Specific metabolic and cellular mechanisms of the vegetative desiccation tolerance in resurrection plants for adaptation to extreme dryness.

MAIN CONCLUSION: Substantial advancements have been made in our comprehension of vegetative desiccation tolerance in resurrection plants, and further research is still warranted to elucidate the mechanisms governing distinct cellular adaptations. Resurrection plants are commonly referred to as a small group of extremophile vascular plants that exhibit vegetative desiccation tolerance (VDT), meaning that their vegetative tissues can survive extreme drought stress (> 90% water loss) and subsequently recover rapidly upon rehydration. In contrast to most vascular plants, which typically employ water-saving strategies to resist partial water loss and optimize water absorption and utilization to a limited extent under moderate drought stress, ultimately succumbing to cell death when confronted with severe and extreme drought conditions, resurrection plants have evolved unique mechanisms of VDT, enabling them to maintain viability even in the absence of water for extended periods, permitting them to rejuvenate without harm upon water contact. Understanding the mechanisms associated with VDT in resurrection plants holds the promise of expanding our understanding of how plants adapt to exceedingly arid environments, a phenomenon increasingly prevalent due to global warming. This review offers an updated and comprehensive overview of recent advances in VDT within resurrection plants, with particular emphasis on elucidating the metabolic and cellular adaptations during desiccation, including the intricate processes of cell wall folding and the prevention of cell death. Furthermore, this review highlights existing unanswered questions in the field, suggests potential avenues for further research to gain deeper insights into the remarkable VDT adaptations observed in resurrection plants, and highlights the potential application of VDT-derived techniques in crop breeding to enhance tolerance to extreme drought stress.

Dynamics of chromatin accessibility and genome wide control of desiccation tolerance in the resurrection plant Haberlea rhodopensis.

BACKGROUND: Drought is one of the main consequences of global climate change and this problem is expected to intensify in the future. Resurrection plants evolved the ability to withstand the negative impact of long periods of almost complete desiccation and to recover at rewatering. In this respect, many physiological, transcriptomic, proteomic and genomic investigations have been performed in recent years, however, few epigenetic control studies have been performed on these valuable desiccation-tolerant plants so far. RESULTS: In the present study, for the first time for resurrection plants we provide evidences about the differential chromatin accessibility of Haberlea rhodopensis during desiccation stress by ATAC-seq (Assay for Transposase Accessible Chromatin with high-throughput sequencing). Based on gene similarity between species, we used the available genome of the closely related resurrection plant Dorcoceras hygrometricum to identify approximately nine hundred transposase hypersensitive sites (THSs) in H. rhodopensis. The majority of them corresponds to proximal and distal regulatory elements of different genes involved in photosynthesis, carbon metabolism, synthesis of secondary metabolites, cell signalling and transcriptional regulation, cell growth, cell wall, stomata conditioning, chaperons, oxidative stress, autophagy and others. Various types of binding motifs recognized by several families of transcription factors have been enriched from the THSs found in different stages of drought. Further, we used the previously published RNA-seq data from H. rhodopensis to evaluate the expression of transcription factors putatively interacting with the enriched motifs, and the potential correlation between the identified THS and the expression of their corresponding genes. CONCLUSIONS: These results provide a blueprint for investigating the epigenetic regulation of desiccation tolerance in resurrection plant H. rhodopensis and comparative genomics between resurrection and non-resurrection species with available genome information.

Proteomics Evidence of a Systemic Response to Desiccation in the Resurrection Plant Haberlea rhodopensis.

Global warming and drought stress are expected to have a negative impact on agricultural productivity. Desiccation-tolerant species, which are able to tolerate the almost complete desiccation of their vegetative tissues, are appropriate models to study extreme drought tolerance and identify novel approaches to improve the resistance of crops to drought stress. In the present study, to better understand what makes resurrection plants extremely tolerant to drought, we performed transmission electron microscopy and integrative large-scale proteomics, including organellar and phosphorylation proteomics, and combined these investigations with previously published transcriptomic and metabolomics data from the resurrection plant Haberlea rhodopensis. The results revealed new evidence about organelle and cell preservation, posttranscriptional and posttranslational regulation, photosynthesis, primary metabolism, autophagy, and cell death in response to desiccation in H. rhodopensis. Different protective intrinsically disordered proteins, such as late embryogenesis abundant (LEA) proteins, thaumatin-like proteins (TLPs), and heat shock proteins (HSPs), were detected. We also found a constitutively abundant dehydrin in H. rhodopensis whose phosphorylation levels increased under stress in the chloroplast fraction. This integrative multi-omics analysis revealed a systemic response to desiccation in H. rhodopensis and certain targets for further genomic and evolutionary studies on DT mechanisms and genetic engineering towards the improvement of drought tolerance in crops.

Heterologous Expression of MfWRKY7 of Resurrection Plant Myrothamnus flabellifolia Enhances Salt and Drought Tolerance in Arabidopsis.

Drought and salinity have become major environmental problems that affect the production of agriculture, forestry and horticulture. The identification of stress-tolerant genes from plants adaptive to harsh environments might be a feasible strategy for plant genetic improvement to address the challenges brought by global climate changes. In this study, a dehydration-upregulated gene MfWRKY7 of resurrection Plant Myrothamnusflabellifolia, encoding a group IId WRKY transcription factor, was cloned and characterized. The overexpression of MfWRKY7 in Arabidopsis increased root length and tolerance to drought and NaCl at both seedling and adult stages. Further investigation indicated that MfWRKY7 transgenic plants had higher contents of chlorophyll, proline, soluble protein, and soluble sugar but a lower water loss rate and malondialdehyde content compared with wild-type plants under both drought and salinity stresses. Moreover, the higher activities of antioxidant enzymes and lower accumulation of O2- and H2O2 in MfWRKY7 transgenic plants were also found, indicating enhanced antioxidation capacity by MfWRKY7. These findings showed that MfWRKY7 may function in positive regulation of responses to drought and salinity stresses, and therefore, it has potential application value in genetic improvement of plant tolerance to abiotic stress.

A WRKY Protein, MfWRKY40, of Resurrection Plant Myrothamnus flabellifolia Plays a Positive Role in Regulating Tolerance to Drought and Salinity Stresses of Arabidopsis.

WRKY transcription factors (TFs), one of the largest transcription factor families in plants, play an important role in abiotic stress responses. The resurrection plant, Myrothamnus flabellifolia, has a strong tolerance to dehydration, but only a few WRKY proteins related to abiotic stress response have been identified and functionally characterized in M. flabellifolia. In this study, we identified an early dehydration-induced gene, MfWRKY40, of M. flabellifolia. The deduced MfWRKY40 protein has a conserved WRKY motif but lacks a typical zinc finger motif in the WRKY domain and is localized in the nucleus. To investigate its potential roles in abiotic stresses, we overexpressed MfWRKY40 in Arabidopsis and found that transgenic lines exhibited better tolerance to both drought and salt stresses. Further detailed analysis indicated that MfWRKY40 promoted primary root length elongation and reduced water loss rate and stomata aperture (width/length) under stress, which may provide Arabidopsis the better water uptake and retention abilities. MfWRKY40 also facilitated osmotic adjustment under drought and salt stresses by accumulating more osmolytes, such as proline, soluble sugar, and soluble protein. Additionally, the antioxidation ability of transgenic lines was also significantly enhanced, represented by higher chlorophyll content, less malondialdehyde and reactive oxygen species accumulations, as well as higher antioxidation enzyme activities. All these results indicated that MfWRKY40 might positively regulate tolerance to drought and salinity stresses. Further investigation on the relationship of the missing zinc finger motif of MfWRKY40 and its regulatory role is necessary to obtain a better understanding of the mechanism underlying the excellent drought tolerance of M. flabellifolia.

Chromosome-Scale Genome Assembly of the Resurrection Plant Acanthochlamys bracteata (Velloziaceae).

Acanthochlamys bracteata (Velloziaceae) is a resurrection plant with cold tolerance. Herein, a chromosome-level reference genome of A. bracteata based on Nanopore, Illumina, and Hi-C data is reported. The high-quality assembled genome was 197.97 Mb, with a scaffold N50 value of 8.64 Mb and a contig N50 value of 6.96 Mb. We annotated 23,509 protein-coding genes. Eight contracted gene families and three expanded gene families were detected. Repeat sequences accounted for approximately 28.63% of the genome. The LEA1 and Dehydrin gene families, which are involved in desiccation resistance, expanded in A. bracteata. We identified genes involved in chilling tolerance, COLD1.

Analysis of the complete mitochondrial genome sequence of the resurrection plant Haberlea rhodopensis.

Haberlea rhodopensis is a paleolithic tertiary relict species that belongs to the unique group of resurrection plants sharing remarkable tolerance to desiccation. When exposed to severe drought stress, this species shows an ability to maintain structural integrity of its deactivated photosynthetic apparatus, which easily reactivates upon rehydration. In addition to its homoiochlorophyllous nature, the resurrection capability of H. rhodopensis is of particular importance to the global climate change mitigation. In this study, we sequenced, assembled, and analyzed the mitochondrial (mt) genome of H. rhodopensis for the first time. The master circle has a typical circular structure of 484 138 bp in length with a 44.1% GC content in total. The mt genome of H. rhodopensis contains 59 genes in total, including 35 protein-coding, 21 tRNAs, and 3 rRNAs genes. 7 tandem repeats and 85 simple sequence repeats (SSRs) are distributed throughout the mt genome. The alignment of 20 plant mt genomes confirms the phylogenetic position of H. rhodopensis in the Lamiales order. Our comprehensive analysis of the complete mt genome of H. rhodopensis is a significant addition to the limited database of organelle genomes of resurrection species. Comparative and phylogenetic analysis provides valuable information for a better understanding of mitochondrial molecular evolution in plants.

Identification and characterization of CTP:phosphocholine cytidylyltransferase CpCCT1 in the resurrection plant Craterostigma plantagineum.

Phosphatidylcholine is a major phospholipid which is shown to be involved in stress adaptation. Phosphatidylcholine increased during dehydration in Craterostigma plantagineum, and therefore we characterized CTP:phosphocholine cytidylyltransferase (CpCCT1), a key regulatory enzyme for phosphatidylcholine synthesis in plants. The CpCCT1 gene from the resurrection plant C. plantagineum was cloned and the amino acid sequence was compared with homologs from other species including yeast and rat. CCT proteins have conserved catalytic and membrane-binding domains while the N-terminal and C-terminal domains have diverged. The tissue specific expression analysis indicated that CpCCT1 is expressed in all tested tissues and it is induced by dehydration and in response to 0.5 M NaCl solutions. In plants exposed to low temperature in the dark, the CpCCT1 transcript increased after 4 h at 4 °C. CpCCT1 expression also increased during mannitol and sorbitol treatments in a concentration dependent manner. Phytohormones such as abscisic acid and indole-3-acetic acid also trigged transcript accumulation. Comparisons of transcript and protein accumulations for different treatments (except for dehydration) suggest transcriptional and translational control mechanisms. Analysis of promoter activity and polysome occupancy suggest that CpCCT1 gene expression is mainly under translational regulation during dehydration.

The dehydration- and ABA-inducible germin-like protein CpGLP1 from Craterostigma plantagineum has SOD activity and may contribute to cell wall integrity during desiccation.

MAIN CONCLUSION: CpGLP1 belongs to the large group of germin-like proteins and comprises a cell wall-localized protein which has superoxide dismutase activity and may contribute towards ROS metabolism and cell wall folding during desiccation. The plant cell wall is a dynamic matrix and its plasticity is essential for cell growth and processing of environmental signals to cope with stresses. A few so-called resurrection plants like Craterostigma plantagineum survive desiccation by implementing protection mechanisms. In C. plantagineum, the cell wall shrinks and folds upon desiccation to avoid mechanical and oxidative damage which contributes to cell integrity. Despite the high toxic potential, ROS are important molecules for cell wall remodeling processes as they participate in enzymatic reactions and act as signaling molecules. Here we analyzed the C. plantagineum germin-like protein 1 (CpGLP1) to understand its contribution to cell wall folding and desiccation tolerance. The analysis of the CpGLP1 sequence showed that this protein does not fit into the current GLP classification and forms a new group within the Linderniaceae. CpGLP1 transcripts accumulate in leaves in response to dehydration and ABA, and mannitol treatments transiently induce CpGLP1 transcript accumulation supporting the participation of CpGLP1 in desiccation-related processes. CpGLP1 protein from cell wall protein extracts followed transcript accumulation and protein preparations from bacteria overexpressing CpGLP1 showed SOD activity. In agreement with cell wall localization, CpGLP1 interacts with pectins which have not been reported for GLP proteins. Our data support a role for CpGLP1 in the ROS metabolism related to the control of cell wall plasticity during desiccation in C. plantagineum.

MfPIF1 of Resurrection Plant Myrothamnus flabellifolia Plays a Positive Regulatory Role in Responding to Drought and Salinity Stresses in Arabidopsis.

Phytochrome-interacting factors (PIFs), a subfamily of basic helix-loop-helix (bHLH) transcription factors (TFs), play critical roles in regulating plant growth and development. The resurrection plant Myrothamnus flabellifolia possesses a noteworthy tolerance to desiccation, but no PIFs related to the response to abiotic stress have been functionally studied. In this study, a dehydration-inducible PIF gene, MfPIF1, was cloned and characterized. Subcellular localization assay revealed that MfPIF1 is localized predominantly in the nucleus. Overexpression of MfPIF1 in Arabidopsis thaliana led to enhanced drought and salinity tolerance, which was attributed to higher contents of chlorophyll, proline (Pro), soluble protein, and soluble sugar, activities of antioxidant enzymes as well as lower water loss rate, malondialdehyde (MDA) content, and reactive oxygen species (ROS) accumulation in transgenic lines compared with control plants. Moreover, MfPIF1 decreased stomatal aperture after drought and abscisic acid (ABA) treatment, and increased expression of both ABA biosynthesis and ABA-responsive genes including NCED3, P5CS, and RD29A. Overall, these results indicated that MfPIF1 may act as a positive regulator to drought and salinity responses, and therefore could be considered as a potential gene for plant genetic improvement of drought and salinity tolerance.

Transcriptional and metabolic changes in the desiccation tolerant plant Craterostigma plantagineum during recurrent exposures to dehydration.

MAIN CONCLUSION: Multiple dehydration/rehydration treatments improve the adaptation of Craterostigma plantagineum to desiccation by accumulating stress-inducible transcripts, proteins and metabolites. These molecules serve as stress imprints or memory and can lead to increased stress tolerance. It has been reported that repeated exposure to dehydration may generate stronger reactions during a subsequent dehydration treatment in plants. This stimulated us to address the question whether the desiccation tolerant resurrection plant Craterostigma plantagineum has a stress memory. The expression of four representative stress-related genes gradually increased during four repeated dehydration/rehydration treatments in C. plantagineum. These genes reflect a transcriptional memory and are trainable genes. In contrast, abundance of chlorophyll synthesis/degradation-related transcripts did not change during dehydration and remained at a similar level as in the untreated tissues during the recovery phase. During the four dehydration/rehydration treatments the level of ROS pathway-related transcripts, superoxide dismutase (SOD) activity, proline, and sucrose increased, whereas H2O2 content and electrolyte leakage decreased. Malondialdehyde (MDA) content did not change during the dehydration, which indicates a gain of stress tolerance. At the protein level, increased expression of four representative stress-related proteins showed that the activated stress memory can persist over several days. The phenomenon described here could be a general feature of dehydration stress memory responses in resurrection plants.

Analysis of pcC13-62 promoters predicts a link between cis-element variations and desiccation tolerance in Linderniaceae.

Reproductive structures of plants (e.g. seeds) and vegetative tissues of resurrection plants can tolerate desiccation. Many genes encoding desiccation-related proteins (DRPs) have been identified in the resurrection plant Craterostigma plantagineum, but the function of these genes remains mainly hypothetical. Here, the importance of the DRP gene pcC13-62 for desiccation tolerance is evaluated by analysing its expression in C. plantagineum and in the closely related desiccation-tolerant species Lindernia brevidens and the desiccation-sensitive species Lindernia subracemosa. Quantitative analysis revealed that pcC13-62 transcripts accumulate at a much lower level in desiccation-sensitive species than in desiccation-tolerant species. The study of pcC13-62 promoters from these species demonstrated a correlation between promoter activity and gene expression levels, suggesting transcriptional regulation of gene expression. Comparison of promoter sequences identified a dehydration-responsive element motif in the promoters of tolerant species that is required for dehydration-induced ß-glucuronidase (GUS) accumulation. We hypothesize that variations in the regulatory sequences of the pcC13-62 gene occurred to establish pcC13-62 expression in vegetative tissues, which might be required for desiccation tolerance. The pcC13-62 promoters could also be activated by salt stress in Arabidopsis thaliana plants stably transformed with promoter::GUS constructs.

Seed desiccation mechanisms co-opted for vegetative desiccation in the resurrection grass Oropetium thomaeum.

Resurrection plants desiccate during periods of prolonged drought stress, then resume normal cellular metabolism upon water availability. Desiccation tolerance has multiple origins in flowering plants, and it likely evolved through rewiring seed desiccation pathways. Oropetium thomaeum is an emerging model for extreme drought tolerance, and its genome, which is the smallest among surveyed grasses, was recently sequenced. Combining RNA-seq, targeted metabolite analysis and comparative genomics, we show evidence for co-option of seed-specific pathways during vegetative desiccation. Desiccation-related gene co-expression clusters are enriched in functions related to seed development including several seed-specific transcription factors. Across the metabolic network, pathways involved in programmed cell death inhibition, ABA signalling and others are activated during dehydration. Oleosins and oil bodies that typically function in seed storage are highly abundant in desiccated leaves and may function for membrane stability and storage. Orthologs to seed-specific LEA proteins from rice and maize have neofunctionalized in Oropetium with high expression during desiccation. Accumulation of sucrose, raffinose and stachyose in drying leaves mirrors sugar accumulation patterns in maturing seeds. Together, these results connect vegetative desiccation with existing seed desiccation and drought responsive pathways and provide some key candidate genes for engineering improved drought tolerance in crop plants.

Angiosperm Plant Desiccation Tolerance: Hints from Transcriptomics and Genome Sequencing.

Desiccation tolerance (DT) in angiosperms is present in the small group of resurrection plants and in seeds. DT requires the presence of protective proteins, specific carbohydrates, restructuring of membrane lipids, and regulatory mechanisms directing a dedicated gene expression program. Many components are common to resurrection plants and seeds; however, some are specific for resurrection plants. Understanding how each component contributes to DT is challenging. Recent transcriptome analyses and genome sequencing indicate that increased expression is essential of genes encoding protective components, recently evolved, species-specific genes and non-protein-coding RNAs. Modification and reshuffling of existing cis-regulatory promoter elements seems to play a role in the rewiring of regulatory networks required for increased expression of DT-related genes in resurrection species.

LEA gene expression, RNA stability and pigment accumulation in three closely related Linderniaceae species differing in desiccation tolerance.

Desiccation-tolerant plants (Craterostigma plantagineum and Lindernia brevidens) evolved a highly efficient strategies to prevent dehydration-induced irreversible damage. The protection system involves synthesis of LEA proteins, decrease of photosynthetic activity and activation of antioxidant systems. The regulation of these processes requires joint action of multiple proteins. Here, we present comparative analyses of accumulation of transcripts encoding components of the protection machinery, such as selected LEA proteins, enzymes of the chlorophyll degradation pathway and anthocyanin biosynthesis enzymes in total and polysomal RNA pools. The analyses revealed that desiccation-tolerant plants recruit mRNAs to ribosomes with higher efficiency than the desiccation-sensitive species L. subracemosa. Desiccation-tolerant species accumulated high amounts of LEA transcripts during dehydration and precisely controlled the amounts of chlorophyll keeping it at a level sufficient to activate photosynthesis after rehydration. In contrast, mRNA of L. subracemosa was prone to dehydration-induced degradation, decomposition of the photosynthetic apparatus and degradation of free chlorophyll. Thus, the results of the studies point to differences in the control of gene expression and degradation of chlorophyll in desiccation-tolerant versus desiccation-sensitive species when the plants were subjected to dehydration.

The Craterostigma plantagineum glycine-rich protein CpGRP1 interacts with a cell wall-associated protein kinase 1 (CpWAK1) and accumulates in leaf cell walls during dehydration.

Craterostigma plantagineum tolerates extreme desiccation. Leaves of this plant shrink and extensively fold during dehydration and expand again during rehydration, preserving their structural integrity. Genes were analysed that may participate in the reversible folding mechanism. Analysis of transcripts abundantly expressed in desiccated leaves identified a gene putatively coding for an apoplastic glycine-rich protein (CpGRP1). We studied the expression, regulation and subcellular localization of CpGRP1 and its ability to interact with a cell wall-associated protein kinase (CpWAK1) to understand the role of CpGRP1 in the cell wall during dehydration. The CpGRP1 protein accumulates in the apoplast of desiccated leaves. Analysis of the promoter revealed that the gene expression is mainly regulated at the transcriptional level, is independent of abscisic acid (ABA) and involves a drought-responsive cis-element (DRE). CpGRP1 interacts with CpWAK1 which is down-regulated in response to dehydration. Our data suggest a role of the CpGRP1-CpWAK1 complex in dehydration-induced morphological changes in the cell wall during dehydration in C. plantagineum. Cell wall pectins and dehydration-induced pectin modifications are predicted to be involved in the activity of the CpGRP1-CpWAK1 complex.

Trehalose Accumulation Triggers Autophagy during Plant Desiccation.

Global climate change, increasingly erratic weather and a burgeoning global population are significant threats to the sustainability of future crop production. There is an urgent need for the development of robust measures that enable crops to withstand the uncertainty of climate change whilst still producing maximum yields. Resurrection plants possess the unique ability to withstand desiccation for prolonged periods, can be restored upon watering and represent great potential for the development of stress tolerant crops. Here, we describe the remarkable stress characteristics of Tripogon loliiformis, an uncharacterised resurrection grass and close relative of the economically important cereals, rice, sorghum, and maize. We show that T. loliiformis survives extreme environmental stress by implementing autophagy to prevent Programmed Cell Death. Notably, we identified a novel role for trehalose in the regulation of autophagy in T.loliiformis. Transcriptome, Gas Chromatography Mass Spectrometry, immunoblotting and confocal microscopy analyses directly linked the accumulation of trehalose with the onset of autophagy in dehydrating and desiccated T. loliiformis shoots. These results were supported in vitro with the observation of autophagosomes in trehalose treated T. loliiformis leaves; autophagosomes were not detected in untreated samples. Presumably, once induced, autophagy promotes desiccation tolerance in T.loliiformis, by removal of cellular toxins to suppress programmed cell death and the recycling of nutrients to delay the onset of senescence. These findings illustrate how resurrection plants manipulate sugar metabolism to promote desiccation tolerance and may provide candidate genes that are potentially useful for the development of stress tolerant crops.

Quantification of expression of dehydrin isoforms in the desiccation tolerant plant Craterostigma plantagineum using specifically designed reference genes.

Craterostigma plantagineum is a desiccation tolerant resurrection plant. Many genes are induced during desiccation. Dehydrins are a group of dehydration-induced genes present in all higher plants. The current study aims at classifying the most abundantly expressed dehydrin genes from vegetative tissues of C. plantagineum and quantifying their expression. To identify variations between dehydrin isoforms at different stages of desiccation and rehydration by RT-qPCR, the target mRNA requires an accurate and reliable normalization. Previously we reported that RNAs from leaves and roots of C. plantagineum are not degraded during desiccation and subsequent rehydration thus allowing the use of RT-qPCR to test the stability of reference genes. The expression stability of eight candidate reference genes was tested in leaves, roots and callus. These genes were ranked according to their stability of gene expression using GeNorm(PLUS) and RefFinder. The most consistently expressed reference genes in each tissue were identified and used to normalize gene expression data. Dehydrin isoforms were divided in three groups based on the expression level during the desiccation process in three different tissues (leaves, roots and callus).

A molecular physiological review of vegetative desiccation tolerance in the resurrection plant Xerophyta viscosa (Baker).

MAIN CONCLUSION: Provides a first comprehensive review of integrated physiological and molecular aspects of desiccation tolerance Xerophyta viscosa. A synopsis of biotechnological studies being undertaken to improve drought tolerance in maize is given. Xerophyta viscosa (Baker) is a monocotyledonous resurrection plant from the family Vellociacea that occurs in summer-rainfall areas of South Africa, Lesotho and Swaziland. It inhabits rocky terrain in exposed grasslands and frequently experiences periods of water deficit. Being a resurrection plant it tolerates the loss of 95% of total cellular water, regaining full metabolic competency within 3 days of rehydration. In this paper, we review some of the molecular and physiological adaptations that occur during various stages of dehydration of X. viscosa, these being functionally grouped into early and late responses, which might be relevant to the attainment of desiccation tolerance. During early drying (to 55% RWC) photosynthesis is shut down, there is increased presence and activity of housekeeping antioxidants and a redirection of metabolism to the increased formation of sucrose and raffinose family oligosaccharides. Other metabolic shifts suggest water replacement in vacuoles proposed to facilitate mechanical stabilization. Some regulatory processes observed include increased presence of a linker histone H1 variant, a Type 2C protein phosphatase, a calmodulin- and an ERD15-like protein. During the late stages of drying (to 10% RWC) there was increased expression of several proteins involved in signal transduction, and retroelements speculated to be instrumental in gene silencing. There was induction of antioxidants not typically found in desiccation-sensitive systems, classical stress-associated proteins (HSP and LEAs), proteins involved in structural stabilization and those associated with changes in various metabolite pools during drying. Metabolites accumulated in this stage are proposed, inter alia, to facilitate subcellular stabilization by vitrification process which can include glass- and ionic liquid formation.

What can we learn from the transcriptome of the resurrection plant Craterostigma plantagineum?

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 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 post-transcriptional level and thus these RNAs may be key players in the rewiring of regulatory networks of desiccation-related genes in C. plantagineum.