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Plant species that exhibit vegetative desiccation tolerance can survive extreme desiccation for months and resume normal physiological activities upon re-watering. Here we survey the recent knowledge gathered from the sequenced genomes of angiosperm and non-angiosperm desiccation-tolerant plants (resurrection plants) and highlight some distinct genes and gene families that are central to the desiccation response. Furthermore, we review the vast amount of data accumulated from analyses of transcriptomes and metabolomes of resurrection species exposed to desiccation and subsequent rehydration, which allows us to build a systems biology view on the molecular and genetic mechanisms of desiccation tolerance in plants.

Summary The resurrection plant Craterostigma plantagineum possesses an extraordinary capacity to survive long‐term desiccation. To enhance our understanding of this phenomenon, complementary transcriptome, soluble proteome and targeted metabolite profiling was carried out on leaves collected from different stages during a dehydration and rehydration cycle. A total of 7348 contigs, 611 proteins and 39 metabolites were differentially abundant across the different sampling points. Dynamic changes in transcript, protein and metabolite levels revealed a unique signature characterizing each stage. An overall low correlation between transcript and protein abundance suggests a prominent role for post‐transcriptional modification in metabolic reprogramming to prepare plants for desiccation and recovery. The integrative analysis of all three data sets was performed with an emphasis on photosynthesis, photorespiration, energy metabolism and amino acid metabolism. The results revealed a set of precise changes that modulate primary metabolism to confer plasticity to metabolic pathways, thus optimizing plant performance under stress. The maintenance of cyclic electron flow and photorespiration, and the switch from C3 to crassulacean acid metabolism photosynthesis, may contribute to partially sustain photosynthesis and minimize oxidative damage during dehydration. Transcripts with a delayed translation, ATP‐independent bypasses, alternative respiratory pathway and 4‐aminobutyric acid shunt may all play a role in energy management, together conferring bioenergetic advantages to meet energy demands upon rehydration. This study provides a high‐resolution map of the changes occurring in primary metabolism during dehydration and rehydration and enriches our understanding of the molecular mechanisms underpinning plant desiccation tolerance. The data sets provided here will ultimately inspire biotechnological strategies for drought tolerance improvement in crops., Significance Statement This study provides a transcriptomic, proteomic and metabolic signature of Craterostigma plantagineum leaves during a dehydration and rehydration cycle. Integrative analysis of all three data sets reveals a set of precise changes that modulate primary metabolism to confer plasticity to metabolic pathways, thus optimizing plant performance under stress. The data provided here are a step towards a systems biology approach to understand desiccation tolerance and will ultimately inspire biotechnological strategies for drought tolerance improvement in crops. ​

Resurrection plants can survive prolonged anhydrobiosis, and desiccation tolerance has evolved recurrently across land plants as a common adaptation for survival in regions with seasonal drying. Craterostigma plantagineum was among the first model resurrection plants, and many of the genetic mechanisms underlying desiccation tolerance were discovered in this important system. Here, we analyzed the complex, octoploid Craterostigma (C. plantagineum) genome and surveyed spatial and temporal expression dynamics to identify genetic elements underlying desiccation tolerance. Homeologous genes within the Craterostigma genome have divergent expression profiles, suggesting the subgenomes contribute differently to desiccation tolerance traits. The Craterostigma genome contains almost 200 tandemly duplicated early light induced proteins (ELIPs), a hallmark trait of desiccation tolerance, with massive upregulation under water deficit. We identified a core network of desiccation responsive genes across all tissues but observed almost entirely unique expression dynamics in each tissue during recovery. Roots and leaves have differential responses related to light and photoprotection, autophagy, and nutrient transport, reflecting their divergent functions. Our findings highlight a universal set of likely ancestral desiccation tolerance mechanisms to protect cellular macromolecules under anhydrobiosis, with secondary adaptations related to tissue function.

Introduction Craterostigma plantagineum and Lindernia brevidens are resurrection plants, so these plants can tolerate desiccation of their vegetative tissues. Different components and mechanisms contribute to desiccation tolerance and secondary plant metabolites, like phenolic compounds, may play a role during these processes. Objectives Secondary plant metabolites of the two resurrection plants, C. plantagineum and L. brevidens as well as the closely related desiccation sensitive species, L. subracemosa, were investigated regarding the polyphenol profile. Material and methods Secondary plant compounds were extracted with acidified methanol and analysed with ultra-high-performance liquid chromatography electrospray ionisation mass spectrometry (UHPLC-ESI-MS). Phenolic compounds were identified by comparing of ultraviolet (UV) and MSn -spectra with published data. All compounds were quantified as verbascoside equivalents by external calibration at the compound specific wavelength. Results In total, eight compounds that belong to the subclass of phenylethanoid glycosides and one flavone, luteolin hexoside pentoside, were identified. Two of these compounds exhibited a fragmentation pattern, which is closely related to phenylethanoid glycosides. The predominantly synthesised phenylethanoid in all of the three plant species and in every stage of hydration was verbascoside. The total content of phenolic compounds during the three stages of hydration, untreated, desiccated, and rehydrated revealed differences especially between C. plantagineum and L. brevidens as the latter one lost almost all phenolic compounds during rehydration. Conclusion The amount of verbascoside correlates with the degree of desiccation tolerance and verbascoside might play a role in the protective system in acting as an antioxidant.

Root and tuber crops are of great importance. They not only contribute to feeding the population but also provide raw material for medicine and small-scale industries. The yield of the root and tuber crops is subject to the development of stem/root tubers, which involves the initiation, expansion, and maturation of storage organs. The formation of the storage organ is a highly intricate process, regulated by multiple phytohormones. Gibberellins (GAs) and abscisic acid (ABA), as antagonists, are essential regulators during stem/root tuber development. This review summarizes the current knowledge of the roles of GA and ABA during stem/root tuber development in various tuber crops.

Reactive oxygen species (ROS) signalling is crucial in modulating stress responses in plants, and NADPH oxidases (NOXs) are an important component of signal transduction under salt stress. The goal of this research was to investigate whether the regulation of NOX-dependent signalling during mild and severe salinity differs between the halophyte Eutrema salsugineum and the glycophyte Arabidopsis thaliana. Gene expression analyses showed that salt-induced expression patterns of two NOX genes, RBOHD and RBOHF, varied between the halophyte and the glycophyte. Five days of salinity stimulated the expression of both genes in E. salsugineum leaves, while their expression in A. thaliana decreased. This was not accompanied by changes in the total NOX activity in E. salsugineum, while the activity in A. thaliana was reduced. The expression of the RBOHD and RBOHF genes in E. salsugineum leaves was induced by abscisic acid (ABA) and ethephon spraying. The in silico analyses of promoter sequences of RBOHD and RBOHF revealed multiple cis-acting elements related to hormone responses, and their distribution varied between E. salsugineum and A. thaliana. Our results indicate that, in the halophyte E. salsugineum, the maintenance of the basal activity of NOXs in leaves plays a role during acclimation responses to salt stress. The different expression patterns of the RBOHD and RBOHF genes under salinity in E. salsugineum and A. thaliana point to a modified regulation of these genes in the halophyte, possibly through ABA- and/or ethylene-dependent pathways.

Craterostigma plantagineum belongs to the desiccation‐tolerant angiosperm plants. Upon dehydration, leaves fold and the cells shrink which is reversed during rehydration. To understand this process changes in cell wall pectin composition, and the role of the apoplastic glycine‐rich protein 1 (CpGRP1) were analysed. Cellular microstructural changes in hydrated, desiccated and rehydrated leaf sections were analysed using scanning electron microscopy. Pectin composition in different cell wall fractions was analysed with monoclonal antibodies against homogalacturonan, rhamnogalacturonan I, rhamnogalacturonan II and hemicellulose epitopes. Our data demonstrate changes in pectin composition during dehydration/rehydration which is suggested to affect cell wall properties. Homogalacturonan was less methylesterified upon desiccation and changes were also demonstrated in the detection of rhamnogalacturonan I, rhamnogalacturonan II and hemicelluloses. CpGRP1 seems to have a central role in cell adaptations to water deficit, as it interacts with pectin through a cluster of arginine residues and de‐methylesterified pectin presents more binding sites for the protein−pectin interaction than to pectin from hydrated leaves. CpGRP1 can also bind phosphatidic acid (PA) and cardiolipin. The binding of CpGRP1 to pectin appears to be dependent on the pectin methylesterification status and it has a higher affinity to pectin than its binding partner CpWAK1. It is hypothesised that changes in pectin composition are sensed by the CpGRP1−CpWAK1 complex therefore leading to the activation of dehydration‐related responses and leaf folding. PA might participate in the modulation of CpGRP1 activity.

The study of lipid-protein interactions is crucial for understanding reactions of proteins involved in lipid metabolism, lipid transport, and lipid signaling. Different detection methods can be employed for the identification of lipid-binding interactions. Isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR) spectroscopy enable real-time monitoring of lipid protein interactions and provide thermodynamic parameters of the interacting partners. However, these technologies depend on the availability of the large equipment, limiting the practicability in many laboratories. Protein-lipid overlay assays are a simple first approach to screen for protein interactions with different lipids or lipid intermediates and are independent of large equipment. Subsequently, specific interactions can be analyzed in detail using protein-liposome association assays.

For more than a decade molecular biology has offered the tools to isolate plant genes and to analyze their structures. In general, knowledge of the gene product is a prerequisite for gene isolation. In the first part of this chapter the methodologies available for plant gene isolation are discussed and the examples described. The strategy for gene isolation depends on the abundancy of the gene product as well as on the knowledge of the expression pattern or primary structure of the gene and encoded protein. The second part will deal with molecular techniques which can be applied to isolate genes of which the gene products are unknown. For many genes isolated by the methods described so far it is often difficult to prove that the clone contains the desired gene. A major interest of plant biology is on development, cell differentiation, and cell response to environmental factors at the molecular level.

The study of lipid-protein interactions is crucial for understanding reactions of proteins involved in lipid metabolism, lipid transport, and lipid signaling. Different detection methods can be employed for the identification of lipid-binding interactions. Isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR) spectroscopy enable real-time monitoring of lipid protein interactions and provide thermodynamic parameters of the interacting partners. However, these technologies depend on the availability of the large equipment, limiting the practicability in many laboratories. Protein-lipid overlay assays are a simple first approach to screen for protein interactions with different lipids or lipid intermediates and are independent of large equipment. Subsequently, specific interactions can be analyzed in detail using protein-liposome association assays.

Aldehyde dehydrogenases (ALDHs) constitute an evolutionary conserved superfamily of oxidoreductases, which convert a large array of aldehydes to carboxylic acids. Plant ALDHs caught attention in the last two decades after the discovery of their central role in the adaptive responses to abiotic stress. Recent advances in next-generation sequencing and genome assembly enabled us to identify many ALDH genes of plants from diverse lineages. This provided valuable clues to trace their evolutionary trajectory. The ALDH superfamily has ancient origins that go back to the chromista kingdom. Major evolutionary events like the conquest of land by plants, and later their vascularization, along with the acquisition of developmental complexity coincide with important changes in the abundance, expansion, and diversification of ALDH genes and proteins. Plant ALDH sequences divergence led to the emergence of functions, absent in their algal ancestors. The most evolved, higher plant ALDHs have functional promiscuity which positioned them as important ‘hubs’ at the crossroads of the primary/basal and the stress-related metabolism. Stress-responsive ALDHs mitigate the harmful effect of cytotoxic aldehydes resulting from lipid peroxidation occurring during oxidative stress and contribute to the synthesis of osmolytes, like glycine betaine. Other isoforms play significant roles in glycolysis, TCA cycle, and amino acid catabolism. Therefore, plant ALDHs are attractive targets for molecular breeding of stress tolerant plants. The overall three-dimensional structures and the catalytic mechanism of ALDHs are conserved in prokaryotes, mammalians, and plants. However, some amino acids at specific locations, underwent progressive changes in the course of the evolution of plantae, yielding shifts in the enzymatic properties, including substrate and cofactor specificities. We explore data related to the evolutionary history of ALDHs, gather information about their biochemical functions, and discuss their physiological relevance.

Main conclusion The cell wall protein CpWAK1 interacts with pectin, participates in decoding cell wall signals, and induces different downstream responses. Abstract Cell wall-associated protein kinases (WAKs) are transmembrane receptor kinases. In the desiccation-tolerant resurrection plant Craterostigma plantagineum, CpWAK1 has been shown to be involved in stress responses and cell expansion by forming a complex with the C. plantagineum glycine-rich protein1 (CpGRP1). This prompted us to extend the studies of WAK genes in C. plantagineum. The phylogenetic analyses of WAKs from C. plantagineum and from other species suggest that these genes have been duplicated after species divergence. Expression profiles indicate that CpWAKs are involved in various biological processes, including dehydration-induced responses and SA- and JA-related reactions to pathogens and wounding. CpWAK1 shows a high affinity for “egg-box” pectin structures. ELISA assays revealed that the binding of CpWAKs to pectins is modulated by CpGRP1 and it depends on the apoplastic pH. The formation of CpWAK multimers is the prerequisite for the CpWAK–pectin binding. Different pectin extracts lead to opposite trends of CpWAK–pectin binding in the presence of Ca2+ at pH 8. These observations demonstrate that CpWAKs can potentially discriminate and integrate cell wall signals generated by diverse stimuli, in concert with other elements, such as CpGRP1, pHapo, Ca2+[apo], and via the formation of CpWAK multimers.

Carotenoid levels in plant tissues depend on the relative rates of synthesis and degradation of the molecules in the pathway. While plant carotenoid biosynthesis has been extensively characterized, research on carotenoid degradation and catabolism into apocarotenoids is a relatively novel field. To identify apocarotenoid metabolic processes, we characterized the transcriptome of transgenic Arabidopsis (Arabidopsis thaliana) roots accumulating high levels of β-carotene and, consequently, β-apocarotenoids. Transcriptome analysis revealed feedback regulation on carotenogenic gene transcripts suitable for reducing β-carotene levels, suggesting involvement of specific apocarotenoid signaling molecules originating directly from β-carotene degradation or after secondary enzymatic derivatizations. Enzymes implicated in apocarotenoid modification reactions overlapped with detoxification enzymes of xenobiotics and reactive carbonyl species (RCS), while metabolite analysis excluded lipid stress response, a potential secondary effect of carotenoid accumulation. In agreement with structural similarities between RCS and β-apocarotenoids, RCS detoxification enzymes also converted apocarotenoids derived from β-carotene and from xanthophylls into apocarotenols and apocarotenoic acids in vitro. Moreover, glycosylation and glutathionylation-related processes and translocators were induced. In view of similarities to mechanisms found in crocin biosynthesis and cellular deposition in saffron (Crocus sativus), our data suggest apocarotenoid metabolization, derivatization and compartmentalization as key processes in (apo)carotenoid metabolism in plants.

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. Abstract 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.

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 (re-)evolution of vegetative desiccation tolerance in Linderniaceae appears to be linked to the presence of dehydration-responsive cis-elements in the promoters of desiccation-related genes., 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 β-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.

Oropetium thomaeum is a desiccation tolerant grass and acquisition of desiccation tolerance is correlated with changes in carbohydrate metabolism. Here we address the question whether the changes in carbohydrate metabolism are specific to the dehydration process or whether other environmental factors such as high temperature, low temperature, hypoxia, salinity or exogenous ABA application trigger the same or different changes in the sugar metabolism. Fifteen different sugar metabolites were identified by GC/MS, including erythritol, arabinose, fructose, galactose, glucose, myo-inositol, sedoheptulose, sucrose, trehalose, galactinol, maltose, raffinose, manninotriose and stachyose. Together with starch, these sugars were placed into the pathways of sucrose metabolism and raffinose family oligosaccharides (RFOs) metabolism, as well as into the group of rare sugars. By comparing the changes of sugars under various stresses, we concluded that the changes in the sugar metabolism are both convergent and divergent in response to different stresses. Except for the general response to stress, such as starch degradation, the changes of specific sugar metabolites reflect a stress-specific response of O. thomaeum. Erythritol seems to be specific for dehydration, myo-inositol for salt stress and trehalose for hypoxia stress. Similar as dehydration, low temperature, salt stress and ABA application resulted in the accumulation of sucrose and RFOs in O. thomaeum, which indicates that these stresses share high similarity with dehydration. Thus it is proposed that sucrose and RFOs have a general protective role under these stresses. In contrast sucrose and RFOs did not accumulate in response to high temperature or hypoxia whose effects tend to be consumptive and destructive. The accumulation of galactose, melibiose and manninotriose demonstrate that RFOs are degraded under stress. The accumulation of these sugar metabolites might result from the reaction of RFOs and stress-produced hydroxyl radicals, which supports a possible role of RFOs in stress defense. In addition, ABA application led to substantial synthesis of stachyose which occurs only in response to dehydration, indicating that stachyose synthesis is possibly closely related to ABA in O. 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.

Phospholipase D (PLD) and its cleavage product phosphatidic acid (PA) are crucial in plant stress-signalling. Although some targets of PLD and PA have been identified, the signalling pathway is still enigmatic. This study demonstrates that the phosphoprotein At5g39570, now called PLD-regulated protein1 (PLDrp1), from Arabidopsis thaliana is directly regulated by PLDα1. The protein PLDrp1 can be divided into two regions with distinct properties. The conserved N-terminal region specifically binds PA, while the repeat-rich C-terminal domain suggests interactions with RNAs. The expression of PLDrp1 depends on PLDα1 and the plant water status. Water stress triggers a pldα1-like phenotype in PLDrp1 mutants and induces the expression of PLDrp1 in pldα1 mutants. The regulation of PLDrp1 by PLDα1 and environmental stressors contributes to the understanding of the complex PLD regulatory network and presents a new member of the PA-signalling chain in plants.

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.

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.

Plant cell walls define the shape of the cells and provide mechanical support. They function as osmoregulators by controlling the transport of molecules between cells and provide transport pathways within the plant. These diverse functions require a well-defined and flexible organization of cell wall components, i.e., water, polysaccharides, proteins, and other diverse substances. Cell walls of desiccation tolerant resurrection plants withstand extreme mechanical stress during complete dehydration and rehydration. Adaptation to the changing water status of the plant plays a crucial role during this process. This review summarizes the compositional and structural variations, signal transduction and changes of gene expression which occur in cell walls of resurrection plants during dehydration and rehydration.

Highlight Transketolase 7 and 10 of Craterostigma plantagineum participate in the synthesis of octulose phosphate in an alternative Calvin cycle. Octulose is the main transport sugar in fully hydrated plants., Phylogenetic analysis revealed that Craterostigma plantagineum has two transketolase genes (transketolase 7 and 10) which are separated from the other transketolase genes including transketolase 3 from C. plantagineum. We obtained recombinant transketolase 3, 7, and 10 of C. plantagineum and showed that transketolase 7 and 10 of C. plantagineum, but not transketolase 3, catalyse the formation of octulose-8-phosphate in vitro. Transketolase 7 and 10 of C. plantagineum performed the exchange reaction that produces octulose-8-phosphate using glucose-6-phosphate and fructose-6-phosphate as substrates. Octulose is localized in the cytosol and phloem exudate analysis showed that octulose was the dominant sugar exported from the leaves to the roots.

Lipids are one of the major components of biological membranes including the plasma membrane, which is the interface between the cell and the environment. It has become clear that membrane lipids also serve as substrates for the generation of numerous signalling lipids such as phosphatidic acid, phosphoinositides, sphingolipids, lysophospholipids, oxylipins, N-acylethanolamines, free fatty acids and others. The enzymatic production and metabolism of these signalling molecules are tightly regulated and can rapidly be activated upon abiotic stress signals. Abiotic stress like water deficit and temperature stress triggers lipid-dependent signalling cascades, which control the expression of gene clusters and activate plant adaptation processes. Signalling lipids are able to recruit protein targets transiently to the membrane and thus affect conformation and activity of intracellular proteins and metabolites. In plants, knowledge is still scarce of lipid signalling targets and their physiological consequences. This review focuses on the generation of signalling lipids and their involvement in response to abiotic stress. We describe lipid-binding proteins in the context of changing environmental conditions and compare different approaches to determine lipid-protein interactions, crucial for deciphering the signalling cascades.

Nitric oxide (NO) is an intracellular messenger that mediates stress responses. Several plant aldehyde dehydrogenase (ALDH) genes are expressed during abiotic stress conditions to reduce the level of cytotoxic aldehydes. We investigated a possible interference between NO and ALDHs, using the isoform ALDH3H1 of Arabidopsis thaliana as model. The physiological NO donor; S-nitrosoglutathione (GSNO), inhibits ALDH3H1 in a time- and concentration-dependent manner. Mutagenesis and ESI-MS/MS analyses show that all Cys residues of ALDH3H1 are targets of GSNO-mediated S-nitrosation. Chemical labelling indicates that the deactivation is due to the conversion of the catalytic thiol into a catalytically non-active nitrosothiol. GSNO has the same effect on the chloroplastic ALDH3I1, suggesting that susceptibility of the catalytic Cys to NO is a common feature of ALDHs. S-Nitrosation and enzymatic inhibition of ALDH were reverted by reducing agents. Our study proves that the function of ALDHs does not exclusively depend on transcriptional regulation, with stress-induced expression, but may be also susceptible to posttranslational regulation through S-nitrosation. We discuss the potential involvement of S-nitrosoglutathione reductase (GSNOR), binding specific cofactors and reducing partners in a protective system of ALDHs in vivo, which will be experimentally corroborated in our forthcoming study.

Desiccation tolerance was a critical adaptation for the colonization of land by early nonvascular plants. Resurrection plants have maintained or rewired these ancestral protective mechanisms, and desiccation-tolerant species are dispersed across the land plant phylogeny. Although common physiological, biochemical, and molecular signatures are observed across resurrection plant lineages, features underlying the recurrent evolution of desiccation tolerance are unknown. Here we used a comparative approach to identify patterns of genome evolution and gene duplication associated with desiccation tolerance. We identified a single gene family with dramatic expansion in all sequenced resurrection plant genomes and no expansion in desiccation-sensitive species. This gene family of early light-induced proteins (ELIPs) expanded in resurrection plants convergent through repeated tandem gene duplication. ELIPs are universally highly expressed during desiccation in all surveyed resurrection plants and may play a role in protecting against photooxidative damage of the photosynthetic apparatus during prolonged dehydration. Photosynthesis is particularly sensitive to dehydration, and the increased abundance of ELIPs may help facilitate the rapid recovery observed for most resurrection plants. Together, these observations support convergent evolution of desiccation tolerance in land plants through tandem gene duplication.

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.

Although several resurrection plant genomes have been sequenced, the lack of suitable dehydration-sensitive outgroups has limited genomic insights into the origin of desiccation tolerance. Here, we utilized a comparative system of closely related desiccation-tolerant (Lindernia brevidens) and -sensitive (Lindernia subracemosa) species to identify gene- and pathway-level changes associated with the evolution of desiccation tolerance. The two high-quality Lindernia genomes we assembled are largely collinear, and over 90% of genes are conserved. L. brevidens and L. subracemosa have evidence of an ancient, shared whole-genome duplication event, and retained genes have neofunctionalized, with desiccation-specific expression in L. brevidens. Tandem gene duplicates also are enriched in desiccation-associated functions, including a dramatic expansion of early light-induced proteins from 4 to 26 copies in L. brevidens. A comparative differential gene coexpression analysis between L. brevidens and L. subracemosa supports extensive network rewiring across early dehydration, desiccation, and rehydration time courses. Many LATE EMBRYOGENESIS ABUNDANT genes show significantly higher expression in L. brevidens compared with their orthologs in L. subracemosa. Coexpression modules uniquely upregulated during desiccation in L. brevidens are enriched with seed-specific and abscisic acid-associated cis-regulatory elements. These modules contain a wide array of seed-associated genes that have no expression in the desiccation-sensitive L. subracemosa. Together, these findings suggest that desiccation tolerance evolved through a combination of gene duplications and network-level rewiring of existing seed desiccation pathways.

Resurrection plants, which are the "gifts" of natural evolution, are ideal models for studying the genetic basis of plant desiccation tolerance. Here, we report a high-quality genome assembly of 301 Mb for the diploid spike moss Selaginella tamariscina , a primitive vascular resurrection plant. We predicated 27 761 protein-coding genes from the assembled S . tamariscina genome, 11.38% (2363) of which showed significant expression changes in response to desiccation. Approximately 60.58% of the S . tamariscina genome was annotated as repetitive DNA, which is an almost 2-fold increase of that in the genome of desiccation-sensitive Selaginella moellendorffii . Genomic and transcriptomic analyses highlight the unique evolution and complex regulations of the desiccation response in S . tamariscina , including species-specific expansion of the oleosin and pentatricopeptide repeat gene families, unique genes and pathways for reactive oxygen species generation and scavenging, and enhanced abscisic acid (ABA) biosynthesis and potentially distinct regulation of ABA signaling and response. Comparative analysis of chloroplast genomes of several Selaginella species revealed a unique structural rearrangement and the complete loss of chloroplast NAD(P)H dehydrogenase (NDH) genes in S . tamariscina , suggesting a link between the absence of the NDH complex and desiccation tolerance. Taken together, our comparative genomic and transcriptomic analyses reveal common and species-specific desiccation tolerance strategies in S . tamariscina , providing significant insights into the desiccation tolerance mechanism and the evolution of resurrection plants.

Aldehyde dehydrogenase enzymes (ALDHs) catalyze the oxidation of aliphatic and aromatic aldehydes to their corresponding carboxylic acids using NAD+ or NADP+ as cofactors and generating NADH or NADPH. Previous studies mainly focused on the ALDH role in detoxifying toxic aldehydes but their effect on the cellular NAD(P)H contents has so far been overlooked. Here, we investigated whether the ALDHs influence the cellular redox homeostasis. We used a double T-DNA insertion mutant that is defective in representative members of Arabidopsis thaliana ALDH families 3 (ALDH3I1) and 7 (ALDH7B4), and we examined the pyridine nucleotide pools, glutathione content, and the photosynthetic capacity of the aldh mutants in comparison with the wild type. The loss of function of ALDH3I1 and ALDH7B4 led to a decrease of NAD(P)H, NAD(P)H/NAD(P) ratio, and an alteration of the glutathione pools. The aldh double mutant had higher glucose-6-phosphate dehydrogenase activity than the wild type, indicating a high demand for reduced pyridine nucleotides. Moreover, the mutant had a reduced quantum yield of photosystem II and photosynthetic capacity at relatively high light intensities compared to the wild type. Altogether, our data revealed a role of ALDHs as major contributors to the homeostasis of pyridine nucleotides in plants.

The relation between water status and expression profiles of desiccation -related genes has been studied in the desiccation tolerant (DT) aeroterrestrial green microalga Trebouxia gelatinosa, a common lichen photobiont. Algal colonies were desiccated in controlled conditions and during desiccation water content (WC) and water potential (Ψ) were measured to find the turgor loss point (Ψtlp). Quantitative real-time PCR was performed to measure the expression of ten genes related to photosynthesis, antioxidant defense, expansins, heat shock proteins (HSPs), and desiccation related proteins in algal colonies collected during desiccation when still at full turgor (WC > 6 g H2O g−1 dry weight), immediately before and after Ψtlp (−4 MPa; WC∼1 g H2O g−1 dry weight) and before and after complete desiccation (WC < 0.01 g H2O g−1 dry weight), quantifying the HSP70 protein levels by immunodetection. Our analysis showed that the expression of eight out of ten genes changed immediately before and after Ψtlp. Interestingly, the expression of five out of ten genes changed also before complete desiccation, i.e. between 0.2 and 0.01 g H2O g−1 dry weight. However, the HSP70 protein levels were not affected by changes in water status. The study provides new evidences of the link between the loss of turgor and the expression of genes related to the desiccation tolerance of T. gelatinosa, suggesting the former as a signal triggering inducible mechanisms.

Orphan crops are crops with little significance at the global scale but they play a vital role in the food and nutrition security as well as the livelihood of resource-poor farmers and consumers in the developing world. The term ‘orphan’ refers to the neglect of the crop by the international research community. Orphan crops are also known as indigenous-, lost-, minor-, promising-, and underutilized-crops, among other names (Tadele 2019). Although little scientific research has been done on most orphan crops, a limited number of them have enjoyed advanced studies. This has mainly been due to committed scientists and institutions in developing countries as well as financial and technical support from developed nations. Most orphan crops are resilient to extreme environmental conditions. Due to this adaptability to marginal and low input environments, orphan crops offer opportunities for low greenhouse gas emissions (Mabhaudhi et al. 2019). In addition, these indigenous crops provide nutrient-rich biodiversity and healthier diets to resource-poor consumers (Hunter et al. 2019). Due to multiple dietary benefits and their tolerance to extreme environmental conditions, some orphan crops are considered to be crops for the future.

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.

Photosynthesis is the key process that is affected by dehydration in plants. Desiccation-tolerant resurrection plants can survive conditions of very low relative water content. During desiccation, photosynthesis is not operational, but is recovered within a short period after rehydration. While homoiochlorophyllous resurrection plants retain their photosynthetic apparatus during desiccation, poikilochlorophyllous resurrection species dismantle chloroplasts and degrade chlorophyll but resynthesize them again during rehydration. Dismantling the chloroplasts avoids the photooxidative stress in poikilochlorophyllous resurrection plants, whereas it is minimized in homoiochlorophyllous plants through the synthesis of antioxidant enzymes and protective proteins or metabolites. Although the cellular protection mechanisms in both of these species vary, these mechanisms protect cells from desiccation-induced damage and restore photosynthesis upon rehydration. Several of the proteins synthesized during dehydration are localized in chloroplasts and are believed to play major roles in the protection of photosynthetic structures and in recovery in resurrection species. This review focuses on the strategies of resurrection plants in terms of how they protect their photosynthetic apparatus from oxidative stress during desiccation without membrane damage and with full recovery during rehydration. We review the role of the dehydration-induced protection mechanisms in chloroplasts and how photosynthesis is restored during rehydration.

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).

Inhibition of photosynthesis is a central, primary response that is observed in both desiccation-tolerant and desiccation-sensitive plants affected by drought stress. Decreased photosynthesis during drought stress can either be due to the limitation of carbon dioxide entry through the stomata and the mesophyll cells, due to increased oxidative stress or due to decreased activity of photosynthetic enzymes. Although the photosynthetic rates decrease in both desiccation-tolerant and sensitive plants during drought, the remarkable difference lies in the complete recovery of photosynthesis after rehydration in desiccation-tolerant plants. Desiccation of sensitive plants leads to irreparable damages of the photosynthetic membranes, in contrast the photosynthetic apparatus is deactivated during desiccation in desiccation-tolerant plants. Desiccation-tolerant plants employ different strategies to protect and/or maintain the structural integrity of the photosynthetic apparatus to reactivate photosynthesis upon water availability. Two major mechanisms are distinguished. Homoiochlorophyllous desiccation-tolerant plants preserve chlorophyll and thylakoid membranes and require active protection mechanisms, while poikilochlorophyllous plants degrade chlorophyll in a regulated manner but then require de novo synthesis during rehydration. Desiccation-tolerant plants, particularly homoiochlorophyllous plants, employ conserved and novel antioxidant enzymes/metabolites to minimize the oxidative damage and to protect the photosynthetic machinery. De novo synthesized, stress-induced proteins in combination with antioxidants are localized in chloroplasts and are important components of the protective network. Genome sequence informations provide some clues on selection of genes involved in protecting photosynthetic structures; e.g. ELIP genes (early light inducible proteins) are enriched in the genomes and more abundantly expressed in homoiochlorophyllous desiccation-tolerant plants. This review focuses on the mechanisms that operate in the desiccation-tolerant plants to protect the photosynthetic apparatus during desiccation.

The expression of selected Arabidopsis ALDH genes is induced in response to heat and stress combination, and ALDH mutant lines are more sensitive to heat stress and stress combinations., Aldehyde dehydrogenases (ALDH) are a family of enzymes that are involved in plant metabolism and contribute to aldehyde homeostasis to eliminate toxic aldehydes. The ALDH enzymes produce NADPH and NADH in their enzymatic reactions and thus contribute to balancing redox equivalents. Previous studies showed that Arabidopsis ALDH genes are expressed in response to high salinity, dehydration, oxidative stress, or heavy metals, suggesting important roles in environmental adaptation. However, the role of ALDH genes in high temperature and stress combinations (heat stress combined with dehydration, wounding, or salt stress) is unclear. Here, we analysed expression patterns of selected ALDH genes on the transcript and protein level at different time points of heat stress, basal and acquired thermotolerance, and stress combination treatments. Our results indicate that ALDH3I1 and ALDH7B4 are strongly induced by heat stress. Higher levels of ALDH7B4 accumulated in response to dehydration–heat, heat–salt and wounding–heat combination stress than in response to single stressors. The comparison of physiological and biological parameters in T-DNA double mutants of ALDH genes and wild-type plants demonstrated that mutant lines are more sensitive to heat stress and stress combinations than wild-type plants.

Genotype variation in selected Kenyan barley breeding lines was determined by differential profiles of seed hordein proteins using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Consequently, a phylogenetic relationship derived from unweighted pair group method with arithmetic mean Jaccard clustering matrix was generated based on absence or presence of hordein bands. In parallel, physiological and molecular responses to abiotic stresses were evaluated in seedlings. For physiological assays seedlings were exposed to 0, 300, 600 mM NaCl. Variation in physiological parameters including ionic conductance, lipid peroxidation, chlorophyll content, accumulation of proline, glycine betaine, sucrose was determined. Response to abiotic stress was found to be genotype dependent and varied with the stress magnitude. A low ionic conductance, lipid peroxidation and increased proline, sucrose, GB, chlorophyll were associated with stress tolerance. The lines Nguzo, MN-24 and MN-8 were tolerant while Karne, and Sabini were susceptible to abiotic stress. Transcript analysis of selected members of the dehydrin (Dhn) superfamily (LEA II) genes in root and shoot tissue were evaluated. Expression of Dhn genes was found to be genotype dependent, tissue specific and was affected by type and duration of stress. Dehydrin Dhn1 and Dhn9 genes were exclusively dehydration responsive while Dhn3, Dhn4 and Dhn7 were induced by both dehydration and increased salt treatments. Immunoblot analysis using polyclonal anti-sera detecting the K segment consensus peptide TGEKKGIMDKIKEKLPGQH showed a direct correlation between transcript level and accumulation of corresponding Dhn proteins in response to stress. These screening assays may be potential selection markers to aid rapid screening in breeding programs.

Due to the ability to tolerate extreme dehydration, desiccation-tolerant plants have been widely investigated to find potential approaches for improving water use efficiency or developing new crop varieties. The studies of desiccation-tolerant plants have identified sugar accumulation, specific protein synthesis, cell structure changes, and increased anti-oxidative reactions as part of the mechanisms of desiccation tolerance. However, plants respond differently according to the severity of water loss, and the process of water loss affects desiccation tolerance. A detailed analysis within the dehydration process is important for understanding the process of desiccation tolerance. This review defines dehydration and desiccation, finds the boundary for the relative water content between dehydration and desiccation, compares the molecular responses to dehydration and desiccation, compares signaling differences between dehydration and desiccation, and finally summarizes the strategies launched in desiccation-tolerant plants for dehydration and desiccation, respectively. The roles of abscisic acid (ABA) and reactive oxygen species (ROS) in sensing and signaling during dehydration are discussed. We outline how this knowledge can be exploited to generate drought-tolerant crop plants.

Incluye 3 ficheros de datos Imazamox and glyphosate represent two classes of herbicides that inhibit the activity of acetohydroxyacid synthase in the branched-chain amino acid biosynthesis pathway and the activity of 5-enolpyruvylshikimate-3-phosphate synthase in the aromatic amino acid biosynthesis pathway, respectively. However, it is still unclear how imazamox and glyphosate lead to plant death. Both herbicides inhibit amino-acid biosynthesis and were found to induce ethanol fermentation in plants, but an Arabidopsis mutant deficient in alcohol dehydrogenase 1 was neither more susceptible nor more resistant than the wild-type to the herbicides. In this study, we investigated the effects of the amino acid biosynthesis inhibitors, imazamox and glyphosate, on the pyruvate dehydrogenase bypass reaction and fatty acid metabolism in A. thaliana. We found that the pyruvate dehydrogenase bypass was upregulated following the treatment by the two herbicides. Our results suggest that the Arabidopsis aldehyde dehydrogenase 7B4 gene might be participating in the pyruvate dehydrogenase bypass reaction. We evaluated the potential role of the aldehyde dehydrogenase 7B4 upon herbicide treatment in the plant defence mechanism. Plants that overexpressed the ALDH7B4 gene accumulated less soluble sugars, starch, and fatty acids and grew better than the wild-type after herbicide treatment. We discuss how the upregulation of the ALDH7B4 alleviates the effects of the herbicides, potentially through the detoxification of the metabolites produced in the pyruvate dehydrogenase bypass. Miriam Gil-Monreal received funding from fellowships through Universidad Pública de Navarra. This work was financially supported by two grants from the Ministerio Español de Economía y Competitividad (AGL-2013-40567R and AGL-2016-77531R).