dehydration. Indeed, an increase in
that amphiphilic probes partition into the molecular mobility at the membrane surface
lipid phase with drying, in proportion to was observed early during dehydration,
the reduction of the cytoplasmic volume, probably brought about by endogenous
and vice versa with rehydration (Golovina amphiphiles. While this mobility decreases
et al., 1998; Hoekstra and Golovina, 2000). in desiccation-tolerant organisms on further
A similar partitioning behaviour with drying, it remains high in desiccation-sensi-
drying is plausible for endogenous cyto- tive organisms until almost all water has
plasmic amphiphiles. Extracted endoge- disappeared (Golovina and Hoekstra, 2001).
nous amphiphiles reversibly partitioned Endogenous biologically relevant
into liposomal membranes with drying, amphiphiles that might undergo partition-
which made the liposomes transiently ing similar to the spin probes are phenolic
leaky for entrapped polar compounds acids and flavonoids. These molecules are
(Golovina et al., 1998). Thus, partitioning abundantly present in dry seeds, pollen and
into membranes is possible and has been resurrection plants (Larson, 1988; Oliver et
used to explain the transient leakage of al., 1998; Shirley, 1998) and may play sev-
cytoplasmic solutes from rehydrating anhy- eral roles in relation to desiccation toler-
drobiotes. Plasma membrane permeability ance. A wide array of amphiphiles present
of pollen, for example, was indeed elevated in plants are known to be potent antioxi-
as long as the amphiphiles resided in the dants (Larson, 1988; Saija et al., 1995; Rice-
lipid phase. On sufficient rehydration, Evans et al., 1997). Partitioning of such
when the amphiphiles had mainly returned amphiphilic antioxidants from the cyto-
to the aqueous cytoplasm, permeability plasm into membranes during drying might
returned to the low level observed before prevent desiccation-induced oxidative
dehydration, and leakage rates became low. damage. A dehydration test with the antiox-
This transiently increased permeability idant flavonoid rutin on liposomes indi-
upon imbibition lasted approximately 10 s cated that rutin depresses the average
in the case of pollen (Hoekstra et al., 1999). phase-transition temperature of the lipo-
There are more indications that in dry somes (Hoekstra and Golovina, 2000). From
anhydrobiotes endogenous amphiphiles this it has been inferred that amphiphiles
reside in membranes. In situ Fourier trans- might have a role in controlling the mem-
form infrared (FTIR) spectroscopy data has brane-phase transition temperature (Tm),
indicated that the average rigidity of mem- along with the non-reducing di- and
branes in the gel phase is less when the oligosaccharides (see Section 10.5.3).
organism is dry than when it is hydrated. Although partitioning seems beneficial,
When comparing dried isolated membranes as indicated above, it appears that the trans-
fer must be controlled in order to confer It has been shown in model experi-
desiccation tolerance (Buitink et al., 2000d). ments that these substances stabilize pro-
This is because amphiphiles can perturb tein structure and activity against thermal
membrane functions (Herbette et al., 1983; denaturation (Arakawa and Timasheff,
Takahashi et al., 1998), which might be par- 1985). Further, they protect isolated pro-
ticularly detrimental at high water contents. teins (Carpenter and Crowe, 1988;
For example, if mitochondrial respiration is Carpenter et al., 1990) and (functional)
affected, there is the likelihood of an vesicles (Rudolph and Crowe, 1985;
enhanced production of free radicals. Anchordoguy et al., 1987, 1988) during
freeze–thawing by minimizing denatura-
tion and the vesicles by preventing fusion.
10.5. Macromolecule Stabilization Coming from chemically dissimilar
classes, these substances have in common
Macromolecule stabilization, such as the that they are preferentially excluded from
retention of phospholipid bilayers or contact with the surface of proteins in
proper folding of proteins, depends on the aqueous solution, which makes it thermo-
presence of water. Upon dehydration, these dynamically unfavourable for proteins to
macromolecules need to be protected from unfold (Arakawa and Timasheff, 1985;
the deleterious effects that are normally Carpenter and Crowe, 1988; Carpenter et
associated with the removal of water. The al., 1992). In other words, these substances
stabilization of the molecules in the cells is keep the macromolecules preferentially
realized through one or more of the mecha- hydrated. In contrast, compounds such as
nisms that are discussed below, in the urea and guanidine–HCl, which preferen-
order of the mechanisms that are func- tially bind with proteins, destabilize pro-
tional at high hydration levels to those that teins in solution. This means that when
are operational at low water contents. the bulk water is removed (below 0.3
H2O g1 dw), this mechanism would fail to
work because there is no water left for
10.5.1. Preferential hydration preferential hydration (Crowe et al., 1990).
Indeed, most of the compatible solutes,
Many plants and microorganisms accumu- except a few sugars, are unable to protect
late organic osmolytes in response to envi- proteins and membranes against air-drying
ronmental stresses that cause cellular or freeze-drying. In the case of sugars, it is
dehydration, such as drought, freezing and envisaged that, after the bulk water is lost,
osmotic shock. This accumulation corre- hydrogen bonding and glass formation are
lates with improved stress tolerance. the mechanisms by which proteins and
Evidence of a causal relationship between membranes are structurally and function-
elevated levels of these so-called compati- ally preserved (see Sections 10.5.3 and
ble solutes and stress tolerance has come 10.5.4).
from the results of enrichment experiments One has to realize that dehydrating
(e.g. Saranga et al., 1992) and from the anhydrobiotes pass through stages of
behaviours of transgenic organisms engi- drought during which the mechanism of
neered to accumulate these compounds preferential exclusion may be effective. It
(e.g. Takagi et al., 1997; Strom, 1998). is, therefore, not surprising that anhydro-
Among these compatible solutes are pro- biotes are endowed with large amounts of
line, serine, glutamate, glycine-betaine, car- compatible solutes, e.g. proline and
nitine, mannitol, sorbitol, fructans, polyols, sucrose in pollen (Zhang et al., 1982;
trehalose, sucrose and oligosaccharides. Hoekstra et al., 1992), di- and oligosaccha-
The absolute osmolyte concentrations, rides in seeds (Amuti and Pollard, 1977),
however, are unlikely to mediate osmotic and proline, glycine-betaine and trehalose
adjustment (Hare et al., 1998). in microorganisms and yeast.
10.5.2. DNA integrity and chromatin undergo reversible changes in condensation

condensation states, indicating that certain protective
substances are necessary to confer stability
The maintenance of genetic information is to the chromatin during drying in desicca-
central to survival upon dehydration and tion-tolerant tissues (Leprince et al., 1995a).
rehydration (see Chapter 12). As a dynamic While the stability of DNA and chro-
and hydrated molecule in vivo, DNA can matin in the dehydrated state must be a
assume different conformational structures, prerequisite for desiccation tolerance in
depending on the water activity, the base seeds and resurrection plants, much
sequence and the presence of specific bind- remains to be ascertained about factors
ing proteins (Osborne and Boubriak, 1994). contributing to the genetic stability and the
Determination of the integrity of extracted responses to dehydration in both desicca-
DNA in embryos of seeds and in wind-dis- tion-tolerant and sensitive material.
persed pollen during the transition from
the desiccation-tolerant to the sensitive
stage showed that only DNA from desicca- 10.5.3. Water replacement hypothesis
tion-tolerant cells retained integrity when
cells were subjected to drying regimes. It Studies on model liposome systems com-
was further proposed that the attainment of posed of pure phospholipids (PLs) show
stable secondary structures that are resistant that drying induces a phase transition in
to degradation in vivo at low water poten- the membranes from the fluid liquid-crys-
tials is a likely accessory to desiccation tol- talline phase to the solid gel phase (Crowe
erance (Osborne and Boubriak, 1994). et al., 1997a; see Chapter 9). The rise of the
Another genetic factor that changes as a membrane-phase transition temperature
function of hydration is the structure of (Tm) with drying commences with the dis-
chromatin. In the desiccation-tolerant sipation of the last 10–12 water molecules
phase of developing and germinating ortho- per PL molecule, i.e. below 0.2–0.3 g
dox seeds, the chromatin is in a condensed H2O g1 dw. The removal of water mole-
state. This state appears to be retained dur- cules from the head groups leads to a reduc-
ing early rehydration, while seeds are still tion in the lateral spacing between the PL
desiccation-tolerant (Leprince et al., 1995a; molecules and, consequently, to the forma-
Pammenter and Berjak, 1999). During ger- tion of a gel phase. Thus, the Tm of model
mination, the rehydration stage correspond- membranes composed of phosphatidyl-
ing to the resumption of DNA replication is choline increases by as much as 70°C during
associated both with chromatin deconden- dehydration. A liquid-crystal-to-gel-phase
sation and loss of desiccation tolerance transition of membrane PLs during dehy-
(Deltour, 1985). Orderly, reversible chro- dration has been detected in intact pollen,
matin compaction and rehydration-induced using FTIR spectroscopy (Crowe et al.,
redispersion of condensed chromatin thus 1989a). Similar to the model liposome sys-
appear to be associated with desiccation tems, the average Tm values of membranes
tolerance. Evidence for this comes from in pollen increased with dehydration.
electron microscope observations on seeds Comparison of the Tm value in dried cattail
(Crèvecoeur et al., 1976; Deltour, 1985) and pollen (32°C) with that of dry membranes
resurrection plants (Hallam and Luff, 1980), isolated from this pollen (58°C) indicated
and from a biophysical characterization of that the Tm in intact pollen was depressed
isolated chromatin from germinating maize by 26°C (Hoekstra et al., 1991). Apparently,
embryos (Leprince et al., 1995a). Electron in the dry, intact organism there is a mech-
microscopy studies show that irreversible anism that depresses the dehydration-
chromatin compaction accompanies injuri- induced increase of Tm, which is lost when
ous dehydration of desiccation-sensitive the membranes are isolated. Upon adding
material. Extracted chromatin from dried, sucrose to the isolated membranes before
desiccation-sensitive tissues was unable to drying, the Tm of the dried membranes was
again depressed from 58 to 31°C. The In the presence of trehalose (a common

mechanism by which this depression is disaccharide often found in anhydrobiotic
considered to work is discussed below. microscopic animals and yeasts (Crowe et
Experiments on model systems have al., 1984)) polar compounds trapped
given some insight into the mechanism by inside the liposomes do not leak during
which sugars depress the Tm of dry mem- drying and rehydration (Crowe et al.,
branes. It has been established that during 1986). Also, other disaccharides (sucrose)
desiccation soluble sugars interact with the and oligosaccharides such as stachyose
polar head groups and replace the water and raffinose are effective at retaining
molecules. Phospholipid molecules thus compounds trapped inside the liposomes.
largely retain the original spacing between It is thought that the leakage does not
one another, and the desiccation-induced occur because a phase change is prevented
increase in Tm is circumvented (Fig. 10.1). (Fig. 10.1). In the light of these in vitro
Interaction with sugars can even lead to studies, the presence of sucrose and
depression of Tm to values considerably oligosaccharides in desiccation-tolerant
below the Tm of the hydrated specimens organisms is thought to be similarly
(Crowe et al., 1996). As the size of the solu- involved in the depression of Tm in situ
ble carbohydrate molecule increases, the and the prevention of leakage.
chance of interaction between the phos- From air-drying experiments with lipo-
phate and the carbohydrate decreases, and somes in the presence of disaccharides, it
the depression of Tm is less. Thus, it has has become clear that a mass ratio of 5 : 1,
been demonstrated that hydrogen-bonding sugar : PL, satisfies the head group hydrogen-
between the head group PO and sugar OH bonding capacity (Hoekstra and Golovina,
groups is pivotal for the depression of Tm 1999). Such mass ratios often occur in
in dry membranes (Crowe et al., 1996). anhydrobiotic pollen and seeds, but excep-
Water
Sugars
Rehydration
Hydrated No leakage,
liquid crystal no fusion
+ sugars
Desiccation
Hydrated,
liquid crystal
– sugars
Rehydration
Extensive
leakage + fusion
Dry gel
Fig. 10.1. Schematic representation of the phase behaviour of phospholipids in a bilayer as influenced by
desiccation in the presence or absence of a disaccharide. The disaccharide maintains the original spacing
between the phospholipid molecules and, thus, prevents the transition from the liquid crystalline phase to
the gel phase.
tions with lower mass ratios have been during drying before the formation of a
found, which are nevertheless desiccation- glass, thereby increasing the chance of
tolerant (Hoekstra et al., 1997). The effec- solute loss. Thus, depression of Tm may
tive availability of sugars to PLs in situ prevent a loss of membrane permeability
might be less than expected, because sug- during drying. Furthermore, depression of
ars also interact with other constituents of the Tm in dry anhydrobiotes is also impor-
the cells and with one another. This tant to reduce the risk of leakage during
restricted availability of the sugars for rehydration (also called imbibitional leak-
interaction with the head groups would be age), apart from evading leakage during
in line with the observation that depres- drying (see Chapter 12).
sion in situ of Tm in dry organisms is often Another role for sugars in relation to
not to the low value measured in the desiccation tolerance is their stabilizing
hydrated specimens (Hoekstra and effect on dried proteins (see Chapter 9).
Golovina, 1999). It has been suggested that, Sugars are special in that they allow the
besides sugars, other molecules might aid removal of the closely associated water
in the depression of Tm in desiccation-tol- from proteins without this leading to con-
erant organisms, for example by the trans- formational changes and loss of enzymatic
fer of fluidizing compounds from the function. Above 0.3–0.5 g H2O g1 dw,
cytoplasm into membranes (see Section other (compatible) solutes can also be
10.4). effective (see also Section 10.5.1).
The question remains as to why post- Employing phosphofructokinase (PFK), an
poning or preventing the rise in Tm with extremely labile protein when dried or
dehydration might be beneficial. The fact freeze-dried, Carpenter and co-workers
that dry pollen and seeds can be safely have shown that the disaccharides sucrose,
stored below 20°C (i.e. under conditions maltose and trehalose are very effective sta-
that make the gel-phase formation particu- bilizing agents, particularly in the presence
larly likely) indicates that the presence of of certain divalent cations (Carpenter et al.,
the gel phase per se is not detrimental. A 1987a,b, 1990). FTIR spectroscopy studies
phase change leads to transiently increased indicate that sugars act as a water substi-
permeability, and a loss of solutes may be tute by satisfying the hydrogen-bonding
expected under these conditions. The requirement of polar groups on the surface
extent of this loss will depend on the vis- of the dried protein (Carpenter and Crowe,
cosity of the surrounding cytoplasmic envi- 1989; Prestrelski et al., 1993; Wolkers et
ronment. Below 0.8 g H2O g1 dw, the al., 1998b). This mechanism is analogous
viscosity increases exponentially and to the water replacement hypothesis men-
becomes extremely high when the cyto- tioned above for membranes, although fixa-
plasm enters a glassy state (Leprince and tion of the protein’s secondary structure
Hoekstra, 1998) (see Section 10.5.4). No also occurs via glass formation (see Section
loss of solutes is to be expected when the 10.5.4) (Franks et al., 1991). Thus, protein
phase change of the membranes occurs unfolding and aggregation during dehydra-
while the cytoplasm is close to reaching a tion are prevented. In dry, orthodox seeds,
glassy state. However, leakage may result proteins retain their native secondary
when the phase change occurs when the structure for decades, long after the seeds
cytoplasm is in the liquid state. Data on have died (Golovina et al., 1997). Recently,
cattail pollen have indicated that, during Wolkers et al. (1998b) contributed to the
dehydration at 20°C, the gel phase and the question of whether glass formation or
glassy state are formed simultaneously hydrogen bonding of sugar with protein is
(Hoekstra and Golovina, 1999). Any uplift involved in the stabilization during slow
of the phase diagram (i.e. the curve repre- air-drying. The sugars having the best
senting the relationship between Tm and hydrogen-bonding properties give the best
water content) by ineffective depression of structural protection, but have the lowest
the Tm would result in gel-phase formation glass transition temperature (Tg), which
supports the hydrogen-bonding mechanism state (Franks et al., 1991). A glass is a

of protection. highly viscous solid liquid. Its high viscos-
ity has been shown to slow down severely
molecular diffusion and decrease the prob-
10.5.4. Vitrification ability of chemical reactions (see Slade and
Levine, 1991; Roos, 1995, for reviews).
Upon drying of desiccation-tolerant tis- Glass formation has been detected in seeds
sues, as the concentration of solutes (Williams and Leopold, 1989; Bruni and
increases there is an increase in the viscos- Leopold, 1992; Leopold et al., 1994;
ity of the cytoplasm and a decrease in mol- Leprince and Walters-Vertucci, 1995),
ecular mobility of molecules. For example, pollen (Buitink et al., 1996) and the resur-
in embryonic tissues below 0.8 g H2O g1 rection plant C. plantagineum (J. Buitink,
dw, the cytoplasm becomes increasingly unpublished results). Apparently, glass for-
viscous (Leprince and Hoekstra, 1998; mation is a characteristic typical of all des-
Buitink et al., 2000d). Drying below 0.3 g iccation-tolerant tissues.
H2O g1 dw leads to a decrease in the mol- The presence of a glassy state is depen-
ecular mobility in the cytoplasm of over dent on three factors: water content, temper-
five orders of magnitude (Buitink et al., ature and chemical composition. A decrease
1999). Finally, at around 0.1 g H2O g1 dw, in the water content of the tissue results in
the cytoplasm vitrifies and exists in a so- an increased glass transition temperature
called glassy state. A glass is defined as an (Tg), as demonstrated by a state diagram,
amorphous metastable state that resembles which depicts the relationship between the
a solid, brittle material, but retains the dis- water content and the Tg (Fig. 10.2). The
order and physical properties of the liquid magnitude of Tg is also dependent on the
Fast drying
60 Slow drying
Glass transition temperature (C)
30
–30
–60
0.00 0.08 0.16 0.24 0.32

Water content (g H2O g–1 dry weight)
Fig. 10.2. State diagram depicting the relationship between water content and glass transition temperature
of developing bean axes (O. Leprince and C. Walters, unpublished data). Seeds were harvested near mass
maturity. After slow drying (i.e. 3 days), isolated axes were found to elongate and grow when cultured in
vitro on agar. They were considered to be desiccation-tolerant. In contrast, isolated axes failed to elongate
after fast drying (5 h) and were considered to be desiccation-sensitive. The different water contents were
obtained by drying isolated axes for different times. Glass transition temperatures were measured at the mid-
point using a differential scanning calorimeter (DSC) during heating at a scanning rate of 10°C min1
according to Leprince and Walters-Vertucci (1995). Data points represent three to five axes in the DSC pan.
composition of the amorphous state. For the glassy state can be effective. Although
one-component model systems, Tg is known glass formation is not a mechanism that
to vary with Mr in a characteristic and theo- initially confers tolerance to desiccation
retically predicted fashion (Slade and during drying, its formation is indispens-
Levine, 1991). For instance, a sugar of a high able for surviving the dry state, as dis-
Mr (like stachyose) exhibits a higher Tg over cussed below.
the entire range of water contents than a An important consequence of the forma-
small Mr sugar such as glucose. tion of the glassy state in tissues is the
absence of crystallization (Leopold et al.,
1994; Sun and Leopold, 1997). It has been
10.5.4.1. Role of vitrification in desiccation
suggested that loss of viability could be
tolerance and longevity
due to time-dependent crystallization lead-
Intracellular glasses were, originally, sug- ing to loss of membrane structure and cel-
gested to play a role in desiccation tolerance. lular integrity (Caffrey et al., 1988; Sun and
Considering the glass-forming capability of Leopold, 1993). However, so far there is no
sugars, several studies have appeared experimental evidence that demonstrates
attempting to link changes in sugar composi- crystallization events in desiccation-
tion during the acquisition of desiccation sensitive tissues (Sun and Leopold, 1993).
tolerance with the existence of glasses upon It is surmised that the complex cytoplas-
drying (Koster, 1991; Williams and Leopold, mic composition is most probably respon-
1995). For instance, sugars similar to those sible for preventing crystallization
found in desiccation-tolerant seeds (sucrose, (Walters, 1998; Buitink et al., 2000e). The
raffinose) are capable of forming glasses at major function of intracellular glasses in
ambient temperatures, whereas sugar mix- dry seeds may be their contribution to the
tures similar to those found in axes that do stability of macromolecular and structural
not tolerate desiccation were found only to components during storage.
form glasses at sub-zero temperatures One of the most studied functions of
(Koster, 1991). Williams and Leopold glasses is maintenance of the structural and
(1995) showed that, after 50 h of imbibition, functional integrity of macromolecules (see
the Tg of desiccation-sensitive pea embry- Slade and Levine, 1991, 1993; Roos, 1995,
onic axes was remarkably lower than that for reviews). Glasses are known to slow
of desiccation-tolerant axes imbibed for 14 down detrimental reactions, such as the rate
h, accompanying the loss of oligosaccha- of browning reactions (Karmas et al., 1992),
rides and replacement by monosaccha- to increase the stability of enzymes (Chang
rides. However, in other studies, glasses et al., 1996) and to prevent conformational
have also been found in desiccation-sensi- changes in proteins (Prestrelski et al., 1993).
tive tissues (Sun et al., 1994; Buitink et al., It has also been shown in model systems
1996). For instance, the state diagrams of that glasses are capable of preventing the
developing embryonic axes of bean after fusion of membranes. Leakage from lipo-
fast and slow drying (which renders them somes can be the result of a phase change
desiccation-sensitive and -tolerant, respec- (Section 10.5.3), but also because of fusion
tively) were found to be identical (Fig. 10.2). between the liposomes. In the former case,
This questions the exclusive role of glasses the size of the liposomes remains the same,
in desiccation tolerance. It is important to but, in the latter case, the size increases
realize that the water content at which glass considerably. Protection of liposomes has
formation occurs during drying at room also been shown to depend on how effec-
temperature in seeds (~ 10% moisture; Fig. tively sugars can form glasses (Crowe et al.,
10.2) is much lower than the critical water 1998). In this respect, monosaccharides are
content most desiccation-sensitive species poor protectants, despite their generally
exhibit. Apparently, dehydration-induced excellent capability to interact with the
damage in these seeds occurs at water con- polar head groups (Section 10.5.3).
tents far above those at which protection of Monosaccharides differ from the di- and
oligosaccharides in having low Tgs (below water contents (Buitink et al., 2000c). All
room temperature). This means that during these arguments suggest that ageing rates
drying the liposomes remain in a liquid and, consequently, life span of germplasm
environment, which results in fusion- are influenced by the molecular stability of
induced release of the entrapped contents. the cytoplasm, signifying the pivotal func-
The importance of both the ability to form tion of intracellular glasses in conferring
glasses at ambient temperature and direct such stability during storage.
interaction with the polar head groups to Recently, the consequences of being in
protect membranes (see Section 10.5.3) has or near the glassy state have gained further
been demonstrated by drying liposomes in physiological significance. A study on the
the presence of two different compounds, molecular mobility in desiccation-tolerant
hydroxy-ethyl starch and glucose (Crowe et tissues indicated that the special composi-
al., 1997b). Hydroxy-ethyl starch has a tion of biological glasses might have a role
high Tg, thus forming a glass at ambient in survival (Buitink et al., 2000e). Based on
temperature, but it does not depress Tm saturation transfer-electron paramagnetic
because it is too large to fit between the resonance (ST-EPR) spectroscopy measure-
head groups to efficiently interact. Glucose, ments, Buitink et al. (2000e) observed a sec-
by contrast, depresses Tm in dry lipids but ond kinetic change in mobility at a definite
will not easily form a glass at ambient tem- temperature above Tg, referred to as the crit-
perature. Thus, hydroxy-ethyl starch pre- ical temperature (Tc). The occurrence of Tc
vents the leakage associated with fusion has been coupled to the collapse tempera-
(below 1.5 g H2O g1 dw), but cannot pre- ture of sugar glasses (Tg + 15°C; Fig. 10.3), a
vent the leakage associated with the phase well-documented phenomenon, which is
transition (below 0.25 g H2O g1 dw). attributed to a reduction in viscosity such
Together, however, hydroxy-ethyl starch that a flow on a practical time scale is
and glucose protect the dry liposomes, but observed. Although the viscosity decreases
neither compound is effective alone. Both around Tg, it is not until the temperature Tc
properties – timely glass formation during is reached that the viscosity abruptly drops
drying and interaction with the polar head (see Fig. 10.3). This is contrary to the
groups – appear to be required for preser- belief that the viscosity decreases abruptly
vation. Disaccharides combine these two at Tg, as is often assumed. The Tc in desic-
essential properties within one compound. cation-tolerant organisms occurs at temper-
The effect of glasses on the stability of atures as high as 55°C above Tg (Fig. 10.3).
macromolecular and structural components A high Tc implies high stability as a result
during storage has led to the concept that of high viscosity (> 108 Pa s) far above Tg.
glasses play an essential role in the This high Tc in biological tissues has
longevity of seeds and pollen. For example, important implications for the survival of
the storage stability of Arabidopsis thaliana germplasm in its natural environment.
seeds has been found to correlate with the Under ambient conditions, for example
molecular density of the cellular matrix 20°C and 50% relative humidity (RH), seed
(Wolkers et al., 1998a). Sun and Leopold tissues are around their Tg. This means that
(1994) have found that seed deterioration any environmental fluctuation that results
appears to be accelerated when seeds are in an increase in RH or temperature will
not in the glassy state, as estimated through bring the tissue above its Tg. However, the
the viability equation of Ellis and Roberts unique properties of the intracellular glass
(1980). Probably the most compelling evi- protect the tissue from dramatic changes
dence to suggest that ageing rates are caused by environmental fluctuations. If the
affected by the viscosity of the intracellular intracellular glass were composed of
glass has come from the linear relationship sucrose alone, a small increase in RH or
between ageing rate and cytoplasmic mole- temperature would bring the sucrose glass
cular mobility found for many different tis- above its Tc (Fig. 10.3), resulting in crystal-
sues over a wide range of temperatures and lization and loss of macromolecule function
Fast
Sucrose
Molecular mobility
Poly-L-lysine
Bean embryonic axis
Slow
–20 0 20 40 60 80 100 120
T – Tg (C)
Fig. 10.3. The effect of melting the glassy state on the molecular mobility of a guest molecule (spin probe)
incorporated in dry glasses composed of sucrose or poly-L-lysine, and in the intracellular glass of dried
embryonic axes of bean. Molecular mobility was measured by electron paramagnetic resonance
spectroscopy (Buitink et al., 2000e). The indicates the critical temperature, Tc, where the dynamics of the
system changed from solid-like to liquid-like (signified by an abrupt drop in viscosity).
and integrity (see Roos, 1995, for review). role in intracellular glass formation. Sun
Therefore, the characteristically high Tc of and Leopold (1993) found that the magni-
intracellular glasses serves as an ecological tude of the glassy signal and Tg, both mea-
and physiological advantage. sured by the thermally stimulated
depolarization current (TSDC) method,
decreased during accelerated ageing of soy-
10.5.4.2. Composition of glasses in
bean seeds. Yet no differences were
desiccation-tolerant organisms
observed in sucrose, raffinose and stachyose
When the concept of glasses was intro- contents during the same period of time.
duced in seed science, sugars were thought Despite different sugar compositions in soy-
to play an important role in the composi- bean axes compared with oak cotyledons,
tion of the glass. This assumption was their state diagrams are similar (Sun et al.,
based on the fact that sugars are present in 1994). Also, the state diagrams of immature
large amounts in desiccation-tolerant and mature soybean axes are similar,
tissues and are known to be excellent glass- despite the accumulation of oligosaccha-
formers. The correlation between oligosac- rides during maturation. Another indication
charides and longevity (Horbowicz and that sugars alone are not sufficient to
Obendorf, 1994; Bernal-Lugo and Leopold, explain the formation of the vitreous state in
1995; Steadman et al., 1996; Sun and seeds came from Sun and Leopold (1997),
Leopold, 1997) and the knowledge that who showed that the state diagram of maize
oligosaccharides increase Tg in model sys- embryos is different from that of a represen-
tems (Slade and Levine, 1991; Roos, 1995) tative carbohydrate mix. An extensive
added to the notion that sugars are impor- calorimetric study on the glass transition in
tant for in vivo glass formation. bean axes revealed the complexity of intra-
However, several reports do not support cellular glasses. Correspondence of differ-
the contention that sugars play a dominant ential scanning calorimetry (DSC) data
from beans with a model that predicts the Leprince et al., 1990a; Blackman et al.,
effects of glass components on Tg has sug- 1992) and/or galactosyl cyclitols
gested that intracellular glasses could be (Horbowicz and Obendorf, 1994; Obendorf,
composed of a highly complex oligomeric 1997). Also accumulating during the acqui-
sugar matrix, such as, for instance, malto- sition of desiccation tolerance are different
dextrin (Leprince and Walters-Vertucci, members of the LEA protein family (Ingram
1995). Buitink et al. (2000a,b) observed and Bartels, 1996). The current under-
that a change in sugar composition upon standing is that these molecules seem to
priming did not change Tg or the molecular act as stabilizing agents through one or
mobility in the intracellular glass. All these more different mechanisms. Furthermore, a
data suggest that, besides sugars, other role for HSPs in desiccation tolerance has
molecules play a crucial role in intracellu- recently been put forward (Wehmeyer and
lar glass formation. Vierling, 2000). The (putative) functions of
In this respect, the role of proteins in these protective molecules will be dis-
intracellular glass formation has received cussed in the following sections.
recent attention. Wolkers et al. (1998a,b)
have found that the molecular density (i.e.
hydrogen-bonding strength) of dry seeds of 10.6.1. Sucrose/oligosaccharides
A. thaliana was quite different from that of
a sugar glass, but much more comparable to The roles of sucrose and trehalose in pref-
that of a protein–sugar glass. Investigations erential exclusion, water replacement and
on the glass properties in biological sys- vitrification have been implicated as dis-
tems using EPR spectroscopy also point to cussed above. However, the general abun-
a role for proteins in intracellular glass for- dance of oligosaccharides in anhydrobiotic
mation (Buitink et al., 2000e). The temper- higher plant systems raises the question as
ature dependence of molecular mobility in to why they too are preferentially accumu-
intracellular glasses is much more compa- lated (see Chapters 1 and 5).
rable to that in protein glasses than that in The accumulation of oligosaccharides
sugar glasses (see Fig. 10.3). Although more and cyclitols during seed maturation and
research is needed to elucidate what types their disappearance during germination
of protein may play a role in the glass for- has led to the hypothesis that these sugars
mation, LEA proteins are possible candi- are important for desiccation tolerance.
dates (Sun and Leopold, 1997). Wolkers et However, it has been shown that desicca-
al. (1999) suggested that LEA proteins that tion tolerance in seeds can occur in the
are embedded in the glassy cellular matrix absence of oligosaccharides (Hoekstra et
confer stability on slowly dried carrot al., 1994; Lin and Huang, 1994; Bochicchio
somatic embryos. Indeed, LEA proteins et al., 1997; Black et al., 1999; Buitink et
change the hydrogen-bonding properties of al., 2000a). Moreover, the accumulation of
model sugar systems comparable to those oligosaccharides does not necessarily lead
of intracellular glasses, pointing to a possi- to the establishment of desiccation toler-
ble participation of LEA proteins in intra- ance (Still et al., 1994; Black et al., 1999).
cellular glass formation (Wolkers et al., Although oligosaccharides do not appear to
2000). be pivotal for desiccation tolerance, there
seems to be a significant correlation
between the oligosaccharide:sucrose ratio
10.6. Roles of Specific Compounds in and storage longevity of dry seeds
Stability (Horbowicz and Obendorf, 1994; Steadman
et al., 1996; Sun and Leopold, 1997). For
A specific feature of all anhydrobiotic example, Horbowicz and Obendorf (1994)
organisms is the accumulation of non- calculated that orthodox seeds of species
reducing sugars, particularly of the raffi- with a sucrose:oligosaccharide ratio of
nose series (Koster and Leopold, 1988; < 1.0 have storability half-viability periods
of > 10 years, whereas those with a ratio oligosaccharides accumulate during matu-
> 1.0 have storability half-viability periods ration, it could be possible that oligosac-
of < 10 years. None the less, there are charides are linked indirectly with storage
exceptions where there is no relation stability, being merely an indicator of seed
between the oligosaccharide content and maturity. Future research will have to
longevity (Steadman et al., 1996; Buitink et focus on roles for oligosaccharides in seed
al., 2000a), indicating that molecules other maturation other than cellular protection.
than oligosaccharides might have a similar
function and take over the role of these
sugars in their absence. 10.6.2. Late embryogenesis abundant
How oligosaccharides mediate the (LEA) proteins
increase in longevity is not yet resolved.
Because of their intrinsic high Tg it has Generally, the presence of LEA proteins
been suggested that they play a role in the correlates well with desiccation tolerance
protection of cytoplasmic components dur- (see Chapters 1, 5, 11). They accumulate
ing storage by elevating the Tg of the intra- during late maturation of developing seeds
cellular glass, thereby increasing viscosity (Galau et al., 1986; Bartels et al., 1988;
(see Section 10.5.4). Yet there is no evidence Baker et al., 1995; Blackman et al., 1995;
that oligosaccharides have an effect on the Ingram and Bartels, 1996; Kermode, 1997;
Tg or viscosity of intracellular glasses Oliver and Bewley, 1997; Cuming, 1999),
(Buitink et al., 2000a,b). Considering that in dehydrating vegetative tissues of the
oligosaccharides often make up only 4% of desiccation-tolerant grass Sporobolus stap-
the dry weight in seeds like Impatiens or fianus (Kuang et al., 1995), and in the res-
bell pepper (Buitink et al., 2000a), it is per- urrection plant C. plantagineum
haps unsurprising that no effect on intra- (Piatkowski et al., 1990). However, lea
cellular glass properties could be transcripts have also been detected in
measured. An obvious role for sugars in recalcitrant seeds and in desiccation-sensi-
seeds is the protection of macromolecular tive tissues submitted to water and/or tem-
structures in the dry state, especially mem- perature stress (Kermode, 1997). LEA
branes. Hydrogen bonding of sugar mole- proteins represent a broad class of highly
cules with macromolecules will increase conserved genes expressed in a wide range
their stability (see Crowe et al., 1998, for a of plants. Comparisons of the deduced
review; see Section 10.5.3). However, this polypeptide sequences of the various lea
explanation does not provide a clue to the genes have led to the establishment of five
role of oligosaccharides in particular. In subclasses of LEA proteins, simply desig-
fact, there are indications that oligosaccha- nated Group 1, Group 2 (dehydrins), Group
rides are less effective than disaccharides 3, Group 4 and Group 5 LEA proteins (see
at hydrogen bonding with the polar head Cuming, 1999, for a review, and Chapters
groups, particularly in the case of saturated 1, 5 and 11). Spatial patterns of LEA pro-
phospholipids (Crowe et al., 1986, 1996). tein accumulation indicate that they are
Also, oligosaccharides in seeds have been primarily localized in the cytosol and
suggested to be able to prevent crystalliza- nucleus (Asghar et al., 1994; Goday et al.,
tion. Notwithstanding the argument that 1994; Blackman et al., 1995; Egerton-
oligosaccharides are indeed capable of pre- Warburton et al., 1997). In the leaves of the
venting crystallization in model sugar resurrection plant C. plantagineum, LEA
glasses (Caffrey et al., 1988), this character- proteins were found to be both cytosolic
istic would most probably not be required and present in the chloroplasts of the
in vivo because of the complex mixture of leaves (Schneider et al., 1993).
all the different compounds in the cyto- Furthermore, the presence of LEA proteins
plasm, as mentioned before (see Section has been detected at the membranes of pro-
10.5.4). Because the maturation stage of tein and lipid bodies of Zea mays kernels
seeds correlates with storage stability, and (Egerton-Warburton et al., 1997) and at the
plasmalemma of Saccharomyces cerevisiae lar macromolecules, coating these macro-

(Sales et al., 2000). Whereas the molecular molecules with a cohesive water layer and
genetics of LEAs will be discussed else- preventing their coagulation during desic-
where (Chapter 11), this section will be cation (Section 10.5.1; Close, 1996). Upon
limited to the hypothetical mechanisms of removal of their own hydration shell, these
how LEA proteins act in stabilization and proteins would still be capable of playing a
protection during desiccation. role in stabilizing macromolecular struc-
The strongest support for a primary des- tures, as they could provide a layer of their
iccation-protecting role comes from the own hydroxylated residues to interact with
observation that the accumulation of the the surface groups of other proteins, acting
proteins coincides with the acquisition of as ‘replacement water’ (see Section 10.5.3;
desiccation tolerance. Bartels et al. (1988) Cuming, 1999). It has been shown that
demonstrated that desiccation tolerance purified maize dehydrin has potent cryo-
could be induced precociously in imma- protective activity in vitro in a rabbit lac-
ture barley embryos by the application of tate dehydrogenase freeze–thaw assay,
abscisic acid (ABA), coinciding with the especially in combination with solutes
accumulation of LEA proteins. Similarly, including compatible solutes such as
Blackman et al. (1995) used exogenous sucrose, proline and glycine-betaine (Close,
ABA to elevate the level of heat-soluble 1996). Rinne et al. (1999) suggested that, in
LEA-like proteins in axes from immature cold-acclimatized apices of birch, dehy-
seeds of soybean. As the LEA-like proteins drins might create local pools of water in
accumulated in response to ABA, solute otherwise dehydrated cells, thereby main-
leakage from the dried soybean embryos taining enzyme function. Under conditions
upon rehydration markedly declined. Both of low water activity (20% polyethylene
factors were apparently dependent on the glycol (PEG), corresponding to approxi-
presence of ABA. These data are consistent mately –0.5 MPa), the activity of -amylase
with the hypothesis that the LEA-like pro- was greater in the presence of a partially
teins contribute to the increase in desicca- purified dehydrin fraction than in the pres-
tion tolerance in response to ABA. ence of bovine serum albumin (BSA) as a
Since the earliest identification of LEA control (Rinne et al., 1999).
proteins, hypotheses regarding their func- For the Group 2 and 3 LEA proteins,
tions in desiccation tolerance have hypotheses concerning their biological
revolved around physiological and bio- function are based on their potential for
chemical experiments and predictions on amphipathic helix formation. It has been
the structure–function relationship that suggested that the amphipathic helices of
can be deduced from the amino acid the Group 3 LEA proteins could form bun-
sequence. The fact that LEA proteins accu- dles through hydrophobic interactions,
mulate to a much higher cellular concen- thereby exposing a highly charged surface
tration than is typically the case for to the exterior to which ions can bind
enzymes, together with their predicted (Dure, 1993). Thus, sequestration of ions
structural flexibility (of Group 2 and 3 LEA can take place, whose intracellular concen-
proteins), appears to rule out an enzymatic trations might otherwise become unaccept-
role. For the Group 1 LEA proteins, it has ably high within the dehydrating cell.
been calculated that these polypeptides The nuclear localization of dehydrins
have a tremendously high potential for raises the possibility of a dehydrin–
hydration, several times greater than that chromatin alliance or an association with
for ‘normal’ cellular proteins (McCubbin et the nuclear matrix, since the matrix is the
al., 1985). This is a function of the remark- chromatin-organizing structure (Nickerson
ably high number of charged and et al., 1989). In this regard, it is interesting
uncharged polar residues within the struc- to note that many dehydrins contain a tract
ture. Because of these specific properties, of serine residues (the S segment). In maize
LEA proteins potentially bind to intracellu- RAB17 and tomato TAS14, it has been
demonstrated that the serine residues in Savage et al., 1994). It seems that the sole
the S segment can be phosphorylated, and ability, or lack thereof, to express LEAs or
it has been proposed that phosphorylation dehydrin-like proteins cannot be taken as
is related to the binding of nuclear localiza- an indication that the seeds of a particular
tion signal peptides and, therefore, to species can or cannot withstand dehydra-
nuclear transport (Goday et al., 1994; tion, reflecting the earlier views of
Godoy et al., 1994). Because the matrix is Blackman et al. (1991, 1992) and Leprince
associated with regulatory processes, dehy- et al. (1993) that desiccation tolerance
drins may protect genetically sensitive must be the outcome of the interplay of
areas. As in the nucleus, nucleolar dehy- more than one (and probably many) mech-
drins may have a structural role via an anisms or processes.
association with the nucleolar filament
matrix, or a functional role in protecting
transcriptionally active regions (Godoy et 10.6.3. Heat-shock proteins (HSPs)
al., 1994).
It has also been speculated that LEA Another class of proteins that have recently
proteins might play a role in glass forma- been associated with desiccation tolerance
tion (Sun and Leopold, 1997; Wolkers et are the HSPs (see also Chapters 1 and 5). In
al., 1999; Buitink et al., 2000e). Wolkers et contrast to those of other eukaryotes, the
al. (1999) suggested that LEA proteins that most prominent HSPs of plants are small
are embedded in the glassy matrix might heat-shock proteins (sHSPs). They have
confer stability on slowly dried carrot monomeric molecular masses of 15–42
somatic embryos. Indeed, LEA proteins kDa, but assemble into oligomers of nine to
changed the hydrogen-bonding properties over 20 subunits, depending on the protein
of model sugar systems toward those of (Waters et al., 1996, and references
intracellular glasses, pointing to a possible therein). Their implication in desiccation
participation of LEA proteins in intracellu- tolerance has been inferred from studies on
lar glass formation (Wolkers et al., 2000; gene expression in developing seeds and in
see Section 10.5.4). resurrection plants. In pea, Arabidopsis
Despite numerous studies on the gene and sunflower seeds, sHSP expression is
expression of LEA proteins in plants, at always observed significantly before dis-
present, their biological function remains cernible seed desiccation, and sHSPs are
to be assessed in vivo. None the less, it abundant in the dry seeds (Coca et al.,
appears that, based on their observed local- 1994; DeRocher and Vierling, 1994;
ization at a number of sites, coupled with Wehmeyer et al., 1996). During germina-
their accumulation in response to desiccation, the developmentally regulated sHSPs
tion stress, dehydrins have a general role in are relatively abundant for the first few
protection during drought and/or desicca- days and then decline quickly. However,
tion stress. Recently, Black et al. (1999) the precise timing corresponding to the
found that, in wheat embryos, dehydrin acquisition and loss of desiccation toler-
accumulation is not regulated by factors ance was not assessed in the above studies.
that specifically control the induction of In vegetative tissues of C. plantagineum,
tolerance. Instead, it would appear that the constitutive expression of sHSPs has been
dehydrin-like protein is produced in detected (Alamillo et al., 1995).
response to grain detachment, even when Further evidence that HSPs may be impli-
this is not followed by dehydration, as was cated in desiccation tolerance comes from
also concluded for soybean (Blackman et the observation that there appears to be a
al., 1991). Remarkably, recalcitrant seeds coordinated expression of lea and sHSP tran-
were also found to accumulate dehydrins scripts during embryo development in
during seed development and in response response to ABA, indicating the existence of
to dehydration or to ABA treatment common regulatory elements of both LEA
(Bradford and Chandler, 1992; Finch- proteins, sHSPs and desiccation tolerance
(Almoguera and Jordano, 1992; Wehmeyer et HSPs are thought to offer a general protec-
al., 1996). Also, in desiccation-sensitive cal- tive role in dry anhydrobiotes, based on the
lus tissue of Craterostigma, no sHSP-related observation that HSPs are molecular chap-
polypeptides could be detected, but sHSP erones (i.e. they interact with other pro-
expression and the concurrent acquisition of teins and, in doing so, minimize the
desiccation tolerance in the callus were initi- probability that these other proteins will
ated by exogenous ABA treatment (Alamillo interact inappropriately with one another
et al., 1995). Recently, a reporter-gene tran- (Waters et al., 1996; Gething, 1997; Feder
scription assay showed that sHSP expression and Hofmann, 1999)).
exhibits little tissue specificity, but instead The function and working mechanisms
spreads throughout the embryo during of HSPs are well investigated in mam-
development until essentially all cells are malian systems, although direct evidence
stained in the mature seeds prior to com- originates only from in vitro studies (see
plete desiccation (Wehmeyer and Vierling, Feder and Hofmann, 1999, for a review). It
2000). The activity of the reporter gene was is currently known that HSPs recognize
strongly reduced in fus3-3, lec1-2 and was and bind to other proteins when these pro-
almost nil in abi3-6, all desiccation- teins are in non-native conformations,
intolerant mutants of Arabidopsis seeds. whether because of protein-denaturing
This would suggest an overall protective stress or because the peptides they com-
effect of HSPs in the cells during drying. As prise have not yet been fully synthesized,
for all protective mechanisms reviewed so folded, assembled or localized to an appro-
far, sHSPs may be necessary for desiccation priate cellular compartment. Binding
tolerance, but they are unlikely to be suffi- and/or release of these other proteins is
cient. often regulated by association with and/or
In contrast to other seeds, which typi- hydrolysis of nucleotides. Typically, HSPs
cally accumulate low to moderate levels of function as oligomers, or as complexes of
sHSPs, recalcitrant chestnut (Castanea several different chaperones, co-chaper-
sativa) seed cotyledons contain a highly ones, and/or nucleotide exchange factors.
abundant sHSP (Collada et al., 1997). This Interaction with chaperones is variously
sHSP was shown to exhibit molecular responsible for: (i) maintaining HSP part-
chaperone activity in vitro, as demon- ner proteins in a folding-competent, folded
strated by the ability of the sHSP to main- or unfolded state; (ii) organellar localiza-
tain soluble cytosolic proteins in their tion, import and/or export; (iii) minimizing
native formation during both heat and cold the aggregation of non-native proteins; and
stress (Soto et al., 1999). A model liposome (iv) targeting non-native or aggregated pro-
system with the encapsulated fluorescent teins for degradation and removal from the
dye calcein was used to investigate the pro- cell. Presumably, the last two functions are
tection of membranes by the LEA-like pro- the most important in coping with environ-
tein HSP 12 from S. cerevisiae during mental stress (Feder and Hofmann, 1999).
desiccation (Sales et al., 2000). This LEA-
like HSP was found to act in an analogous
manner to trehalose and protect liposomal 10.7. Conclusion and Outlook
membrane integrity against desiccation.
The interaction between HSP 12 and the From the above evidence amassed so far it is
liposomal membrane was judged to be clear that more than one mechanism acts in
electrostatic, as membrane protection was conferring desiccation tolerance on plant
only observed with positively charged lipo- organisms. Often, the involvement of a spe-
somes and not with either neutral or nega- cific substance in desiccation tolerance is
tively charged liposomes. difficult to establish, because different sub-
So far, there is no direct experimental stances may substitute for one another.
evidence that points to a specific role of Decreasing water potential appears to neces-
sHSPs in desiccation tolerance. Small sitate successive mechanisms of protection
during drying. In the early stages of dehy- as preferential hydration and enzymatic
dration, partitioning of amphiphiles into antioxidant activity ineffective. Then,
membranes might cause signalling of the immobilization in a glassy matrix via
stress, leading to a variety of responses. hydrogen bonding with water-replacing
Among these responses are the production substances gains importance. This prevents
of antioxidants, compatible solutes, dehy- excessive ordering, e.g. crystallization, and
dration proteins and, probably, sHSPs. protects the structure of macromolecules.
Whereas in seeds these substances are pro- There is an important role for sugars in this
duced as a part of the developmental pro- hydrogen-bonding process – from interac-
gramme, in vegetative plants that are tion with proteins to interaction with other
sensitive to complete dehydration these sugar molecules in the formation of a
responses often occur upon exposure to glassy matrix. Some of the dehydration
moderate levels of water loss (= drought). proteins may help improve the stability of
Since these substances appear to improve the glassy matrix. Glasses are considered as
tolerance to drought, it can be implied that particularly important in slowing molecu-
the mechanisms involved give protection lar mobility and chemical reaction rates.
under conditions where there is still bulk Consequently, they are important in
water left. Among these mechanisms could depressing the rate of ageing in the dry
be depression of metabolism, improved free- state.
radical scavenging and preferential hydra- The majority of investigations concern-
tion of macromolecules. Oxidative damage, ing anhydrobiosis in plants have been
membrane fusion and protein denaturation focused on phenomena in the dry state.
are thus prevented. It has to be realized that Considering the fact that desiccation-sensi-
it is often in these high water content tive organisms usually die at relatively
ranges, i.e. when bulk water is present, that high water contents of, for example, 2–0.5
desiccation-sensitive organisms die. g H2O g1 dw, future research should be
When most of the bulk water has disap- addressed more towards mechanisms of
peared, the interaction between molecules protection that operate in this particular
increases, which renders mechanisms such range of water contents.
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11 Molecular Genetics of Desiccation and

Tolerant Systems
Jonathan R. Phillips,1 Melvin J. Oliver2 and Dorothea Bartels3

1Max-Planck-Institute
for Plant Breeding Research, Carl-von-Linné-Weg 10,
D-550829 Köln, Germany; 2USDA-ARS Plant Stress and Germplasm
Development Unit, 3810 4th Street, Lubbock, Texas 79415, USA; 3Institute of
Botany, University of Bonn, Kirschallee 1, D-53115 Bonn, Germany
11.1. Definition of Desiccation Tolerance 320

11.2. Resurrection Plants: Definition and Distribution 320
11.3. Metabolic Changes During the Dehydration–Rehydration Cycle in
Resurrection Plants 321
11.4. Molecular Studies with Resurrection Plants 321
11.4.1. LEA proteins 323
11.4.2. Carbohydrate metabolism 324
11.5. Regulation of Gene Expression During the Desiccation Process in
Resurrection Plants 324
11.6. Desiccation-tolerant Bryophytes 326
11.7. Constitutive Cellular Protection 327
11.8. Cellular Damage and Recovery Following Rehydration 328
11.9. Gene Expression During Recovery 329
11.9.1. Rehydrins 330
11.10. Transgenic Approaches towards Improving Plant Dehydration/
Desiccation Tolerance 330
11.10.1. Compatible solutes or osmolytes 331
11.10.2. Oxygen-scavenging proteins 333
11.10.3. LEA proteins 334
11.10.4. Regulatory genes 334
11.11. Conclusions and Perspectives 335
320 J.R. Phillips et al.
11.1. Definition of Desiccation tive tissues during drying. By using differen-

Tolerance tial screening approaches, cDNAs corre-
sponding to transcripts expressed only in
Desiccation is the drying out of an organ- response to water deficit have been isolated
ism that is exposed to air. Under these con- and characterized (Ingram and Bartels,
ditions, most of the protoplasmic water is 1996; Bockel et al., 1998). However, it
lost and a very low amount of tightly should be emphasized that, due to the
bound water remains in the cell. purely descriptive nature of the data, the
Desiccation tolerance apparently depends functional role of many gene products is
on the ability of the cells to maintain the unclear. Using examples from resurrection
integrity of the cell membranes and to pre- plants and the moss Tortula ruralis, this
vent denaturation of proteins. Tolerance in chapter will review what is currently
organs such as seeds and pollen is wide- known about the molecular responses that
spread among higher plants and in fact par- occur during the acquisition of desiccation
tial desiccation is a prerequisite for the tolerance and how this knowledge may be
completion of the life cycle in most species applied to improve plant tolerance to limit-
producing seeds. In contrast, only a few ing water conditions.
plants possess mature foliage or vegetative
tissue that is desiccation-tolerant. These
include a small group of angiosperms, 11.2. Resurrection Plants: Definition
termed resurrection plants (Gaff, 1971), and Distribution
some ferns and fern allies (Bewley and
Krochko, 1982), and several species of Desiccation tolerance in vegetative tissues of
algae, lichens and bryophytes (Oliver, higher plants has been most studied in the
1996; Oliver and Bewley, 1997; Oliver et so-called resurrection plants, which possess
al., 2000; see Chapters 1 and 7). the unique ability to revive from an air-dried
Desiccation has to be distinguished from state (Gaff, 1971). Such plants are often
a mild water deficit, which is a condition poikilohydrous and their water content cor-
where the water status of plants undergoes relates with fluctuations in the relative
relatively small changes (Bray, 1997). Many humidity (RH) of the local environment.
plants are able to cope with this challenge Broadly two types of desiccation-tolerant
either by reducing water flux through the plants have been reported: those that lose
plant or by increasing their water uptake. chlorophyll during dehydration and those
Water loss can be avoided by various that retain chlorophyll (see Chapters 1
mechanisms such as stomatal closure, and 7).
reduction of leaf growth or production of Resurrection plants colonize ecological
specialized leaf surfaces to avoid transpira- niches with restricted seasonal water avail-
tion, whereas water uptake can only be ability. Most species are found preferen-
increased by the development of specialized tially on rock outcrops at low to moderate
root structures. Many of these mechanisms elevations below 2000 m in tropical and
are common to both desiccation-tolerant subtropical zones and to a lesser extent in
and non-tolerant plants; however, little is temperate climates (Porembski and
known about those mechanisms that, for Barthlott, 2000). The geographic locations
example, allow a resurrection plant to sur- where resurrection plants have been identi-
vive equilibrium with 0% air humidity. fied are southern Africa (including
Desiccation tolerance has been studied at Madagascar), Australia, India and parts of
the molecular level by examining tolerant South America (Gaff, 1977, 1987; Gaff and
systems such as seeds, resurrection plants Bole, 1986). Although rarely found in
and mosses. As a result of these studies, it Europe, a few species have been observed
has become apparent that tightly regulated in the western Balkan mountains (Stefanov
programmes of gene expression, both at the et al., 1992).
spatial and temporal levels, occur in vegeta- Desiccation-tolerant plants comprise
Molecular Genetics of Desiccation and Tolerant Systems 321
monocotyledonous and dicotyledonous physiological and morphological studies,

species within the angiosperms. To date, has been recently reviewed in some detail
however, no desiccation-tolerant plants (Oliver and Bewley, 1997; Hartung et al.,
have been reported that belong to the gym- 1998; Scott, 2000). The emphasis in this
nosperms. The desiccation-tolerant chapter will be on summarizing the results
dicotyledonous species appear to be repre- of biochemical and molecular studies.
sented mainly in the Gesneriaceae,
Myrothamnaceae and Scrophulariaceae
plant families, in contrast to their mono- 11.4. Molecular Studies with
cotyledonous counterparts, which are more Resurrection Plants
widely distributed throughout evolution.
Many of the described dicotyledonous res- To date, molecular studies of resurrection
urrection plants belong to the plants are limited to several species: the
Scrophulariaceae, which include ten dicotyledonous species Craterostigma
Craterostigma and 15 Lindernia species plantagineum (Bartels et al., 1990; Frank et
that are indigenous to Africa (Fischer, al., 2000), the monocotyledonous species
1992) (see Chapter 7). Sporobulus stapfianus (Neale et al., 2000)
The acquisition of desiccation tolerance and Xerophyta villosa (Mundree et al.,
in resurrection plants is complex. This is 2000), and the moss T. ruralis (Wood et al.,
due to the multiple stresses that are 1999). In C. plantagineum and S. stapfi-
imposed on plant tissues during severe anus the majority of changes in gene
dehydration. Consequently, tolerant plants expression occur during dehydration, not
must overcome several problems, including during the rehydration phase of the resur-
minimization of mechanical damage asso- rection process, which in turn leads to a
ciated with turgor loss, maintenance of the very efficient protection system against
functional integrity of macromolecules desiccation. This contrasts with what is
such as proteins and nucleic acids, mini- observed for T. ruralis and may be linked to
mization of toxin accumulation and free- differing mechanisms of desiccation toler-
radical damage, and initiation of repair ance that exist in vascular higher plants
mechanisms upon rehydration. The speed versus bryophytes.
of water loss and the events before dehy- Dehydration induces the expression of a
dration appear to be critical for the sur- large number of transcripts in both C. plan-
vival, such that, if the speed of dehydration tagineum and S. stapfianus (see Table
is too fast, plants do not acquire tolerance 11.1). Homology analyses reveal a broad
to desiccation. This observation suggests spectrum of differentially regulated genes
that the acquisition of desiccation toler- with diverse putative functional identities,
ance is an active process and requires spe- which underlines the fact that desiccation
cific biochemical changes and the tolerance is the result of a complex interac-
synthesis of desiccation-related molecules. tion of different cellular processes. It has
The nature of these molecules has recently been suggested that the dehydration-
been described for some species by molec- induced gene products can be associated
ular and biochemical studies. with signal transduction pathways and reg-
ulation of stress-specific transcription, with
carbohydrate metabolism or with cellular
11.3. Metabolic Changes During the protection (Phillips and Bartels, 2000).
Dehydration–Rehydration Cycle in Comparison between the dehydration-
Resurrection Plants induced genes identified in C. plan-
tagineum and S. stapfianus has revealed
Physiological, morphological, biochemical that several genes encode homologous pro-
and molecular studies have been per- teins and thus belong to the same func-
formed with several resurrection plants. tional group. These include gene products
This information, largely derived from with a putative protective function such as
Table 11.1. Desiccation-related genes from resurrection plants.
322
Dessication 11
Putative function Gene Origin Gene product Acc. No.
Signal transduction
Secondary messenger production via CpPLD-2 Craterostigma Phospholipase D AJ133000
phospholipid cleavage
18/3/02
Regulation of gene expression via CpPK1 Craterostigma Serine/threonine protein kinase AJ005373
phosphorylation/dephosphorylation SDG37c Sporobolus Serine/threonine phosphatase type 2C AJ242803
Transcriptional activator CpVP1 Craterostigma VP-1/ABI3 AJ000552
1:58 pm
Transcriptional regulators CpHB-1/2 Craterostigma Homoeodomain leucine zipper proteins AJ005820

AJ005833
CpM-7/10 Craterostigma myb-related proteins U33917
U33915
Translation initiation factor SDG134c Sporobolus eIF1 protein AJ242801
Page 322
Carbohydrate metabolism
Conversion of sucrose uridine-diphosphate CpSS-1/2 Craterostigma Sucrose synthase AJ131999
into fructose and UDP-glucose AJ132000
Synthesis of sugar phosphate intermediates CpTKT-7/10 Craterostigma Transketolase Z46648
Z46647
J.R. Phillips et al.
Synthesis of sucrose 6-phosphate from fructose CpSPS-1/2 Craterostigma Sucrose-phosphate synthase Y11821
6-phosphate and uridine 5’-diphosphate-glucose Y11795
Reversible oxidation and phosphorylation of CpGAPDH Craterostigma Cytosolic glyceraldehyde-3-phosphate dehydrogenase X78307
glyceraldehyde-phosphate to 1,3-bisphosphoglycerate
Cellular protection
Small molecule/water transporters CpPIP Craterostigma Major intrinsic proteins AJ001292
AJ001293
AJ001294
SDG50c Sporobolus AJ242805
Protection of macromolecular structures Cp6-19 Craterostigma Late embryogenesis abundant proteins X74067
Cp27-45 Craterostigma X69883
Protection of photosystem II DSP22 Craterostigma Early light inducible protein X66598
SDG69c Sporobolus AJ242806
late embryogenesis abundant (LEA) pro- tolerant and also in desiccation-sensitive

teins, the thylakoid membrane-associated plants (Close, 1997; Bartels, 1999; Cuming,
early light-induced protein (ELIP) or 1999; see Chapters 1, 5 and 10). LEA pro-
tonoplast intrinsic protein (TIP). Homology teins comprise a large family of plant pro-
also extends to cDNAs isolated from T. teins that accumulate to high levels during
ruralis (Wood et al., 1999). Expression pat- late stages of embryo development (Galau
terns of the isolated genes have been et al., 1986). Expression studies show that
described at the mRNA level. Three basic LEA proteins are generally associated with
patterns of gene expression are observed: cellular dehydration in seeds and in
class 1 transcripts accumulate to high lev- response to water deficit in vegetative tis-
els from an initial low level during dehy- sues. Treating plant tissues with the plant
dration and disappear during rehydration; hormone ABA can also induce the expres-
class 2 transcripts accumulate transiently sion of lea genes. A common feature of
during low-level dehydration; class 3 tran- most LEA proteins is their high
scripts are down-regulated during dehydra- hydrophilicity, which permits solubility
tion. Most of the class 1 transcripts and after boiling. Correlative studies and bio-
some of the class 2 transcripts also accu- chemical features strongly suggest a protec-
mulate following the application of exoge- tive role in the plant cell during
nous abscisic acid (ABA), which indicates dehydration.
a central role for ABA in mediating gene LEA proteins from different plant species
expression during dehydration. have been divided into groups based on pre-
It has been hypothesized that the tempo- dicted biochemical properties and sequence
ral and spatial expression patterns would similarities (Dure et al., 1989; Ingram and
provide information concerning the func- Bartels, 1996; Cuming, 1999). The strong
tion of a gene. Data on spatial expression conservation of motifs in LEA proteins dur-
patterns and subcellular localization as ing evolution points to domains with func-
investigated by RNA in situ hybridization tional constraints. One such motif, which is
and cell fractionation are only available characteristic for group 1 LEA proteins, is 20
from C. plantagineum. The analyses amino acids in length and was first found in
revealed that RNAs and proteins show a the wheat Em protein. Group 2 LEA pro-
specific tissue and cellular distribution teins, also referred to as dehydrins, are the
(Schneider et al., 1993). LEA-like proteins, most widely studied LEA proteins (Close,
which may have a general protective func- 1997). Many homologues have been isolated
tion, are found in most tissues and cell from species ranging from gymnosperms to
types, and accumulate in the cytoplasm or dicotyledonous and monocotyledonous
in chloroplasts. Water channel-like pro- angiosperms. Dehydrins are characterized by
teins, which are predicted to be involved a lysine-rich 15-amino-acid motif (termed
in water flux or transport of small mole- the K-segment), which is predicted to form
cules, are associated with membranes an amphipathic -helix, a tract of contiguous
(Mariaux et al., 1998). Transcripts encoding serine residues and a conserved motif con-
sucrose synthase are only detected in the taining the consensus sequence DEYGNP,
external phloem of vascular bundles, sug- which is found close to the N-
gesting an association with transport terminus of the protein. Group 3 LEA pro-
processes within the plant (Kleines et al., teins share a characteristic repeat motif of 11
1999). amino acids, which appears to have under-
gone duplication and some substitution
events. Dure et al. (1989) predicted that the
11.4.1. LEA proteins 11-amino-acid peptide forms an amphi-
pathic -helix with possibilities for intra-
LEA proteins represent one major group of and intermolecular interactions. In relation
proteins that are reported to be expressed to the others, LEA proteins belonging
in response to dehydration in desiccation- to groups 4 and 5 are less frequently
represented in the literature. Group 4 is function may be to protect the cell via glass
characterized by a conserved N-terminus formation rather than solutes crystallizing
predicted to form -helices and a diverse C- (see Chapter 10). Through the presence of
terminal part with a random coil structure. sugars, a supersaturated liquid is produced
Group 5 LEA proteins contain more at desiccation with the mechanical proper-
hydrophobic residues than groups 1 to 4 and ties of a solid. Secondly, sucrose may main-
consequently are not soluble after boiling, tain hydrogen bonds within and between
leading to the suggestion that they probably macromolecules and maintain the structure.
adopt a globular conformation. The lea genes This property has been shown in in vitro
from all five groups have been isolated and experiments for trehalose, a non-reducing
characterized at the molecular level from the disaccharide of glucose (-D-glucopyranosyl
resurrection plant C. plantagineum, and a (1-1) -D-glucopyranoside) (Crowe et al.,
remarkably high level of LEA proteins has 1992). Trehalose is found in desiccation-tol-
been found to accumulate in desiccated veg- erant lower organisms such as yeast or
etative tissues. This observation leads to the Selaginella, but only in small amounts in the
hypothesis that cellular desiccation toler- resurrection plants Myrothamnus flabelli-
ance depends on a relatively high concentra- folia and S. stapfianus (Bianchi et al., 1993;
tion of a number of different LEA proteins, Drennan et al., 1993; Albini et al., 1994).
which are simultaneously expressed in When the carbohydrate levels in hydrated
response to water deficit. To test this, a quan- and dehydrated tissues in resurrection
titative comparison of LEA protein accumu- plants are compared, it is clear that in some
lation in tolerant and sensitive vegetative species large qualitative and quantitative
tissues of two closely related species may be changes occur during the dehydration–
informative. rehydration cycle but in others only small
Despite extensive studies, biochemical differences are observed (see Table 11.2).
knowledge of the function of LEA proteins One well-documented change in drying
is scarce. Functional studies have used two leaves is the conversion of the highly abun-
approaches: in vitro protection assays with dant C8-sugar 2-octulose into sucrose,
purified proteins, and in vivo studies over- which comprises up to 40% of dry weight
expressing LEA proteins in plants or yeast. in desiccated leaves, as first reported for C.
Results from both types of experiments plantagineum (Bianchi et al., 1991a).
support a role for LEA proteins in the
acquisition of desiccation tolerance.
11.5. Regulation of Gene Expression
During the Desiccation Process in
11.4.2. Carbohydrate metabolism Resurrection Plants
In addition to de novo synthesis of proteins, Knowledge of regulatory pathways is of par-

major changes in carbohydrate metabolism ticular importance because they determine
take place during the resurrection process. the expression of a set of genes in a multi-
While the nature of dehydration-induced genic trait. Despite the large body of infor-
proteins is broadly similar among different mation concerning genes that are induced
resurrection species, the abundant carbo- during dehydration, knowledge of the regu-
hydrate molecules in the hydrated tissues latory network(s) is scarce. Most informa-
appear to be diverse. The accumulation of tion concerning the regulation of gene
sucrose in dehydrated tissues is, however, expression is derived from promoter analy-
a common theme (see Table 11.2). Taken ses and comes from desiccation-sensitive
together, resurrection plant species appear species, in particular Arabidopsis. Mutants
to possess different metabolic pathways have yet to be fully exploited as a potential
that result in the synthesis of sucrose. This tool for dissecting regulatory pathways.
supports a role for sucrose in desiccation This is mainly due to the facts that desicca-
tolerance, which may be twofold. One tion tolerance is a polygenic character and
Dessication 11
18/3/02
Table 11.2. Contents of abundant carbohydrates in hydrated and dehydrated leaves of some resurrection plants.
Sugar content (mol g1 dry weight)

1:58 pm
Species Hydrated leaves Dehydrated leaves Reference
Craterostigma plantagineum (Scorophulariaceae) Octulose 620 Sucrose 520 Bianchi et al., 1991a
Craterostigma lanceolatum (Scorophulariaceae) Octulose 1120 Sucrose 304 A. Richter and D. Bartels, unpublished data
Craterostigma hirsutum (Scorophulariaceae) Octulose 810 Sucrose 59 A. Richter and D. Bartels, unpublished data
Page 325
Lindernia acecularis (Scorophulariaceae) Octulose 373 Sucrose 198 A. Richter and D. Bartels, unpublished data
Lindernia brevidens (Scorophulariaceae) Octulose 914 Sucrose 195 A. Richter and D. Bartels, unpublished data
Myrothamnus flabellifolia (Myrothamnaceae) Glucose-glycerol 8a Glucose-glycerol 14a Bianchi et al., 1993
Trehalose 148 Trehalose 136 Drennan et al., 1993
Sucrose 383 Sucrose 463
Boea hygroscopica (Gesneriaceae) Sucrose 14a Sucrose 90a Bianchi et al., 1991b
Ramonda myconi (Gesneriaceae) Sucrose 96 Sucrose 204 Müller et al., 1997
Haberlea rhodopensis (Gesneriaceae) Sucrose 59.7 Sucrose 165 Müller et al., 1997
Sporobulus stapfianus (Poaceae) Sucrose 236 Sucrose 772 Albini et al., 1994
Trehalose 29 Trehalose 17.5
Molecular Genetics of Desiccation and Tolerant Systems
Oropetium thomaeum (Poaceae) Sucrose 116 Sucrose 156 A. Richter and D. Bartels, unpublished data
Xerophyta villosa (Velociaceae) Sucrose 43 Sucrose 83 Ghasempour et al., 1998
aThese values are expressed as % sugar in aqueous extracts.
325
that many resurrection plants are polyploid whereas pcC27-45 and pcC11-24 promoters
and consequently do not lend themselves are not active in vegetative tissues. The
to mutational studies. Here, regulation of ectopic expression of the Arabidopsis
gene expression in C. plantagineum will be ABI-3 gene product did, however, lead to
discussed with reference to the general ABA-inducible promoter activity in leaves
field, and some recent discoveries concern- of Arabidopsis, suggesting that the activity
ing Arabidopsis will be described later (see of transcription factors in the leaves of C.
Section 11.10). plantagineum is absent from the leaves of
The plant hormone ABA is associated Arabidopsis or tobacco (Furini et al., 1996).
with dehydration-regulated gene expres- The promoter analysis approach led to the
sion. Exposure to exogenous ABA causes identification of cis-elements that have
the induction of genes that otherwise are since been used to isolate DNA-binding
activated by dehydration. Mutants with proteins.
altered sensitivity to ABA or a modified Molecules with putative transcriptional-
ABA biosynthesis pathway provide evi- activating activities or putative signalling
dence for the role of ABA in mediating molecules have been obtained from differ-
gene expression in response to water ential screening experiments. These
deficit (Leung and Giraudat, 1998). It has, include members of the myb transcription
however, also become apparent that ABA- factor family (Iturriaga et al., 1996), a heat-
independent regulatory systems function shock transcription factor (Bockel et al.,
in gene expression under the same stress 1998), members of the homoeodomain
(Frank et al., 1998; Shinozaki and leucine zipper family (Frank et al., 1998),
Yamaguchi-Shinozaki, 2000). phospholipase D (Frank et al., 2000) and a
The regulation of gene expression by novel C. plantagineum gene cDT-1 (Furini
dehydration and ABA in vegetative tissues et al., 1997). Transcripts encoding these
of C. plantagineum involves several sig- molecules are induced by dehydration,
nalling pathways. Different types of cis- suggesting an involvement of the gene
acting elements are required for stress- products in the dehydration process. A dif-
responsive, coordinated gene expression. ficult challenge is to identify the target
Promoters from different groups of stress- genes of these putative regulatory mole-
inducible genes have been analysed and cules. The findings from C. plantagineum
compared. The most extensively studied are extended mainly by studies of the
promoters from C. plantagineum regulate response to dehydration in Arabidopsis,
the expression of three ABA-responsive which involves transcription factors
lea-like genes, pcC6-19, pcC27-45 and belonging to the Myb, Myc, AP2/EREBP
pcC11-24 (Michel et al., 1993, 1994; and bZip classes, protein kinases and pro-
Velasco et al., 1998). Despite the fact that tein phosphatases. Although the exact role
dehydration and ABA regulate all three of most factors in gene regulation is
genes, no common sequence motifs were unknown, it is interesting to note that
apparent in the promoter sequences. members of different regulator families are
Promoter analyses were performed in part of the regulatory dehydration network.
transgenic tobacco and Arabidopsis plants
to determine the functional cis elements.
All three promoters were found to be 11.6. Desiccation-tolerant Bryophytes
highly active in seeds and pollen.
However, the pcC6-19 promoter differs Desiccation-tolerant bryophytes are found
from pcC27-45 and pcC11-24 since no worldwide and inhabit a variety of habi-
protein synthesis is required for ABA- tats, most of which could, during some
mediated transcription. A second differ- period of the year, be considered as
ence is that the pcC6-19 promoter is extreme, either on a macro or micro level.
inducible by dehydration or ABA in vege- In most cases, the extremes that these
tative tissues of tobacco or Arabidopsis, plants experience are both in water avail-
ability and temperature (Clausen, 1952; Lee the first hour or two following rehydration.
and Stewart, 1971; Norr, 1974; Dilks and This has led to the suggestion that a major
Proctor, 1976; Alpert and Oechel, 1987; see component of the mechanism of desiccation
Chapter 7). tolerance in bryophytes is a rehydration-
Desiccation-tolerant bryophytes, because induced cellular repair response (see
of their simple architecture, have little or Bewley and Oliver, 1992; Oliver and
no morphological (or indeed physiological) Bewley, 1997, for reviews). The implication
characteristics or adaptations that can limit is that, although cellular protection and
water loss or regulate plant temperature. hence desiccation tolerance are constitutive,
As a result of this, the internal water con- it is not sufficient to prevent some damage
tent of their photosynthetic tissues rapidly from occurring (or being manifested) upon
equilibrates to the water potential of the rehydration, and thus repair processes are
environment once free water is lost from needed and induced when water returns to
the surface of the plant. This in turn means the protoplasm of the cells. Much of the evi-
that these plants experience drying rates dence for these hypotheses comes from the
that are much faster than those experi- study of a family of desiccation-tolerant
enced by their more complex pteridophyte mosses, the Tortula complex, and in partic-
or angiosperm counterparts. In fact, the ular the species T. ruralis (synonymous with
drying rates that desiccation-tolerant Syntrichia ruralis).
bryophytes experience are in the main
lethal to desiccation-tolerant ferns and
flowering plants (Bewley and Krochko, 11.7. Constitutive Cellular Protection
1982). The fact that bryophyte tissues
rapidly equilibrate to the water potential of Observational experiments confirm a pro-
the environment means that, in the major- tective component to the mechanism of
ity of cases where temperatures become desiccation tolerance in bryophytes.
extreme, hot or cold, these plants are dry. It Freeze–fracture studies of dried T. ruralis
is in the dried state that desiccation-toler- cells (both rapidly and slowly dried)
ant bryophytes (and most desiccation-toler- demonstrate that cellular integrity is
ant plants of the less complex clades) maintained during drying (Platt et al.,
tolerate temperature extremes (Norr, 1974; 1994) and plasma membranes, internal
Malek and Bewley, 1978). membranes and structures remain
The rapid equilibration of protoplasmic undamaged. Physiological experiments
water potential with that of the environ- designed to elucidate the effect of desic-
ment in bryophyte tissues appears to cation on photosynthesis suggest that the
demand a type of desiccation tolerance that protection mechanisms that are operating
is significantly different from that exhib- in Tortula are very effective in protecting
ited by the resurrection plants so far the photosynthetic apparatus and in
described (Oliver and Bewley, 1997). allowing for the rapid recovery of photo-
Rather than acquiring desiccation tolerance synthetic activity (Tuba et al., 1996;
in response to a dehydration event as seen Proctor et al., 1998; Csintalan et al., 1999;
in Craterostigma, Sporobolus and other Proctor, 2000). The almost instantaneous
desiccation-tolerant angiosperms, desicca- photosystem recovery and the relatively
tion-tolerant bryophytes appear to express short time needed (20 min) to reach a
this trait constitutively (Bewley and Oliver, positive carbon balance, as described by
1992; Oliver and Bewley, 1997). This form these authors, occur at a time when
of desiccation tolerance is considered the chloroplast structure is substantially dis-
most primitive of those that have received rupted (see below). How this is achieved
attention so far (Oliver et al., 2000). In this is enigmatic but ecologically it makes
type of tolerance, the major genetic response sense for these opportunistic bryophytes
to a desiccation event, at least at the level of selectively to protect their photosynthetic
gene expression, occurs after the fact, during capability.
Metabolically, desiccation of gameto- recently been reported. Protein analyses

phytic tissues of T. ruralis results in a rapid using purified antibodies raised against the
decline in protein synthesis, as in all desic- common C-terminus of maize seedling
cation-tolerant and sensitive mosses tested dehydrins (Close et al., 1993) show that T.
so far (see Bewley and Oliver, 1992; Oliver ruralis produces two major dehydrins
and Bewley, 1997, for reviews). In T. ruralis, (80–90 kDa and 35 kDa). These are present
this loss of protein synthetic capacity is in the hydrated state and do not appear to
manifested by a loss of polysomes resulting increase during rapid or slow drying
from the run-off of ribosomes from mRNAs, (Bewley et al., 1993). A similar result was
concomitant with their failure to reinitiate obtained with the desiccation-tolerant
protein synthesis (see Bewley, 1979; moss Thuidium delicatulum (T.L.
Bewley and Oliver, 1992, for reviews). The Reynolds, M.J. Oliver and J.D. Bewley,
rapid loss of polysomes during drying and unpublished data).
the apparent sensitivity of the initiation
step of protein synthesis to protoplasmic
drying lead to the conclusion that the pro- 11.8. Cellular Damage and Recovery
tection component of the mechanism of Following Rehydration
tolerance for these plants does not involve
the synthesis of proteins induced by the Following rehydration, gametophytic cells
onset of a water deficit. This is borne out of desiccation-tolerant mosses undergo
by the observation that no new mRNAs are substantial and universal disruption of cel-
recruited into the protein synthetic com- lular integrity including breaches of all
plex, even if the rate of water loss is slow membrane systems (see Oliver and Bewley,
(Oliver, 1991, 1996). The fact that the moss 1984a, for review). Internal organelles
survives rapid desiccation (even when des- swell and distort and their internal mem-
iccation is achieved in a few minutes in a brane systems become dispersed.
lyophilizer) also indicates that an Nevertheless, the cells do not die, as do
inducible protection mechanism is not cells of sensitive species, but return to a
necessary for survival. normal appearance within 12–24 h. The
As discussed above, LEA proteins and amount of cellular disruption that occurs
carbohydrates are important components of during rehydration clearly depends upon
protective mechanisms in desiccation- the rate at which water was lost during
tolerant plants and plant tissues. In desiccation. Chloroplasts of T. ruralis dried
desiccation-tolerant mosses, sucrose is the to air dryness over 4–6 h (a natural rate) are
only free sugar available for cellular protec- swollen when rehydrated but retain more
tion (Bewley et al., 1978; Smirnoff, 1992). of their normal internal structure and
The amount of this sugar in gametophytic exhibit fewer clefts in their membranes
cells of T. ruralis is approximately 10% of than do the chloroplasts in rehydrated cells
dry mass, which is sufficient to offer mem- of gametophytes dried within an hour
brane protection during drying, at least in (Tucker et al., 1975). The greater retention
vitro (Strauss and Hauser, 1986). Moreover, of chloroplast structure allows slow-dried
neither drying nor rehydration in the dark T. ruralis to effect a more rapid recovery of
or light results in a change in sucrose con- photosynthesis, achieving a positive car-
centration, suggesting that it is important bon balance within 20 min following rehy-
for cells to maintain sufficient amounts of dration (Bewley, 1979; Tuba et al., 1996).
this sugar (Bewley et al., 1978). The lack of The time required for full photosynthetic
an increase in soluble sugars during drying recovery upon rehydration, however, varies
appears to be a common feature of desicca- considerably among species, depending on
tion-tolerant mosses (Smirnoff, 1992). The their degree of desiccation tolerance
existence of dehydrin-type LEA proteins in (Proctor et al., 1998). Electrolyte leakage
desiccation-tolerant vegetative tissues of upon rehydration, a measure of membrane
desiccation-tolerant bryophytes has only damage, is also affected by the speed at
which desiccation occurs. After slow dry- in T. ruralis, Oliver (1991) demonstrated
ing, leakage in moss is less than half as that during the first 2 h of hydration the
great as after rapid desiccation and is simi- synthesis of 25 proteins is terminated, or
lar to leakage of hydrated controls, indicat- substantially decreased, and the synthesis
ing minimal membrane damage (Bewley of 74 proteins is initiated, or substantially
and Krochko, 1982). It is these observa- increased. Controls over changes in synthe-
tional studies that first led to the hypothesis of these two groups of proteins, the for-
sis that the mechanism for tolerance in mer termed hydrins and the latter
desiccation-tolerant bryophytes includes a rehydrins, are not mechanistically linked.
repair-based strategy to recover from the It takes a certain amount of prior water loss
damage manifested upon rehydration. to fully activate the synthesis of rehydrins
upon rehydration. This may in turn indi-
cate that there is also a mechanism by
11.9. Gene Expression During Recovery which the amount of water loss is ‘sensed’
and ‘translated’ into a protein synthetic
The repair aspect of the mechanism of des- response upon rehydration. Such a sce-
iccation tolerance in these plants, although nario was also proposed for the novel pat-
demonstrated to be a major component of tern of protein synthesis associated with
tolerance, is difficult to detail and charac- the drying of S. stapfianus (Kuang et al.,
terize. Most work has focused on the pro- 1995). Perhaps this is a strategy that has
teins whose synthesis is induced evolved to link the amount of energy
immediately upon rehydration of desic- expended in repair to the amount of dam-
cated gametophytic tissue. Early work (see age potentiated by differing extents of dry-
Bewley, 1979, for review) established the ing.
ability of T. ruralis and other mosses Since rehydrins appear to be synthe-
rapidly to recover synthetic metabolism sized from a transcript pool that is qualita-
when rehydrated. The speed of this recov- tively unaffected by desiccation or
ery was dependent upon the rate of prior rehydration (Oliver, 1991), it was of inter-
desiccation: the faster the rate of desicca- est to determine what are the translational
tion, the slower the recovery. In addition, controls of gene expression that operate
although the pattern of protein synthesis in during the initial phases of recovery in T.
the first 2 h of rehydration of T. ruralis is ruralis. A partial answer was gained from
distinctly different from that of hydrated the use of rehydrin cDNA clones isolated
controls, novel transcripts were not made from differential screening of a T. ruralis
in response to desiccation (Oliver and rehydration cDNA library (Scott and
Bewley, 1984b; Oliver, 1991). Hence it was Oliver, 1994). RNA blots revealed that sev-
suggested that T. ruralis responds to desic- eral rehydrin transcripts accumulate dur-
cation by an alteration in protein synthesis ing slow drying (Oliver and Wood, 1997;
upon rehydration that is in large measure Wood and Oliver, 1999) at a time when it is
the result of a change in translational con- assumed that transcriptional activity is
trol. Changes in transcriptional activity rapidly declining. These transcripts do not
were observed for nearly all transcripts accumulate during rapid desiccation, nor is
studied (Scott and Oliver, 1994) but did not their accumulation during slow drying
result in a qualitative change in the tran- associated with an increase in endogenous
script population during desiccation or ABA accumulation. ABA is undetectable in
rehydration. It thus appears that T. ruralis this moss (Bewley et al., 1993; M.J. Oliver,
relies more upon the activation of pre- unpublished data), and T. ruralis does not
existing repair mechanisms for desiccation synthesize specific proteins in response to
tolerance than it does on either pre-estab- applied ABA. The accumulation of these
lished or activated protection systems. transcripts was postulated to be the result
In a detailed study of the changes in of an increase in mRNA stability brought
protein synthesis initiated by rehydration about by the removal of water from the
cells (Scott and Oliver, 1994). Recent stud- protein contains 15 15-amino-acid repeats
ies clearly demonstrate that these tran- predicted to form amphipathic helices
scripts are sequestered in the dried (Velten and Oliver, 2001), which is very
gametophytes in messenger ribonucleopro- reminiscent of the predicted structural
tein particles (mRNPs) (Wood and Oliver, characteristics of group 3 LEA proteins
1999) and that this results in the change in (Dure et al., 1989). The interesting aspect
their stability. The implication from this of this protein is that it appears to be syn-
work is that the sequestration of mRNAs thesized during the rehydration event and
required for recovery hastens the repair of not at all during drying, indicating that
damage induced by desiccation or rehydra- LEA-like proteins may play a role in dam-
tion and thus minimizes the time needed age repair as well as cellular protection. It
to restart growth upon rehydration. These is also possible that some LEA proteins
findings may also explain the ability of T. play a role in protecting the cell from dam-
ruralis to ‘harden’ during recurring desic- age initiated during rehydration.
cation events in the absence of inducible More recently, Wood et al. (1999)
dehydrin or sugar responses (Schonbeck reported the establishment of a small
and Bewley, 1981a,b). Expressed Sequence Tag (EST) database
from a cDNA library constructed from
slow-dried gametophyte polysomal RNA
11.9.1. Rehydrins (in an attempt to target sequences
sequestered in mRNPs – see above). Of 152
Eighteen rehydrin cDNAs, isolated by Scott ESTs that were generated and partially
and Oliver (1994), have been sequenced sequenced, only 30% showed significant
(Oliver et al., 1997; Wood et al., 1999). homology to previously identified nucleic
Only three exhibit significant sequence acid and/or polypeptide sequences.
homology to known genes in the Genbank Interestingly, several ESTs showed signifi-
databases. Tr155 has a strong sequence cant similarity to unidentified desiccation-
similarity to an alkyl hydroperoxidase tolerance genes isolated from C.
linked to seed dormancy in barley (Aalen plantagineum (Bockel et al., 1998).
et al., 1994) and Arabidopsis embryos
(Haslekas et al., 1998), and in rehydrated
but dormant Bromus secalinas L. seeds 11.10. Transgenic Approaches towards
(Goldmark et al., 1992). Tr213 exhibits a Improving Plant Dehydration/
high degree of similarity to polyubiquitins Desiccation Tolerance
from several plant sources. The finding that
polyubiquitin is a rehydrin is indicative of The relevance of desiccation tolerance in
an increased need for protein turnover dur- determining productivity under moisture-
ing recovery from desiccation, an idea that limited environments is debatable as, agri-
is not new (Ingram and Bartels, 1996). In culturally, desiccation represents a small
Tortula there are three detectable ubiquitin proportion of the total instances of drought
transcripts, two appear to be constitutively (Subbarao et al., 1995). Furthermore, yield
expressed but the third is responsive to reduction due to water deficit becomes
desiccation and rehydration (O’Mahony important before desiccation occurs.
and Oliver, 1999). This is in contrast to However, improvement in yield in relation
Sporobolus, where only two classes of to limiting water supply is agronomically
ubiquitin transcripts are evident and both desirable. In environments where water
respond to desiccation and rehydration deficits can occur at any stage of growth,
(O’Mahony and Oliver, 1999). Tr288 has a knowledge of the mechanisms of desiccation
dehydrin-like K box sequence at the tolerance should play a role in survival of
C-terminus of the predicted protein but lit- the crop until soil moisture levels improve.
tle other sequence similarity to known Transgenic approaches offer a powerful
dehydrins. However, the predicted Tr288 means of gaining valuable information
towards a better understanding of the Overexpression of a gene encoding for

mechanisms that govern stress tolerance. moth bean P5CS in transgenic tobacco
They also open up new opportunities to plants resulted in accumulation of proline
improve stress tolerance by incorporating up to 10–18-fold over control plants and
genes involved in stress protection from better growth under dehydration stress
any source into agriculturally important (Kavi-Kishor et al., 1995). The transgenic
crop plants. To date, the ‘transgenic plants demonstrated enhanced biomass
approach’ has been to transfer a single gene production and flower development, as
into plants and then observe the pheno- determined by increased root length, root
typic and biochemical changes before and dry weight, capsule number and seed num-
after a specific stress treatment. A limita- ber per capsule. The same gene was also
tion of this strategy is that the functions of introduced into rice under the control of an
very few genes involved in desiccation tol- ABA-responsive promoter (Zhu et al.,
erance have been established, or not 1998). The transgenic plants accumulated
enough is known about the regulatory up to 2.5-fold more proline than control
mechanisms. Molecular marker analysis plants under stress conditions. This study
has been used to study dehydration toler- showed that the stress-inducible expres-
ance, principally in cereals (Quarrie, 1996). sion of the p5cs transgene in rice plants
Based on results from this type of resulted in an increase in biomass as
approach, it is mainly viewed that toler- reflected by higher fresh shoot and root
ance to water deficit is a complex quantita- weight under salt- and water-stress condi-
tive trait, since no single diagnostic marker tions compared with untransformed plants.
for tolerance has been found. However, As previously mentioned, trehalose
transgenic plants with improved tolerance accumulates in a large number of organ-
to water deficit have been produced using isms in response to different stress condi-
various genes (see Table 11.3) and will be tions. With the exception of two
described in the following text. resurrection plants (see Section 11.4.2.),
trehalose is generally not accumulated in
plants. However, genes for trehalose metab-
11.10.1. Compatible solutes or osmolytes olism have been identified in higher plants
and characterized by expression studies
Many plants respond to water deficit by and functional complementations of corre-
accumulating organic compounds of low sponding yeast mutants (Müller et al.,
molecular weight, known as compatible 1999). The data obtained from plants exter-
solutes or osmolytes. Therefore, engineer- nally supplied with trehalose or from
ing-increased osmolyte content in trans- transgenic plants expressing trehalose
genic plants is a rational strategy for biosynthesis genes from microorganisms
protecting plants against dehydration point towards a role for trehalose in dehy-
stress. Transgenic plants harbouring genes dration tolerance. For example, the yeast
encoding enzymes involved in the produc- trehalose 6-phosphate synthetase gene
tion of proline, fructans and trehalose (TPS1) was introduced into tobacco and
show a reduction of dehydration stress. the resulting trehalose-accumulating plants
Most of these studies have been carried out showed improved dehydration tolerance,
in tobacco and Arabidopsis because the although a decrease of 30–50% in growth
transformation technology is very well rate in conditions optimal for growth was
established; however, an improvement in also reported (Holmström et al., 1996). In a
stress tolerance has also been reported in second study, Pilon-Smits et al. (1998)
other species such as rice and sugar beet. introduced bacterial trehalose 6-phosphate
The enzyme 1-pyrroline-5-carboxylate synthase (otsA) and trehalose 6-phosphate
synthetase (P5CS) catalyses the conversion phosphatase (otsB) into tobacco. The leaves
of glutamate to pyrroline-5-carboxylate, of the transgenic plants were larger and
which is then reduced to proline. showed better growth, in terms of dry
332
Dessication 11
18/3/02
Table 11.3. Transgenic plants with altered tolerance to water deficit.
Gene Origin Gene product Host Tolerance phenotype Reference

1:58 pm
Adc Oat Arginine decarboxylase Rice Lower chlorophyll loss Capell et al., 1998
cdt-1 Craterostigma Regulatory RNA or short polypeptide Craterostigma Survival of callus without ABA pretreatment Furini et al., 1997
DREB1a Arabidopsis DRE-binding trans-factor Arabidopsis Higher survival rate Kasuga et al., 1999
HVA1 Barley Group 3 LEA protein Rice Higher growth rate and delay in damage Xu et al., 1996
Wheat symptoms Sivamani et al., 2000
Page 332
otsA Escherichia coli Trehalose-6-phosphate synthase Tobacco Higher photosynthetic rate and increased Pilon-Smits et al., 1998
otsB E. coli Trehalose-6-phosphate phosphatase Tobacco dry weight
p5cs Moth bean 1-pyrroline-5-carboxylate synthetase Tobacco Enhanced root biomass and flower Kavi-Kishor et al., 1995
Rice development Zhu et al., 1998
J.R. Phillips et al.
sacB Bacillus subtilis Fructosyl transferase Tobacco Higher growth rate Pilon-Smits et al., 1995
Fructosyl transferase Sugarbeet Pilon-Smits et al., 1999
TPS1 Saccharomyces Trehalose-6-phosphate synthetase Tobacco Increase in leaf survival Holmström et al., 1996
cerevisiae
IMT1 Mesembryanthemum Myo-inositol O-methyltransferase Tobacco Higher photosynthetic rate Sheveleva et al., 1997
crystallinum
Sod Nicotiana Manganese-superoxide dismutase Lucerne Higher survival rate McKersie et al., 1996
plumbaginifolia
weight, under dehydration stress. Detached transgenic plants was inhibited less during
leaves from young, well-watered transgenic dehydration and salt stress, and the plants
plants showed better capacity to retain recovered faster than wild type.
water when air-dried than the wild-type Furthermore, preconditioning of plants
plants. These transgenic plants also had expressing the imt1 cDNA in low-salt
more efficient photosynthetic activity. media increased D-ononitol amounts and
Although the trehalose protective effect is resulted in increased protection when the
unclear at the molecular level, correlative plants were stressed subsequently with a
evidence suggests that trehalose stabilizes higher salt concentration. Unlike solutes
proteins and membrane structures under such as proline and sucrose, D-ononitol
stress (Colaco et al., 1995; Iwahashi et al., levels did not show significant diurnal
1995). fluctuations This led the authors to suggest
Fructans are polyfructose molecules that that stress-inducible solute accumulation
are produced by many plants and bacteria. may provide better protection under
Owing to their high solubility, they may drought conditions than do strategies using
help plants survive periods of osmotic osmotic adjustment by metabolites that are
stress. Pilon-Smits et al. (1995) introduced constitutively present.
a gene encoding a bacterial fructan syn- Polyamines are small nitrogenous cellu-
thase (sacB) isolated from Bacillus subtilis lar compounds that have being implicated
into tobacco. Under unstressed conditions, in a variety of stress responses in plants.
the presence of fructans had no significant Polyamines accumulate under several abi-
effect on growth rate and yield. The trans- otic stress conditions including drought.
genic plants performed significantly better Cultivars demonstrating a higher degree of
under osmotic stress than wild-type salt tolerance contain higher levels of
tobacco, and the stress resistance correlated polyamines. Furthermore, exogenous
with the amount of fructan accumulated. application of polyamines results in pro-
The same sacB gene was introduced into tection against osmotic stress. Transgenic
sugarbeet to produce bacterial fructans rice cell lines and plants have been pro-
(Pilon-Smits et al., 1999). The transgenic duced that express an oat arginine decar-
sugarbeets accumulated fructans to low lev- boxylase cDNA, the gene product of which
els in both roots and shoots. Two indepen- converts ornithine to the diamine
dent transgenic lines of fructan-producing putrescine, under the control of the cauli-
sugarbeets showed significantly better flower mosaic virus (CaMV) 35S promoter
growth under dehydration stress than did (Capell et al., 1998). A four- to sevenfold
untransformed beets. Dehydration-stressed increase in arginine decarboxylase activity
fructan-producing plants attained higher was observed in transformed plants com-
total dry weights than wild-type sugarbeet, pared with wild-type controls. Biochemical
due to higher biomass production of analysis of cellular polyamines indicated
leaves, storage roots and fibrous roots. up to a fourfold increase in putrescine lev-
Again, no significant differences were els in transgenic plants. Although the
observed between the transgenic and wild- plants had improved drought tolerance in
type plants under well-watered conditions. terms of chlorophyll loss under drought
The introduction of fructan biosynthesis in stress, constitutive expression of this gene
transgenic plants is therefore a promising slowed down growth severely.
approach to improving crop productivity
under dehydration stress.
Expression of a cDNA encoding myo- 11.10.2. Oxygen-scavenging proteins
inositol O-methyltransferase (imt1) in
tobacco during salt and dehydration stress One consequence of dehydration and many
resulted in the accumulation of methylated other stresses is the production of activated
inositol D-ononitol (Sheveleva et al., 1997). oxygen molecules that cause cellular injury
Photosynthetic carbon dioxide fixation in (see Chapters 9 and 10). The protection of
sensitive metabolic reactions by stabiliza- expressing transgenic and non-transgenic

tion of protein complexes or membrane controls under moderate water-deficit con-
structures by increasing the capacity for ditions. The two homozygous transgenic
hydroxyl radical scavenging in plants should plant lines also had significantly greater
provoke improved performances under non- total dry mass, root fresh and dry weights,
lethal stress conditions. Transgenic lucerne and shoot dry weight compared with the
expressing Mn-superoxide dismutase cDNA two controls under soil water-deficit condi-
tended to have reduced injury from water- tions. As is the case for all LEAs, the pre-
deficit stress as determined by chlorophyll cise mode of action of HVA1 under drought
fluorescence, electrolyte leakage and conditions remains unclear.
regrowth from crowns (McKersie et al., In contrast, attempts to introduce three
1996). A 3-year field trial indicated that lea-like genes from the resurrection plant
both yield and survival of transgenic plants C. plantagineum into tobacco did not
was significantly improved, supporting the result in a drought-tolerant phenotype
hypothesis that tolerance of oxidative (Iturriaga et al., 1992). However, this result
stress is important in adaptation to field is perhaps less surprising considering that
environments. drought stress does induce an array of dif-
ferent LEA-related proteins in plants (see
Section 11.4.1). It is also likely that other
11.10.3. LEA proteins factors are required for the expression of
tolerance where LEA-type proteins are
A barley group 3 LEA protein termed involved.
HVA1 is specifically expressed in the aleu-
rone layer and the embryo during the late
stages of seed development, correlating 11.10.4. Regulatory genes
with the acquisition of seed desiccation
tolerance. ABA and several stress condi- The machinery leading to the expression of
tions including dehydration also induce dehydration-responsive genes is expected
HVA1 expression in young seedlings. Xu et to conform to a general cellular model. In
al. (1996) produced transgenic rice plants general, signal transduction cascades can
expressing the barley HVA1 gene, driven be divided into the following basic steps:
by a constitutive promoter. This led to the perception of stimulus; processing, includ-
constitutive accumulation of HVA1 protein ing amplification and integration of the sig-
in both leaves and roots of transgenic rice nal; and a response reaction in the form of
plants. The second-generation transgenic de novo gene expression. As mentioned
rice plants showed increased tolerance to earlier (see Section 11.5), studies of dehy-
water deficit and salinity. In a second dration-activated signalling cascades have
study, HVA1 was introduced into spring resulted in the identification of potential
wheat (Sivamani et al., 2000). High levels regulatory genes, such as transcription fac-
of expression of the HVA1 gene, regulated tors. The transformation of plants using
by a maize ubiquitin promoter, were regulatory genes is an attractive approach
observed in leaves and roots of indepen- for producing dehydration-tolerant plants.
dent transgenic wheat plants. Progenies of Since the products of these genes regulate
four selected transgenic wheat lines were gene expression and signal transduction
tested under greenhouse conditions for tol- under stress conditions, the overexpression
erance of soil water deficit. Potted plants of these genes can activate the expression
were grown under moderate water deficit of many stress-tolerance genes simultane-
and well-watered conditions, respectively. ously.
Two homozygous and one heterozygous Expression of many Arabidopsis dehy-
transgenic lines expressing the HVA1 gene dration-responsive and cold-regulated
had significantly higher water-use effi- (COR) genes is mediated by a DNA regula-
ciency values as compared with the non- tory element termed the dehydration-
responsive element/C-repeat (DRE/CRT) contrast, calluses transformed with control

(Yamaguchi-Shinozaki and Shinozaki, vectors did not survive dehydration. The
1994). A major breakthrough was made desiccation-tolerant transformants accumu-
when a transcriptional activator, CBF1, that lated anthocyanins and their phenotype
binds to the DRE/CRT was identified was indistinguishable from the original
(Stockinger et al., 1997). In a second report transfer DNA (T-DNA)-tagged mutant line.
it was demonstrated that overexpression of RNA hybridization analysis confirmed that
CBF1 in transgenic Arabidopsis plants at the desiccation-tolerant transformants con-
non-acclimatizing temperatures induces tained high levels of the 0.9 kb transcript
COR gene expression and increases plant and constitutively expressed the ABA-
freezing tolerance (Jaglo-Ottosen et al., responsive marker genes. This regulatory
1998). More recently, Kasuga et al. (1999) gene has a unique structure, but does share
transformed Arabidopsis with a cDNA some features with mammalian retrotrans-
encoding DREB1a, a homologue of CBF1, posons. The function of the cDT-1 gene
driven by either the constitutive CaMV 35S product is not immediately obvious
promoter or an abiotic stress-inducible pro- because it encodes a transcript with no
moter. The overexpression of this gene acti- large open reading frame. It is possible that
vated the expression of many stress- the biologically active product of cDT-1 is a
tolerance genes such as lea genes and P5CS. regulatory RNA or a short polypeptide.
In all cases, the transgenic plants were
more tolerant to drought, salt and freezing
stresses. However, the constitutive overex- 11.11. Conclusions and Perspectives
pression of DREB1a also resulted in severe
growth retardation under normal growth Molecular analyses of desiccation-tolerant
conditions. In contrast, the stress-inducible systems use a variety of strategies and
expression of this gene had minimal effects involve different plant species. Initial
on plant growth and provided greater toler- research has been largely descriptive and
ance to stress conditions than genes driven many genes have been isolated that play a
by a strong constitutive promoter. potential role in desiccation tolerance.
Although C. plantagineum can tolerate Furthermore, major themes in the molecu-
extreme dehydration, in vitro-propagated lar response have been established such as
callus derived from this plant has a strict changes in sugar metabolism and the
requirement for exogenously applied ABA expression of lea genes. Studies have
in order to survive a severe dehydration. begun to examine mechanisms that control
This property has been exploited for isola- gene expression and regulatory pathways
tion of dominant mutants by activation tag- are being established. Attempts to under-
ging, in which high expression of resident stand gene function have used transgenic
genes activated by insertion of a foreign plants, the results of which are of clear
promoter would confer desiccation toler- biotechnological importance.
ance to the transformed cells without prior In order to address further the question
ABA treatments. One gene was identified of gene function and ultimately to under-
(cDT-1), whose high expression did confer stand the molecular basis of desiccation
the expected phenotype in calli and led to tolerance, other experimental approaches
constitutive expression of several ABA- are required. One strategy is the develop-
and dehydration-inducible genes (Furini et ment of a genetic model system to study
al., 1997). In a second experiment, trans- desiccation tolerance in vegetative tissue.
genic calluses that constitutively express Insertional mutagenesis via T-DNA or
cDT-1 under the control of two different transposon tagging could then be employed
promoters were produced. When calluses – both are proven methods to deduce the
from both lines were dehydrated, transfor- function of genes in genetic model systems
mants from both lines were able to with- such as Arabidopsis. Secondly, natural
stand desiccation in the absence of ABA. In allelic variation has proved successful for
identifying genes involved in plant devel- habit can occur via minor gene alterations
opment (Swarup et al., 1999). Quantitative (Doebley and Stec, 1991).
trait locus (QTL) analysis of plant acces- The existence of ‘common genomes’
sions that exhibit extensive variation for suggests that genes required for any path-
desiccation tolerance may be a means of way are present in all plant species.
identifying genes in complex regulatory However, the genes involved in pathways
networks. may differ in their spatial expression pat-
In mosses, where desiccation-tolerant terns and locations, due to minor changes
tissues are haploid, the possible use of in regulatory regions. For example, differ-
gene replacement, either directed or by ences in tissue specificity and/or control of
random tagging, utilizing efficient homolo- gene expression among members of a tran-
gous recombination techniques, offers a scription factor gene family in desiccation-
novel and powerful technology for func- tolerant and desiccation-sensitive species
tional gene analysis (Puchta, 1998; Reski, may account for differences in desiccation
1999). tolerance. Therefore, it may be possible to
Comparative mapping studies have alter significantly multigenic traits such as
demonstrated that closely related plant desiccation/dehydration tolerance, by the
species have highly conserved gene con- identification and transfer of single genes
tent and chromosomal positions of genes that account for the physiological differ-
are also maintained, even though chromo- ence between the species.
somal rearrangements differ. The similarity
in gene content of, for example, grasses
indicates that genes are rarely created 11.12. Acknowledgements
within individual species and variation
between species is likely to arise from gene The work in the laboratory of D. Bartels
duplication and/or minor sequence modifi- was supported by the DFG Schwerpunkt
cations of existing genes (Bennetzen and ‘Molekulare Analyse der Phytohormon-
Freeling, 1993). This hypothesis is sup- wirkung’. We thank A. Richter, Vienna, for
ported by evidence that major modifica- communicating data on sugar analysis
tions in plant morphology and growth before publication.
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12 Rehydration of Dried Systems:

Membranes and the Nuclear Genome
Daphne J. Osborne,1 Ivan Boubriak1 and Olivier Leprince2

1TheOxford Research Unit, Open University, Foxcombe Hall, Boars Hill OX1 5HR,
UK; 2UMR Physiologie Moléculaire des Semences, Institut National d’Horticulture,
16 Bd Lavoisier, F49045 Angers, France

12.2. The Dangers of Rehydration and Membrane Changes 344
12.2.1. Factors governing imbibitional injury 344
12.2.2. Causes for solute leakage as mechanisms of imbibitional
injury: the fate of plasma membranes during rehydration 346
12.2.2.1. Conformational properties of plasma membranes 346
12.2.2.2. Mechanical stress 347
12.2.3. Protections against imbibitional injury 347
12.2.3.1. Seed coat 347
12.2.3.2. Protection at the molecular level 349
12.3. Maintaining Integrity of the Genome 350
12.3.1. Hydration-determined changes in DNA 350
12.3.2. Seeds 350
12.3.2.1. Survival after rehydration reflects the dry state
experience 351
12.3.2.2. First events of seed rehydration 352
12.3.2.3. Partial rehydration: priming 353
12.3.2.4. The recalcitrant seeds 354
12.3.3. Pollen 354
12.3.3.1. First events of pollen rehydration 355
12.3.4. Whole plants 356
12.3.5. Requirements for successful rehydration of a genome 356
344 D.J. Osborne et al.
12.1. Introduction erance has not always been fully appreci-

ated. For example, Kovach and Bradford
The period of water uptake by a dry anhy- (1992) showed that the loss of viability in
drobiote is one of hazard and sensitivity to wild rice was due to imbibitional damage
stresses. Imbibitional injury can occur in a that had previously been interpreted as
wide range of desiccated organisms includ- desiccation intolerance. A similar conclu-
ing seeds (Vertucci, 1989; Hoekstra and sion was reached by Ellis et al. (1990) with
Golovina, 1999), pollens (Hoekstra et al., pea seeds and by Sacandé et al. (1998) with
1992, 1999), algae, mosses and ferns seeds of neem, a tropical recalcitrant
(Bewley, 1979), and yeasts (van Stevenick species, which exhibits an orthodox behav-
and Ledeboer, 1974). iour when stored seeds are rehydrated
The embryos of most seeds, certain pol- above 20–25°C. Vegetative anhydrobiotic
lens and spores, many lower plants and a tissues (mosses, lichens and resurrection
diverse, but limited, selection of whole plants) also appear to be sensitive to the
vascular plants – the so-called resurrection rehydration phase (Oliver et al., 1997,
plants (Oliver et al., 1998) – have devel- 1998; Quartacci et al., 1997).
oped remarkable mechanisms to permit Essential components for this mainte-
water loss without irreparable damage to nance of cellular integrity throughout the
their cytoplasmic integrity and without processes of drying and rehydration are the
imperilling their future survival. But those structural organization and composition of
cell types that successfully pass through intracellular membranes and the fidelity
the hazard of dehydration must undergo and conformation of the DNA in nuclear
preparation for this event ahead of time, chromatin and probably also that in mito-
and it is critical to the success of dehydra- chondria and plastids.
tion and to the subsequent orderly accom- This chapter addresses what is currently
plishment of rehydration that both cellular known and speculated upon for these two
compartmentation and genetic information major determining factors in cell survival
are maintained unimpaired. The restora- under the temporal and successive condi-
tion of cytoplasmic organization and functions of changing water availability.
tion within minutes of water being
readmitted to these same dehydrated cells
remains to be properly understood, partic- 12.2. The Dangers of Rehydration and
ularly since all dry tissues show some leak- Membrane Changes
age of cytoplasmic contents on their first
impact with free water. 12.2.1. Factors governing imbibitional injury
Several important crops, particularly
those of tropical and subtropical origins, The sensitivity of seeds and pollen to imbi-
are known to suffer when the seeds take up bitional stress is controlled by at least three
water at chilling temperatures. These crops factors: the initial moisture content of the
include soybean, maize, sorghum, cotton, tissues, the temperature of the imbibition
bean, cowpea and rice. Injury may also medium and the rate of water uptake.
occur at room temperature in species such Chilling temperatures, very low moisture
as pea and Brassica sp. when the seeds are contents and rapid water uptake generally
very dry before imbibition. Upon imbibi- result in greater injury (Fig. 12.1). Slowing
tional stress, dry anhydrobiotes leak down the rate of water uptake by soaking
solutes and macromolecules, leading to seeds in polyethylene glycol solutions
faulty metabolism, loss of cellular integrity improves the quality of seedlings of vari-
and/or infection by opportunistic ous species (Woodstock and Taylorson,
pathogens, which consequently lead to the 1981; Vertucci and Leopold, 1983; Priestley
death of the tissues. Despite its widespread and Leopold, 1986; Chern and Sung, 1991).
occurrence, the impact of imbibitional While the low water potential of dry anhy-
stress on the expression of desiccation tol- drobiotes is the driving force for imbibi-
Rehydration of Dried Systems 345
100
25C
80
% Germinated seeds
60
5C
40
20
0
0 5 10 15 20 25
Initial water content (% dw)
Fig. 12.1. The effect of initial water content of axes on the final percentage of germination of cowpea
(Vigna unguiculata). After equilibration at various relative humidities, seeds were soaked in water at 5°C ()
or 25°C () for 1 h, then rolled in filter papers and incubated at 20°C for 7 days. At both temperatures, there
is a threshold water content below which seeds suffer from imbibitional injury. Least-squares linear
regressions were determined for the data below the threshold water content. Water contents are expressed
on a dry weight (dw) basis.
tion, the permeability of the seed regulates ture content at which the seed is most sen-
the rate of water uptake. The permeability sitive to imbibition (Vertucci and Leopold,
of seed tissues to water is a complex func- 1983). Wolk et al. (1989) suggested that the
tion of the seed morphology, structure, wettability depends on the water-binding
composition and water content. It is note- characteristics of the dry tissues. In dry tis-
worthy that, during ageing, seeds and sues a glassy state is presumed to prevail
pollen become increasingly sensitive to (Chapter 10), which has certain peculiar
imbibition (Woodstock and Taylorson, physical properties. Whether the condition
1981; Bruggink et al., 1991; van Bilsen et of the glassy state plays a role in the initial
al., 1994; Sacandé et al., 1998). wettability of the tissues remains to be
Experimental evidence (Vertucci and ascertained. Large increases in seed vol-
Leopold, 1983; Wolk et al., 1989) and theo- ume are usually observed during imbibi-
retical considerations about water penetration (Vertucci, 1989), and these changes in
tion in dry materials (reviewed by Vertucci, volume have been interpreted as an unfold-
1989) suggest that the water uptake occurs ing of biopolymers of unknown nature as
in two phases: an initial wetting phase and the seed takes up water (Vertucci, 1989).
a subsequent hydraulic flow. It has been This author calculated that the difference
suggested that for seeds imbibitional dam- between the rate of water uptake and rate
age is imposed by the initial wetting of volume change during imbibition is
because the moisture content at which this greatest at the time when the seed is most
phase is observed corresponds to the mois- sensitive to imbibitional damage, suggest-
ing that structural factors may contribute to rehydration, a transition from a gel to a
imbibitional damage. It is likely that the liquid phase occurs under those conditions
volume increase during imbibition also of temperature and moisture content that
interferes with the wettability of the tissues promote solute leakage and loss of viability
(Vertucci and Leopold, 1983). upon imbibition (Hoekstra et al., 1992,
The considerable volume of literature on 1999; Hoekstra and Golovina, 1999). Earlier
imbibitional injury points to plasma mem- experiments on liposomes had shown that
branes as the target of the rehydration during a thermotropic phase transition
stress. Indeed, imbibitional injury interferes from gel to liquid crystalline, membranes
with the rapid re-establishment of mem- become leaky (Crowe et al., 1989). Leakage
branes, as has been shown from the exten- is thought to occur because of the struc-
sive leakage of cytoplasmic solutes and the tural defects at the boundary between the
disorganization and ruptured appearance of coexisting phases. Analogous to model sys-
membranes following rehydration tems, it was suggested that the transiently
(Vertucci, 1989; Bedi and Basra, 1993; coexisting phases allow leakage of solutes
Hoekstra et al., 1999). One can argue that on the penetration of liquid water in the
plasma membranes are the first macromole- dry plant tissues (Crowe et al., 1989;
cular structures to be affected during imbi- Hoekstra et al., 1992). This hypothesis
bition because solute leakage may occur received support from pollen experiments
within a few seconds to minutes, which is showing that treatments prior to imbibition
before the resumption of metabolism. that reduce leakage (such as exposure to
humid air or an increase in soaking tem-
perature) also promote the return of the
12.2.2. Causes for solute leakage as phospholipid bilayer to the liquid-
mechanisms of imbibitional injury: the fate of crystalline state (Crowe et al., 1992;
plasma membranes during rehydration Hoekstra et al., 1992). Impatiens pollen is
comparatively tolerant to imbibition at low
In the past, several hypotheses have been temperature (Hoekstra and Golovina,
promoted to explain the mechanisms of 1999). It appears that the phase transition
leakage during the imbibition of seeds and temperature of plasma membranes in dry
pollen. Successive hypotheses (e.g. the dis- Impatiens pollen is very low, suggesting
ruption of membranes in the dry state, or that a gel phase barely forms, thus prevent-
the formation of hexagonal phases), which ing leakage during imbibition. Thus, these
first prevailed and were then abandoned, data would imply that the transition tem-
have been recently reviewed by Crowe et perature (Tm) at which membranes undergo
al. (1997), Hoekstra and Golovina (1999) a phase change can determine imbibitional
and Hoekstra et al. (1999). Our current injury.
understanding points to two causes that Recent evidence, however, now suggests
lead to solute leakage during rehydration that changes in membrane phase per se are
as a result of imbibitional injury: the physi- insufficient to explain the permanent loss of
cal and/or conformational properties of membrane integrity in rehydrating tissues.
membranes in the dry state (see Chapter 9; In Typha latifolia pollen, the permeability
Crowe et al., 1992) and mechanical stresses after a membrane-phase change during rehy-
imposed during rehydration (Spaeth, 1987; dration appears to exhibit both a transient
Vertucci, 1989; Hoekstra et al., 1999). and permanent character (Hoekstra et al.,
1999). Using an electron paramagnetic reso-
nance (EPR) spin-probe technique that per-
12.2.2.1. Conformational properties of
mits a detailed analysis of the kinetics of
plasma membranes
leakage during imbibition of pollen, it was
Imbibitional injury in anhydrobiotes has shown that plasma membranes are highly
been linked to the occurrence of mem- permeable within the first 10 s of imbibi-
branes in gel phase in the dry state. During tion. Furthermore, this permeability persists
in conditions that permit plasma mem- membranes of dry mosses, pollen and seeds
branes to pass through a phase transition appear intact (Platt et al., 1994, 1997;
(Hoekstra et al., 1999). To explain this obser- Hoekstra et al., 1999; Claessens et al., 2000).
vation, another mechanism leading to
increased permeability has been proposed.
12.2.2.2. Mechanical stress
Golovina et al. (1998) showed that amphi-
pathic compounds such as flavonoids, Whether the ultrastructural damage to
which are present in the cytoplasm, parti- membranes upon imbibition results from
tion into the lipid phase during drying and mechanical stresses and/or changes in
vice versa during rehydration. Endogenous physical properties of the membrane phase
amphipathic compounds extracted from remains to be fully ascertained. Several
various dry anhydrobiotes were found to lines of evidence suggest that membranes
fluidize membranes isolated from these will undergo considerable mechanical
organisms and also from prepared lipo- stress during imbibition. Vertucci (1989)
somes (Hoekstra et al., 1997; Golovina et al., estimated that the rate of volume increase
1998). Results on liposomes and in situ on imbibition is larger than the rate of
experiments on pollen using an amphiphilic water uptake. Thus, the resulting cellular
spin probe suggest that an anhydrobiote expansion may stretch the plasma mem-
would leak as long as the amphiphilic com- brane beyond its extensibility limit and
pounds resided in the plasma membrane. induce lesions, as demonstrated for cucum-
Since the increase in water volume during ber cotyledons (Willing and Leopold,
imbibition eventually induces the partition- 1983). Spaeth (1987) suggested that inter-
ing back into the cytoplasm (Golovina et al., nal pressure during imbibition may be a
1998), leakage should then cease. The tran- driving force for membrane damage. His
sient nature of such leakage was confirmed suggestion was based on the observations
with leakage experiments using a fluores- of proteins and starch grains that were
cent dye (Hoekstra et al., 1999). The tran- extruded through blisters formed on the
sient leakage is thought to be responsible for surface of imbibed cotyledons of bean and
some loss of germinative vigour of the pea. The process of extrusion resembled a
pollen but not of viability. The above obser- process in which viscous fluids are forced
vations lead to the conclusion that the per- by internal pressure through irregular ori-
manent nature of the leakage that leads to fices (Spaeth, 1987). Further evidence sup-
loss of viability must originate from an irre- porting the role of internal pressure in
versible damage that is other than an imbibitional injury comes from studies on
amphipath partition phenomenon. In con- the formation of cracks during imbibition.
trast to liposome systems, where the transi- Cracks in cotyledons originate from tensile
tion from the gel to liquid crystalline phase stresses in the dry interior of partially
during rehydration is accompanied by a hydrated tissues (Spaeth, 1987, and refer-
transient leakage, it is possible that a phase ences therein). Since tensile stresses are
transition in anhydrobiotes may induce due to compressive strains (i.e. a form of
much more severe and irreversible changes pressure), it implies that there is an inter-
during rehydration (Hoekstra et al., 1999). nal pressure that is applied on partially
Indeed, electron microscope observations of imbibed tissues. However, this contention
freeze–fracture images of imbibitionally has not received further attention.
injured pollen and seeds show folding irreg-
ularities and holes in the plasma mem-
12.2.3. Protections against imbibitional
branes (Hoekstra et al., 1999; Claessens et
injury
al., 2000). Similar damage has been widely
reported in earlier electron microscope
12.2.3.1. Seed coat
studies in which dry tissues were fixed in
cold aqueous fixatives (Buttrose, 1973). The seed coat or testa is extremely impor-
When anhydrous fixation is used, plasma tant in protecting the seed from imbibi-
tional injury. Differences in vigour and via- In certain crops, the seed coat semi-per-
bility of seeds have been associated with meability or impermeability to water has
the pigmentation of the testa. been attributed to the presence of waxy
Characteristically, seeds of the same materials embedded in the epidermis
species with a light-coloured or translucent (McDonald et al., 1988) and high levels of
testa are more sensitive to imbibition than phenolics or hydroxyphenolics that are
seeds with a darker testa. The relationship oxidized (Marbach and Mayer, 1974)
between the presence of a coloured testa and/or polymerized into insoluble lignin
and the absence of imbibition injury has polymers (Marbach and Mayer, 1974; Egley
been established in a wide range of crops et al., 1983, and references therein). In soy-
(e.g. pea, dwarf French bean, faba bean, bean, hydroxyproline-rich glycoproteins
cowpea; Legesse and Powell, 1992; Kantar accumulate in large amounts in the seed
et al., 1994; Demir, 1996) and weeds (proso coat during late maturation. They could be
millet, Khan et al., 1996). A cause–effect involved in regulating the water entry in
relationship between seed-coat pigmenta- the embryonic tissues (Cassab et al., 1985).
tion and imbibition stress has been con- It is not yet understood how these proteins
firmed by comparing the imbibitional or polymeric substances interact with
damage in five pairs of isogenic lines in water molecules to influence the rate of
pea, differing only in the A gene for seed imbibition. According to Powell (1989), the
colour (Powell, 1989). presence of anthocyanins in coloured seed
Several lines of evidence suggest that the coat is thought to decrease the wettability
role of the seed coat against imbibitional of the inner surface of the seed coat.
injury is to act as a physical barrier that reg- However, in pea, this cannot explain why
ulates water movement both temporally coloured seeds suffered less imbibitional
and spatially. The kinetics of water uptake damage than white seeds. Indeed, the dif-
(Powell, 1989) in pea seeds indicated that ferences in imbibition kinetics between
pigmented seeds imbibed more slowly than coloured and white seeds were maintained
those with completely or partially light- when the seed coat was removed (Powell,
coloured or white testas. Harvesting prac- 1989). In resurrection plants, there is no
tices that damage the seed coat such as the direct evidence indicating that protective
formation of epidermal cracks (Duke et al., layers may regulate the rehydration of
1986; McCormac and Keefe, 1990; Bruggink dried tissues. However, several studies
et al., 1991) or the actual removing of the report that leaves of Craterostigma sp.,
testa (Abdel Samad and Pierce, 1978; Duke Xerophyta viscosa and Sporobolus stapfi-
and Kakefuda, 1981; McDonald et al., 1988) anus are covered with waxes or cuticular
resulted in a higher rate of water uptake by coatings or hairy structures, which are
the dry seeds and higher leakage rate dur- thought to control the loss of water during
ing imbibition. Even imbibitional damage- drying (Dalla Vecchia et al., 1998; Sherwin
resistant seeds become sensitive to chilling and Farrant, 1998). Thus, it may be possi-
stress after testa damage (Tully et al., 1981; ble that these epicuticular waxes and other
Prusinski and Borowska, 1996). These epidermal features may also act to regulate
seeds also showed increased hydration water entry during rehydration.
rates and solute leakage. In the particular Decoated seeds suffer more from imbibi-
case of Arabidopsis thaliana, the rate of tional damage than do intact seeds.
water uptake is likely to be mainly con- However, in groundnut, the seed coat does
trolled by a mucilage layer surrounding the not appear to pose a physical barrier to
seed coat without the interference of the rehydration of the embryonic tissues
seed coat tannins and anthocyanins (Albert (Abdel Samad and Pierce, 1978). This
et al., 1997; Debeaujon et al., 2000). This observation leads to the suggestion that fac-
may explain the lack of correlation between tors other than regulating the water uptake
imbibitional stress and testa colour in may be involved in protecting the seed tis-
Arabidopsis seeds. sues from imbibitional stress. Considering
that the seed coat contains phenolics, and/or water uptake is repaired by mole-
which are amphiphilic substances cules that are synthesized during drying
(Marbach and Mayer, 1974), and that these and/or rehydration. Work on Tortula ruralis
substances can alter the conformational and two resurrection plants (Craterostigma
state of the membranes (Hoekstra et al., plantagineum and Sporobolus stapfianus)
1997; Golovina et al., 1998), it would be has identified a series of transcripts, which
interesting to investigate whether are referred to as rehydrins (Ingram and
amphiphilic substances migrate from the Bartels, 1996; Oliver et al., 1997; see
seed coat to the surface cells of the embryo Chapters 1 and 11). So far, three of the rehy-
with the water front, thereby modifying the drin transcripts (Tr155, Tr213 and Tr288)
physical properties of membranes con- have been studied. Tr213 shows a high
comitant with the water entry. The degree of similarity to polyubiquitins. The
swelling properties of the seed coat during main function of ubiquitin is to eliminate
imbibition may also contribute to the regu- undesirable proteins that are damaged or
lation of water uptake. For example, the being recycled. The degradation involves
testa of coloured pea seeds were found to the conjugation of ubiquitin with targeted
remain closely associated with the cotyle- proteins and degradation via a reaction cas-
dons whereas the white seed coats were cade involving the proteosome (Jentsch and
loosened and water was held between the Schlenker, 1995). The presence of polyubiq-
seed tissues and the testa (Powell, 1989). uitins during rehydration points to an
increased removal of proteins during imbi-
bitional stress. Thus, repair machinery
12.2.3.2. Protection at the molecular level
appears to play an active role during rehy-
Have anhydrobiotes evolved protecting dration of vegetative tissues in eliminating
strategies at the molecular level against the damaged proteins, which are accumulated
dangers of rehydration? Studies on pollen during drying. The mechanisms that regu-
membranes during rehydration have late the synthesis of this machinery appear
demonstrated that depressing the phase to be complex. Rehydrins can be constitu-
transition temperature of dry tissues may tively expressed in the plant or both quali-
be beneficial in reducing the risk of leakage tatively and quantitatively transcribed
during water uptake. This depression is during dehydration and/or rehydration
thought to be achieved by altering the (Ingram and Bartels, 1996; Oliver et al.,
phospholipid composition, as in Impatiens 1997; O’Mahony and Oliver, 1999). The
pollen, and/or synthesis of fluidizing question remains as to whether a ubiquitin-
amphipaths that partition into the mem- based mechanism of repair is also essential
brane during drying (Hoekstra and to alleviate imbibitional stress in seeds. In
Golovina, 1999). From results of in vitro this respect, Tr288 is an interesting tran-
experiments using liposomes, it has been script because it has similarity to a tran-
suggested that disaccharides (sucrose and script specifically expressed during
trehalose) can reduce the risk of imbibi- rehydration of dormant embryos of barley
tional injury by suppressing the transition and Bromus secalinas (Oliver et al., 1997).
temperature of the membrane during dry- In addition to repair mechanisms, dry
ing and increasing fluidity (Crowe et al., anhydrobiotes appear to be endowed with
1997). However, the concentrations of sug- specific proteins that protect macromolecu-
ars are not sufficient to protect fully in vivo lar structures from the rapid entry of water,
membranes of dry anhydrobiotes, even if although data supporting this idea are
all sugar molecules would form hydrogen scant. Dry oily seeds contain large amounts
bonds with the phospholipid head groups of oleosins, the interfacial proteins that
(Hoekstra et al., 1997). surround the oil bodies. The main function
Another strategy has evolved in mosses of oleosins is thought to be to maintain the
and resurrection plants. In these tissues, the integrity of the oil bodies as discrete
extensive damage that occurs during drying organelles during rehydration (Leprince et
al., 1998). In those recalcitrant seeds that Currently we have no information as to

are devoid of oleosins (cocoa), the oil bod- whether such hydration-driven changes
ies remain relatively stable after slow or occur during the successful desiccation
fast drying, but, during rehydration, they and rehydration of any plant cell or how
fuse to form large droplets, resulting in the far chromatin topology might be altered by
loss of cellular integrity. So far, no struc- even small changes in the overall water sta-
tural proteins other than the oleosins have tus. However, the variable and decreasing
been detected in dry anhydrobiotes that longevity of seeds held under different
could similarly protect cytoplasmic conditions of even small levels of increas-
organelles during imbibition. ing humidity tells us at once that the cyto-
plasm of embryo cells is in a physically
dynamic state with intracellular molecular
12.3. Maintaining Integrity of the interactions undergoing constant change.
Genome Since so little is known of DNA integrity in
the mitochondria or plastids in seeds or
12.3.1. Hydration-determined changes pollen, this part of the rehydration chapter
in DNA is essentially confined to what is known of
nuclear DNA.
Cells in the dry state are never wholly dry.
Molecules of water are closely associated
with specific chemical groups on the 12.3.2. Seeds
charged proteins and nucleic acids within
the cytoplasmic matrix. In the nuclear Perhaps the earliest evidence for nuclear
DNA, water molecules are intrinsic to the DNA changes in stored dry seeds has come
phosphate backbone of the fully hydrated from microscopic studies showing the
B-form DNA that exists in most living cells. increase in chromosomal aberrations that
Loss of water molecules from the DNA appear in nuclei of embryo cells (Navashin,
backbone can, in vitro, successively con- 1933). That embryos could still germinate
vert B-form to A-form and Z-form confor- after such DNA damage and that cells did
mations and there is evidence for such not necessarily perpetuate the initial chro-
conformational changes during dehydra- mosomal aberrations through subsequent
tion in prokaryotes (Setlow, 1992a) and meristematic cell divisions were the first
during differentiation in specialized clear evidence for DNA repair processes
eukaryote cells (Nordheim et al., 1986). As operating early during the biochemical
Setlow (1992a) has shown for Bacillus events of rehydration (Nichols, 1941).
subtilis, the A-form is maintained by the However, the extent of change during stor-
binding of small acidic soluble proteins age also depends upon the condition of the
(SASPs) synthesized during dehydration, seed when it is shed from the parent plant.
and the dry spores, with their A-form DNA, The maternal history during seed develop-
are remarkably resilient to high or low tem- ment and maturation is partly determined
peratures and to chemical stress. genetically and partly determined by the
Furthermore, the A-form is converted back environmental conditions (temperature,
to the normal B-form when the spores are humidity, sunlight) that the mother plant
rehydrated through the action of an SASP- experiences. Thus, when the seed is har-
specific protease, at which point the spores vested or shed, factors such as potential
lose their tolerance to desiccation and dormancy, seed-coat restrictions and
other stresses (Setlow, 1992b). This indi- embryo vigour are already predetermined
cates the critical part played by available variables. Given the signal inputs that an
water and DNA conformation in determin- embryo receives during development, the
ing the survival of the bacterial spore uncertainties of the maturation and final
through both dehydration and rehydration desiccation phases, and the continuous
processes. molecular changes that progress through-
out the period of seed storage, it can be 1986 and later in plant mitochondria and
seen that the biochemical processes of plastids (for review, see Benne, 1996) and
rehydration and germination do not repre- involves the insertion or deletion of uri-
sent a single or simple system. dine residues in a complex of several
enzymes (the editosome), or direct conver-
sions of cytosine to uridine (Smith et al.,
12.3.2.1. Survival after rehydration reflects
1997). The conversion of L-aspartyl
the dry state experience
residues in ageing proteins into abnormal
Continued storage, even under optimum L-isoaspartyl groups can also be achieved
conditions, leads to a progressive deteriora- in many plant and animal tissues by the
tion, and all the events listed timewise for action of L-isoaspartyl methyl transferase
rye (see following section) are reduced in without total protein degradation, and this
activity and extended in time when enzyme has been shown to be present in
retested as the period of storage becomes seeds (Mudgett and Clarke, 1993). All the
longer. The progressive lessening of the other nucleic acids, proteins, lipids and
ability to incorporate amino acids into pro- polysaccharides are subject to synthesis
tein in the early hours of imbibition is a and degradation and are not, as far as we
measure of an embryo’s being no longer know, subject to an enzymic, energy-
able to germinate quickly. The time to root requiring process of molecular repair. This
emergence, the first critical round of S- makes DNA and the genomic information it
phase DNA synthesis and the first cytoki- carries of special importance in embryo
nesis can be delayed for hours or even days cell survival in the dry state and critical to
(Osborne, 1983). Eventually, the embryo the competence for DNA repair when the
can no longer synthesize protein at all on cells first imbibe water on rehydration.
rehydration, but, interestingly, there is a There is no precise moisture content
late stage in the detrimental programme of that determines what we call the ‘dry state’
change in the dry state when transcription of a seed. When embryos reach maturation
of short oligonucleotides can still take dehydration to moisture contents below
place in the nucleus; however, neither 10%, the cytoplasm of the embryo enters
stored messages nor these new small tran- into a glassy state in which molecular
scripts are translated by the embryo (Bray movement is strictly limited (Williams and
and Dasgupta, 1976; Sen and Osborne, Leopold, 1989; Sun and Leopold, 1993;
1977). Currently, we do not know what Buitink et al., 2000; see Chapter 10). But as
these small transcripts might code for. different parts of a seed can be at slightly
They might, of course, just represent the different levels of hydration and the attain-
most stable sequences in the heterochro- ment of the glassy state in different species
matin of genomic DNA and do not there- depends upon the molecular composition
fore code for anything that is critical to of the cells (Leopold et al., 1994), the levels
embryo survival. of hydration that provide a glassy state for
The detrimental changes in an ageing all the different tissues of a seed can differ.
embryo are multiple and much work has As far as current detection methods can
been directed towards determining the bio- reveal, there is no respiration or ATP gen-
chemical and physical nature of these eration, no transcription and no translation
events. Of all the macromolecules of the in dry seeds; synthetic events of all kinds
living cell, only one, DNA, is known to be are therefore excluded (Bewley, 1979).
routinely repaired. A caveat may be added Non-energy-requiring processes are not,
here that the post-transcriptional editing of however, excluded, so free radicals can be
mRNA has also been considered as a repair generated as local events and non-energy-
process as the molecule is not degraded requiring enzyme activities such as those
before bases are removed and replaced. of nucleases and proteases can occur where
Such editing was first detected in mito- they are in close molecular juxtaposition
chondrial transcripts of trypanosomes in with their substrates. The progressive loss
of DNA integrity to increasing levels of ran- and, using autoradiography, incorporation

dom-sized small-molecular-weight frag- of tritium(3H)-labelled precursors into
ments without loss of total DNA (Cheah nucleoli was evidence of rRNA synthesis
and Osborne, 1978) and the loss of activity within 10 min. Incorporation of 3H-thymi-
in many enzymes without loss of the actual dine into nuclei of embryo cells has shown
protein molecules are evidence of this that DNA repair (but not replication) is also
(Osborne, 1983). But, whereas some activated within minutes of imbibition in
hydrolytic enzymic proteins are remark- rye, and incorporation of labelled amino
ably stable (DNases, RNases) and long out- acids into protein is apparent in the cyto-
live in function the life span of the embryo plasm within 15 min (Osborne et al., 1977).
(Osborne et al., 1977), others start to lose The initiation of S-phase DNA synthesis
enzymatic activity soon after harvest and and DNA replication from 2C to 4C levels,
on storage they become progressively less first evident in nuclei of root-tip meris-
efficient with time. In this group are the tems, occurs late in the process of rehydra-
enzymes concerned with DNA repair: tion and the first cytokinesis may be hours
nuclear DNA polymerases (Yamaguchi et or days after RNA transcription and protein
al., 1978), -type polymerase (Castroviejo synthesis are fully established. Whether
et al., 1990), DNA polymerase- (Coello- the start of replication requires a particular
Coutiño et al., 1994) and DNA ligase (Elder physical state of hydration of the nucleus
et al., 1987), so that, when a fresh or a is not known. Embryos become highly
stored seed is rehydrated from the dry metabolically active on imbibition even
state, the extended time required to repair though they may remain dormant; those of
fragmentation lesions in the DNA and to Avena fatua can reach a moisture content
synthesize new repair enzymes is immedi- as high as 40% without a sign of growth.
ately evident (Elder et al., 1987). This can These embryos may defer amplification of
account for the delays in germination that nuclear DNA from 2C to 4C levels for
are observed between freshly harvested months or years before initiating the entry
seeds and those that have been stored. to cell cycling and attendant germination.
The control factors that hold the cells of
imbibed embryos in this homoeostatic but
12.3.2.2. First events of seed rehydration
metabolic state, while also preventing pro-
Assuming that seeds have been matured gression into cell cycling, cell expansion
under satisfactory conditions on the and cell division, have still to be deter-
mother plant and that they have not been mined, and this is still a key question in
held after harvest in a way that would seed biology. It is of special interest to
impair their ability to germinate, then the studies of desiccation tolerance because,
events of rehydration from the dry state fol- throughout their dormancy, imbibed seeds
low an essentially similar pathway of can be dehydrated back to their original
nuclear reactivation for all the embryos weight without loss of viability or cell
that have been studied in detail. death. A clue to the survival of dormant
On imbibition in water, there is first a embryos is their ability to maintain an active
physical hydration of the cytoplasm, which DNA repair from the time of rehydration and
is usually completed by 60 min throughout the duration of imbibition.
(Obroucheva, 1999). This will occur in Although replicative DNA synthesis is
embryos of dead as well as living seeds blocked, DNA repair synthesis is as effective
(Hallam et al., 1972, 1973) but in living in dormant as in germinating embryos. As
seeds further uptake then proceeds contin- experiments with A. fatua have shown, even
uously. The transcription of all classes of the repair of -irradiation-induced single-
RNA within 20–40 min has been demon- and double-strand breaks occurs as effi-
strated by electrophoretic fractionation of ciently and in the same time in dormant and
newly synthesized radiolabelled nucleic non-dormant imbibed material (Elder and
acids in rye embryos (Sen et al., 1975), Osborne, 1993).
12.3.2.3. Partial rehydration: priming visible cell division has not started and the
embryo has not been held at G2M for a pro-
It might be concluded that even a short longed period, imbibed or primed seed can
period of imbibition that is then followed be dried back safely and the seedling will
by dehydration back to the dry state with- emerge rapidly on planting (Fig. 12.2).
out damage or loss of any of the newly syn- The secret of successful priming, there-
thesized components might lead to an fore, is to establish the stage before the first
improvement in subsequent seed germina- cell cycle division when the nucleus has
tion when water is again available. In fact, reinitiated transcription, has fully repaired
this is generally true and forms the basis of any DNA damage from the dry state and
priming seed (Heydecker and Coolbear, the optimal spectrum of newly translated
1977; van Pijlen et al., 1996). Successful but desiccation-stable proteins has been
priming achieves and conserves both DNA translated and they are present in active
repair and the early events of germination, form in the cytoplasm.
including a significant level of mitochon- However, this is not necessarily the
drial DNA synthesis. These are events that whole story, for, although primed seeds
are stable to desiccation and take place will germinate quickly, they frequently
before the embryo cells pass into the condi- store less well (Tarquis and Bradford, 1992;
tion of desiccation sensitivity, when Nascimento and West, 2000), and in some
embryo cells will die if dehydrated (Ashraf species they lose viability more readily
and Bray, 1993). The conversion from a than those of unprimed seeds. The reasons
state of tolerance to cytoplasmic water loss for this are uncertain, but would appear to
to one of intolerance is still not fully be linked to the changes that take place
understood, but it coincides with the stage during priming in the physical state of the
in embryo cells, particularly those of the nucleus and cytoplasm and to the progres-
root tip, when they approach the first sion in the stage of the cell cycle at which
cytokinesis at G2M and are on the border of the nuclei are dried back. A low level of
undergoing the first cell division. Provided replicative DNA synthesis continues
Desiccation- Desiccation-
sensitive sensitive
90
Desiccation-
tolerant
% Moisture content
50
2M
Replication Cytokinesis
G
arrest
→1
G
G1
10
Seed Dry Rehydration

maturation state
Fig. 12.2. Progression of hydration-linked cell cycle events in a seed embryo during water restriction at
maturation and the renewal of free water at rehydration, showing the relation with desiccation tolerance.
throughout priming, progressing from 2C ther low molecular weight DNA disintegra-
towards 4C though not to cytokinesis. tion (Fig. 12.3d).
There is evidence that cells will accumu-
late at G2M and this may represent a topol-
ogy for nuclear DNA that is particularly 12.3.3. Pollen
accessible to nuclease fragmentation. Not
all imbibed or primed seeds from different Certain pollens can stay dry and alive for
species will reach this same condition of prolonged periods of time, depending on
cytoplasmic or nuclear organization after the ambient relative humidity. The level of
the same duration of rehydration, and moisture in dry pollen can vary and is low-
therefore on drying back they will not est (c. 8%) for wind-dispersed pollen.
reach similar stages of vulnerability to stor- Damage to these dry pollen cells can be
age. A comparison of primed seeds (ach- more severe than that for the embryos of
enes) of Ranunculus sceleratus and seeds. This is because the individual wind-
Ranunculus arvensis is one such example. blown pollen cells are unprotected from
With R. sceleratus, longevity of the dried- ultraviolet light (UV) and other environ-
back seeds is increased, whilst that of R. mental stresses from the moment they are
arvensis is reduced (Probert et al., 1991). shed from the anther. Since these pollen
cells are haploid and lack the resource that
doubled (diploid) genetic information pro-
12.3.2.4. The recalcitrant seeds
vides, damage to the single genome can
Many seeds, particular those of tropical result in serious genetic consequences to
species, and many large-seeded species of the next generation. In these special cir-
temperate climates maintain a high per- cumstances, DNA repair in pollen plays a
centage of water in both axes and cotyle- crucial role and both photoreactivation and
dons. Although they can accommodate a dark DNA repair systems operate together
small amount of water loss without harm, in a germinating pollen grain (Ikenaga and
desiccation to levels of 10–26% are lethal Mabuchi, 1966; Jackson and Linskens,
and embryos do not restore synthetic activ- 1978). Incorporation of 3H-thymidine in
ity when rehydrated. A feature that has germinating pollen grains of Petunia starts
now been explored is whether or not the immediately water becomes available and
DNA repair function is lost on dehydration continued incorporation in the presence of
of these seeds below a certain critical level. hydroxyurea (which blocks replication)
In Avicennia marina, a mangrove species confirmed active DNA repair synthesis in
indigenous to the tropics and subtropics, a germinating pollen. Hydration of birch
water loss from the seed that exceeds pollen at 100% humidity is not of itself
20–30% normally leads to seed death. sufficient to permit full excision repair,
Experimental samples of the excised axes although the germination rate is improved,
were dehydrated under a cool air stream to but, on transfer to free water, fully imbibed
different levels of water loss, then irradi- pollen will then complete repair within 2 h
ated from a -source to introduce a similar (Grodzinsky and Bubryak, 1985). During
level of single- and double-strand breaks in rehydration, the excision DNA repair sys-
each, and then rehydrated for a period of tem of pollen can remove a number of dif-
2 h (Boubriak et al., 2000). Results have ferently induced lesions incurred in the
shown that fully hydrated embryos (Fig. dry state, including those of chemical
12.3a and b) permit almost full recovery of mutagenesis (Jackson and Linskens, 1978,
fragmented DNA to levels of that in unirra- 1979), heavy metal damage and -irradia-
diated controls. However, generating a 22% tion (Jackson and Linskens, 1982; Bubryak
dehydration of the axes prior to irradiation et al., 1991). Efficiency of such repair can
led to the complete failure of DNA repair differ in pollens depending upon the dif-
(Fig. 12.3c) and, by 46% dehydration, the ferent levels of environmental stress under
fragmentation of DNA continued into fur- which they were formed, i.e. lowland or
1.2
750 Gy (fully hydrated) 1.2 750 Gy (fully hydrated)
2 h Imbibed
(a) (b)
1.0
Absorbance at 260 nm (relative units)
1.0
1.2 750 Gy (22% dehydrated) 750 Gy (46% dehydrated)

2 h Rehydrated 1.2 2 h Rehydrated
(c) (d)
1.0 1.0
Low Mol. Wt. High Mol. Wt.

Migration
Fig. 12.3. Loss of DNA integrity with loss of DNA repair capability in embryo axes of Avicennia marina
seeds following different levels of dehydration, shown by competence to repair a -irradiation damage of
750 Gy during 2 h rehydration. Molecular weight profiles of scans of DNA fractionated by electrophoresis
on neutral agarose gels: (a) fully hydrated axes after -irradiation ((Low Mol. Wt.) DNA strand breaks
induced); (b) fully hydrated axes, -irradiated, then rehydrated 2 h ((Low Mol. Wt.) DNA repaired); (c) 22%
dehydrated axes, -irradiated, then rehydrated 2 h (no DNA repair); and (d) 46% dehydrated axes, -
irradiated, then rehydrated 2 h (further DNA disintegration).
high-altitude mountains (Bubryak and essential to retain genetic fidelity. This is

Grodzinsky, 1985; Grodzinsky and not so if pollen is shed as a three-nuclei
Bubryak, 1988). cell in which the generative nucleus has
already divided to produce the two haploid
sperm cells (as in maize). For a long time it
12.3.3.1. First events of pollen rehydration
has been puzzling that there was no evi-
Efficient DNA repair during the imbibition dence for excision repair during rehydra-
of pollen grains is an essential physiologi- tion of maize pollen even though this is
cal event only for two-nuclei pollen grains also wind-distributed and can therefore be
(e.g. birch) where, at shedding, each damaged by UV. Maize pollen is viable for
nucleus is held at G2 (2C DNA values). a few days only, so it is possible that no
During germination, the generative nucleus DNA repair system is maintained in these
of the pollen grain divides to produce two haploid sperm cells since they have
haploid sperm cells, so it is at this G2 already passed their last G2 checkpoint.
checkpoint that DNA repair is absolutely An efficient DNA repair system is not
confined to wind-pollinating pollens but now known of the genes induced during
has been shown to occur also in the two- dehydration processes in these desiccation-
cell pollens of certain insect-pollinated tolerant plants (Neale et al., 2000; see
plants such as Petunia and Amaryllis. Chapter 11) and of mechanisms involved
These pollens can repair induced UV and in transcription and translation (Oliver et
-irradiation damage despite the fact that al., 1997, 1998). However, we have little
they rarely receive a high radiation dose evidence yet that links the successful rehy-
during the pollination process. Although dration of these plants to their competence
such repair can occur, it is less efficient either to sustain fidelity of the nuclear,
than in wind-pollinated species (Bubryak mitochondrial or plastid genomes on dehy-
and Grodzinsky, 1985). dration or, perhaps more importantly, to
It is quite possible that, in pollen, check- the restoration the overall fidelity of the
point repair (in G2) for generative nuclei different genomes on rehydration.
requires time and, therefore, a repair capa- Current knowledge indicates that pro-
bility to withstand DNA damaging factors in gressive changing states of gene expression
the environment is maintained for longer. occur at distinct thresholds of hydration
This may include desiccation tolerance of throughout a dehydration process (Neale et
the two-nuclear pollen both for wind-polli- al., 2000; Chapter 11). The possibility then
nated and insect-pollinated species. Before arises that there could be alterations in the
shedding from the hydrated enclosure of the conformational state of DNA as it becomes
catkin, pollen is killed if it is dehydrated, progressively dehydrated, so perhaps this
but at shedding (when the moisture content area should be investigated for evidence of
is reduced to 8–14%) pollen becomes stable a transition to A-form DNA. What occurs
to dry storage for long periods of time (years on rehydration is even less sure, and how
for hazel and birch). Such pollen can be far cell survival depends upon the mainte-
hydrated in moist air and dehydrated back nance of genomic fidelity during the period
several times without losing the capability of desiccation or upon the repair of DNA
for excision repair and for germination. lesions at rehydration, or both, remains to
However, if exposed to liquid water, be discovered.
hydrated pollen grains germinate and then, At least it now seems sure that water
like the embryos of seeds, the germinated deficits can lead to a rise in abscisic acid
pollen loses tolerance to drying and (ABA) levels and thence to the induction of
becomes, once again, desiccation-sensitive ABA-directed new gene expressions. It is
(Osborne and Boubriak, 1994). What we do also now sure, however, that desiccation
not know is the topological organization of tolerance is not always controlled by ABA
pollen nuclear DNA throughout these in all plants. In certain mosses and ferns,
changing levels in water status. for example, ABA is either not detected or
the levels may actually fall during desicca-
tion (Reynolds and Bewley, 1993).
12.3.4. Whole plants Considering the diverse groups of plants
and tissues that have acquired desiccation
Much interest is currently directed towards tolerance, it seems likely that more than
the survival mechanisms of vascular plants one survival mechanism will have evolved.
under drought conditions, and a relatively For all, however, preservation of genetic
small group of plants including mono- information will be paramount.
cotyledon grasses, dicotyledon ‘resurrec-
tion plants’, many liverworts, mosses and
ferns are sufficiently drought-tolerant to 12.3.5. Requirements for successful
survive a 95% water loss for months or rehydration of a genome
years, then recover and become metaboli-
cally active when free water is again avail- There would appear to be two aspects of
able (Bewley, 1979; Gaff, 1980). Much is the rehydration of a dry but living cell that
are essential for the successful re-establish- (Zarain et al., 1987), an early synthesis of
ment of organized metabolic activity. One DNA that did not correspond in character
is the physical rehydration of dry to either repair or replication was found.
organelles and the rehydration of a dry One possibility was that this represented
cytoplasm. This involves the molecular mitochondrial DNA synthesis (Vazquez-
reorganization of the membranes defining Ramos and Osborne, 1986) but further
each region of compartmentation, the rehy- studies by Bucholc and Buchowicz (1992)
dration of the protein–polysaccharide using oligonucleotide-hybridizing tech-
structures of the cell walls and the free- niques showed that part of the DNA repair
water access to folded macromolecules of synthesis that occurs after imbibition of the
proteins and nucleic acids (see Section embryo can be attributed to a new synthe-
12.2). Before information exchange can sis of telomeric DNA. Furthermore, they
take place between nucleus and cytoplasm, found that one of the degradative changes
the nuclear chromatin must itself be rehy- in DNA of stored wheat embryos is the pro-
drated and the DNA made available for reg- gressive cleavage and removal of the telom-
ulated transcription. For accurate transfer eric repeats.
of genomic information to the cytoplasm, This shortening of telomeric DNA cap-
any damage present in DNA must first be ping repeats on storage of seed embryos is,
replaced. Only if this second and metabolic like the loss that occurs in mammalian
aspect of rehydration is achieved can a cell cells, a measure of their age and may repre-
re-establish directed metabolic activity sent a commonality in the progress to
with an opportunity for renewed cell senescence and death. Delay or failure to
growth and development. Only if the mito- reinitiate these DNA end groupings may
chondrial genome is also fully restored can therefore be another critical factor in deter-
an integrated informational exchange prop- mining success in the rehydration that pre-
erly operate. cedes germination. The stability of the
Another aspect of DNA degradation, the telomere polymerase may be found to play
recovery from which could be considered a key role in restoring the overall integrity
as a DNA repair operation, is the loss of of the embryo genome.
repetitive telomeric sequences from the ter- Although dry seeds appear to accumu-
mini of DNA molecules within the double late most DNA damage in the form of single-
helix. Ageing human fibroblasts show a strand DNA breaks, presumably from
shortening of these telomeric regions enzymatic endonuclease cleavage or possi-
(Harley et al., 1990) and convincing exten- bly also from free-radical attack and by
sions of the life span in retinal pigment spontaneous base loss (Dandoy et al.,
epithelial cells has been achieved by trans- 1987), the effects of the accumulation of all
fecting cells obtained from telomerase- types of damage before rehydration can be
negative cell types with vectors encoding linked to the progressive failure of DNA
the human telomerase catalytic subunit repair functions when water is again avail-
(Bodnar et al., 1998); telomere-expressing able. Such events have led to the proposi-
clones have elongated telomeres, contin- tion that loss of DNA repair is a major
ued cell divisions and an absence of senes- factor in determining the loss of genomic
cence. integrity and poor survival of an embryo on
Telomeres and telomerases are present rehydration (Elder and Osborne, 1993;
on the chromosomes in nearly all plant Osborne and Boubriak, 1994; Boubriak et
cells (Adams et al., 2000; Leitch, 2000). al., 1997).
The role of telomerases in seeds is there- Evidence that competent DNA repair is
fore of considerable interest with respect to indeed an essential factor for successful
ageing and the events of early rehydration. rehydration comes from experiments with
In studies of DNA repair in embryos of rye the embryos of rye (Boubriak et al., 1997).
(Vazquez-Ramos and Osborne, 1986), of If isolated embryos are -irradiated from a
wheat (Marciniak et al., 1987) and of maize caesium source (750 Gy) whilst in the dry
state in order to introduce DNA damage, still in the desiccation-tolerant period

(Fig. 12.4a,b) subsequent rehydration in when rehydrated (Boubriak et al., 1997). It
water for 2.5 h leads to complete restora- would be of considerable interest now to
tion of the fragmented DNA to high molec- assess if there is a similar requirement for
ular weight (Fig. 12.4c). These embryos functional DNA repair during the rehydra-
survive. However, if DNA repair is blocked tion of other desiccation-tolerant and leafy
by inhibitors of both - and -polymerase species such as mosses and resurrection
(aphidicolin and dideoxythymidine tri- plants.
phosphate, respectively), DNA integrity is Although nuclear transcription in the
not restored, DNA becomes even more frag- embryos of fresh, dry seeds starts almost
mented over 2.5 h and the embryos die (see immediately on imbibition, it is unclear at
DNA scans in Fig. 12.4d). The inhibition of what level of hydration the mitochondria
DNA repair has prevented the successful first become active and ATP first becomes
rehydration and survival of these DNA- available for synthetic processes (Attucci et
damaged embryos, even though they were al., 1991). It may be as low as 14% (apple
Control (dry) 750 Gy (dry)

1.5 1.6
(a) (b)
Absorbance at 260 nm (relative units)
1.1
1.0
1.5
750 Gy (rehydrated 2.5 h) 750 Gy (rehydrated 2.5 h
1.5 in AP + dTTP)
(c) (d)
1.1 1.0
Low Mol. Wt. Migration High Mol. Wt.
Fig. 12.4. Restoration of DNA integrity on rehydration requires DNA repair, shown by the effect of blocking
DNA repair in -irradiated (750 Gy) embryos of rye, Secale cereale. Molecular weight profiles of scans of DNA
fractionated by electrophoresis on neutral agarose gels from: (a) untreated dry embryos (controls); (b) dry
embryos -irradiated ((Low Mol. Wt.) DNA strand breaks induced); (c) irradiated embryos rehydrated for 2.5 h
(DNA repaired); and (d) irradiated embryos rehydrated for 2.5 h in presence of aphidicolin (AP) -polymerase
inhibitor and dideoxythymidine triphosphate (dTTP) β-polymerase inhibitor (DNA repair blocked).
embryos) or as high as 25% in pea seeds tion of these positions and re-establishment
(Leopold and Vertucci, 1989). Because of the of a fully functional genome are early and
low levels of early ATP synthesis at rehydra- necessary events upon rehydration of both
tion, an alternative non-mitochondrial embryo and pollen cells. The speed and
cytosolic source has been sought to fuel the fidelity at which these processes are
earliest synthetic events through a glycolic accomplished dictate the lag period to the
fermentation of starch or lipid (Raymond et initiation of the first cell cycle after the dry
al., 1985; Perl, 1986). How important this state and hence determine the eventual
might be and at what levels of hydration it success of germination. Not only does DNA
might operate are not yet known. Certainly, repair in seeds have the essential role of
continued mitochondrial ATP generation restoring fidelity to DNA fragmented dur-
has a critical function in providing the ing dry storage, it also plays a critical part
essential energy generation for new protein in maintaining desiccation tolerance
synthesis, including that for new DNA throughout the early hours of rehydration.
repair enzymes and for the mitochondrial Efficient DNA repair is thus a critical
dehydrogenases, both of which lose activity component of the cell machinery, which
with time in a stored seed (Throneberry and may act immediately on rehydration of all
Smith, 1955; Elder et al., 1987). The extent desiccation-tolerant dry cells, and the
of the DNA repair function when an embryo extent of the success of DNA repair can be
first becomes hydrated from the dry state seen as a major factor determining the fate
depends not only upon the remaining activ- of plant cells following a dehydration/
ity of the repair enzymes stored within the rehydration cycle. It will therefore be of
dry cells but also upon the available ATP for much interest to discover the facts that will
this repair to take place in restoring integrity be revealed when resurrection plants are
to fragmented nuclear DNA molecules and scrutinized for the status of genomic, plas-
to the DNA of the mitochondria. Only on tid or mitochondrial DNAs following desic-
the re-establishment of intact coding cation and rehydration, and to learn
sequences in both nucleus and mitochon- whether telomere lengths are subject to
dria can new repair-enzyme mRNAs be tran- change in similar circumstances.
scribed in either organelle.
These experiments raise intriguing ques-
tions in seed and pollen survival, which 12.4. Acknowledgements
remain to be properly resolved. How stable
are the circles of mitochondrial DNA? Are O. Leprince is grateful to the Netherlands
the DNA sequences that code for the DNA Organization for Scientific Research, the
repair enzymes specially labile to nuclease Netherlands Technological Foundation for
or free-radical assault? Is the conforma- Scientific Research and the French Ministry
tional folding of DNA, the state of methyla- of Agriculture and Fisheries for support. Dr
tion or acetylation and the nature of F.A. Hoekstra is acknowledged for his con-
DNA-binding proteins at the specific DNA structive comments on Section 12.2.
repair sites on these genomes critical to D.J. Osborne and I. Boubriak acknowl-
successful DNA repair on rehydration? edge support from Hortlink MAFF (Scottish
Whether or not the sites of DNA cleav- Office), Framework IV FAIR5–3711 and the
age are specific hypersensitive sites, religa- Royal Society, London.
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Part V
Retrospect and Prospect

13 Damage and Tolerance in Retrospect

and Prospect
Michael Black,1 Ralph L. Obendorf2 and Hugh W. Pritchard3

1Division of Life Sciences, King’s College, Franklin Wilkins Building, 150 Stamford
Street, London SE1 6NN, UK; 2Seed Biology, Department of Crop and Soil Sciences,
Cornell University, Ithaca, New York, USA; 3Seed Conservation Department, Royal
Botanic Gardens Kew, Wakehurst Place, Ardingly, West Sussex RH17 6TN, UK
The preceding chapters have provided a ability to complete germination is often

wide perspective on desiccation in plants. employed as the indicator of desiccation tol-
In this brief, concluding chapter we will erance in seeds. Since the completion of
select some information from these germination, i.e. expansion of the axis to
accounts, which point to generalizations rupture the enclosing structures, normally
that can be made. This is not intended as a occurs before cells begin to divide, germina-
comprehensive overview or evaluation but tion as an index of tolerance may not reflect
rather an attempt to identify some areas the integrity of cell division. Some authori-
that offer prospects for further research and ties therefore insist that unequivocal desic-
increase in our understanding. cation tolerance is shown only by seeds that
The ability to tolerate desiccation obvi- germinate to produce normal seedlings,
ously lies at the heart of the desiccation and where the capacity for cell division has not
survival scenario. But there are problems been compromised. Moreover, since germi-
implicit in this statement: how do we define nation in the strict sense is a response spe-
and measure tolerance and survival? We can cific to the axis, the elongation ability of the
see from foregoing chapters in this book that previously desiccated axis may not neces-
tolerance, as considered by authorities, cov- sarily indicate the level of tolerance in the
ers a wide spectrum of properties from, for remainder of the embryo, i.e. the cotyledons
example, a rigorously defined, single bio- or scutellum. Considerations of a similar
physical event (Chapters 4 and 10), such as type might also apply to recalcitrance,
the partitioning of an amphiphile into a which is often taken as the ‘archetype’ of
membrane, to the ability of a system (such desiccation sensitivity in seeds. Yet such
as a seed or a whole plant) to resume nor- seeds possess or exhibit many features of
mally the whole gamut of its growth and tolerant seeds, such as the occurrence of
metabolic abilities. Between these two abscisic acid (ABA) and late embryogenesis
extremes there is a range of parameters abundant (LEA) proteins, and degrees of
taken by researchers to indicate tolerance, subcellular integrity maintained after dehy-
and, in some cases, where a high degree of dration. The characteristics that are critical
tolerance exists, some measure of intoler- to intolerance in recalcitrant seeds indeed
ance nevertheless remains. For example, the prove difficult to identify.
368 M. Black et al.
Similar complications exist in respect of requires further study. An interesting find-

vegetative tissues. For example, some resur- ing is that the majority of desiccation-toler-
rection plants (the homoiochlorophyllous ant bryophytes age most rapidly in the
types) exhibit a high degree of desiccation range 9 to 22 MPa and have greatest
tolerance in that the chloroplasts retain longevity at between 150 and 300 MPa
their organization and chlorophyll after (Chapter 7); this is also generally true for
dehydration, whereas the poikilochloro- orthodox seeds and pollen (Chapters 5 and
phyllous types, though in almost all other 6). Thus, the water relations of survival and
respects desiccation-tolerant, are intolerant longevity appear to be similar in material
so far as chloroplast and chlorophyll that is physiologically, biochemically and
integrity are concerned. These few exam- morphologically diverse. Moreover, develop-
ples illustrate that desiccation tolerance ing seeds that are detached from the parent
must be carefully defined and that indica- plant readily become desiccation-tolerant
tors of tolerance and intolerance can exist when held at the upper range, whereas
side by side in a single biological system. here most recalcitrant seeds succumb to
This complexity might arise partly irreversible damage. These approximate
because of the multiplicity of damaging water-potential ranges thus appear to be
events that occur as cells dry out (Chapter 9) crucial for tolerance and longevity, and
and the relative readiness for different kinds research aimed at understanding these
of damage to be repaired (Chapter 12). In processes should, perhaps, be focused there.
respect of the former, it is clear that different At the same time, we must address the prob-
levels of arrest or damage occur as lem in mechanistic terms of why seeds of
decreases, though the biophysical or molecu- various species show such differences in
lar reasons for this are not clear in all cases. longevity around two orders of magnitude
It is interesting to speculate that a similar under identical storage conditions. The
scale of response to might also occur in the practical benefits with regard to storage and
repair processes during rehydration but conservation arising from such knowledge
more information on this is needed. are obviously extremely important.
Another factor that might contribute to The multiplicity of factors participating
the coexistence of tolerance and intoler- in desiccation tolerance have been exten-
ance indicators as mentioned above is the sively discussed in several chapters. As bio-
simultaneous occurrence, as dehydration physical, biochemical, cell biological and
progresses, of protective mechanisms (i.e. molecular technologies have become more
conferring tolerance) as well as damage sophisticated and adapted for use in seeds,
processes. The inception of the protective pollen, spores and vegetative tissues, our
mechanisms occurs in response to initial understanding of the processes involved in
dehydration and develops to completion tolerance has gathered momentum. But
provided that water loss is not too precipi- uncertainty (verging on controversy) has
tous, while damage increases progressively developed in respect of some topics. For
as falls The rate of drying (Chapter 3) example, while the evidence shows that
therefore determines the relative progress glass formation is an important element in
of protective and damaging events, i.e. the the protective mechanisms in ‘dry’ cells, it is
development of both tolerance and intoler- not clear what the components of the vitrifi-
ance characteristics and their presence in cation system are (Chapter 10). Sugars, espe-
the final desiccated system. cially sucrose, are certainly involved, and
One important point that has been possibly also proteins (e.g. LEA proteins) but
touched on in the preceding chapters (e.g. the role of oligosaccharides, which once
Chapter 10) is the relationship between exercised a strong claim for participation
desiccation tolerance and longevity in the (in longevity also), has been questioned
dry state: are the same or similar regulatory (Chapter 10; Bentsink et al., 2000). Most
mechanisms involved? We are far from of the evidence for the participation of
resolving this question, which clearly oligosaccharides in tolerance and longevity
Damage and Tolerance in Retrospect and Prospect 369
comes from studies of correlations between therefore have a spurious appearance of a

the oligosaccharide content and the appro- causal relationship. Longevity is measured
priate physiological property. But, since des- by the ability to germinate, which might
iccation tolerance and presumably longevity also reflect reserve oligosaccharide content.
are likely to depend on multi-component Both drought stress and desiccation in
action (e.g. carbohydrates, LEA proteins, plants have been widely studied at a molec-
antioxidants, free-radical removal, etc.), cor- ular level, especially in vegetative tissues.
relative evidence may be difficult to inter- In the former, numerous genes have been
pret if one or more components other than, identified whose expression is regulated by
for example, oligosaccharides become limit- water stress. Much has also been learned
ing: in such a case, the oligosaccharide con- about signal transduction mechanisms
tent would be scarcely relevant. In addition operating in the stress syndrome, and, in
to the raffinose-series oligosaccharides the case of many, but not all genes, ABA
(galactosides of sucrose), other galactosides – participates at an early stage in signalling.
of cyclitols – are commonly present in seeds. In the desiccation phenomenon too, expres-
Maturing seeds of some species accumulate sion of many genes occurs in response to
fagopyritols, for example, which have been early water loss from vegetative tissues,
suggested to substitute for the role of some of which are, again, ABA-regulated
oligosaccharides, so knowledge restricted to (Chapter 11). But, in contrast, our under-
the content of the latter might not provide a standing of molecular events involved in
full picture (e.g. Steadman et al., 1996). seed desiccation is less advanced.
But, although there is persuasive evi- Expression of several genes in developing
dence against a determinative role for seeds is certainly affected by drying, the
oligosaccharides in desiccation tolerance most intensively studied being those for
and longevity, including on genetic grounds several reserve proteins in which the capac-
(Bentsink et al., 2000), the accumulation of ity for expression is ‘switched off’ by the
these compounds during seed maturation, dehydration experience (Jiang et al., 1995),
apparently in response to the incipient dry- an event that is unlikely to contribute to
ing signal (Blackman et al., 1992; Black et desiccation tolerance. There is some evi-
al., 1999) (and similarly stimulated in dence, though, that certain reserve proteins
hypocotyls (Brenac et al., 2002)), demands or close relatives might be involved in cell
an explanation. After its maturation, two desiccation processes (Chapter 5). Several
major developmental steps await the seed – positive effects of drying on gene expres-
quiescence and then germination. For the sion or enzyme production have been
completion of germination, easily utilizable recorded; but many of these are connected
reserves should be immediately available in with reserve mobilization (e.g. Cornford et
the extending organ itself, usually the radial., 1986) and are therefore unlikely to be
cle, and oligosaccharides such as raffinose involved in desiccation tolerance. The pro-
often fulfil this need (see review by motive effects of incipient dehydration on
Peterbauer and Richter, 2001). It might be, oligosaccharide accumulation in maturing
therefore, that the maturing seed uses the seeds (see above) may possibly operate
same signal, incipient drying, to register the through positive action on expression of
imminence of two different processes, the genes involved in their biosynthesis (see
first being quiescence in the ‘dry’ state, for review by Peterbauer and Richter, 2001),
which desiccation tolerance is necessary, such as galactinol synthase or stachyose
and the second being germination, for synthase, but this has not been determined.
which readily utilizable reserves (relatively The loss of water can also convert a seed
low-molecular-weight carbohydrates such from the developmental to the germinative
as the raffinose-series oligosaccharides) mode in respect of patterns of synthesized
must be prepared. The coincidence proteins, an effect that has been confirmed
between oligosaccharide accumulation and as operating at gene level (for review, see
the onset of desiccation toleration may Kermode, 1995), but again it is not likely
370 M. Black et al.
that these observed changes are related to course, desiccation-sensitive (Chapter 5).
desiccation tolerance. The essential point In general, then, there is a paucity of
is, however, that gene expression in devel- information about gene expression associ-
oping seeds can be modified by water status, ated with desiccation in seeds, especially
so this effect could participate in the estab- as compared with what is known in respect
lishment of desiccation tolerance. The of vegetative parts. Another aspect of the
major relevant genes that might be affected desiccation scenario for which we lack a
by drying are those encoding the LEA pro- balanced perspective concerns post-drying
teins and certain heat-shock proteins, both rehydration. During this process a discrete
of which may aid in the protection against set of genes and proteins is expressed in
some of the damage that desiccation would mosses that are likely to be involved in var-
inflict. None the less, it is not certain that ious repair and protective phenomena. We
expression of these genes is universally up- know little about this so far as seeds (and
regulated by the hydration state of develop- vegetative tissues) of angiosperms are con-
ing seeds; on the contrary, certain lea genes cerned, and it seems important to explore
do not appear to respond to desiccation this area if we are to advance our knowl-
while others are down-regulated, at least in edge of survival mechanisms.
developing seeds that have been dried pre- One problem inherent in the study of
maturely (Han et al., 1996). gene activity during seed desiccation is that
ABA and LEA proteins are almost cer- several processes are occurring during mat-
tainly involved in the medium to long term uration/drying when tolerance is expressed
in the desiccation scenario in seeds and veg- (induced) in planta. Some of these have
etative tissues. In the latter, concentrations been mentioned above and in addition there
of hormone increase in response to drought are those associated with the establishment
stress and impending desiccation. In seeds, of dormancy and with the alterations in pro-
however, the highest concentrations of ABA duction, destruction and sensitivity to hor-
are normally reached in mid-development mones, for example ABA. It may be difficult
and are declining at the time when dehydra- to identify those changes in gene expression
tion commences; there is no evidence that that are specifically associated with the syn-
loss of water generally provokes ABA syndrome of desiccation damage and/or toler-
thesis, as it does in leaves, for example. ance but, none the less, experimental
None the less, application of ABA can con- protocols could be designed to minimize
fer desiccation tolerance on developing such extraneous complications. For exam-
embryos (Chapter 5), so it is conceivable ple, one way to separate operationally the
that the hormone participates in the in specific desiccation-related processes from
planta situation. In vegetative tissues, ABA others is to use very young embryos (prior
up-regulates the expression of several lea to dormancy inception and reserve accumu-
genes, including the dehydrins. There is cir- lation), as was done in early experiments
cumstantial evidence that some LEA pro- with barley (Bartels et al., 1988) and more
teins are also up-regulated by ABA in recently in the examination of amphiphile
developing seeds but the association among partitioning in wheat (Golovina et al., 2001).
dehydration, ABA and LEA proteins at the There is mounting evidence that the
stage of seed development when desiccation responses to drying and the early events in
tolerance is initiated is actually fairly the desiccation scenario in seeds and vegeta-
obscure. Though the implication of LEA tive tissues are initiated by the first changes
proteins and ABA in the desiccation phe- in water content at the beginning of dehydra-
nomena of seeds may be undeniable, the tion (see above). But what actually fires the
precise details of their involvement remain starting signal of the dehydration transduc-
unresolved. One confusing point is that the tion chain remains to be clarified. A plausi-
developing embryos of several types of ble assumption is that the detection of early
recalcitrant seeds are relatively rich in both water loss in vegetative tissues is the same
LEA proteins and ABA and yet they are, of whether only relatively mild water stress is
Damage and Tolerance in Retrospect and Prospect 371
suffered or whether the loss continues into tions where water loss can be strictly regu-
severe desiccation. Detection of changes in lated (such as by osmotic means) might be
turgor are likely to be responsible, involving an approach that will allow the application
trans-membrane sensors such as the putative of advanced biological techniques, but cau-
histidine kinase osmosensor in Arabidopsis tion must be exercised to minimize
(Urao et al., 1999). An intriguing point, how- mechanical damage by rapid dehydration
ever, is that the release of the initial signal or rehydration of tissues not protected by a
sets in motion, in one case, a series of events testa or pericarp.
that culminates in protection against In summary, then, there is still much to
extreme dehydration (e.g. in resurrection be learned about the extent to which the
plants) but, in others, only the ability to cope very early, medium- and longer-term events
with, in comparison, mild drought stress. are shared by seeds, pollen and vegetative
One of the earliest events in drought tissues.
stress is the generation of transient calcium How do we take our understanding for-
signals (for review, see Knight and Knight, ward? Clearly, identifying marker molecules
2001), and these are also likely to occur even for desiccation stress tolerance is highly desir-
when water loss continues to desiccation. able; and quantifying the products of gene
Whether or not calcium signalling partici- expression and how they interact or comple-
pates in all the multiple aspects of the desic- ment each other’s effect needs to be
cation syndrome has to be resolved. In seeds addressed. As has been noted in Chapter 11, a
and pollen almost nothing is known about quantitative comparison of LEA proteins in
the early signalling events set in motion by tolerant and sensitive tissues would be partic-
the incipient dehydration that initiates the ularly informative; in the same context, heat-
acquisition of desiccation tolerance. Is turgor shock proteins also qualify for further
loss by the embryo the first perception of investigation. Ultimately, we should know the
drying and is this, too, followed shortly by intracellular location of these proteins and
transient calcium signalling? examine the possibility of a redistribution of
Recent research indicates that in pollen these molecules and others as part of the tol-
and seeds an early detectable intracellular erance scenario (Chapter 10). Quantification
response to dehydration is the partitioning and identification of free radicals in biological
of amphiphiles from the aqueous cytoplasm systems is a similar challenge, as short half-
to the membrane lipid phase (Chapter 10). lives means that determination by electron
This has been suggested to occur in the paramagnetic resonance is difficult. There is a
establishment of desiccation tolerance, pos- need to identify new techniques and methods
sibly exerting a protective effect on mem- in pursuit of the causes of desiccation sensi-
branes by virtue of the properties (e.g. tivity as, until then, we may well run the risk
antioxidant) of certain endogenous of measuring mainly the consequences of des-
amphiphiles. It is important to determine iccation stress. The other side of the coin is, of
what the endogenous amphiphiles are and if course, recovery from such stress upon rehy-
the same partitioning phenomenon occurs dration and the repair processes involved
in desiccation-tolerant vegetative tissues. therein. This must surely be an area meriting
Investigation of the cell and molecular great attention. Desiccation damage, protec-
biology of signal perception and transduction against it, and repair all involve a multi-
tion in seeds and the early events that plicity of components but we might ask if all
closely follow will require the utilization play equally important roles or if the effects
of experimental systems that are rigorously and actions of some are more critical than
controllable. It is doubtful if the methods others.
that have been employed hitherto – drying Work on lower plants (Chapters 1, 7 and
whole seeds, either in planta or in vitro at 11) and angiosperm seeds (Chapters 5 and
different rates and relative humidities, etc. 8) has revealed some interesting general
– will be satisfactory. The use of isolated associations with habitat, i.e. likely adapta-
embryos (or parts thereof) under conditions to local conditions. Continuing to
372 M. Black et al.
screen biodiversity will undoubtedly sug- Looking to the not-too-distant future,

gest some interesting paradigms on which to one can see the use of our knowledge to
work. As the power of geographical infor- manipulate desiccation tolerance and seed
mation systems increases, in terms of layers longevity. Rapid advances in genomics and
of information that can be analysed and the related sciences will increasingly provide
precision of the data, it will be easier in the opportunities for the development of
future to consider biological data in an eco- unique tools and reporter systems to
logical context. Such a holistic approach to understand and regulate the mechanisms
the issue of desiccation sensitivity will be by which seeds and other organs respond
important if we are to capitalize on the to internal and external signals. The appli-
effort made since the 1980s in genomics and cation of our knowledge to enhance or con-
to gain the most from emerging work in this fer tolerance of desiccation and to increase
area. Biomolecules contribute to the ‘body longevity has important potential in agri-
plan’ of organisms, and form has an impact culture for the improvement of the quantity
on function in specific desiccating environ- and quality of the food supply and will add
ments. Proteomics will have an important to the success with which we can con-
part to play in this context. tribute to plant conservation.
References
Bartels, D., Singh, M. and Salamini, F. (1988) Onset of desiccation tolerance during development of
Bentsink, L., Alonso-Bianco, C., Vreugdenhil, D., Tesnier, K., Groot, S.P.C. and Koornneef, M. (2000)
Genetic analysis of seed-soluble oligosaccharides in relation to seed storability of Arabidopsis.
Brenac, P., Horbowicz, M., Dickerman, A.M., Miseray, F., Smith, M.E. and Obendorf, R.L. (2002) Up-
regulation of raffinose and stachyose accumulation in buckwheat (Fagopyrum esculentum
Moench) seedling hypocotyls during drying. Planta (submitted).
Cornford, C.A., Black, M., Chapman, J.M. and Baulcombe, D.C. (1986) Expression of -amylase and other
gibberellin-regulated genes in aleurone tissue of developing wheat grains. Planta 169, 420–428.
Golovina, E., Hoekstra, F.A. and van Aelst, A.C. (2001) The competence to acquire cellular desicca-
tion tolerance is not dependent on seed morphological development. Journal of Experimental
Botany 52. 1015–1027.
Han, B., Hughes, D.W., Galau, G.A., Bewley, J.D. and Kermode, A.R. (1996) Changes in late-embryo-
genesis abundant (LEA) messenger RNAs and dehydrins during maturation and premature dry-
ing of Ricinus communis L. seeds. Planta 201, 27–35.
Jiang, L., Downing, W.L., Baszczynski, C.L. and Kermode, A.R. (1995) The 5 flanking regions of
vicilin and napin storage protein genes are down-regulated by desiccation in transgenic tobacco.
Kermode, A.R. (1995) Regulatory mechanisms in the transition from seed development to germina-
tion: interactions between the embryo and the seed environment. In: Kigel, J. and Galili, G. (eds)
Seed Development and Germination. Marcel Dekker, New York, pp. 273–332.
Knight, H. and Knight, M.R. (2001) Abiotic stress signalling pathways: specificity and cross talk.
Trends in Plant Science 6, 262–267
Peterbauer, T. and Richter, A. (2001) Biochemistry and physiology of raffinose family oligosaccha-
rides and galactosyl cyclitols in seeds. Seed Science Research 11, 185–197.
Urao, T., Yakubov, B., Satoh, R., Yamaguchi-Shinozaki, K., Seki, M., Hiroyami, T. and Shinozaki, K.
(1999) A transmembrane hybrid-type histidine kinase in Arabidopsis functions as an osmosen-
sor. Plant Cell 11, 1743–1754.
Dessication Glossary 18/3/02 2:00 pm Page 373
Glossary
ABA: Abscisic acid a sesquiterpenoid plant hormone.

adsorption: The attraction of gas or liquid molecules to active surfaces. The rehydration
of dry biological tissues in humidified atmosphere is through water adsorption.
ageing: Time-dependent deterioration of cells and biomolecules. Ageing reactions are
often water-content-dependent.
algae: An informal term covering photosynthetic organisms (largely aquatic) other than
the green land plants (bryophytes, pteridophytes, gymnosperms, flowering plants).
Algae embrace red, green and brown seaweeds and unicellular, colonial and filamen-
tous organisms from a taxonomically diverse range of groups, variously defined by
different authors.
amphiphilic: Having an affinity for both aqueous and non-aqueous phases. Typically used
to describe the behaviour of a molecule with a polar group that can interact with the
cytoplasm and a non-polar, hydrophobic group that interacts with the membrane.
Molecules with two such different groups are amphipaths.
angiosperms: Flowering plants, consisting of two groups, the monocotyledons and the
dicotyledons, characterized by having one or two seedling leaves, respectively.
anhydrobiosis: Suspended life in the dried state.
anthophyte hypothesis: This hypothesis refers to the evolutionary relationships of seed
plants. It proposed that the gnetophytes are the sister group (closest living relatives) to
the angiosperms and thus they shared a common ancestor. Though formerly strongly
supported, recent molecular data have generated considerable doubt about the idea, and
there is strong evidence for the gnetophytes being most closely related to the conifers.
antioxidants: Molecules that remove activated oxygen species from cells by serving as cat-
alysts (enzymes such as catalase, superoxide dismutase and glutathione reductase) or
substrates (ascorbate or tocopherol) in reactions that donate unpaired electrons.
ascospore: A meiospore borne in an ascus (saclike structure formed by the Ascomycota).
bicellular (also binucleate): Refers to the developmental stage of the male gametophyte of
higher plants that has undergone one nuclear division after meiosis. One cell (the
vegetative cell) is involved in directing pollen tube growth; the other cell later
divides into two sperm cells and is located inside the vegetative cell. After division,
the tricellular (or trinucleate) pollen consist of one vegetative cell with two sperm
embedded in it. Depending on whether anther dehiscence occurs after the first or the
second mitosis, mature pollen is designated bicellular or tricellular.
373
374 Glossary
bitegmic: Of ovules (cf. unitegmic or ategmic). Ovules having both outer and inner integu-
ments prior to fertilization and seed development. Where both persist in the mature
seed, the inner integument becomes the tegmen, and the outer integument becomes
the testa sensu stricto.
blastospore: A spore arising by budding such as a conidium arising from a narrow region
of the conidiogenous cell with elongation and swelling and then delimitation of the
conidium by a septum.
Boltzman distribution law: The expression for the ratio of populations of molecules at
any two levels of energy.
bound water: A concept to describe hydration of macromolecules based on interactions of
water molecules with a macromolecular surface and with other water molecules.
Bound or vicinal water has sufficient interactions with the macromolecular surface to
cause changes in its thermodynamic properties and molecular mobility compared
with water in a dilute solution. The concept of bound water was originally based on
simple sorption theory, but has evolved to consider adsorption sites with different
characteristics and relationships between bound water and the structure and activity
of macromolecules.
brachycytes: Spherical, thick-walled cells that are formed on moss protonemata after
extended periods of culture.
Bryophyta (bryophytes): Green land plants with an alternation of haploid and diploid gen-
erations, in which the diploid sporophyte (capsule) remains dependent on the haploid
gametophyte for water and at least in part for nutrition (mosses, liverworts – q.v.).
C3 plant: Plant in which the first detectable photosynthetic products are three-carbon
molecules, e.g. 3-phosphoglyceric acid. Includes most plants of wet or mesic habitats
at all latitudes.
C4 plant: Plant in which the first detectable photosynthetic products are four-carbon
organic acids, e.g. oxaloacetic acid, malic acid. Most characteristically plants of semi-
arid situations in warm climates.
callose: 1-4 glucan; substance often formed in pollen upon stress.
carbon balance: The cumulative net uptake of carbon by a plant; cumulative gross photo-
sythesis minus respiration.
cavitate : To form an air-filled space in water-filled xylem.
chalaza: The chalaza is the tissue at the base of the ovule, from which the integuments
arise, and is probably involved in nutrient transfer from the funicle to the developing
embryo and endosperm. In some species it becomes heavily developed, surrounding
and overtopping the integuments, a state known as pachychalazy.
chaperones: Proteins that modify folding of other (newly formed) proteins and assist
assembly of protein oligomers.
chemical shift in NMR: The shift in the position of the resonance line of the nucleus
because of the local electronic structure, which makes the local magnetic field
slightly different from the external static field.
chlorophyll fluorescence: Excitation energy absorbed by chlorophyll may: (i) bring about
photochemical processes; (ii) be dissipated as heat; or (iii) be re-emitted as red fluo-
rescence, measurement of which provides a basis for non-invasive measurements of
various aspects of photosynthesis.
compatible solutes (osmolytes): Osmotically active, non-toxic, low-molecular-mass mole-
cules (such as quaternary amines, amino acids or sugar alcohols) which may accumu-
late in cells in response to water or freezing stress.
conidium: In fungi a non-motile asexual spore usually formed at tips or sides of a sporoge-
nous cell; in some cases a pre-existing hyphal cell may be converted into a conidium.
constitutive: Occurring constantly rather than being induced in response to a particular
stimulus.
Glossary 375
continuous-wave NMR: A field-swept technique, when the field is swept through the res-
onance frequency and a frequency domain absorption spectrum is obtained.
cooperative stress: Stress that is not associated with water loss per se, but exacerbates the
damaging effects of water loss, possibly because it has a similar mechanism of dam-
age.
crassinucellate: Of ovules (cf. tenuinucellate). Ovules in which the nucellus is massively
developed, rather than scantily so. The nucellus is the megasporangial tissue sur-
rounding the embryo sac, and it persists only relatively rarely in the mature seed,
where it forms the perisperm.
critical minimum surface area: The minimum membrane surface area that can be lost
from the plasmalemma or tonoplast upon contraction during water stress without
causing cells to burst upon rehydration.
critical minimum volume: The minimum volume to which cells or vacuoles can contract
during water stress without bursting upon rehydration. Most non-acclimatized cells
can contract no more than to 50% of their original volume.
critical water content: The minimum water content to which cells or macromolecules can
be dried without imposing irreparable damage. This water content reflects the critical
water potential.
critical water potential: The minimum water potential to which cells or macromolecules
can be dried without imposing irreparable damage. This water potential corresponds
to the critical water content.
cryopreservation: The technique to preserve living organisms or cells at subzero tempera-
tures, usually below –100°C (e.g. in liquid nitrogen –196°C), which includes pretreat-
ment in cryoprotective solutes (if required), cooling to below 0°C, storage at low
temperature, thawing and preparation for resumption of growth.
dehiscence: Opening of anthers in higher plants, which allows pollen to be dispersed: or,
of some fruits, to allow seeds to be dispersed.
dehydrins: Group 2 LEA proteins synthesized in association with dehydration, character-
ized by a lysine-rich 15-amino-acid motif (K-segment), contiguous serine residues
and the consensus sequence DEYGNP.
demixing: Rearrangement and consolidation of similar-type molecules that increases
packing efficiency in severely dehydrated systems. Demixing will disturb the original
configuration of proteins and lipids within bilayers of membranes and aggregation of
molecules can result in irreversible structural changes.
desiccation: The extreme form of water loss, in which most of the protoplasmic water is
lost and a very low amount of tightly bound water remains in the cell.
desiccation avoidance: A protective strategy against drought stress where cells remain
hydrated using adaptive structures that scavenge water. This strategy prevents water
loss. See also desiccation resistance.
desiccation damage (sensu stricto): Mechanical or structural damage to cells and cellular
constituents directly resulting from water removal.
desiccation resistance: A protective strategy against drought stress where cells reduce the
rate of water loss by using adaptive structures that form barriers to water loss or by
accumulating solutes that lower the water potential difference between the cell and
the environment. This strategy prevents water loss.
desiccation tolerance: The ability to recover biological functions after drying to a point at
which no liquid phase remains in the cells (e.g. water content down to 5% or less of
dry weight, in equilibrium with water potential down to 200 MPa or less).
desorption: Opposite of adsorption; the dehydration of biological tissues in dry atmos-
phere is water desorption.
diaspore: Any spore or other plant part able to form a new organism, e.g. the haploid
structure formed out of a part of the moss gametophyte.
376 Glossary
diffusional correlation time: The average time between jumps in position for water mole-
cules in the system.
Dollo’s law: Dollo’s law or rule suggests that, for complex characters or traits, parallel or
multiple origin is unlikely, but that reversal or loss may be easy. The assumption is
that many genes must change to create a morphological structure or physiological
trait, but only one of them needs to change in order to lose it.
dormancy: An endogenous mechanism that prevents viable hydrated plant parts (e.g.
seeds, spores, buds) from resuming growth and full metabolic activity.
drought: In plants, the partial limitation of water content, often for a prolonged period;
usually when cell water potentials (w) are ≤ 3 MPa in non-transpiring cells. Water
contents may reach 20–25% (fresh weight basis), 0.25–0.33 g water g1 dry weight.
drought tolerance: The ability to survive drought.
dry: To remove water (verb) or without water (adjective). Since achieving and measuring
truly dry material is logistically difficult, ‘dry’ is often used as a relative term to
describe material that is drier than its undried counterpart.
electron paramagnetic resonance (EPR): The absorption of electromagnetic energy during
transition of electrons between two energy levels of Zeeman splitting (q.v.).
endocytotic vesicles: Invaginations of plasma membrane into the cytoplasm observed dur-
ing osmotic contraction of protoplasts from non-acclimatized plants. These are
believed to be deleted from the membrane surface area.
enthalpy (H): A defined thermodynamic variable of state that consists of internal energy
of the system (E), specified pressure (P) and volume (V). H = E +PV, where the PV
units are converted to calories, ergs or joules. Different enthalpy, H, describes the
change of energy status.
entomopathogenic: Pathogens (e.g. fungi, bacteria) that feed on insects.
entropy (S): A thermodynamic term that quantifies the randomness or disorder of the sys-
tem.
epilithic: Growing on the surface of rocks.
EPR imaging: Visualizing the distribution of paramagnetic centres in a sample.
eukaryotic: Of cells having a nucleus.
exocytotic extrusions: Folding of the plasma membrane to the cell exterior observed dur-
ing osmotic contraction of protoplasts from acclimatized plants. These are reincorpo-
rated into the plasma membrane upon expansion and so are believed to be a
mechanism to retain overall membrane surface area in contracting cells.
ferns: The largest group of pteridophytes, having leaves with branching veins (mega-
phylls).
flash drying: Drying by rapid flow of dry air over excised embryonic axes, somatic
embryos or small tissue pieces.
Fourier self-deconvolution: Transformation of the absorption bands in the Fourier trans-
form infrared spectrum to line shapes with narrower peaks (resolution enhancement
technique).
Fourier transformation: The mathematical method to convert the time dependence of the
NMR signal (FID) into the frequency dependence of the NMR signal.
free energy (Gibbs free energy, G): A quantity that is used to describe the energetics of
chemical, physical and biological processes. Gibbs free energy in a system is the max-
imum amount of energy for work. It decreases for a spontaneous process such as
rehydration of dry tissues, in which free energy of water decreases.
free induction (FID) signal: The measure of the NMR signal over time.
freeze–fracture: A method of preparing specimens for electron microscopy by freezing
and then splitting them.
freezing point depression: One of the four colligative properties of solutions in which the
freezing point is depressed below that of the pure solvent.
frequency: Number of cycles per second.
Glossary 377
gametophyte: The structure that forms the haploid, gamete-forming part of a plant’s life
cycle.
gas exchange: Usually refers in plant-physiological contexts primarily to uptake or output
of CO2 and O2 in the course of photosynthesis and respiration.
Gauss: The unit of strength of the magnetic field.
g-factor: The ratio of the magnetic moment to the spin angular moment of an electron
(determines the position of the EPR spectrum).
glass (glassy state): A fluid that is so viscous that it acquires mechanical properties
(strength) of a solid. The viscosity results from numerous intermolecular interactions
in a random array forming an irregular matrix of pores. Because there is no regular
pattern to the arrangement of molecules in this fluid (similar to a liquid), the struc-
ture is considered amorphous relative to the defined arrangement of molecules in a
crystalline solid. Because molecular mobility of molecules within the glass is
restricted, physical and chemical reactions are slowed but not stopped. Thus, the
glass is considered kinetically, but not thermodynamically, stable. Glass transitions
are considered second-order state changes (as opposed to phase transitions, which
are first-order) because of a continuous change in enthalpy, entropy and volume (first
derivative of chemical potential) throughout the transition but a discontinuous
change in heat capacity (second derivative of chemical potential).
glass formation: A change from a fluid to a semi-solid state in the protoplasm of desiccat-
ing cells, also known as vitrification. Carbohydrates and proteins are the main com-
pounds that can contribute to glass formation in the cytoplasm.
heat-shock proteins: Proteins generally synthesized in response specifically to relatively
high temperatures (e.g. 40°C): often act as chaperones (q.v.) and protein protectants.
Höfler diagram: The plot of cellular water potential components against protoplast vol-
ume.
homoiochlorophyllous: Retaining most or all chlorophyll through a drying–rewetting cycle.
homoiohydrous: Maintaining a high water potential and active metabolism during times
of low water availability.
hydration force explanation: A hypothesis used to describe how compression of macro-
molecules can lead to their deformation. Compression of cell volume by water
removal can lead to repulsive forces as molecules with similar charges come within
close proximity. The repulsion between surfaces can lead to lateral tensions within
structures that cause phase changes or deformations. The hydration force explanation
invokes non-specific mechanisms for the protection of macromolecules by carbohy-
drates (as opposed to the water replacement hypothesis, which invokes specific inter-
actions), first as osmotica that resist water loss, and then as glass formers that provide
mechanical resistance to the compression.
hydration levels: Ranges of water contents or water potentials that define different struc-
tures/functions of molecules or physiological activities of cells. Also, ranges of water
contents or water potentials that define changes in thermodynamic or motional prop-
erties of water.
hydraulic conductivity: A measure of the diffusional resistance of a water transport path-
way within the tissue.
hyperfine interaction: The interaction between nuclei and unpaired electrons in EPR.
hyperfine splitting constant: The distance between lines of an EPR spectrum originating
from hyperfine splitting.
hyperfine splitting of the EPR spectrum: Separation between lines of an EPR spectrum
due to the hyperfine interaction.
hysteresis: The difference in the equilibrium water between desorption and adsorption
isotherms.
378 Glossary
image contrast: The differences in signal intensity in NMR imaging between different
regions of the sample.
imbibitional stress: Stress imposed on a dehydrated organism by imbibition of water. The
injury that ensues usually encompasses the loss of plasma membrane integrity. The
injury is severe when imbibition occurs at low temperature and when the
organism/cell is very dry: avoidance can be achieved by prehydration in humid air
and warm imbibition.
in vitro: Literally ‘in glass’, referring to tissues cultured in a sterile container on an artifi-
cial, sterile nutrient medium.
inhomogeneous broadening: The broadening of the EPR lines because of unresolved
hyperfine structure.
integrated intensity: The area beneath an absorption peak.
interfacial region: The boundary between two phases.
intermediate seeds: Seeds that tolerate considerable (at least to 30% RH) but not complete
drying. Life spans of stored seeds progressively increase as the storage RH is
decreased to about 50% and then a reversed trend is observed with storage RH <
50%. Seeds with intermediate characteristics cannot be stored using standard recom-
mended storage protocols: though they appear to survive low water contents, they do
not survive the added stress of exposure to 18°C. See orthodox, recalcitrant.
IRGA (infrared gas analysis): Methods using gas-phase absorption bands in the near
infrared to measure concentration changes in CO2 (or other gases).
isotropic motion: Uniform tumbling of a spin probe in all directions; the isotropic EPR
spectrum originates from the averaging of the spectral anisotropy.
LEA (late embryogenesis abundant) proteins: A broad family of universal plant proteins
with conserved amino acid motifs which accumulate to high levels during late stages
of embryo development or in response to osmotic stress in vegetative tissues. They
are very hydrophilic, remain soluble at T 90°C, and are believed to protect cells
during water stress through an, as yet, unknown mechanism. See also dehydrins.
lichenized fungus: A fungus that has formed a lichen in association with an alga.
line width: The width at half-height of absorption peak.
liverworts: Bryophytes (q.v.) with leafy or (less commonly) thalloid, usually dorsiventral
gametophytes and short-lived sporophytes (Hepaticae; Hepaticopsida).
magic angle: The angle (54° 55) of the axis of mechanical rotation of the sample in high-
resolution NMR to eliminate line broadening.
magnetic susceptibility: The ratio between magnetization and magnetic field strength.
magnetogyric ratio: The ratio of magnetic dipole moment to the spin angular moment of a
specific nucleus.
manometric methods: Techniques using change in pressure (Warburg) or volume (e.g.
Gilson) in a gas space over the reaction mixture or material to follow a metabolic
process (e.g. respiration or photosynthesis).
minimum critical volume: See critical minimum volume.
monolayer hydration: The level of hydration at which only polar sites are bounded by
water.
mosses: Bryophytes (q.v.) typically with (+ or ) radial leafy shoots, and sporophyte cap-
sules borne on a long-lived seta (Musci, Bryopsida).
NMR imaging: Imaging of spatial distribution of water or water properties based on pro-
ton magnetic resonance.
nuclear magnetic resonance (NMR): The absorption of electromagnetic energy during the
transition of a nucleus between two discrete energy levels of Zeeman splitting.
oligosaccharide: A carbohydrate whose molecules are composed of a few (< 20) generally
mixed (e.g. glucose, fructose, galactose) monosaccharide units: three, four and five in
raffinose, stachyose, verbascose, respectively.
Glossary 379
order parameter: Quantity calculated from the shape of the EPR spectrum, which indi-
cates the degree of motional anisotropy.
orthodox seeds: Seeds that tolerate the immediate effects of severe water loss (i.e. desicca-
tion-tolerant). Life spans of stored seeds progressively increase as the storage RH is
decreased to about 20% and then a reversed trend may be observed with storage RH
< 20% (decreasing life span with decreasing RH). Seeds with orthodox characteristics
can be stored using standard recommended storage protocols of drying to 0.05 ± 0.02 g
water g1 dry mass and storing in a freezer at about 18°C. Storage longevity can be
predicted from temperature and seed water content. See intermediate, recalcitrant.
osmolytes: See compatible solutes.
osmotic adjustment: The net accumulation of solutes after the plant tissue has been
exposed to a predetermined rate of water deficit.
osmotic excursions: Reversible shrinking and swelling of cells and protoplasts during
exposure to cycles of low and high water potentials.
osmotically inactive: Apoplastic water present in very small pores and strong water-
binding sites of biological surfaces in plant tissues.
osmotically unresponsive: Membrane vesicles that fail to swell when water stress is
relieved because the lumen lacks osmotically active constituents. This most probably
occurs when different membrane systems compress together during dehydration and
fuse, excluding formerly contained constituents.
ovule: The female (mega) spore and gametophyte of higher plants: becomes the seed after
fertilization.
pachychalazy: See chalaza.
paramagnetic: Atom or molecule containing an unpaired electron.
partitioning: Distribution of molecules, e.g. between lipid and aqueous phases.
permanent wilting point: Minimum water potential tolerated by non-transpiring cells.
Similar in concept to critical water potential.
phase separation: A consequence of demixing. The aggregation of similar-type molecules
into enriched domains leads to higher localized chemical potentials and greater like-
lihood of phase changes.
phase transition: Change of state between solid, liquid and vapour phases. Phase changes
in lipids are complex because of the diverse crystalline states of pure lipid and lipid
mixtures. For polar lipids, phase changes occur when a fluid gel converts to a liquid
crystalline or hexagonal phase. Phase changes in polar lipids can be induced by alter-
ing the water status (drying favours gel and hexagonal phases) or temperature (low
temperatures favour gel phases). Phase changes are termed first-order transitions
because of an abrupt, discontinuous change in the enthalpy, entropy and volume
(first derivatives of chemical potential) and a consequent infinite heat capacity (sec-
ond derivative of chemical potential).
phycobiont: In lichens, the alga that is associated with the fungus, or mycobiont.
phylogenetic classification: Phylogenetic classifications attempt to generate systems that
reflect as closely as possible the evolutionary relationships and history of a group of
organisms. They recognize only those groupings of species that are monophyletic, i.e.
all the members of the group are likely to be descended from a single common ances-
tor.
phytochrome: Chromoproteins that undergo a reversible conformational change maxi-
mally upon absorption of red or far-red light. They regulate many aspects of plant
function.
plasma membrane: The membrane that envelops a cell protoplast.
plasmolysis: Withdrawal of the cytoplasm from the cell wall when the cell is placed in a
solution of lower osmotic potential than the cell sap.
poikilochlorophylly: The ability to reversibly lose chlorophyll during desiccation.
380 Glossary
poikilohydrous: Of plants whose water content closely follows fluctuations of humidity in

their environments (in contrast to homoiohydrous plants), typically suspending
metabolism during periods of drought.
pollen: Male gametophyte of higher plants, which functions to deliver its haploid sperm
cells to the ovules in order to bring about fertilization.
population (of molecules): The number of molecules occupying a particular energy level.
prothallus: Gametophyte (haploid) in ferns, horsetails, Selaginella and Lycopodium that is
formed from spore germination and which produces gametes. After fertilization the
diploid sporophyte grows out of the prothallus.
protonema: The structure (immature gametophyte) that develops from spore germination,
e.g. in mosses.
Pteridophyta (pteridophytes): Green plants with an alternation of haploid (gametophyte,
prothallus) and diploid (sporophyte) generations, both (at least potentially) capable of
living independently, the sporophyte being the dominant plant (ferns, horsetails, club-
mosses, spikemosses).
pulsed NMR: NMR technique based on the recording of NMR signals with time after excit-
ing nuclei by a short intense pulse of radio-frequency radiation.
PV curve: The relationship between the reciprocal of water potential and relative water
content of a tissue.
radical adduct: The product of the interaction of a primary free radical with a spin-trap
molecule.
reactive oxygen species (ROS): Molecules containing oxygen with an unpaired electron or
a pair of electrons with parallel spin (singlet oxygen). These molecules, often resulting
from free-radical reactions, seek an additional electron and so are highly reactive with
biomolecules which are electron-rich. Reacting with ROS, biomolecules are peroxi-
dized to become reactive themselves, initiating a cascade of degradative reactions.
recalcitrant seeds: Mature seeds that do not survive if desiccated to water potentials less
than about 15 MPa (about 90% RH) and hence cannot be stored in the ‘dry’ state.
Hydrated storage at either cryogenic or supra-freezing temperatures appears to be the
best storage option.
receptivity of the isotope: The product of the sensitivity of the isotope for NMR experi-
ments and its natural abundance.
rehydrins: Proteins synthesized specifically in association with rehydration in desiccation-
tolerant plants.
relative humidity: Water activity multiplied by 100.
relative water content (RWC): Tissue water content relative to (fraction or percentage)
water content at full turgor.
relaxation process: The return of magnetization to thermal equilibrium.
resurrection plants: A small group of poikilohydrous higher plants which tolerate almost
complete water loss in their vegetative tissues and resume normal functional activity
after rehydration: occur in specific ecological niches with seasonal water availability.
RH (relative humidity): Water content of air expressed as percentage (or fraction) of satu-
rated water content at the same temperature.
rotational correlation time: The time it takes for a molecule to rotate one radian around its
axis.
rubber: A fluid that is more viscous than a syrup but less viscous than a glass. There are
fewer intermolecular interactions and larger pore sizes in rubbers compared with
glasses and this allows for greater elasticity in the structure.
RWC: Relative water content.
second-derivative analysis: Mathematical procedure used for increasing the resolution of
an FTIR spectrum.
self-incompatibility: In higher plants the phenomenon of pollen being unable to establish
fertilization within the same plant or some individuals of the same plant species.
Glossary 381
sensitivity (spectral): The minimal number of spins (electrons, nuclei) which can be mea-
sured by a method (EPR, NMR, respectively).
sensitivity of the isotope for NMR experiments: The quantity depending on magnetogyric
ratio () and spin quantum number (I).
spatial resolution: The precision in the determination of the location of a signal source
(or the measure of the minimal distance between two signal sources within the sam-
ple which still allows them to be distinguished).
spectral anisotropy: The dependence of EPR spectra on the orientation of the spin probes,
originating from the anisotropy of the interaction of an electron with the externally
applied magnetic field.
spectral resolution: Quantity that expresses how the lines in a spectrum are separated
from one another.
spectroscopy: The measurement of the energy differences between discrete energetic lev-
els of atoms or molecules.
spin label (spin probe): Stable free radical that contains a nitroxide fragment with an
unpaired electron.
spin trap: Diamagnetic molecule forming a nitroxide radical when interacting with a pri-
mary free radical.
spin-echo technique: Pulse NMR based on applying a second pulse after a set of delay
times to eliminate the effect of the inhomogeneity of the magnet.
spin-lattice (or longitudinal) relaxation time (T1): The time constant of magnetization
decay because of the interaction of nuclear/electron magnetic moments (spin) with
the environment (lattice).
spin–spin (or transverse) relaxation time (T2 ): The time constant of magnetization decay
because of the interaction of nuclear/electron magnetic moments (spin) with each
other.
sporangia: Spore-producing structures, e.g. in pteridophytes.
spore: In mosses, ferns, horsetails, Lycopodium and Selaginella, a haploid, stress-resistant
cell formed by meiosis; in fungi, spores may not necessarily be the result of meiosis
and also may be diploid.
spore bank: Layer of accumulated spores in the soil.
sporocarp: Structure in certain ferns that contains the sporangia.
sporophyte: The diploid (asexual) phase of the alternation of generations in plants.
structural water: Water required to maintain the configuration of macromolecules nor-
mally observed under aqueous conditions, and so is most often identified in drying
systems as the minimum amount of water required to prevent a conformational
change. Water, at water contents where conformational changes in macromolecules
occur, has unusual thermodynamic properties or restricted mobility. Consequently,
structural water is a component of bound or vicinal water in hydration models using
the bound-water concept. In alternative solution-based models of hydration, struc-
tural water is a likely component of the super-viscous solutions with rubbery or
glassy characteristics.
sucrose: A disaccharide; molecules each contain one glucose and one fructose unit.
symbiosis: A regular association between two organisms characterized by mutual benefit
and interdependence.
Tg: Glass–liquid transition temperature.
Tm: Gel–liquid crystalline temperature of membranes.
teliospore: A thick-walled resting spore of fungi belonging to the rusts and the smuts, in
which karyogamy occurs.
trehalose: A disaccharide in which each molecule contains two glucose units with an
1→1 linkage: often associated with desiccation tolerance in animals and some plants.
tricellular (or trinucleate): See bicellular.
turgid: Swollen or firm because of the pressure of water within the cell or tissue.
382 Glossary
turgor: Cellular pressure generated from the movement of water into a cell. Pressure
exerted by the cell wall, balancing the difference between the osmotic potential of the
cell contents and the water potential of the surroundings.
urediniospore: A binucleate spore of Uredinales (rust fungi).
vascular plants: Green land plants which possess vascular tissues for water conduction,
comprising pteridophytes and seed plants (gymnosperms and angiosperms).
viability: Potential ability of a cell, tissue, organ or plant part to resume full metabolism
and growth under favourable conditions (e.g. of seeds).
vitrification: A change from a fluid to a semi-solid state in the protoplasm of desiccating
cells, also known as glass formation.
volumetric elasticity module: An expression to quantify the relationship between the
change in volume and the applied pressure.
water activity: Proportion of water available compared with pure water. Water activity is
usually measured in the vapour above a condensed phase and ranges from 0 (no
water in the condensed phase) to 1 (pure water in the condensed phase). At equilib-
rium, the water activities of the vapour and condensed phases are the same. The nat-
ural log of water activity directly relates to the chemical potential of water. See water
potential.
water content: A measure of the concentration of water that is usually expressed by mass
ratios on either an absolute scale of 0 to ∞ (no water to pure water) by dividing mass
of water by mass of dry material, or on a relative scale of 0–1 (no water to full hydra-
tion). The denominator for the relative expression is either total mass or mass of
water in fully hydrated tissues, an empirically derived value that varies with species,
tissue and tissue development (a.k.a. relative water content or RWC).
water potential: A measure of availability of water in terms of pressure that decreases
from 0 (pure water) to ∞ as water content decreases. Units are usually expressed as
MPa. The related parameter, chemical potential of water, which describes the avail-
ability of water or its potential for effecting reactions in energy terms (units are usu-
ally joules), is the difference between the molar free energy of pure water at standard
temperature and pressure (STP) and the water potential times the molar volume of
pure water at STP (18 cm3 mol1). Chemical potential of water inversely correlates
with chemical potentials of solutes, which in turn are components of the free-energy
difference that drive reactions.
water replacement hypothesis: A mechanism of protecting macromolecules from struc-
tural changes during dehydration by inserting hydrophilic groups of specific solutes
(usually carbohydrates) on to hydrophilic sites of macromolecules. This substitution
prevents aggregation of molecules by van der Waals forces and the separation pre-
vents deleterious interactions.
water sorption isotherm: A plot that describes the relationship between water content
and RH (RH ≤ 93%) or water content and water potential (w ≥ 20 MPa) for a par-
ticular material at a particular temperature.
water-clustering function: A volumetric analysis of water relation for the formation of
water clusters (e.g. water–water self-association).
wave number (spectroscopy): The number of waves per centimetre.
xeromorphic: Having morphological characteristics particularly adapted to conserving
water under dry conditions.
Zeeman splitting: The splitting of the energy levels of the electron/nucleus due to the
interaction of its magnetic moment with the external static magnetic field.
zygospore: A resting spore that results from the fusion of two gametangia in
Zygomycotina (fungi).
(Terms included do not necessarily reflect the collective views of the authors)
Dessication Taxonomic Index 3/4/02 2:30 pm Page 383
Taxonomic Index
Authorities for names are not included and synonymy is not rationalized, but can be cross-checked
via the relevant chapters.
Acanthaceae Adiantaceae
vegetative desiccation tolerance 222 desiccation tolerant species 218
Acer Adiantum
platanoides desiccation tolerant species
cell cycle 174 incisum 218
oligosaccharides 172 gametophyte cryopreservation
respiration 173 tenerum 225
seed anatomy 252 trapeziforme 225
seed development 156–157 spore desiccation tolerance
seed drying time 159 capillus-veneris 192
see also Norway maple Aesculus hippocastanum
pseudoplatanus axis water stress 267
cell cycle 174 critical water potential and water content 50
desiccation tolerance, and seed drying seed development 159
time 159
seed dormancy 158, 245
oligosaccharides 172
water loss curve 68
respiration 173
water and seed longevity 103
seed anatomy 252
Afrotrilepis pilosa
seed development 156–157
in situ/natural habitat 12, 224
seed dormancy 158
longevity when dry 225
see also sycamore
sp. specialized structures/velamen 224
free-radical scavenging 174 vegetative desiccation tolerance 220
variation in desiccation tolerance 155, Agathis
244 robusta
Aceraceae seed storage classification 241
angiosperm phylogeny 242 sp.
recalcitrant seeds 247 orthodox and recalcitrant seeds 244–245
seed storage classification 248 seed weight 245
Acinetobacter radioresistans Aglaonema sp.
survival at 31% RH 10 desiccation sensitive pollen 187
Acorales Agrostemma githago
seed storage classification 247 seed development 153
Actiniopteris Alismatales
desiccation tolerant species seed storage classification 247
dimorpha 218 Allium sp.
radiata 218 vegetative propagules 228
383
384 Taxonomic Index
Aloina aloides apple

ABA-induced tolerance 216 orthodox seed 252
Alternaria TBARS assay 119
desiccation tolerant spores 195 see also Malus
porri 196 Aquifoliales
Amaranthaceae seed storage classification 247
angiosperm phylogeny 242 Arabidopsis
seed storage classification 248 sp.
Amaryllis sp. aba, abi-3 and other mutants 164–165
pollen DNA repair 356 abi-3 and other gene products 163, 332
Amborellaceae alkyl hydroperoxidase 330
angiosperm phylogeny 246 AtPer1 expression 170
Amomyrtus luma desiccation intolerant mutants 25, 253,
seed storage classification 241 310, 324
Anacardiaceae ‘dormancy’ gene 29
recalcitrant seeds 247 HSP 165, 309
Anacystis sp. osmosensor 371
desiccation-tolerant cysts 17 protein denaturation 169
Anamodon viticulosus transgenic plants 326, 331–332, 334–335
chlorophyll fluorescence 212 thaliana
desiccation tolerance 209, 216 1H-NMR of seeds for betaine 132
photosynthesis rate 210 EST collections 30
recovery processes 214
homologues of lea cDNAs 162
Andira inermis
lea genes 164
critical water potential and water content
protein–sugar glass 306
50
seed mucilage 348
Andreaea
seed storage stability 304
rothii
trehalose synthesis 168
CO2 uptake 214
Araceae
desiccation tolerance 213
desiccation sensitive pollen 9, 187
sp.
seed storage classification 248
Andreaeales Araucaria
desiccation tolerance 209 angustifolia
Anemia recalcitrant seeds 245
phyllitidis seed respiration 173
spore desiccation tolerance 192 araucana
tomentosa recalcitrant seeds 245
desiccation tolerant species 220 bidwillii
Anemone coronaria recalcitrant seeds 245
tuber desiccation tolerance 228 seed size and weight 246
angiosperms brownii
vegetative desiccation tolerance 220 seed size 246
Annonaceae cunninghamii
seed storage classification 247–248 seed weight 245
Anomodon viticulosus heterophylla
dark respiration 215 seed weight 245
in situ, desiccated and hydrated 14 hunsteinii
photosynthesis and water potential 229 recalcitrant seeds 245
Anthurium seed weight 246
seed storage classification 240 water and seed longevity 103
Apiaceae mirabilis
seed storage classification 248 seed size 246
see also Umbelliferae Section of Araucariaceae 245–246
Apiales sp.
seed storage classification 247 orthodox and recalcitrant seeds 244
Apocynaceae sphaerocarpa
seed storage classification 248 seed size 246
Taxonomic Index 385
Araucariaceae sp.
habitat and seed storage 252 desiccation sensitive pollen 187
seed desiccation sensitivity 244–245 Avicennia marina
Archidium alternifolium ABA and seed development 171
spore germination 194 axis drying curve 99
Arecaceae cell vacuolation 174
habitat and seed storage 252 desiccation sensitivity 159, 268
recalcitrant seeds 247 DNA repair 174, 354–355
seed anatomy 252 respiration 172
seed storage classification 248–249, 253 seed desiccation 153
see also Palmae seed development 157
Arecales stachyose 172
number of desiccation sensitive seeded sub-cellular de-differentiation 173
species 246–247 viviparous germination 158
Artemia Aylthonia blackii
desiccation tolerant cysts 281 vegetative desiccation tolerance 222
water difussion coefficient in cysts 130 Azadirachta indica
Arthropteris orientalis axis drying curve 99
desiccation tolerant species 220 EPR of chilling stress 122
Artocarpus heterophyllus imbibitional damage 102
axis drying curve 99 seed storage classification 241
Asclepiadaceae
variation in desiccation tolerance 157, 266
see also neem
Asparagales
Azolla
spore dormancy 192
Aspergillus japonicus
sporocarp storage
desiccation tolerant spores 196
filiculoides 193
Aspleniaceae
desiccation tolerant species 219
Asplenium
Bacillus subtilis
desiccation tolerant species
DNA form 350
aethiopicum 219
bourgaei 219 gene product 332–333
pringlei 219 Balsaminaceae
ruta-muraria 220 pollen storage life 190
rutifolium var. bipinnatum 219 Barbacenia
sandersoni 219 vegetative desiccation tolerance
trichomanes 220 flava 222
vegetative desiccation tolerance longifolia 222
septentrionale 220 riedeliana 222
Asteraceae sellovii 222
seed storage classification 248 Barbaceniopsis
tricellular pollen 188 vegetative desiccation tolerance
see also Compositae boliviensis 222
Asterales humahuagensis 222
seed storage classification 247 Barbula sp.
Athyrium filix-femina temperature and survival 213
spore bank 193 barley
spore storage 193 alkyl hydroperoxidase 330
Atrichum androgynum chemical shift imaging of seeds 131
partial dehydration 216 ‘dormancy’ gene 29, 349
Austrobaileyaceae gene product 332
angiosperm phylogeny 246 lea gene 25
Avena LEA proteins 24, 334
fatua PER1 protein 170
seed DNA repair 353 pulsed (spin-echo) NMR of developing
see also oat seeds 130
386 Taxonomic Index
barley continued Brassicaceae

seed development 153 seed storage classification 248
see also Hordeum species coverage 246
Bauhinia see also Cruciferae
seed anatomy 252 Brassicales
bean seed storage classification 247
axis water stress 267 Bromeliaceae
cell contraction 270 seed storage classification 248
chilling damage 344 Bromus secalinas
DSC data 306 ‘dormancy’ gene 29, 330, 349
glass formation in axes 302, Bryaceae
state diagram 303, 305 induced desiccation tolerance unexplored
seed imbibition damage 334 216
Beauveria Bryum
desiccation tolerant spores predrying
bassiana 196 caespiticium 216
brongniarti 196 capillare 216
sp. 195 pseudotriquetrum 216
bell pepper buckwheat
oligosaccharides 307 fagopyritol 168
birch see also Fagopyrum esculentum
DNA repair in pollen 354, 355 Bunya
Blechnum spicant Section of Araucariaceae 245–246
spore storage 193
Blossfeldia liliputana
vegetative desiccation tolerance 222 cabbage
Boea TBARS assay 119
hygroscopica Cactaceae
carbohydrates 325 seed storage classification 248
forest understorey 17 Calophyllum sp.
non-xeromorphic 10 orthodox and recalcitrant seeds 244
predrying 226 Caltha palustris
vegetative desiccation tolerance 222 seed storage classification 241, 244, 253
water content and survival 101 Camellia sinensis
sp. seed drying curve 99
single desiccation tolerant species 217 variable seed desiccation tolerance 266
Bombacaceae see also tea
seed storage classification 248 Capparaceae
Boraginaceae seed storage classification 248
seed storage classification 248 Cardamine sp.
Borya vegetative propagules 228
inopinata Carex physodes
vegetative desiccation tolerance 221 vegetative desiccation tolerance 217, 220
nitida carrot
chloroplasts 272 somatic embryos and LEA proteins 309
predrying 226 Caryophyllaceae
tolerant and sensitive individuals 10 seed storage classification 248
vegetative desiccation tolerance 221 tricellular pollen 188
xeromorphic characteristics 224 Caryophyllales
septentrionalis seed storage classification 247
vegetative desiccation tolerance 221 Castanea
sp. sativa
in situ 12 electron transport chain 173
leaf desiccation tolerance 9 HSP 166, 171, 310
Brachyachne patentiflora sp.
vegetative desiccation tolerance 221 desiccation sensitive seeds 249
Brassica sp. Castaneoideae
seed imbibition damage 344 subfamily phylogeny 249
Dessication Taxonomic Index 9/4/02 9:34 am Page 387
Taxonomic Index 387
Castanopsis vellea 219

orthodox and recalcitrant seeds 244, 249 wrightii 219
seed weight 249 Cheilothela chloropus
Castanospermum desiccation tolerance 209
australe Chenopodiaceae
axis drying curve 99 angiosperm phylogeny 242
sp. seed storage classification 248
seed anatomy 252 tricellular pollen 188
cattail Chenopodium quinoa
pollen membrane Tm 299, 301 seed storage classification 241
Celastraceae Cibotum glaucum
seed storage classification 248 gametophyte cryopreservation 225
Ceratodon purpureus Citrus
predrying 216 limon
spore storage 194 variable seed desiccation tolerance 266
Ceratophyllales sp.
orthodox seeds 246 orthodox and recalcitrant seeds 240, 244
seed storage classification 247 see also lemon
Ceratophyllum demersum Cladonia
orthodox seeds 246 dark respiration
Ceterach convoluta 215
desiccation tolerant species furcata 215
cordatum 220 Clusiaceae
officinarum 220 seed storage classification 247–248
Chamaegigas intrepidus cocoa
drying in situ 226 oil body fusion 350
leaf desiccation tolerance 9 oligosaccharide:sucrose ratio 172
morphology 10 see also Theobroma cacao
natural habitat 224 Cocos nucifera
vegetative desiccation tolerance 223 seed anatomy 252
Cheilanthes Coffea
desiccation tolerant species arabica
albomarginata 218 development and desiccation tolerance
bonariensis 218 159
buchtiennii 218 effect of drying 266
capensis 218 seed water sorption 64
depauperata 218 sp.
dinteri 218 compilation of desiccation sensitive seeds
distans 219 240
eckloniana 218 habitat and seed storage 252
farinosa 218 seed moisture content at dispersal 252
fragillima 219 variation in desiccation tolerance 155,
glauca 218 253, 266
hirta 218 water and physiological activity 52
inaequalis 218 Colletotrichum gloeosporioides
integerrima 218 desiccation tolerant spores 196
lasiophylla 219 Coleochloa
lendigera 219 pallidior
marginata 218 vegetative desiccation tolerance 221
marlothii 218 setifera
multifida 218 longevity when dry 225
myriophylla 218 vegetative desiccation tolerance 221
parviloba 218 velamen 224
paucijuga 219 Commelinales
pringlei 219 seed storage classification 247
sieberi 219 Compositae
sp. 10, 217 pollen germination time 188
tenuifolia 219 see also Asteraceae
388 Taxonomic Index
Commelinaceae post-germination response 254

pollen storage life 190 tolerance of rapid desiccation 223
Convolvulaceae vegetative desiccation tolerance 60
seed storage classification 248 Cruciferae
Coprosma sp. tricellular pollen 188
orthodox and recalcitrant seeds 244 see also Brassicaceae
Cordia alliodora Ctenopteris heterophylla
seed storage classification 241 desiccation tolerant species 220
Cornales cucumber
seed storage classification 247 axes CO2 production 295
Corylus avellana metabolic imbalance 169
seed storage classification 241 plasma membrane lesions 347
cotton Cucurbita
lea mRNAs 171 desiccation sensitive pollen 187
LEA proteins 24 pollen proline content 190
seed imbibition damage 344 Cucurbitaceae
cowpea desiccation sensitive pollen 9, 187
seed imbibition damage 344–345, 348 pollen storage life 190
Craterostigma seed storage classification 248
hirsutum Cucurbitales
lanceolatum Cupressus macrocarpa
monroi Cyathea
vegetative desiccation tolerance 223 spore storage
nanum delgadii 193
tolerance of rapid desiccation 224 spinulosa 193
vegetative desiccation tolerance 223 Cyperaceae
plantagineum in situ 188
ABA 25 seed storage classification 248
carbohydrates 28, 325 vegetative desiccation tolerance 220, 243
desiccation tolerance 217 Cyperus bellis
drying in situ 226 vegetative desiccation tolerance 221
gene expression 30–31, 326, 330
glass formation 27, 302
homologues to TIPs and PIPs 166 Dactylis glomerata
HSP 26, 165, 307, 309 pollen storage 192
2-octulose 26 Davallia fejeensis
LEA proteins 25, 162 gametophyte cryopreservation 225
lipoxygenase inhibitor 296 Davalliaceae
molecular studies 321 desiccation tolerant species 220
water uptake 227–228 Dendrographa minor
rehydrins 349 photosynthesis and water potential 229
RNA 323 dicotyledons
tolerance of rapid desiccation 224 vegetative desiccation tolerance 222
transgenic calli 335 Dicranales
vegetative desiccation tolerance 223 desiccation tolerance 209
sp. Dicranoweisia
control of water loss 348 cirrata
gene product 332 desiccation tolerance 209
molecular studies 322 crispula
transgenic plants 332 spore storage 194
vegetative desiccation tolerance 321, 327 Dicranum elongatum
wilmsii temperature and photosynthesis 213
anthocyanin levels 224, 296 Diffenbachia
ascorbate peroxidase activity 296 desiccation sensitive pollen 187
desiccated and hydrated state 11 Dioscoreales
folded cell walls 25 seed storage classification 247
Taxonomic Index 389
Diospyros sp. brachyphylla 221

orthodox and recalcitrant seeds 244 nardioides 221
Diphyscium foliosum Eragrostis
ABA-induced tolerance 216 vegetative desiccation tolerance
Dipsacales hispida 221
seed storage classification 247 invalida 221
Dipterocarpaceae nindensis 221
habitat and seed storage 252 paradoxa 221
recalcitrant seeds 247 post-germination response
Dipterocarpus nindensis 254
seed anatomy Ericaceae
alatus 252 seed storage classification 248
tuberculatus 252 Ericales
variation in seed desiccation tolerance number of desiccation sensitive seeded
sp. 155 species 246–247
Doryopteris Erythrina caffra
desiccation tolerant species respiratory enzymes 173
concolor 219 Escherichia coli
kitchingii 219 gene product 332
pedata 219 LEA-like proteins 162
triphylla 219 Euphorbiaceae
Dovyalis hebecarpa seed storage classification 248
seed storage classification 241 Eurhynchium pulchellum
Drymaria quercifolia vegetative desiccation tolerance 209
gametophyte cryopreservation 225
Eutacta
Dryopteris
Section of Araucariaceae 245–246
filix-mas
Exormotheca holstii
spore desiccation tolerance 192
ABA-induced vegetative desiccation
paleacea
tolerance 216
chlorophyll fluorescence and germination
192
spore desiccation tolerance 192
faba bean
Dumortiera hirsuta
seed imbibition damage 348
negative turgor pressure 57
Fabaceae
dwarf French bean
seed anatomy 252
seed imbibition damage 348
see also Leguminosae
Ekebergia capensis Fabales
seed drying curve 99 seed storage classification 247
Elaeis guineensis Fagaceae
seed storage classification 241 seed storage classification 246, 248–249,
see also oilpalm 253
Encalypta species coverage 246
sp. Fagales
desiccation tolerance 209 seed storage classification 247
streptocarpa (contorta) Fagoideae
predrying 216 subfamily phylogeny 249
Encalyptales Fagopyrum esculentum
desiccation tolerance 209 seed storage classification 241
Equisetum see also buckwheat
arvense Fagus sp.
spore germination 194 desiccation tolerant seeds 249
hyemale seed weight 249
spore longevity 194 Fimbristylis
Eragrostiella vegetative desiccation tolerance
vegetative desiccation tolerance dichotoma 221
bifaria 221 sp. 221
390 Taxonomic Index
Fissidens adiantoides Gymnocarpium dryopteris

predrying 216 spore bank 193
Flacourtia indica Gymnosperm
seed storage classification 241 desiccation tolerance 220
Fragraea fragrans pollen germination time 188
Funaria hygrometrica
ABA-induced tolerance 216 Haberlea
rhodopensis
carbohydrates 325
Garcinia sp. vegetative desiccation tolerance 222
orthodox and recalcitrant seeds 244 sp.
Garryales
Hamamelidales
Gentianaceae
vegetative desiccation tolerance 243
hazelnut
Gentianales
Geraniales Hedera helix
seed storage classification 247 seed storage classification 241
Gesneriaceae Hedwigia
carbohydrates 325 ciliata (albicans)
desiccation tolerant species 10 predrying 216
vegetative desiccation tolerance 220, 243, sp.
321 desiccation tolerance 209
Gramineae Hedwigiales
cryogenic storage of pollen 191 desiccation tolerance 209
desiccation sensitive pollen 187–188 Helminthosporium
desiccation tolerance and seed oryzae 196
development 155 sp. 195
pollen germination time 188 Hippocastanaceae
pollen shape 188 seed storage classification 248
tricellular pollen 188 Homalothecium lutescens
vegetative desiccation tolerance 192 photosynthesis and water potential 229
see also Poaceae Hookeria lucens
Grammitidaceae chlorophyll fluorescence 211
desiccation tolerant species 220 Hookeriales
Grimmia desiccation sensitivity 209
apocarpa Hopea sp.
in situ 13 variation in seed desiccation tolerance 155
desiccation tolerance 209 Hordeum sp.
laevigata
desiccation sensitive pollen 187
see also barley
in situ 13
Hymenophyllaceae
longevity 209
survival after storage 7
Hymenophyllum
pulvinata
chlorophyll fluorescence 212 desiccation tolerant species
CO2 uptake 214 tunbridgense 220
predrying 216 wilsonii 220
recovery of carbon fixation 230 vegetative desiccation tolerance
temperature and survival 213 sanguinolentum 220
Grimmiales Hypnales
desiccation tolerance 209 desiccation tolerance 209
groundnut Hypnobryales
seed coat and rehydration 348 desiccation studies needed 216
Guifoylia monostylis Hypnum sp.
respiratory enzymes 173 desiccation tolerance 209
Taxonomic Index 391
Illicales lemon
angiosperm phylogeny 246 variable seed desiccation tolerance 266
Ilysanthes see also Citrus
vegetative desiccation tolerance lettuce
purpurascens 223 EPR imaging of seeds 126
wilmsii 223 Leucondon sciuroides
Impatiens sp. desiccation tolerance 209
desiccation tolerant pollen 346 Liliaceae
oligosaccharides 307 vegetative desiccation tolerance 10, 221, 243
pollen membrane phase transition 349 Liliales
thermal events in seeds 136 seed storage classification 247
Indian wild rice Limosella
seed storage classification 241 gradiflora
see also Zizania desiccation tolerant corms 9
Inga sp. sp.
seed anatomy 252 vegetative desiccation tolerance 223
Intermedia Lindernia
Section of Araucariaceae 245–246 carbohydrates
Iridaceae acecularis 325
seed storage classification 248 brevidens 325
Isoetaceae vegetative desiccation tolerance
desiccation tolerant species 218 sp. 223, 321
Isoetes australis Litchi chinensis
desiccation tolerant species 218 desiccation tolerance and seed
development 159
Lobaria pulmonaria
Juglans sp. high-light damage to tissue 229
compilation of desiccation sensitive seeds lucerne
240 transgenic plants 332, 334
Juncaceae Lunularia cruciata
tricellular pollen 188 lunularic acid 216
Lycopsida (clubmosses)
Kyllinga alba Lygodium japonicum
vegetative desiccation tolerance 221 spore desiccation tolerance 192
Labiatae Magnolia sp.

vegetative desiccation tolerance 223, 243 orthodox and recalcitrant seeds 244
Lamiaceae Magnoliales
seed storage classification 248 orthodox and recalcitrant seeds 246–247
Lamiales maize
seed storage classification 247 ABA-deficient mutants 163–164
Landolphia kirkii antioxidants and desiccation tolerance 296
development and desiccation tolerance 160 chemical shift imaging of kernels 131
seed drying curve 99 chromatin 278, 299
Lauraceae dehydrin 328
recalcitrant seeds 246 DNA repair 357
seed storage classification 248 LEA proteins 24, 308, 334
Laurales pollen DNA repair 355
number of desiccation sensitive seeded pollen proline content 190
species 246–247 pollen shape 189
Leguminosae pollen storage 192
seed anatomy 252 protein secondary structure 280
see also Fabaceae seed imbibition damage 344
Lemnaceae sugars and vitreous state 305
seed storage classification 248 see also Zea mays
392 Taxonomic Index
Malpighiales Mnium
seed storage classification 247 hornum
Malus sp. recovery time 230
compilation of desiccation sensitive seeds marginatum
240 predrying 216
see also apple Mohria caffrorum
Malvaceae desiccation tolerant species 220
seed storage classification 248 monocotyledons
Malvales vegetative desiccation tolerance 220
number of desiccation sensitive seeded Moraceae
species 246–247 seed storage classification 247–248
Mariscus capensis moth bean
vegetative desiccation tolerance 221 gene product 332
Mauritia sp. mung bean
compilation of desiccation sensitive seeds chemical shift imaging of seeds 131
240 1H-NMR spectra of water 77
Melastomataceae Muntingia calabura

species coverage 246 Myrothamnaceae
Meliaceae carbohydrates 325
habitat and seed storage 252 vegetative desiccation tolerance 10, 223,
multiple criteria 250 321
recalcitrant seeds 247 Myrothamnus
Mesembryanthemum crystallinum flabellifolius (flabellifolia)
gene product 332 anthocyanin levels 227
Metarhizium carbohydrates (flabellifolia) 325
desiccation tolerant spores desiccated and hydrated state 11
anisopliae 196 desiccation tolerance 217
sp. 195 natural habitat 224
flavoviride negative turgor pressure 57
desiccation tolerant spores 195–196, 242 rehydration 227
drying rate and survival 195 trehalose (flabellifolia) 168, 324
spore imbibitional injury 197 vegetative desiccation tolerance
Michelia champaca (flabellifolia) 220, 223
seed storage classification 241 moschata
Micraira vegetative desiccation tolerance 223
vegetative desiccation tolerance Myrtaceae
adamsii 221 seed storage classification 247–248
spinifera 221 Myrtales
subulifolia 221 seed storage classification 247
tenuis 221
Microchloa
vegetative desiccation tolerance
caffra 221 Najas flexilis
indica 221 seed desiccation tolerance 253
kunthii 221 Nanuza plicata
Microdracoides squamosa vegetative desiccation tolerance 222
vegetative desiccation tolerance 221 neem
Mielichhoferia elongata EPR of axis chilling stress 122
predrying 216 imbibitional stress 121, 344
Millettia sp. seed storage classification 241
seed anatomy 252 thermal events in seeds 136
Mimordica variable seed desiccation tolerance 266
desiccation sensitive pollen 187 see also Azadirachta indica
Mniaceae nematodes
desiccation tolerance studies needed 216 trehalose 168
Taxonomic Index 393
Neosartorya fischeri Orthotrichales

desiccation tolerant spores 196 desiccation tolerance 209
heat resistant spores 197 Orthotrichum
Nicotiana plumbaginifolia anomalum
gene product 332 in situ, desiccated and hydrated 14
Norway maple desiccation tolerance 209
seed development 156 Osmunda japonica
see also Acer platanoides spore proline and arginine content 193
Nostoc sp. Oxalidales
desiccation-tolerant cysts 17 seed storage classification 247
water and physiological activity 52 Oxalis sp.
Nothochlaena bulbils/vegetative propagules 228
marantae
vegetative desiccation tolerance 217, 219
parryi
Paecilomyces
temperature and photosynthesis 224
desiccation tolerant spores
vegetative desiccation tolerance 219
farinosus 195–196
Nothofagus sp. fumosoroseus 196
desiccation tolerant seeds 249 Palmae
seed weight 249 recalcitrant seeds 247
Nymphaea seed anatomy 252
gigantea see also Arecareae
orthodox seeds 249 Pandanales
sp. seed storage classification 247
angiosperm phylogeny 246 Papaveraceae
Nymphaeaceae pollen shape 188
recalcitrant seeds 247 Papaver
Nymphaeales dubium
angiosperm phylogeny 246 pollen development 189
Nyssa aquatica rhoeas
seed storage classification 241 pollen shape 189
papaya
intermediate seed 268
oak Paraceterach muelleri
state diagram for cotyledon 305 desiccation tolerant species 219
see also Fagaceae and Quercus pea
oat axis water stress 267
gene product 332 chemical shift imaging of seeds 131
plasmalemma fusion in leaves 274 carbon dioxide production in axes 294
see also Avena fatua desiccation tolerance 280
glass formation in axes 303
oilpalm
lipid peroxidation in axes 118
metabolic imbalance 169
see also Elaeis guineensis
mitochondrial activity on seed
Oligotrichum hercynicum
rehydration 359
spore storage 194
seed imbibition damage 344, 348
Onoclea sensibilis sHSP 309
spore desiccation tolerance 192 TBARS assay 119
Orchidaceae see also Pisum
seed storage classification 248 Pellaea
Oropetium desiccation tolerant species
carbohydrates atropurpurea 219
thomaeum 325 boivinii 219
vegetative desiccation tolerance calomelanos 219
capense 221 falcata 219
roxburghianum 221 glabella 219
thomaeum 221 hastata 219
394 Taxonomic Index
Pellaea continued Plagiomnium rostratum

desiccation tolerant species continued predrying 216
longimucronata 219 Plagiothecium undulatum
ovata 219 predrying 216
quadripinnata 219 water potential and survival 211
rotundifolia 219 Platycerium stemaria
sagittata var. cordata 219 vegetative desiccation tolerance 217, 220
ternifolia 219 Platyhypnidium rusciforme
viridis 219 predrying 216
Penicillium bilaji Pleurochaete squarrosa
desiccation sensitive conidia 195 chlorophyll fluorescence 212
spore storage 195 Pleurosorus rutifolius
Pennisetum desiccation tolerant species 220
desiccation sensitive pollen Pleurostima sp.
purpureum 188 vegetative desiccation tolerance 222
sp. 187 Pleurozium schreiberi
desiccation tolerant pollen predrying 216
americanum 188 Poa bulbosa
typhoides 188 vegetative desiccation tolerance 221
Pentaclethra sp. Poaceae
seed anatomy 252 carbohydrates 325
Pentagramma triangularis desiccation sensitive pollen 9
forest understorey 17 desiccation tolerant species richness 10
in situ, desiccated and hydrated 13 seed storage classification 248
Petunia sp. vegetative desiccation tolerance 221, 243
pollen DNA repair 354, 356 see also Gramineae
Phacelia tanacetifolia Poales
31P NMR and seed metabolism 132 seed storage classification 247
Phaseolus vulgaris Podocarpaceae
ABA and seed development 171 seed desiccation sensitivity 244
NMR imaging of seeds 131 Podocarpus
premature drying 161 henkelii
seed development 152–153, 159 radical tip and axis drying curves 99
Phegopteris connectilis usambarensis
spore bank 193 orthodox seed 244
Philonotis seriata Pohlia elongata
predrying 216 predrying 216
Phycomyces blakesleeanus Polygonaceae
desiccation tolerant spores 196 seed storage classification 248
Phyllisis scolopendrium Polypodiaceae
spore storage 193 desiccation tolerant species 220
Pilotrichella ampullacea Polypodium
recovery time on remoistening 214 desiccation tolerant species
Piper hispidum cambricum 220
seed storage classification 241 vulgare 220
Piperaceae polypodioides
seed storage classification 248 illuminated drying 226
Piperales specialized structures 224
orthodox seeds 246–247 vegetative desiccation tolerance 217, 220
Pisum sp. virginianum
seed anatomy 252 desiccation tolerant species 220
see also pea repair processes 28
Pithecellobium sp. Polystichum setiferum
seed anatomy 252 spore storage 193
Pittosporum sp. Polytrichales
orthodox and recalcitrant seeds 244 desiccation tolerance 209
Taxonomic Index 395
Polytrichum desiccation tolerance 213, 216

formosum recovery processes 215
recovery time 230 temperature and photosynthesis 213
piliferum water potential and survival 211
desiccation tolerance 209 sp.
Porphyra sp. desiccation tolerance 209
large scale drying 97 survival pattern 213
Pottiales Ramalina maciformis
desiccation tolerance 209 water storage and gas difussion 20
proso millet wetting and drying 18
testa colour and imbibition 348 Rammondia sp.
Proteaceae desiccation tolerant species 10
seed storage classification 248 Ramonda
Proteales carbohydrates
seed storage classification 247 myconi 325
Pseudopezicula vegetative desiccation tolerance
desiccation tolerant spores 195 myconi 223
Pteridaceae nathaliae 223
desiccation tolerant species richness 10 pyrenaica 223
Pteropsida (ferns) serbica 223
desiccation tolerant species 218 Ranunculaceae
Puccinia seed storage classification 248
desiccation tolerant spores Ranunculales
graminis 196 seed storage classification 247
recondita 196 Ranunculus
seed priming
arvensis 354
sceleratus 354
Quercus
tuber desiccation tolerance
robur
asiaticus 228
ABA and seed development 171
vegetative propagules
critical water content 66
ficaria 228
critical water potential 50
red rice
cytoskeleton 174, 273 13C labelling and seed metabolism 132
matrix-bound water 53
Rhizocarpon geographicum
protectant against oxidative stress 174
adaptation to climate 230
seed development and desiccation
Rhizophoraceae
tolerance 158–9
recalcitrant seeds 247
soluble sugars 172
Rhynchostegium riparioides
volatiles and unregulated respiration 173
predrying 216
rubra
Rhytidiadelphus
critical water potential or water content loreus
50 chlorophyll fluorescence 211
pressure–volume curve 58 dark respiration 215
sorption isotherm of cotyledon tissue 67 recovery time 230
soluble sugars 172 sp.
water hydration sites 66 predrying 216
sp. Riccia
desiccation sensitive seeds 249 fluitans
variation in desiccation tolerance 155 effect of ABA 216
see also oak macrocarpa
gametophyte longevity 209
survival after storage 7
Racomitrium rice
aciculare lea gene 25
spore storage 194 non-detection of Tg by DSC 136
lanuginosum seed chilling injury 344
chlorophyll fluorescence 212 transgenic plants 331–334
396 Taxonomic Index
Ricinus communis Schizophyllum commune

desiccation intolerance 155 survival after storage 8
seed development 152–153 Scleropodium tourretii
Rosaceae desiccation tolerance 209
seed storage classification 248 Sclerotinia sclerotinum
Rosales desiccation tolerant spores 196
seed storage classification 247 Scrophulariaceae
Roystonea carbohydrates 325
Rubiaceae vegetative desiccation tolerance 223, 243,
seed storage classification 248, 253 321
Rutaceae Secale
seed storage classification 247–248 cereale
rye DNA repair 358
DNA repair in embryos 357–358 see also rye
pollen cryogenic storage 192 sp.
transcription in embryos 352 desiccation sensitive pollen 187
see also Secale cereale Selaginella
desiccation tolerant species
caffrorum 218
Sabal sp. convoluta 218
seed storage classification 240 digitata 218
Saccharomyces imbricata 218
cerevisiae njam-njamensis 217–218
desiccation tolerant cells 196 peruviana 218
HSP 310 pilifera 218
LEA-like proteins 162, 308 sartorii 218
mutants 196 lepidophylla
see also yeast desiccation tolerant species 218
uvarum drying rate 6
desiccation tolerant cells 196 folded cell walls 60
Saccharum sp. illuminated drying 226
desiccation sensitive pollen 187 membrane organization 22
Salix sp. predrying 226
seed storage classification 240 resurrection 217
Santalales sellowii
seed storage classification 247 desiccation tolerant species 218
Santalum album in situ 12
seed storage classification 241 sp.
Sapindaceae desiccation tolerant species richness 10,
angiosperm phylogeny 242 217
seed storage classification 248 evolution of desiccation tolerance 243
Sapindales heat tolerance 8
seed storage classification 247 photosynthesis 17, 227
Sapotaceae trehalose 324
seed storage classification 247–248 Selaginellaceae
Satureja gilliesii desiccation tolerant species 218
desiccation tolerant organ/tissue 9, 223 Septoria nodorum
Saxifraga sp. hydrated storage of spores 197
vegetative propagules 228 Shorea
Saxifragales robusta
seed storage classification 247 free-radical scavenging 174
Schistidium rivulare sp.
spore storage 194 variation in seed desiccation tolerance
Schizaea sp. 155
desiccation tolerant species 220 Solanaceae
Schizaeaceae pollen shape 188
desiccation tolerant species 220 seed storage classification 248
Taxonomic Index 397
Solanales sugarbeet
seed storage classification 247 transgenic plants 332–333
Sordaria sunflower
desiccation tolerant spores ABA-deficient mutants 164
macrospora 196 sHSP 309
survival of cavitation 57 Swietenia
sorghum seed storage classification 240
desiccation sensitive pollen 187 sycamore
seed imbibition damage 344 seed development 156
soybean see also Acer pseudoplatanus
antioxidant in seed membranes 296 Syntrichia
glass composition in axes 305 desiccation tolerance 209
glycoproteins in seed coat 348 Syzigium guiniense
LEAs in axes 308 axis drying curve 99
lipid-soluble antioxidants 169
13C NMR and seed metabolism 132
seed development and desiccation Talaromyces flavus

tolerance 153 desiccation tolerant spores 196
seed imbibition 155 heat resistant spores 197
seed imbibition damage 344 Talbotia elegans
water clustering in axes 66 vegetative desiccation tolerance 222
Sphagnum sp. Taxus brevifolia
net photosynthesis 17 orthodox seed 252
Spondias sp. tea
orthodox and recalcitrant seeds 244 axis viability loss 102
Sporobolus metabolic imbalances in seeds 280
vegetative desiccation tolerance variable seed desiccation tolerance 266
see also Camellia sinensis
atrovirens 221
Telphairia occidentalis
elongatus 221
viviparous germination 158
festivus 221
Theobroma cacao
fimbriatus 222
ABA and seed development 171
lampranthus 222
drying curves of axes 69–71, 99
pellucidus 222
free-radical scavenging 174
sp.
see also cocoa
Thrinax sp.
gene expression 330
molecular studies 322
Thuidium delicatulum
stapfianus
protein analysis 328
carbohydrates 325
Tiliaceae
control of water loss 348
desiccated and hydrated 15 Timmia austriaca
EST collections 30 predrying 216
LEA proteins 307 tobacco
molecular studies 321 non-detection of Tg by DSC 136
protein synthesis 329 transcription factors 326
rehydrins 349 transgenic plants 326, 331–334
repair processes 28 Todea barbara
trehalose 168, 324 spore storage 193
vegetative desiccation tolerance 220 tomato
xeromorphic characteristics 224 ABA-deficient mutants 163
Stagonospora convolvuli fruit shedding 252
desiccation tolerant spores 196 Tortella
spore longevity 197 desiccation tolerance 209
Sterculiaceae Tortula
seed storage classification 248 latifolia
Streptocarpus sp. in situ, desiccated and hydrated 14
desiccation tolerant vegetative tissue 223 (ruralis subspecies) ruraliformis
398 Taxonomic Index
Tortula continued Typha

(ruralis subspecies) ruraliformis continued latifolia
sucrose 26 membrane permeability 346
CO2 uptake 214 31P NMR and phospholipids 133
(syn Syntrichia) ruralis pollen imbibitional leakage and EPR

ABA 216 spectra 123
cellular integrity 327 pollen storage life 191
cellular protection and dehydrins 25 sp.
chlorophyll fluorescence 211–212 pollen monolayer hydration 65
chloroplast 328
dark respiration 215
desiccation and hydration in situ 15 Ulota
desiccation tolerance 216 crispa
drying rate 216 chlorophyll fluorescence 212
EST collections 30–31 sp.
growth in dark 229 desiccation tolerance 209
leaf longevity 209 Umbelliferae
metabolism and protein synthesis tricellular pollen 188
328–329 see also Apiaceae
metabolism on de- and rehydration 29 Uromyces appendiculatus
molecular studies 321, 327, 329–330 desiccation tolerant spores 195–196
phosphorus and potassium content and Urticaceae
nitrate reductase activity 19 seed storage classification 248
predrying 216 Ustilago scitaminea
rapid adaptation 230 desiccation tolerant spores 195–196
recovery time on remoistening 214–215 spore longevity 197
rehydrins 349
sucrose 27
TEM of leaf cells 16
temperature and survival 213 Vellozia sp.
water potential and longevity 211 vegetative desiccation tolerance 222
sp. Velloziaceae
desiccation tolerance 209 carbohydrates 325
Trichilia dregeana desiccation tolerant species 222
axis drying curve 99 desiccation tolerant species richness 10
axis viability loss 102 in situ 12
cytoskeleton 273 vegetative desiccation tolerance 243
water and seed longevity 103 Venturia inaequalis
Trichoderma harzianum desiccation tolerant spores 195–196
desiccation tolerant spores 196 Vicia narbonensis
Trilepis sp. ADP-glucose pyrophosphorylase activity
vegetative desiccation tolerance 221 112
Trimeniaceae Vitellaria paradoxa
angiosperm phylogeny 246 habitat and seed storage 252
Tripogon Vitex sp.
vegetative desiccation tolerance orthodox and recalcitrant seeds 244
capillaris 222 Vochysia honurensis
curvatus 222 seed storage classification 241
filiformis 222
jacquemontii 222
lolioformis 222 walnut
lisboae 222 serotonin accumulation 170
minimus 222 Washingtonia sp.
polyanthus 222 seed anatomy 252
spicatus 222 Welwitschia mirabilis
Triticum sp. foliage desiccation tolerance 217, 220, 225
desiccation sensitive pollen 187 Welwitschiaceae
see also wheat desiccation tolerant species 220
Taxonomic Index 399
wheat villosa
amphiphile partitioning 370 carbohydrates 325
dehydrins in embryos 309 molecular studies 321
DNA repair 357 viscosa
EM protein 25 anthocyanin levels 227
EPR imaging of kernels 126 ascorbate peroxidase activity 296
EPR of seed tissues 124 control of water loss 348
LEA transgene 332, 334 desiccated and hydrated state 11
NMR imaging of kernels 131
proembryo desiccation tolerance and EPR
121 yeast
seed development 152 trehalose 168, 324
T2 water relaxation 129 see also Saccharomyces cerevisiae
see also Triticum
Wollemia nobilis
desiccation tolerant seeds 245 Zea mays
seed size 245 desiccation sensitive pollen 187–188
Woodsia ilvensi LEA proteins 307
desiccation tolerant species 220 see also maize
Zingiber sp.
desiccation sensitive pollen 187
Xerophyta Zingiberaceae
humilis desiccation sensitive pollen 187–188
chloroplast 272 seed storage classification 248
pinnifolia Zingiberales
velamen 224 seed storage classification 247
retinervis Zizania
desiccated and hydrated state 11 aquatica
scabrida seed storage classification 241
CO2 and photosynthesis 19 palustris
rehydration and respiration 18 drying temperature 98
sp. imbibitional damage 102, 344
adaptation to habitat 230 post-germination response 266
desiccation tolerant species richness 217 tetrazolium test 104
vegetative desiccation tolerance 222 see also Indian wild rice
squarrosa Zygodon
longevity when dry 225 desiccation tolerance 209
Dessication Subject Index 3/4/02 2:30 pm Page 401
Subject Index
ABA see Abscisic acid Anoxia 8

ABRE see Abscisic acid Antarctica 17, 18
Abscisic acid (ABA) 24 et seq, 154 et seq, 190, Anthesis 189
194, 216, 226, 308, 309, 323 et seq, 334, Anthocyanins 18, 118, 165, 227, 265, 296, 348
356, 367 et seq, Antioxidants 167, 169, 228, 264, 294 et seq,
analog 164 311, 371
mutants 25, 163 et seq, 310, 326 et seq, 335 Antisense 112
response element (ABRE) 164 Aquaporins 166
Abscission 172 Aquatic species 241, 249, 250, 253
Accelerated ageing 113 Arthropods 7
Acetaldehyde 114, 115, 169, 173, 295 Ascopore 57, 196, 197
Activity, water 50 et seq Ascorbate 296
ADP 294 Ascorbate peroxidase 296
ADP-glucose pyrophosphorylase 112 Ascorbic acid 174, 280
Arginine 190, 195, 196
Adsorption 53
Arginine decarboxylase 333
Ageing 229, 304, 311, 345, 351, 357
Aspartyl protein methyl transferase 28, 170, 351
Aldehydes 114
Aspartyl residues 28, 170, 351
Algae 4 et seq, 320
ATP 351, 358, 359
Aleurone layer 135, 163, 170, 334
Axes see Embryo
Alkanes 114
Alkenes 114
Alkones 114
Alkyl hydroperoxidase 33 Basal meristems, desiccation tolerance 9
Amino acids 190, 198, 229, 277 Betaine 132
Ammonia 170 Bilayers 133
Amphipaths 280, 296, 308, 347 et seq compression 281
Amphiphiles 22 et seq, 113 et seq, 294, 296 et Boreal zone 17
seq, 311, 347, 370 Bovine serum albumin 308
antioxidant 296 et seq Broad leaved forests 249
endogenous 297, 371 Broadening agents 120 et seq
-Amylase 308 Browning 113, 303
Angiosperms 7 et seq, 150 et seq, 220 et seq Brunauer-Emmet-Teller (BET) model 60 et seq
Anhydrobiotes (anhydrobiosis) 116, 186, 198, Bryophytes 6 et seq, 207 et seq, 320 et seq, 368
293 et seq, 349 et seq Bulbils 228
Annuals, desiccation tolerant 10 Bulbs 228
401
402 Subject Index
C3 plants 117, 213 Compaction, of molecules 273

C4 plants, desiccation tolerant 12 Compartmentation 274, 344
Calcium 168, 278, 371 Compatible solutes 190, 198, 264, 276, 294, 298,
Callus 25, 26, 165, 335 301, 308, 311, 331
Calorimetry 74, 112 et seq, 305 Compensation point 17
Calvin cycle 117 Competition 20
Capillary action 227 Conidia 195, 196, 243
Carbohydrates 296, 321 et seq Conservation 240 et seq
Carbon 18 Corms 9
balance 18, 20, 208, 215, 327 Cotyledons 150, 152, 166, 170, 245, 294, 347,
gain 19 354, 367
loss 211, 217 Crassulacean acid metabolism 10
Carbon dioxide 169, 210, 294 Critical water activity 65
exchange 114 Critical water content 157, 303
Carnitine 298 Critical water potential 241, 268
Carotenoids 18, 23 Crustacea 207
Catalase 295 Cryopreservation 160, 225
Cavitation 12, 20 Cryoprotection 196, 197
Cell Crystallization 54, 347, 303, 311
compartmentation 129 Cuticle 217
compartments 132 Cyanobacteria 4, 115
contraction 270 Cyclitols 369
cycle 215, 352, 359 Cytokinesis 351, 353
damage 328 et seq Cytosine 351
division 122, 150, 215, 279, 352, 367 Cytoskeleton 273
enlargement 122 Cytosol 307
expansion 150, 264, 347, 352
integrity 303, 350
pH 126, 132 Damage 21 et seq, 28, 65, 151, 159 et seq, 263 et
recovery 328 et seq seq, 328 et seq, 344 et seq
shrinkage 271 desiccation induced 113, 263 et seq, 294
size 269 free radical 114 et seq, 321
ultrastructure 16, 22 et seq, 136 and metabolism 295
volume 112, 226, 265 et seq D’Arcy–Watt model 61
Cell walls 21 De-esterification 294
convolution 227, 270 Dehydrins see LEAs
elasticity 59 Dehydroascorbic acid 296
folding 270 Dephosphorylation 322
Chalaza 252 Deserts 17
Chaparral 17 Desiccation-sensitive plants 116
Chaperonin 26, 167, 310 Desiccation tolerance 150 et seq
Chemical potential 51 et seq animals 7, 207
Chlorophyll 18, 23, 165, 208 et seq, 223, 265, and bacterial infections 10
320, 333 constitutive 208 et seq, 226, 327
fluorescence 104, 116, 192, 209 et seq, 334, continuum 151, 242, 246
368 definition 4, 320
Chloroplast 16, 23 et seq, 223, 227, 271 et seq, developmental programme 9
307, 328, 368 vs. drought tolerance 5, 207, 230, 320
Chromatin 166, 308, 344, 357 and drying rates 100
Chromium oxalate 120 ecology 9, 13 et seq, 224, 320, 327
cis-Acting elements 326 environmental induction 9
Cladistics 242 evolution 10, 12, 20, 171, 208, 240 et seq,
Cladogram 253 321
Classification 244 et seq genes 31, 161 et seq, 243, 321 et seq
molecular data 246 geographic range 8, 10, 320
Climbers 250 and germination 9, 37
Cold tolerance 8 glasses and 303
Dessication Subject Index 9/4/02 9:32 am Page 403
Subject Index 403
habitats 8, 17, 208, 209, 217 et seq phosphate 350

heat-shock proteins 306, 309 et seq polymerase 352, 358
higher plant vs. bryophyte 321 repair 28, 174, 215, 350 et seq
induced 216 et seq, 226 replication 352
injury 150 et seq, 263 et seq sequence data 242
LEAs and 24 et seq, 161 et seq, 307 et seq stability 278
level 242 synthesis 351
and longevity 368 et seq and water 350
metabolism and 5, 7 Dormancy 9, 29, 150, 173, 186 et seq, 224, 253,
molecular responses 320 et seq 330, 349
morphological types 10 Drought 298
mutants 163 et seq, 310, 324 avoiders 230
and nutrient availability 10, 18 definition 5
oligosaccharides and 27, 168, 306, 368 evaders 230
physiological types 10 hardening 209, 215
and productivity 9 stress 30, 298, 335, 369
provenance and 157 Drought tolerance 4, 186, 207, 264
quantitative 268 vs. desiccation tolerance 5, 207, 230, 320
seeds 150 et seq, 239 et seq Dry matter
sensitivity 239 et seq accumulation 113
taxonomic range 8 et seq, 207 et seq, 321 and cell shrinkage 271
and temperature 213, 224, 280, 327 Drying 113 et seq, 263 et seq, 294 et seq, 368 et
timing 100 seq
water potential 157, 368 in air 4, 94
Devonian–Mississippian 250 air movement 94 et seq
Dew 13, 18, 20 boundary layer 94 et seq
Dew-point depression 53 curves 68 et seq
Diaspores 193
cycles 6, 9, 14, 17 et seq, 224
Dicotyledons
equilibration to low humidities 7
desiccation sensitivity 249
excised axes 95 et seq
desiccation tolerance 10, 321
fast 68, 208 et seq, 216
LEAs 323
flash 95 et seq
Dictysomes 272
free radicals 116
Dielectric relaxation 74
gene expression 369
Differential respirometer 114
in light 6
Differential scanning calorimetry 54, 74 et seq
methods 96 et seq
Diffusion 113
rapid 6, 19, 195, 225, 328
Diffusional correlation time 72
rate 6, 29, 68 et seq, 79, 94 et seq, 321, 327,
Disaccharides 294 et seq, 300 et seq
328
Dispersal
fruit 254 seed shape 98
pollen 186 seed size 98
seed 254 seeds 152 et seq
DNA 350 et seq in shade 94 et seq
amounts 249 silica gel 95, 152
binding proteins 299 slow 29, 195, 208 et seq, 216, 328
breaks 357, 358 and sucrose 27
conformation 299, 344, 356 in sun 94 et seq
damage 52, 174 surface/volume ratio 94 et seq
dehydration 350 temperature 94, 98, 157
forms 350 time 71, 96, 112, 226
free radical damage 117, 357, 359 tissues 94 et seq
hypersensitive sites 359
levels 352
ligase 352 Editosome 351
mitochondrial 117, 353, 357 Electron microscopy 22
nuclear 117, 350 Electron paramagnetic resonance (EPR) 112 et
nuclease 351, 352, 357 seq, 297, 304 346
404 Subject Index
Electron spin resonance (ESR) 112 et seq, 297, Free energy 51, 61
304 and drying 94
Electron transport 116, 173, 294 Gibbs 50
Em Free radicals 5, 65, 114 et seq, 116 et seq, 173,
gene 163 227, 265, 277, 279, 293 et seq, 351
protein 25 et seq attack 169
Embryo 150, 245, 252, 270 et seq, 299, 334, 344 desiccation tolerance 116 et seq, 293 et
et seq seq, 321
axes 78, 95 et seq, 150 et seq, 166, 169, effects on cells 117
294, 295, 303, 305, 354 et seq generators 119
drying 95 et seq, 150 et seq processing 296
somatic 169, 295, 306 scavenging 174, 265, 279, 295 et seq, 311
Embryogenesis 164, 266, 278 Freezing 53, 225, 265, 270, 295, 298, 335
Endoplasmic reticulum 16, 271, 272, 278 Freezing point depression 53 et seq
Endosperm 121, 135, 252 Freezing stress 48
Enthalpy 61, 74 Freezing tolerance 7, 8, 59
Entropy 61 Fructan synthase 333
Enzymes 5 Fructans 298
activities 113 Fructose 322
lability 277 phosphate 322
repair 263 Fruit structure 243, 244, 252
stabilization 169, 303 Fungal spores 115
Eocene 245 Fungi 4
Ethanol 114, 115, 125, 173, 195, 196, 295 infection 103
Ethylene 115, 131 Funiculus 152
Eudicots 247
Evolution, desiccation tolerance 10, 12, 20, 171,
208, 240 et seq, 264 Galactinol 369
Exotherm 54 Galactinol synthase 369
Expressed sequence tags (ESTs) 30 Galactopinitol 168
Extraction of metabolites 113 Galactosyl cyclitols 306
Extracts 116 Gametophytes 7 et seq, 29, 186, 209, 245, 253
Extrusion Gas analysis 209
protein 347 Gas diffusion 20
starch 347 Gases 20, 114
GCMS 114
Genes
Fagopyritol 168, 369 ABA responsive 25, 163
Fatty acids 167, 294 ABI-3 326
diunsaturated 271 desiccation sensitivity 253
and free radicals 117 desiccation tolerance 31, 175, 254, 321 et
polyunsaturated 188 seq, 356
spin labelled 123 Em 163
Fermentation 114, 169 enzymatic antioxidants 296
Ferns 7 et seq, 217 et seq, 320, 344 expression 157, 320 et seq, 329 et seq, 369,
Ferricyanide 120 370
Fixatives 270, 347 fus3 163
Flavonoids 296, 297, 347 LEA 24, 161 et seq, 309, 326, 335, 336
Fluidizing compounds 310, 347 et seq lec1 165
Fluorescein 196 osem 163
Fluorescence spectroscopy 114 promoters 326
Fog 13 Rab2 28, 163
Forbs 10, 250 replacement 336
Forest tree species 250 Vp 163 et seq
Fourier transformation 128, 161 Genetic engineering 210
Fourier transformation infra-red spectroscopy Genome fidelity 356
(FTIR) 112 et seq, 134, 161, 297, 299, 301 Genomics 372
Subject Index 405
Germinability 152 et seq in desiccation tolerance 165 et seq, 309 et

Germination 122, 162, 253, 266, 306, 309, 353 seq
and desiccation tolerance 9, 174, 367 in seeds 26, 165 et seq
and drying 152 et seq transcription factor 326
precocious 154, 164 in vegetative tissues 26
and repair 28 Heat tolerance 8
test 103, 242 Helices, amphipathic 330
Germplasm preservation 30 -Helix 135, 161, 166, 277, 323
Gibberellins 164, 193 Herbs desiccation tolerant 10
biosynthesis 165, 193 Hexagonal phase 274, 346
Glass 72, 78, 135, 169, 191, 269, 281 298, 301 et Histidine kinase 371
seq, 311, 324, 351, 345 Histodifferentiation 150, 268
composition 302 Homoiochlorophylly 227, 230, 368
definition 302 Homoiohydry 217, 228, 243, 252
in desiccation tolerance 303 Hornworts 10
formation 113, 298, 301 et seq HPLC 114
and longevity 303 Human cells, desiccation tolerance 12
maltodextrin in 306 Humic substances 126
and membranes 303 Hydration 50, 52, 66, 72, 114, 155, 267
proteins in 306 levels 173, 268, 294
Hydraulic conductivity 59
and sucrose 26, 27, 303 et seq
Hydraulic flow 345
and sugars 27, 306
Hydrins 29, 215, 329 et seq
temperature 302, 303
Hydrogen bonds 72, 135, 169, 277, 278, 298,
transitions 27, 75, 136, 301 et seq
300, 301, 307, 311, 324, 349
water content 302
Hydrometer 53
Globulin 163, 166
Hydroperoxidase 29
Glucose 303
Hydroperoxides 296
Glucose-glycerol 325
Hydrophilins 162
-Glucuronidase 165
Hydrophilly 162
Glutamate 190, 196, 298, 331
Hydrostatic pressure 51 et seq, 60, 68
Glutathione 296
Hydroxyl groups 26, 167, 168, 300
Glutathione reductase 174, 295 Hygrometry 51
Glyceraldehyde-3-phosphate 322 Hysteresis 60, 105
dehydrogenase 322
Glycerol solutions 55, 195, 196
Glycine-betaine 298 Imbibition 344 et seq, 351
Glycolysis 117 Imbibitional injury (stress) 136, 191, 194, 197,
Graminoids 10, 250 344 et seq
Grana 16, 227, 272 phase change 346
Grasses 20, 336 temperature 344
Gravitational potential 51 et seq Iminonitroxides 126
Growth, effects of desiccation 14 Infra-red spectroscopy 74
cell 52 Inositol D-ononitol 333
Growth, g.rate 14, 19, 20 Insect larvae 7
GTP – binding protein 28 Insertional mutagenesis 335
Guanidine-HCl 298 Intermediate seeds 150, 172, 198, 241, 266
Guggenheim-Anderson-de Boer (GAB) model 60 International Plant Genetic Resources Institute
et seq 241
Gymnosperms 8, 9, 321, 323 International Seed Testing Association 48
Intracellular gas 57
Invasive techniques 112 et seq
Hairs 19 Ions
Hardening 216 distribution 131
Headspace analysis 114 leakage 215
Heat-shock proteins 26, 167, 265, 294, 309 et NMR 131
seq, 371 sequestration 26, 308
406 Subject Index
Isopleth 64 surfaces 320

Isotherm water content 49 et seq
curves 51 water potential 53
hysteresis 60, 105 waxes 348
sorption 60 et seq, 79, 105, 277 Leeuwenhoek, Anthony von 6
Leucine zipper 165, 322, 326
Lichens 6 et seq, 114, 136, 320
K-segment 25, 323 Light 192, 193, 211, 224, 272
Kernel development 121 damage by 18, 226, 272
Kinetics limiting 20
non-equilibrium 70 Lipid bilayer 22
water loss 114 oxidation 65
peroxidation 117 et seq
Lipid bodies 307
Lactate dehydrogenase 277, 308 Lipids 188, 193
Late embryogenesis abundant proteins see LEAs Liposomes 26 et seq, 274 et seq, 310, 346
Late Palaeozoic 245 membranes 169, 297 et seq
Late Precambrian 263 Lipoxygenase 296
Leakage 18, 21, 153, 160, 191, 214, 216, 223, Liquid helium 213
297, 300 et seq, 308, 334, 344 et seq, 348 Liquid nitrogen 126, 192
damage 104, 328 Longevity 27, 297 et seq, 303 et seq, 368 et seq
viability 104 oligosaccharides and 305 et seq, 368 et seq
vigour 104 pollen 27
vital dyes 104 seeds 27, 241 et seq, 303 et seq
Leathers 268, 280 Lyophilization 192
LEAs 24 et seq, 254, 264, 294, 322 et seq, 367 et Lysine 196, 323
seq Lysozyme 52, 72
actions 308
binding properties 308
Macromolecules 228, 273
dehydrins 24 et seq, 161 et seq, 309, 370
hydration 298
and desiccation tolerance 307 et seq
integrity 321
genes 24 et seq, 161 et seq, 309, 326, 335,
stabilization 294, 298, 303, 307
336
Maillard reaction 172
in glasses 306, 309
Malonyldialdehyde 117, 118
groups 25, 161 et seq, 307, 323
Maltose 301
HVA1 25, 31, 334
Mannitol 298
hydration 308
Marker molecules 371
nuclear 309
Marsh species 253
phosphorylation 309
Matric potential 53 et seq
pollen 190
forces 59
proteins 24 et seq Maturation drying 150 et seq, 296, 370
RAB17 308 Megagametophyte 150
structure 25, 323 Meiosis 192
synthesis 153 et seq Membranes
TAS14 308 and amphiphiles 296 et seq
transcripts 24 et seq, 161 et seq conformation 346
Leaves convolution 227
carbohydrates in 325 damage to 5, 21 et seq, 104, 155 et seq, 173,
curling 226 328
cuticle 348 disruption 59, 155 et seq
desiccation tolerance 9 et seq, 18, 320 et dynamics 133
seq effects of water loss 271 et seq
drying 101, 223 fatty acid domains 273
growth 320 fluidity 124, 134, 191, 279
hairs 348 folding 271 et seq
LEAs in 307 fusion 168, 303, 311
rolling 265 hydration 73
Subject Index 407
integrity 121, 160, 169, 214, 228, 276, 293, Mucilage 348
310, 346 et seq Multigenic traits 336
isolated 124 Mutants 25, 112, 163 et seq, 196, 253, 310, 324
liposomal 26 et seq
nuclear 227 Myo-inositol O-methyltransferase 333
packing 273
partitioning into 113, 296 et seq
phase 265, 274 et seq, 299 et seq Nematodes 7, 168, 207
phospholipid 133, 167 Neoteny 9
physical properties 123, 347 Nitrate 192
plastid 227 Nitrate reductase 19
preservation 298 Nitroxide 120 et seq
protection 26 et seq NMR spectroscopy 74 et seq
rehydration 274 Non-invasive techniques 112 et seq
repair 155, 167 Nuclear magnetic resonance (NMR) 112 et seq
rigidity 274, 297 Nucleases 351, 352, 357
sarcoplasmic reticulum 168 Nucleic acids
stability 160, 168 dehydration 278
stucture 303 hydration 52
and sugars 168 integrity 321
tearing 166 synthesis 150
tonoplast 227 and water 350
transition temperature 169, 195, 299 Nucleolus 170, 309
vesicles 28, 168 Nucleus 16, 170, 307, 309
Metabolic activities Nutrient availability 10, 18
and drying 112 Nutrient capture 208
and water 114
Metabolism, regulation 294 et seq
Metabolites 2-Octulose 26, 28, 324, 325
flux 115 Oil
NMR 131 bodies 124, 130, 349
seeds 115 NMR signals 128
Microsomes 296 in seeds 115 et seq, 242
Microtubules 215 Oleosins 349
Minimum critical volume 270 Oligosaccharides 27, 118, 167, 170, 189, 294 et
Mitochondria 16, 116, 161, 189, 227, 271, 273, seq, 303, 368 et seq
279, 296, 297, 344 and desiccation tolerance 306, 368 et seq
dehydrogenases 359 and longevity 305 et seq, 368
DNA 117, 353, 357 Ordovician 243
genome 357 Ornithine 333
Mitotic division 150, 264 Orthodox seeds see Seeds
Modulus of elasticity 55, 59 Osmole 54
Moist forests 17 Osmolytes 298, 331
Moist tropics 250 Osmometer 54
Moisture content (MC) 158, 197, 241 et seq Osmosensor 371
Molecular marker analysis 331 Osmotic potential 52 et seq, 68 et seq, 264
Molecular movement (mobility) 27, 72, 191, Ovule 186, 252
229, 269, 280, 302, 306 351 Oxidation 5
and ageing 304 Oxidative stress 30, 114, 116, 192, 296
Molecular spin probes 78 damage 293, 297, 311
Monocotyledons Oxygen
desiccation sensitivity 246, 249 availability 169
desiccation tolerance 10, 18, 321 exchange 114
LEAs 323 protection against 198, 333
Monosaccharides 172, 303 reactive species (ROS) 116, 170, 265, 279,
Mosses 29 et seq, 207 et seq, 319 et seq, 344 et 294
seq scavenging 333
408 Subject Index
Oxygen continued phase transitions 346

solubility 113 rehydration stress 346
tolerance of low 8 Plasmadesmata 21, 270 et seq
uptake 5, 211, 294 Plasmalemma 129, 270 et seq, 281
Plasmolysis 56, 209, 270
Plastids 271, 279, 344
Paramagnetism 120 Plastoglobuli 23, 271
Partitioning Pleiotropic effects 112
of amphiphiles, amphipaths 22 et seq, 294 Poikilochlorophylly 18, 23, 104, 208, 227, 230,
et seq, 311, 347 368
within cells 120 Poikilohydry 320
into membranes 113, 280, 371 Polar lipids, effects of drying 273
Pathogens 229, 344 Pollen 7 et seq, 20, 22, 24, 27, 112 et seq, 133,
Pectic substances 152 150, 186 et seq, 297 et seq, 344 et seq,
Perennating structures 9 354 et seq, 368
Perennials, desiccation tolerant 10 ABA 190
Permafrost 194 ageing 113, 187
Permanent wilting point 270 amino acids 190
Permo-Carboniferous 245 bicellular 188
Peroxidases 295 compatible solutes 190
Peroxidation 189, 198, 279, 294 dispersal 186
Peroxide 295 dormancy 186
Peroxiredoxin 170, 296 germination 186 et seq
pH 126, 132 glasses 191
Phase transition 22, 134, 167, 274 et seq, 280, hydration 186
281, 297, 299 et seq, 346 et seq LEAs 190
and sugars 300 longevity 186
Phenolics 296, 297 mitochondria 272
Phenols 113 molecular mobility 72
Phloem 323 recalcitrant 187
Phosphatase 322, 326 shape 188
Phosphatidylcholine 299 sperm cells 186 et seq, 355
Phosphofructokinase 168, 301 storage 191, 198
Phosphoglycerate 322 sucrose 189
Phospholipase D 322, 326 tricellular 188
Phospholipid 123, 167, 188, 271 et seq, 294 tube 186 et seq
bilayers 133, 293, 298, 300, 346 viability 186 et seq, 347
composition 349 vigour 347
hexagonal phase 133 water in 48, 65
and sugars 281, 300 et seq, 307 Pollination 186
vesicles 169, 188 Pollinia 191
see also Polar lipids Polyamines 333
Phospholipid:sterol ratio 270 Polyethylene glycol solutions 55, 104, 295, 344
Phosphorus 19 Poly-L-lysine 305
Phosphorylation 309, 322 Polyols 296, 298
Photo-oxidation 5, 264 Polypeptides 277
Photoprotection 227 Polyphosphates 132
Photosynthesis 5, 17 et seq, 29, 52, 114, 209 et Poly(ribo)somes 161, 272, 278, 327
seq, 268, 327, 333 Polyubiquitin 29, 330, 349
Photosystem II 29, 104, 116, 194, 211 et seq, Potassium 19
226, 272, 322 Preferential exclusion 298
Phytochrome 192 Pressure chamber 53
Pioneers 224 Pressure–volume analysis 49 et seq, 106
Plasma membrane 21 et seq, 153, 189, 196, 226, Priming 353 et seq
270 et seq, 327, 345 et seq Productivity
folding 347 crop 333
permeability 122, 346 and desiccation tolerance 9, 20
Subject Index 409
Proembryonic cells 121 Putrescine 333

Proline 190, 195, 331, 333 Pyrolline carboxylate 331
Promoter analysis 326 Pyrolline carboxylate synthetase 331
Promoters 326
Proteases 167, 351
Protectants 165, 228, 264 et seq, 282, 294, 303 Quantitative trait locus (QTL) 336
Protection 268, 321, 322, 327 et seq, 347 et seq, Quasi elastic neutron scattering 75
368 et seq Quiescence 150, 245, 369
Protein 24 et seq Quinones 296
ABI 165, 322
bodies 161, 307
channel 166 Rab2 protein 28
conformation 113, 135, 277, 301, 303, 322 RAB17 163
denatured 167, 169, 311, 320 Rachis 152
desiccation related 161 Raffinose 27, 167, 168, 172, 300 et seq, 369
early light inducible 322 Rain 13, 20
elF1 322 Raman spectroscopy 74
Em 25 et seq Random coil 135
extraction 113 Recalcitrant seeds see Seeds
extrusion 347 Recovery 228, 328 et seq
and free radicals 117 Regeneration niche 250
folding 293, 298 Rehydration 17 et seq, 22 et seq, 28, 113, 153,
FUS3 165 166, 169, 209 et seq, 227, 242, 269 et
heat shock 26, 167, 265, 294, 309 et seq, seq, 294, 321 et seq, 344 et seq, 368 et
371 seq
heat stable 190, 194, 198 damage 344 et seq
hydration 52, 72, 73, 276, 349 Rehydrins 29, 215, 329 et seq, 349
hydrins 29, 329 et seq Relative humidity 53 et seq, 188, 216, 266 et
integrity 321 seq, 320 et seq
interaction with sugars 135, 301, 311 air 4, 5
kinase 322, 326 and drying 97 et seq
labile 293 equilibrium 51 et seq, 190, 225, 252, 265
LEC1 165 tolerance of 5
L-isoaspartyl residues 28 Relative water content (RWC) 49 et seq, 106,
LEA see LEAs 207 et seq, 225
major intrinsic (MIPs) 166, 322 Relaxation times 127
myb 322, 326 Repair 5, 21, 28 et seq, 151, 173, 215, 229, 263
phosphatase 326 et seq, 294, 321, 327, 350 et seq
preservation 298 Reserve deposition 150, 166, 172
protective 151 Reserve mobilization 369
Rab2, 17, 28, 29, 163 Respiration 19, 52, 114, 150, 173, 209 et seq,
rehydrins 24, 29, 215, 329 et seq, 349 268, 279, 294, 298, 351
repair 28, 167 Resurrection plants 48, 114, 162, 166, 170, 217,
secondary structure 135, 277, 301 225, 226, 244, 268, 281
stabilization 298, 333 drying 101, 296 et seq, 320 et seq, 344 et
storage 164, 165, 193 seq, 367 et seq
synthesis 20, 150, 154, 161, 189, 213, 215, Retrotransposons 335
272, 278, 327 et seq, 351 Ribonucleoprotein
Vp1 322 messenger (mRNP) 29, 320 et seq, 330
Proteomics 175, 372 RNA (mRNA) 329, 330, 351
Proteosome 349 Rock pools 226
Prothalli 192 Root pressure 227
Proton exchanges 277 Roots 163, 264, 331
Protonema(ata) 193, 194, 209, 216 Rosette plants, desiccation tolerant 10
Protoplasts 271 Rotifers 7, 207
Psychrometer 53 Rubbers 269, 280
Pteridophytes 9 et seq, 217 et seq Rutin 297
410 Subject Index
Salinity stress (see salt stress) 25, 48 water in 48 et seq, 151 et seq
Salinity tolerance 8, 334 water loss 151
Salt solutions 53, 55, 97 viability 27, 28, 66, 102, 241, 278
Salt stress 25, 333, 335 weight 249
Savannah 252 Selection pressure 159, 253
Scales 19, 224 Self incompatibility 186
Scutellum 367 Semi-arid grasslands 18
Seed ferns 250 Senescence 152
Seedling 254, 367 Serine residues 25, 308, 323
Seeds 7 et seq, 112 et seq, 149 et seq, 239 et seq, Serotonin 170
320, 344 et seq, 367 et seq Shade plants 17
ageing 113 Shedding 155, 157, 172
banks 241, 253 -Sheet 135
chromosomal aberrations 350 Signalling pathways 326, 334, 369 et seq
coat (see testa) 22, 347 et seq Silica gel 95, 152, 225
colour 348 Silurian 243, 263
desiccation sensitive 239 et seq Solutes 53
desiccation tolerance 150 et seq, 239 et seq leakage 160, 223, 297
development 24, 31, 122, 123, 150 et seq, Sorbitol 298
334, 350 Sorption 269
dormant 150, 253, 330, 349 Sorption properties 242
dry weight 151 Sorption sites 129, 167
drying 152 et seq Spectroscopy 119 et seq
expansion 166 Sperm cells 186, 355
filling 112 Spin labels 120 et seq
free radical damage 117 Spin probes 22, 120 et seq
fresh weight 151 Spin trapping 126
gene expression 369, 370 Spores 4, 7 et seq, 150, 186 et seq
germinating 115, 132 dispersal 186
germination 152, 253 fungal 115
imbibition 166, 344 et seq, 351 Sporocarp 193
imbibitional injury 136, 191, 194, 197, 344 Sporophytes 7 et seq, 253
et seq Stachyose 27, 167, 172, 300 et seq
intermediate 150, 172, 241, 266 synthase 369
longevity 27, 241 et seq Starch extrusion 347
maturation 24, 115, 150 et seq, 253, 266, Steroids 123
294, 295, 307, 350, 369 Stigma 186
maturity 155, 254 Stomata 208, 217, 320
metabolites 115 Storage 155, 242, 297 et seq
moisture content 158, 241 et seq, 250, 252 Stress 264 et seq, 294
non-endospermic 252 chilling 348
oils 115 et seq, 135 drought 30, 298, 335
orthodox 9, 24, 52, 65, 95 et seq, 150 et seq, duration 102, 103
172, 240 et seq, 266 et seq, 344 et seq, freezing 298
368 et seq imbibition 344 et seq
production 30 intensity 102, 103
recalcitrant 9, 50, 53, 66, 95 et seq, 105, mechanical 70, 346 et seq
151 et seq, 172, 240 et seq, 266 et seq, multiple 321
294 et seq, 344 et seq, 367 et seq osmotic 229, 298, 333
reserves 157 oxidative 30, 114, 116, 192, 296
size 98, 244, 250 physico-chemical 70
shape 98, 250 salt 25, 333, 335
storage behaviour 155 et seq, 241, 250 water 25, 154, 162, 167, 172, 226, 266 et
structure 243 seq
sugars 115 Stress strain 69, 266, 347
tree 9 Stress tolerance 30
volume 345 Stroma 227
Subject Index 411
Succulents, desiccation tolerant 10 water uptake 348

Sucrose 298, 324 et seq, 368 et seq waxy 348
alcohols 229 Tetrazolium test
distribution 131 fungi 104
glass formation 26 et seq, 302 et seq viability 104
and membranes 299 Thiobarbituric acid 117, 118
in mosses 26 et seq, 328 Thylakoids 23, 116, 227
phosphate 322 Tocopherol 167, 174, 280, 296
phosphate synthase 322 Tonoplast 129, 132, 272
in pollen 189 Toxin 321
protection 164, 167, 227, 328 Transcription 351
in seeds 26 et seq activator 163, 322, 335
synthase 322, 323 factors 165, 326, 334, 336
Sugars 20, 24, 26 et seq, 115, 118, 168 et seq, regulators 322
294, 335, 368 et seq Transduction see Signalling pathways
alcohols 229 Transgenic plants 112, 330 et seq
hydrophilic 27 Transgenic studies 25, 31, 298, 330 et seq
hydroxyl groups 27 Translation 351
interaction with protein 135, 301, 311 Translation factor 322
and membranes 168, 276 Transpiration 320
phosphates 322 Transposon tagging 31, 335
and phospholipids 281, 300 et seq Tree seeds 9
protective 151, 276, 277, 297 Trehalase 168
Sulphuric acid 225 Trehalose 13, 26, 115, 133, 168, 195, 197, 298 et
Superoxide 295 seq, 310, 324 et seq
Superoxide dismutase 174, 295, 334 stabilization by 333
Syrups 269 Trehalose phosphate phosphatase 168, 331
Trehalose phosphate synthetase 31
Triacylglycerol 118
Tannins 348 Tropics 17, 252
Tardigrades 7, 207 Tubers 228
t-DNA 335 Tundra 17
Teliospores 196 Turgor 49, 55, 106, 226, 267 et seq, 321, 371
Telomeres, telomerase 357, 359 pressure 55 et seq, 264 et seq
Temperature
and desiccation tolerance 213, 224, 225,
327 Ubiquitin 25, 167, 334, 349
evaporation rate 62 see also Polyubiquitin
extremes 265 UDP-glucose 322
and free energy difference 94 Ultrastructure 153
and glasses 113 Umbelliferose 169
and injury 344 Urea 298
and longevity 241 Urediniospores 196
monolayer hydration 65 Uridine 351
and survival time 213 UV radiation 119, 354 et seq
transition 169, 195, 297, 300, 302 et seq, UV-B tolerance 8
346 et seq
TEMPO 123
TEMPONE 121 et seq Vacuoles 21, 227
Testa 252, 347 et seq and cell shrinkage 271
amphiphiles 349 Van der Waals interactions 135, 273
glycoproteins 348 Van’t Hoff relationship 63 et seq
and imbibitional injury 348 Vascular bundle 129, 323
leakage 348 Vascular factors 153
lignin polymers 348 Vascular separation 171, 268
phenolics 348 Vascular system 227
pigmentation 348 Vegetative tissues 207 et seq, 272, 320 et seq
412 Subject Index
Vesicles 168 matrix bound 160

Vesicular trafficking 28 molecular interactions 76, 79
Viability 161, 186 et seq, 241, 278, 303, 353 monolayer 61, 65
Vicilin 166 multimolecular clusters 62
Viscosity 124, 280, 281, 301 et seq non-freezable 73, 151, 160
cytoplasmic 78, 113, 159, 165 et seq osmotic potential 52 et seq, 68, 264
Vitrification see Glass osmotically inactive 73
Vivipary 154, 158, 164, 241, 245, 253 partial molar volume 51
Volatiles 114, 173 potential 49 et seq, 97, 157, 208 et seq, 241
et seq, 267 et seq, 344, 368 et seq
rate of loss 28
Water status 48 et seq, 79
activity 50 et seq, 65 storage 20
apoplastic 55 et seq, 68, 73, 106 stress 25, 154, 162, 226, 266 et seq
binding 25, 59 et seq, 65, 67 strongly bound 128
bulk 73, 126 et seq, 130, 186, 190,298 symplastic 55 et seq, 68, 73, 106
chemical potential 51 et seq transfer routes 131
clustering 65 et seq vapour 18
compartments 130 vapour pressure 51 et seq, 94 et seq
concentration 105 wet weight basis 48 et seq, 105 et seq
conservation 4 Water channel proteins 166, 323
content 48 et seq, 68, 99, 105 et seq, 112 et Water relations 55 et seq
seq, 128 et seq, 151 et seq, 213, 266 et Water replacement hypothesis 12, 276, 280, 299
seq , 345 et seq
diffusion 94 Woody plants 250
dissociation 63
distribution 130
dry weight basis 48 et seq, 105 et seq
Xanthophyll 18, 265
equilibrium water content 48
Xeromorphs, desiccation tolerant 10, 224, 226
exchange 73
X-ray diffraction 75
fractions 130
Xylem cavitation 12, 20
freezable 73, 151, 160
Xylem potential 53
gradient 113,
Xylem refilling 227
gravitational potential 51
hydration levels 74
immobilized 73
intercellular 48, 57 et seq Zeaxanthin 18, 227
and life 4, 48 et seq Zeeman splitting 120, 127
loss 114, 208, 264 et seq Zygospores 196
matric potential 53 et seq Zygote 156

Desiccation and Survival in Plants, Drying Without Dying

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Desiccation prels 19/3/02 1:42 pm Page i

Desiccation and Survival in Plants

Drying Without Dying

Desiccation and Survival in Plants

Drying Without Dying

Royal Botanic Gardens, Kew

CABI Publishing is a division of CAB International

Library of Congress Cataloging-in-Publication Data

Typeset in Melior by Columns Design Ltd, Reading

PART IV. MECHANISMS OF DAMAGE AND TOLERANCE 261

Peter Alpert, Biology Department, University of Massachusetts, Amherst, Massachusetts

Plant survival of desiccation as sporophytic and gametophytic tissues was last

1 Drying Without Dying

Peter Alpert1 and Melvin J. Oliver2

4 P. Alpert and M.J. Oliver

1.1. Introduction Though some desiccation-tolerant plants

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individuals; and there is a continuum of 1.4.2. Vegetative tissues

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organisms depends on the selection and habitats where desiccation-sensitive plants

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Trade-offs between tolerance and growth pensation point for photosynthesis

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(see reviews by Bewley, 1979; Oliver and 1.6.1. Damage

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1.6.2. Protection reach a maximum about three days before

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over 70% of the Em protein is configured in accumulation during desiccation.

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1.6.3. Repair In the desiccation-tolerant fern

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(2KCKMmCK)(2CK22K2)aw expressed as the percentage of the corre-