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 123 428 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- 123 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). 429 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 123 430 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 123 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 431 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 123 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 References Bartels D (2005) Desiccation tolerance studied in the resurrection plant Craterostigma plantagineum. Integr Comp Biol 45:696–701 Bartels D, Salamini F (2001) Desiccation tolerance in the resurrection plant Craterostigma plantagineum. A contribution to the study of drought tolerance at the molecular level. Plant Physiol 127:1346–1353 Bartels D, Schneider K, Terstappen G, Piatkowski D, Salamini F (1990) Molecular cloning of abscisic acid-modulated genes which are induced during desiccation of the resurrection plant Craterostigma plantagineum. Planta 181:27–34 Bartels D, Hanke C, Schneider K, Michel D, Salamini F (1992) A desiccation-related Elip-like gene from the resurrection plant Craterostigma plantagineum is regulated by light and ABA. EMBO J 11:2771–2778 Bernacchia G, Salamini F, Bartels D (1996) Molecular characterization of the rehydration process in the resurrection plant Craterostigma plantagineum. Plant Physiol 111:1043–1050 Bernstein E, Allis CD (2005) RNA meets chromatin. Genes Dev 19:1635–1655 Bertone P, Stolc V, Royce TE, Rozowsky JS, Urban AE, Zhu X, Rinn JL, Tongprasit W, Samanta M, Weissman S, Gerstein M, Snyder M (2004) Global identification of human transcribed sequences with genome tiling arrays. Science 306:2242–2246 Bianchi G, Gamba A, Murelli C, Salamini F, Bartels D (1991) Novel carbohydrate metabolism in the resurrection plant Craterostigma plantagineum. Plant J 1:355–359 Bockel C, Salamini F, Bartels D (1998) Isolation and characterization of genes expressed during early events of the dehydration process in the resurrection plant Craterostigma plantagineum. J Plant Physiol 152:158–166 Planta (2015) 242:427–434 Bond CS, Fox AH (2009) Paraspeckles: nuclear bodies built on long noncoding RNA. J Cell Biol 186:637–644 Burke MJ (1986) The glassy state and survival of anhydrous biological systems. In: Leopold AC (ed) Membranes, metabolism, and dry organisms. Cornell University Press, Ithaca, NY, pp 358–363 Carrieri C, Cimatti L, Biagioli M, Beugnet A, Zucchelli S, Fedele S, Pesce E, Ferrer I, Collavin L, Santoro C, Forrest ARR, Carninci P, Biffo S, Stupka E, Gustincich S (2012) Long non-coding antisense RNA controls Uchl1 translation through an embedded SINEB2 repeat. Nature 491:454–459 Cheng J, Kapranov P, Drenkow J, Dike S, Brubaker S, Patel S, Long J, Stern D, Tammana H, Helt G, Sementchenko V, Piccolboni A, Bekiranov S, Bailey DK, Ganesh M, Ghosh S, Bell I, Gerhard DS, Gingeras TR (2005) Transcriptional maps of 10 human chromosomes at 5-nucleotide resolution. Science 308:1149–1154 Clegg JS, Seitz P, Seitz W, Hazlewood CF (1982) Cellular responses to extreme water loss: the water-replacement hypothesis. Cryobiology 19:306–316 Cooper K, Farrant JM (2002) Recovery of the resurrection plant Craterostigma wilmsii from desiccation: protection versus repair. J Exp Bot 53:1805–1813 Crowe JH, Carpenter JF, Crowe LM (1998) The role of vitrification in anhydrobiosis. Annu Rev Physiol 60:73–103 Cullum R, Alder O, Hoodless PA (2011) The next generation: using new sequencing technologies to analyse gene regulation. Respirology 16:210–222 Cushman JC, Oliver MJ (2011) Understanding vegetative desiccation tolerance using integrated functional genomics approaches within a comparative evolutionary framework. In: Lüttge U, Beck E, Bartels D (eds) Plant desiccation tolerance. Ecological Studies, vol 215. Springer, Berlin Heidelberg, pp 307–338 Doebley J, Lukens L (1998) Transcriptional regulators and the evolution of plant form. Plant Cell 10:1075–1082 Faghihi MA, Wahlestedt C (2009) Regulatory roles of natural antisense transcripts. Nat Rev Mol Cell Biol 10:637–643 Faghihi MA, Modarresi F, Khalil AM, Wood DE, Sahagan BG, Morgan TE, Finch CE, Laurent GS, Kenny PJ, Wahlestedt C (2008) Expression of a noncoding RNA is elevated in Alzheimer’s disease and drives rapid feed-forward regulation of b-secretase. Nat Med 14:723–730 Farrant JM (2000) A comparison of mechanisms of desiccation tolerance among three angiosperm resurrection plant species. Plant Ecol 151:29–39 Farrant JM, Vander Willigen C, Loffell DA, Bartsch S, Whittaker A (2003) An investigation into the role of light during desiccation of three angiosperm resurrection plants. Plant, Cell Environ 26:1275–1286 Farrant JM, Brandt W, Lindsey GG (2007) An overview of mechanisms of desiccation tolerance in selected angiosperm resurrection plants. Plant Stress 1:72–84 Fischer E (1995) Revision of the Lindernieae (Scrophulariaceae) in Madagascar. 1. The genera Lindernia Allioni and Crepidorhopalon E. Fischer. Bulletin du Muséum National d’Histoire Naturelle, section B, Adansonia: Botanique Phytochemie Ser 4, 17:227–257 Furini A, Koncz C, Salamini F, Bartels D (1997) High level transcription of a member of a repeated gene family confers dehydration tolerance to callus tissue of Craterostigma plantagineum. EMBO J 16:3599–3608 Gaff DF (1989) Responses of desiccation-tolerant ‘‘resurrection’’ plants to water stress. In: Kreeb KH, Richter H, Hinckley TM (eds) Structural and functional responses to environmental stresses: water shortages. SPB Academic, The Hague, pp 264–311 433 Gechev TS, Benina M, Obata T, Tohge T, Sujeeth N, Minkov I, Hille J, Temanni MR, Marriott AS, Bergstrom E, Thomas-Oates J, Antonio C, Mueller-Roeber B, Schippers JH, Fernie AR, Toneva V (2013) Molecular mechanisms of desiccation tolerance in the resurrection glacial relic Haberlea rhodopensis. Cell Mol Life Sci 70:689–709 Giarola V, Challabathula D, Bartels D (2015a) Quantification of expression of dehydrin isoforms in the desiccation tolerant plant Craterostigma plantagineum using specifically designed reference genes. Plant Sci 236:103–115 Giarola V, Krey S, Frerichs A, Bartels D (2015b) Taxonomically restricted genes of Craterostigma plantagineum are modulated in their expression during dehydration and rehydration. Planta 241:193–208 Hilbricht T, Varotto S, Sgaramella V, Bartels D, Salamini F, Furini A (2008) Retrotransposons and siRNA have a role in the evolution of desiccation tolerance leading to resurrection of the plant Craterostigma plantagineum. New Phytol 179:877–887 Hoekstra FA, Golovina EA, Buitink J (2001) Mechanisms of plant desiccation tolerance. Trends Plant Sci 6:431–438 Huarte M, Guttman M, Feldser D, Garber M, Koziol MJ, Kenzelmann-Broz D, Khalil AM, Zuk O, Amit I, Rabani M, Attardi LD, Regev A, Lander ES, Jacks T, Rinn JL (2010) A large intergenic noncoding RNA induced by p53 mediates global gene repression in the p53 response. Cell 142:409–419 Ingram J, Bartels D (1996) The molecular basis of dehydration tolerance in plants. Annu Rev Plant Biol 47:377–403 Kapranov P, Cheng J, Dike S, Nix DA, Duttagupta R, Willingham AT, Stadler PF, Hertel J, Hackermuller J, Hofacker IL, Bell I, Cheung E, Drenkow J, Dumais E, Patel S, Helt G, Ganesh M, Ghosh S, Piccolboni A, Sementchenko V, Tammana H, Gingeras TR (2007) RNA maps reveal new RNA classes and a possible function for pervasive transcription. Science 316:1484–1488 Khalturin K, Hemmrich G, Fraune S, Augustin R, Bosch TCG (2009) More than just orphans: are taxonomically-restricted genes important in evolution? Trends Genet 25:404–413 Kotake Y, Nakagawa T, Kitagawa K, Suzuki S, Liu N, Kitagawa M, Xiong Y (2011) Long non-coding RNA ANRIL is required for the PRC2 recruitment to and silencing of p15INK4B tumor suppressor gene. Oncogene 30:1956–1962 Kretz M, Siprashvili Z, Chu C, Webster DE, Zehnder A, Qu K, Lee CS, Flockhart RJ, Groff AF, Chow J, Johnston D, Kim GE, Spitale RC, Flynn RA, Zheng GXY, Aiyer S, Raj A, Rinn JL, Chang HY, Khavari PA (2013) Control of somatic tissue differentiation by the long non-coding RNA TINCR. Nature 493:231–235 Lynch M, Conery JS (2000) The evolutionary fate and consequences of duplicate genes. Science 290:1151–1155 Mercer TR, Dinger ME, Mattick JS (2009) Long non-coding RNAs: insights into functions. Nat Rev Genet 10:155–159 Moore JP, Le NT, Brandt WF, Driouich A, Farrant JM (2009) Towards a systems-based understanding of plant desiccation tolerance. Trends Plant Sci 14:110–117 Moore JP, Nguema-Ona EE, Vicré-Gibouin M, Sørensen I, Willats WG, Driouich A, Farrant JM (2013) Arabinose-rich polymers as an evolutionary strategy to plasticize resurrection plant cell walls against desiccation. Planta 237:739–754 Mutz K-O, Heilkenbrinker A, Lönne M, Walter J-G, Stahl F (2013) Transcriptome analysis using next-generation sequencing. Curr Opin Biotechnol 24:22–30 Ohno S (1970) Evolution by gene duplication. Springer-Verlag, Heidelberg New York Oliver MJ, O’Mahony P, Wood AJ (1998) ‘‘To dryness and beyond’’—preparation for the dried state and rehydration in vegetative desiccation-tolerant plants. Plant Growth Regul 24:193–201 123 434 Oliver M, Tuba Z, Mishler B (2000) The evolution of vegetative desiccation tolerance in land plants. Plant Ecol 151:85–100 Ørom UA, Derrien T, Beringer M, Gumireddy K, Gardini A, Bussotti G, Lai F, Zytnicki M, Notredame C, Huang Q, Guigo R, Shiekhattar R (2010) Long noncoding RNAs with enhancer-like function in human cells. Cell 143:46–58 Pandey RR, Mondal T, Mohammad F, Enroth S, Redrup L, Komorowski J, Nagano T, Mancini-Dinardo D, Kanduri C (2008) Kcnq1ot1 antisense noncoding RNA mediates lineagespecific transcriptional silencing through chromatin-level regulation. Mol Cell 32:232–246 Phillips JR, Hilbricht T, Salamini F, Bartels D (2002) A novel abscisic acid- and dehydration-responsive gene family from the resurrection plant Craterostigma plantagineum encodes a plastid-targeted protein with DNA-binding activity. Planta 215:258–266 Phillips JR, Fischer E, Baron M, van den Dries N, Facchinelli F, Kutzer M, Rahmanzadeh R, Remus D, Bartels D (2008) Lindernia brevidens: a novel desiccation-tolerant vascular plant, endemic to ancient tropical rainforests. Plant J 54:938–948 Prud’homme B, Gompel N, Carroll SB (2007) Emerging principles of regulatory evolution. Proc Natl Acad Sci USA 104:8605–8612 Rodrigo MJ, Bockel C, Blervacq AS, Bartels D (2004) The novel gene CpEdi-9 from the resurrection plant C. plantagineum encodes a hydrophilic protein and is expressed in mature seeds as well as in response to dehydration in leaf phloem tissues. Planta 219:579–589 Rodriguez MC, Edsgärd D, Hussain SS, Alquezar D, Rasmussen M, Gilbert T, Nielsen BH, Bartels D, Mundy J (2010) Transcriptomes of the desiccation-tolerant resurrection plant Craterostigma plantagineum. Plant J 63:212–228 Schneider K, Wells B, Schmelzer E, Salamini F, Bartels D (1993) Desiccation leads to the rapid accumulation of both cytosolic and chloroplastic proteins in the resurrection plant Craterostigma plantagineum Hochst. Planta 189:120–131 Seine R, Fischer E, Barthlott W (1995) Notes on the Scrophulariaceae of Zimbabwean inselsbergs, with the description of Lindernia syncerus sp. nov. Feddes Repertorium 106:7–12 123 Planta (2015) 242:427–434 Sherwin H, Farrant J (1998) Protection mechanisms against excess light in the resurrection plants Craterostigma wilmsii and Xerophyta viscosa. Plant Growth Regul 24:203–210 Tripathi V, Ellis JD, Shen Z, Song DY, Pan Q, Watt AT, Freier SM, Bennett CF, Sharma A, Bubulya PA, Blencowe BJ, Prasanth SG, Prasanth KV (2010) The nuclear-retained noncoding RNA MALAT1 regulates alternative splicing by modulating SR splicing factor phosphorylation. Mol Cell 39:925–938 van den Dries N, Facchinelli F, Giarola V, Phillips JR, Bartels D (2011) Comparative analysis of LEA-like 11-24 gene expression and regulation in related plant species within the Linderniaceae that differ in desiccation tolerance. New Phytol 190:75–88 Wang KC, Chang HY (2011) Molecular mechanisms of long noncoding RNAs. Mol Cell 43:904–914 Wang L, Feng Z, Wang X, Wang X, Zhang X (2010) DEGseq: an R package for identifying differentially expressed genes from RNA-seq data. Bioinformatics 26:136–138 Whitehead J, Pandey GK, Kanduri C (2009) Regulation of the mammalian epigenome by long noncoding RNAs. Biochim Biophys Acta 1790:936–947 Willigen CV, Pammenter NW, Jaffer MA, Mundree SG, Farrant JM (2003) An ultrastructural study using anhydrous fixation of Eragrostis nindensis, a resurrection grass with both desiccationtolerant and -sensitive tissues. Funct Plant Biol 30:281 Wilson GA, Bertrand N, Patel Y, Hughes JB, Feil EJ, Field D (2005) Orphans as taxonomically restricted and ecologically important genes. Microbiology 151:2499–2501 Wilusz JE, Sunwoo H, Spector DL (2009) Long noncoding RNAs: functional surprises from the RNA world. Genes Dev 23:1494–1504 Zhang K, Shi Z-M, Chang Y-N, Hu Z-M, Qi H-X, Hong W (2014) The ways of action of long non-coding RNAs in cytoplasm and nucleus. Gene 547:1–9 Zhou X, Ren L, Meng Q, Li Y, Yu Y, Yu J (2010) The nextgeneration sequencing technology and application. Protein Cell 1:520–536