Planta (2015) 242:427–434
DOI 10.1007/s00425-015-2327-z
REVIEW
What can we learn from the transcriptome of the resurrection
plant Craterostigma plantagineum?
Valentino Giarola1 • Dorothea Bartels1
Received: 30 January 2015 / Accepted: 2 May 2015 / Published online: 22 May 2015
Ó Springer-Verlag Berlin Heidelberg 2015
Abstract
Main conclusion The desiccation transcriptome of the
resurrection plant C. plantagineum is composed of
conserved protein coding transcripts, taxonomically
restricted transcripts and recently evolved non-protein
coding transcripts.
Research in resurrection plants has been hampered by the
lack of genome sequence information, but recently introduced sequencing technologies overcome this limitation
partially and provide access to the transcriptome of these
plants. Transcriptome studies showed that mechanisms
involved in desiccation tolerance are conserved in resurrection plants, seeds and pollen. The accumulation of
protective molecules such as sugars and LEA proteins are
major components in desiccation tolerance. Leaf folding,
chloroplast protection and protection during rehydration
must involve specific molecular mechanisms, but the basis
of such mechanisms is mainly unknown. The study of
regulatory regions of a desiccation-induced C. plantagineum gene suggests that cis-regulatory elements may
be responsible for expression variations in desiccation
tolerant and non-desiccation-tolerant plants. The analysis
of the C. plantagineum transcriptome also revealed that
part of it is composed of taxonomically restricted genes
(TRGs) and non-protein coding RNAs (ncRNAs). TRGs
are known to code for new traits required for the adaptation
Special topic: Desiccation Biology.
Guest editors: Olivier Leprince and Julia Buitink.
& Dorothea Bartels
planta@uni-bonn.de; dbartels@uni-bonn.de
1
Institute of Molecular Physiology and Biotechnology of
Plants (IMBIO), University of Bonn, Kirschallee 1,
53115 Bonn, Germany
of organisms to particular environmental conditions. Thus
the study of TRGs from resurrection plants should reveal
species-specific functions related to the desiccation tolerance phenotype. Non-protein coding RNAs can regulate
gene expression at epigenetic, transcriptional and posttranscriptional level and thus these RNAs may be key
players in the rewiring of regulatory networks of desiccation-related genes in C. plantagineum.
Keywords Desiccation tolerance Linderniaceae
Non-protein coding RNAs Resurrection plants
Taxonomically restricted genes
Abbreviations
CDT-1
Craterostigma desiccation tolerant 1
LEA
Late embryogenesis abundant
ncRNAs Non-protein coding RNAs
TRGs
Taxonomically restricted genes
Introduction
Different strategies have evolved in plants to cope with
water deficit. The majority of land plants use specialised
structures (e.g. seeds) or particular morphological adaptations to overcome dry periods. Most higher plants can cope
with mild water stress, but they die when the cellular water
content is below 60 %. However, a few hundred flowering
plants defined as resurrection plants have the unusual
ability to dehydrate to equilibrium with dry air and then
rehydrate without dying. Thus, resurrection plants represent an excellent resource to study desiccation tolerance in
higher plants. Mechanisms underlying desiccation
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tolerance are complex and involve a combination of genetic and metabolic systems together with molecular stabilising processes (Moore et al. 2009).
Desiccation tolerance may have evolved by ancient
plants to enable land colonisation, but this trait was lost
with the increase of plant complexity in tracheophytes
(Oliver et al. 2000). Lately, this trait apparently independently evolved (or re-evolved) in some clades (Oliver et al.
2000). Molecular studies of resurrection plants support the
idea that each species re-evolved specific mechanisms that
collectively confer plant desiccation tolerance (Farrant
2000; Farrant et al. 2007; Moore et al. 2013). These
mechanisms are inducible rather than constitutive and
mainly consist of the accumulation of protective molecules
during dehydration (Gaff 1989; Oliver et al. 1998).
Cell walls, membranes, macromolecules (DNA and
proteins) and other cellular components are subjected to
denaturation, mechanical and oxidative stress during dehydration. Resurrection plants must use specific mechanisms to protect and repair these structures. It has been
reported that major changes during the desiccation process
in vegetative tissues of resurrection plants are similar to
those occurring in reproductive plant tissues, i.e. seeds and
pollen (Ingram and Bartels 1996; Rodriguez et al. 2010).
Among these changes, the accumulation of non-reducing
sugars and the synthesis of protective proteins, mainly late
embryogenesis abundant (LEA) proteins are believed to
participate in protection during desiccation (Ingram and
Bartels 1996). Besides regulating the water-holding capacity of cells, sugars are thought to protect cells through a
process termed ‘‘glass phase formation’’ or ‘‘vitrification’’
(Burke 1986). In this state chemical reactions are stopped
and conformational changes of proteins and membrane
fusions are prevented (Crowe et al. 1998). It is hypothesised that with the progression of water stress severity, water is replaced by sugar molecules which form
hydrogen bonds with polar groups of macromolecules
stabilising their native conformation (Clegg et al. 1982). At
a particular point during dehydration, molecular mobility
decreases strongly and the cytoplasm vitrifies (Burke
1986). In seeds and resurrection plants various soluble
carbohydrates may be present in fully hydrated tissues, but
sucrose usually accumulates in the dried state. For example, desiccation in the leaves of C. plantagineum is accompanied by conversion of the C8-sugar 2-octulose (90 %
of the total sugars in hydrated leaves) into sucrose, which
accounts for about 40 % of the dry weight in desiccated
leaves (Bianchi et al. 1991). In addition, LEA proteins also
possess properties similar to non-reducing sugars and
likewise may contribute to glass formation (Hoekstra et al.
2001).
Other aspects, such as leaf folding, the protection of
photosynthetic structures and the activation of rehydration-
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Planta (2015) 242:427–434
specific mechanisms appear to be essential for the desiccation tolerance phenomenon but are still poorly understood. Extensive reversible leaf shrinking and folding is
only observed in leaves of resurrection plants and is hypothesised to preserve cellular integrity through the
minimization of the mechanical stress at the cell boundaries (Farrant 2000; Farrant et al. 2003, 2007; Willigen
et al. 2003). Leaf folding was also proposed as a
mechanism to shade chlorophyll and reduce formation of
reactive oxygen species (ROS) in Craterostigma wilmsii
and Myrothamnus flabellifolius (Farrant 2000).
Resurrection plants adopt different strategies to prevent
ROS formation and thus protect photosynthetic structures
of chloroplasts in water-limiting conditions (Sherwin and
Farrant 1998; Moore et al. 2009). Resurrection plants are
classified into two types: plants defined as poikilochlorophyllous degrade chlorophyll pigments during dehydration
and homoiochlorophyllous plants maintain the chlorophyll
during desiccation. Homoiochlorophyllous plants such as
C. plantagineum must use specific mechanisms to protect
the photosynthetic apparatus during water-limiting conditions. In C. plantagineum photosynthetic genes are downregulated, photosynthesis stops and chloroplasts undergo
reversible ultrastructural changes upon dehydration (Schneider et al. 1993). Several proteins which accumulate in
chloroplasts upon dehydration are linked to the protection
of photosynthetic structures, e.g. an early light-inducible
protein (ELIP) (Bartels et al. 1992), a LEA D29 similar
protein (dsp 21), a protein of unknown function (dsp 34)
(Schneider et al. 1993) and the plastid targeted proteins
(CpPTPs) (Phillips et al. 2002). The protection mechanism
may be by interacting with proteins (dsp 34 and dsp 21),
with nucleic acids (CpPTPs) or binding light absorbing
pigments (ELIP).
It is believed that rehydration-induced repair mechanisms do not play a major role in vegetative desiccation
tolerance in angiosperms (Cushman and Oliver 2011).
Most of the studies in these plants have focused on the
characterisation of protection mechanisms in the ‘‘dehydration phase’’ and thus the rehydration process of resurrection plants has been scarcely investigated. However,
several activities are necessary during rehydration to reestablish the original cellular organisation, e.g. leaf unfolding and metabolic activities. These mechanisms are
part of the survival strategy of resurrection plants and must
be coordinated to permit successful recovery from desiccation. The rehydration process in C. plantagineum is a
relatively long process requiring several hours to be accomplished (Bartels et al. 1990). Comparative analysis of
in vitro translated mRNA samples from C. plantagineum
suggested the specific accumulation of some genes in the
rehydration phase, but only two such genes were eventually
identified encoding a chlorophyll a/b binding protein and
Planta (2015) 242:427–434
the transketolase 7 (TKT7) (Bernacchia et al. 1996). This
led to the conclusion that in C. plantagineum the events
occurring during rehydration only contribute to restore the
normal metabolic functions and that molecules may be
already synthesised for rehydration during dehydration
(Bernacchia et al. 1996). However, the requirement of rehydration-induced mechanisms for the recovery from rapid
drying was demonstrated in C. wilmsii thus suggesting the
importance of such mechanisms for plant vitality when
dehydration-induced protection is inadequate (Cooper and
Farrant 2002).
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feature of the C. plantagineum callus is that it can be
switched between a desiccation-sensitive state (normal
condition) to a desiccation-tolerant state by application of
exogenous ABA (Bartels et al. 1990). This observation
together with the fact that ABA levels increase in leaves
during dehydration and many dehydration-induced genes
are also inducible by ABA supports an essential role for
ABA in the acquisition of desiccation tolerance in C.
plantagineum (Bartels 2005). ABA treatment of callus and
dehydration of plants lead to the induction of the same set
of genes (Bartels et al. 1990). Many of those genes encode
LEA or LEA-like proteins.
The experimental system Craterostigma
plantagineum
The Southern African plant C. plantagineum has been extensively studied at the molecular level to understand
mechanisms underlying desiccation tolerance (Bartels and
Salamini 2001). These studies support the notion that
desiccation tolerance in this plant is acquired through the
induction of protection mechanisms during dehydration.
The dehydration process in C. plantagineum is characterised by the accumulation of many dehydration-induced
gene products, which disappear upon rehydration (Bernacchia et al. 1996). A representation of a typical dehydration/rehydration cycle is shown in Fig. 1. One
advantage of using C. plantagineum as an experimental
system resides in the possibility to perform molecular
analysis using both differentiated plant tissues and undifferentiated callus and thus compare gene expression in two
genetically identical systems (Bartels 2005). A remarkable
Fig. 1 Dehydration/rehydration cycle in C. plantagineum (modified
from Rodriguez et al. 2010). The plant suspends all its activities upon
desiccation and rapidly resumes them after rehydration. Early
responses to dehydration are often studied in plants dehydrated to
50–60 % RWC (about 2 days), whereas late responses are analysed in
plants dehydrated to 2 % RWC (1–2 weeks dehydration). Plant
rehydration is achieved by submerging desiccated plants in water for
1 day. Reported dehydration times refer to plants grown and
dehydrated in pots under conditions described by Bartels et al.
(1990). RWC stands for relative water content, a common measure of
the hydration status of C. plantagineum leaves (Bernacchia et al.
1996)
DNA sequencing technologies to study plant
desiccation tolerance
The complete set of transcripts in a cell for a specific developmental stage or physiological condition is defined as
the cell transcriptome. The study of the transcriptome is the
key to understand how organisms grow and respond to
different conditions. The identification and analysis of the
genes and gene isoforms involved in desiccation tolerance
has been hampered by the lack of genome sequence information for resurrection plants. High-throughput RNA
sequencing technologies do not require knowledge of the
genome sequence and thus allow the study of desiccation
tolerance in resurrection plants on a transcriptome-wide
level. RNA-sequencing (RNA-seq) has become the method
of choice for genome-wide expression studies (Wang et al.
2010). RNA-seq enables the identification and quantification of single transcripts and thus detects changes in gene
expression under different physiological conditions (Zhou
et al. 2010; Cullum et al. 2011). RNA-seq overcomes the
limitations observed by microarray analysis and permits to
detect novel transcripts and isoforms, to reveal sequence
variations (e.g. SNPs) and to identify non-protein coding
RNAs and splice variants (Mutz et al. 2013).
cDNA library screenings showed that transcripts
modulated during dehydration stress in C. plantagineum
and in the non-tolerant plant Arabidopsis thaliana belong
to the same functional categories (Bockel et al. 1998;
Bartels 2005). Recently, the transcriptome sequences from
different dehydration and rehydration conditions have been
obtained for C. plantagineum (Rodriguez et al. 2010) and
Haberlea rhodopensis (Gechev et al. 2013). These studies
reinforced previous data that vegetative desiccation tolerance mechanisms are similar to those in seeds and pollen.
Genes involved in vegetative desiccation tolerance are also
present in non-tolerant plants, but expression levels may be
different in desiccation-tolerant and desiccation-sensitive
plants. A general response of both desiccation-tolerant and
desiccation-sensitive plants to dehydration is the
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accumulation of LEA proteins (Bartels et al. 1990; Ingram
and Bartels 1996; Rodriguez et al. 2010). Transcriptome
data from C. plantagineum permit now a comprehensive
view of the identity and the number of stress-related genes
which are supposed to be involved in the desiccation tolerance mechanisms. The access to the gene sequence information allows to design specific primers for the accurate
quantification of expression levels of each transcript isoform by RT-qPCR. Using such an approach the expression
of the most abundant dehydrin isoforms, a class of LEA
genes from C. plantagineum has been quantified (Giarola
et al. 2015a). The precise quantification of the expression
of each dehydrin isoform and of the other dehydrationinduced genes is the prerequisite to understand the functions of these genes in plant desiccation tolerance.
Comparative analyses between closely related
species
It was hypothesised that quantitative differences in the
expression patterns of LEA genes may be responsible for
desiccation tolerance in C. plantagineum and other resurrection plants (Bartels and Salamini 2001). Desiccation
tolerant and sensitive species belonging to the same family
as C. plantagineum exist. For example, all Craterostigma
spp. and several Lindernia spp. from rock outcrops are
desiccation tolerant; however, the majority of Lindernia
spp. are sensitive to desiccation (Fischer 1995; Seine et al.
1995). Interestingly, Lindernia brevidens, a plant of the
montane rain forests of East coastal Africa, was found to
tolerate extreme desiccation, although it never experiences
dry periods in its natural habitat (Phillips et al. 2008).
Conversely, Lindernia subracemosa which shares similar
habitats with L. brevidens is desiccation sensitive. Comparative studies in C. plantagineum and closely related
species can be used as an alternative approach to mutant
analysis to provide support to the ‘‘quantitative’’ hypothesis. Comparative expression studies showed for example that the transcript levels of one abundant
dehydration-induced LEA-like gene, i.e. CDeT11-24 from
C. plantagineum are reduced in the desiccation-sensitive L.
subracemosa plant. The analysis of promoters from C.
plantagineum and L. subracemosa showed different promoter activities suggesting involvement of transcriptional
control of transcript levels (van den Dries et al. 2011). The
low transcript expression in L. subracemosa was correlated
to the reduced promoter activity and to changes in promoter cis-elements (van den Dries et al. 2011). It has been
proposed that the large physiological and morphological
diversity observed in plants and animals is mainly due to
changes in cis-regulatory gene elements (Doebley and
Lukens 1998; Prud’homme et al. 2007) and gene
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Planta (2015) 242:427–434
duplication (Ohno 1970; Lynch and Conery 2000). Adaption to extreme environment may provide genomic stress
which leads to changes in promoter elements and subsequently to changes in gene expression patterns. The use of
RT-qPCR for the analysis of expression levels of other
dehydration-responsive genes in tolerant and sensitive
Linderniaceae provided further support to the ‘‘quantitative’’ hypothesis (Giarola et al. unpublished). Analysis of
the regulatory regions of these genes is required to understand whether changes in cis-regulatory gene elements
play a relevant role in the evolution of the desiccation
tolerance phenotype in C. plantagineum.
Identification and characterisation of taxonomically
restricted genes in C. plantagineum
Besides known genes, transcriptome studies revealed a
large fraction of unknown transcripts. About one-third
(10119 sequences) of the C. plantagineum transcriptome
showed no or scarce similarity to genes in public databanks
thus suggesting the existence of unexplored speciesspecific functions. Similar results were found for H.
rhodopensis where about 40 % of transcripts were reported
to be unknown (Gechev et al. 2013). Every eukaryotic
genome contains about 10–20 % of genes which do not
share any similarity with genes in other species (Wilson
et al. 2005). Such genes are defined as orphan or
taxonomically restricted genes (TRGs) (Khalturin et al.
2009). Studies using the basal metazoan Hydra indicate
that TRGs are required for adaptive species-specific processes (Khalturin et al. 2009). It is likely that TRGs may
have a role together with the variation of regulatory networks in the evolution and adaptation of organisms to
different environmental conditions (Khalturin et al. 2009).
In addition to transcriptome sequencing activation tagging and cDNA library screenings permitted to identify
TRGs linked to desiccation tolerance in C. plantagineum.
The Craterostigma desiccation tolerant 1 (CDT-1) gene
encodes a small regulatory non-coding RNA which is able
to confer constitutive desiccation tolerance to C. plantagineum callus (Furini et al. 1997). This retrotransposonlike gene is thought to reprogramme transcription of desiccation-related genes through a still unknown mechanism
involving a double-stranded 21-bp short interfering RNA
(siRNA) (Hilbricht et al. 2008). The C. plantagineum early
dehydration-induced 9 (CpEdi-9) gene was identified
among genes modulated during early phases of dehydration
(Bockel et al. 1998). CpEdi-9 expression and localisation
analyses support a role of this gene in the protection of
seeds and leaf phloem tissues from desiccation-mediated
damage (Rodrigo et al. 2004). Recently, the analysis of
most abundant and/or most co-varied genes in C. plantagineum transcriptome permitted us to identify additional
Planta (2015) 242:427–434
TRGs linked to desiccation tolerance, i.e. CpCRP1 and
CpEDR1 (Giarola et al. 2015b) (Fig. 2; Table 1). CpCRP1
and CpEDR1 seem to have different functions in the plant.
CpCRP1 codes for a cysteine-rich rehydration-responsive
protein 1 (Fig. 2) which may play a role in the osmotic
stress management at the cell wall prior to dehydration and
upon rehydration. It is hypothesised that this gene may
participate in the rehydration process in C. plantagineum.
The gene CpEDR1 codes for an early dehydration-responsive protein which accumulates in chloroplasts of
desiccated leaves and together with other dehydration-induced chloroplastic proteins it protects these organelles
upon dehydration. How CpEDR1 protects the chloroplast
remains to be established, but the prediction of an amphipathic a-helix structure in the protein sequence (Fig. 2)
suggests that protein–protein and membrane–protein interactions may be involved in the function. Homologs of
CpEDR1 and CpCRP1 were identified in the transcriptome
data from L. brevidens and L. subracemosa (Giarola et al.
2015b) and not in other plants outside the Linderniaceae
thus suggesting that these genes may be restricted to the
Linderniaceae family and may have evolved in this family.
Further characterisation of CpCRP1, CpEDR1 and other
TRG homologs may help to understand whether TRGs are
important for the extreme tolerance to dehydration observed in C. plantagineum.
As suggested by transcriptome studies, a large part of
the transcribed genome of eukaryotes consists of RNAs
without protein coding capacity (Bertone et al. 2004;
Cheng et al. 2005; Kapranov et al. 2007). Thus, part of
unknown transcripts identified in the C. plantagineum
transcriptome are likely non-coding RNAs (ncRNA). One
such long ncRNA (28852 transcript) was identified in the
C. plantagineum transcriptome. This ncRNA is abundantly
expressed during dehydration suggesting a possible role in
plant desiccation tolerance (Giarola et al. 2015b).
BLASTN analysis failed to identify transcripts similar to
28852 in public databanks and in the transcriptome of the
closely related species L. brevidens and L. subracemosa.
This supports a species-specific but still unknown role for
this gene in C. plantagineum. Long ncRNAs are a large and
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distinct class of RNAs with a length of more than 200
nucleotides which can structurally resemble mRNAs, but
do not code for proteins. In contrast to small non-coding
RNAs (e.g. miRNA or siRNA) which are highly conserved
and use a specific mechanism to silence gene expression,
long ncRNAs are poorly conserved and regulate gene expression at the epigenetic, transcriptional or post-transcriptional level by distinct and mainly so far unknown
mechanisms (Bernstein and Allis 2005; Faghihi and
Wahlestedt 2009; Mercer et al. 2009; Whitehead et al.
2009; Wilusz et al. 2009; Wang and Chang 2011; Zhang
et al. 2014). For example, they were found to control RNA
stability (Faghihi et al. 2008; Kretz et al. 2013) and protein
synthesis in the cytoplasm (Huarte et al. 2010; Carrieri
et al. 2012), whereas in the nucleus they were shown to
control the epigenetic state of genes (Pandey et al. 2008;
Kotake et al. 2011), to be involved in transcriptional
regulation (Ørom et al. 2010) and alternative splicing
(Tripathi et al. 2010) and to constitute subnuclear compartments (Bond and Fox 2009). In the past, both the lack
of genome-wide sequence information and the underestimation of the functional value of non-protein coding genes
in general, limited the research efforts towards understanding the contribution of ncRNAs to plant desiccation
tolerance.
Conclusions
Vegetative desiccation tolerance in resurrection plants is a
complex phenomenon which involves the modulation of
several genes. Genome-wide transcript data from resurrection plants represent an important step toward a systems
biology approach which is required to understand the
multitude of mechanisms underlying this trait. The analysis
of these data indicates that desiccation tolerance resides in
the differential regulation of conserved genes and the
contribution of unique possibly species-specific genes.
With respect to the differential regulation of genes the
comparison of regulatory regions of different desiccationrelated genes in closely related species such as Lindernia
Fig. 2 Taxonomically
restricted genes in C.
plantagineum. Protein structure
of CpEDI-9, CpCRP1 and
CpEDR1. Predicted domains
and targeting sequences are
represented with colour boxes
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123
spp. could help to clarify whether regulatory regions are
responsible for expression differences. Non-protein
regulatory molecules such as ncRNAs may also contribute
to modulate expression networks in resurrection plants.
Previous research in C. plantagineum showed that the
CDT-1 ncRNA is able to influence the expression of other
desiccation-related genes (Furini et al. 1997; Hilbricht
et al. 2008), although the precise mechanism of the function is still unclear. Functional characterisation of CDT-1
as well as 28852 ncRNAs from C. plantagineum is required to understand how ncRNAs play a role in the
rewiring of gene expression. The number of predicted
TRGs in resurrection plants is larger than what was expected for eukaryotic organisms. This could be an overestimation due to partial miss-annotation of sequences with
reduced length. Nevertheless, we expect that unknown
TRGs are important in C. plantagineum and other resurrection plants. Such TRGs need to be characterised because they may have species-specific functions which are
required together with conserved protein-coding genes for
establishing desiccation tolerance.
Extended 28852 sequence including 50 and 30 transcript ends (Giarola et al. unpublished)
Author contribution V. Giarola wrote most of the
manuscript, D. Bartels is responsible for the concept and
corrections of the manuscript.
a
Similar genes were retrieved by BLASTN and BLASTX analysis. The expectation value (E) of the best hit is reported
?
0.11
Upstream sequence of Dusp1
ATG start site [Melopsittacus
undulates]
631a
28852
9e-05
Protein notum homolog
[Sesamum indicum]
Chloroplast (Giarola et al.
2015b)
2e-04
BAC clone RP24-198I16 [Mus
musculus]
770
CpEDR1
0.058
Stress response protein
[Sesamum indicum]
Cell wall (Giarola et al. 2015b)
0.021
Complete genome [Geobacter
pickeringii G13]
736
CpCRP1
0.19
Type IV secretion protein Rhs
[Burkholderia oxyphila]
Cytoplasm (Rodrigo et al.
2004)
2e-09
Hypothetical protein
[Erythranthe guttata]
Uncharacterized
LOC105155524 [Sesamum
indicum]
422
CpEdi-9
4e-20
–
No similarity
Genome contig0005186
[Onchocerca ochengi]
911
CDT-1
4e-05
E
BLASTX hit
E
BLASTN hit
Length (bp)
Gene name
Table 1 Examples of BLAST analyses of taxonomically restricted genes from C. plantagineum
?
Planta (2015) 242:427–434
Localisation
432
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