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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.

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.

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.

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-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.

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.

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.

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.

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.

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).

The embryos of some angiosperm taxa contain chlorophyll and this chlorophyllous stage is persisting until the embryo matures (further referred as chloroembryos). Besides being chlorophyllous, these embryos seem to have the ability to photosynthesize. This suggests that the chlorophyllous state of the embryo has an important role in seed development. The photosynthesis of chloroembryos is highly shade adaptive in nature as it is embedded within the supporting tissues (several layers of pod wall, seed coat and endosperm). Moreover, these chloroembryos are developing in a highly osmotic environment, and contain various components of the photosynthetic machinery. Detailed studies were performed in these chloroembryos in order to elucidate the structure of the chloroplasts, pigment composition, the photochemical activities, the rate of carbon assimilation and also the shade adaptive features. It has been shown that the respired CO2 within these chloroembryos is recycled by the efficient photosynthetic components of the chloroembryos and thus potentially influences the seed's carbon economy. Thus, the major role of embryonic photosynthesis is to produce both energy-rich molecules and oxygen, of which the former can be directly used for biosynthesis. During embryogenesis oxygen production is especially important, in a situation wherein the oxygen is limited within the enclosed seed. As these chloroembryos grow in an environment of a sugar rich endosperm, it requires some adaptive mechanisms in this high osmotic environment. The additional polypeptides found in the thylakoids of chloroembryo chloroplasts in comparison to the thylakoids of leaf chloroplast have been suggested to have a role in protecting the photosynthetic components in the chloroembryos in an environment of high osmotic strength. An attempt to understand osmotic stress tolerance existing in these chloroembryos may lead to a better understanding of tolerance of photosynthesis to osmotic stress.

Summary Dehydration leads to different physiological and biochemical responses in plants. We analysed the lipid composition and the expression of genes involved in lipid biosynthesis in the desiccation-tolerant plant Craterostigma plantagineum. A comparative approach was carried out with Lindernia brevidens (desiccation tolerant) and two desiccation-sensitive species, Lindernia subracemosa and Arabidopsis thaliana. In C. plantagineum the total lipid content remained constant while the lipid composition underwent major changes during desiccation. The most prominent change was the removal of monogalactosyldiacylglycerol (MGDG) from the thylakoids. Analysis of molecular species composition revealed that around 50% of 36:x (number of carbons in the acyl chains: number of double bonds) MGDG was hydrolysed and diacylglycerol (DAG) used for phospholipid synthesis, while another MGDG fraction was converted into digalactosyldiacylglycerol via the DGD1/DGD2 pathway and subsequently into oligogalactolipids by SFR2. 36:x-DAG was also employed for the synthesis of triacylglycerol. Phosphatidic acid (PA) increased in C. plantagineum, L. brevidens, and L. subracemosa, in agreement with a role of PA as an intermediate of lipid turnover and of phospholipase D in signalling during desiccation. 34:x-DAG, presumably derived from de novo assembly, was converted into phosphatidylinositol (PI) in C. plantagineum and L. brevidens, but not in desiccation-sensitive plants, suggesting that PI is involved in acquisition of desiccation tolerance. The accumulation of oligogalactolipids and PI in the chloroplast and extraplastidial membranes, respectively, increases the concentration of hydroxyl groups and enhances the ratio of bilayer- to non-bilayer-forming lipids, thus contributing to protein and membrane stabilization.

Classically, lichen phycobionts are described as poikilohydric organisms able to undergo desiccation due to the constitutive presence of molecular protection mechanisms. However, little is known about the induction of cellular responses in lichen phycobionts during drying. The analysis of the lipid composition of the desiccated lichen microalga Asterochloris erici revealed the unusual accumulation of highly polar lipids (oligogalactolipids and phosphatidylinositol), which prevents the fusion of membranes during stress, but also the active degradation of cone-shaped lipids (monogalactosyldiacylglycerol and phosphatidylethanolamine) to stabilize membranes in desiccated cells. The level of phosphatidic acid increased 7-fold during desiccation, implicating a possible role for phospholipase D (PLD) in the response to osmotic stress. Inhibition of PLD with 1-butanol markedly impaired the recovery of photosynthesis activity in A. erici upon desiccation and salt stress (2 M NaCl). These two hyperosmotic stresses caused the phosphorylation of c-Jun N-terminal kinase (JNK) and p38-like mitogen-activated protein kinase (MAPK) and the dephosphorylation of extracellular signal-regulated kinase (ERK). The incubation with 1-butanol reduced the phosphorylation of JNK-like proteins and increased the dephosphorylation of ERK-like proteins, which indicates an upstream control of MAPK cascades by PLD. The phosphoproteome showed that desiccation caused the phosphorylation of several proteins in A. erici, most of them involved in protein turnover. The results demonstrate that lichen phycobionts possess both constitutive and inducible protective mechanisms to acquire desiccation tolerance. Among others, these responses are controlled by the PLD pathway through the activation of MAPK cascades.

The late embryogenesis abundant (LEA)-like protein CDeT11-24 is one of the major desiccation-related phosphoproteins of the resurrection plant Craterostigma plantagineum. In this study, it was shown that CDeT11-24 is mostly intrinsically disordered and protects two different enzymes, citrate synthase and lactate dehydrogenase, against damaging effects caused by desiccation. Lipid-binding assays revealed that CDeT11-24 is able to interact with phosphatidic acid, although electrostatic repulsion was expected due to the overall negative net charge of the protein under the tested physiological conditions. CDeT11-24 carries an N-terminal lysine-rich sequence, which is predicted to form an amphipathic α-helix. Analysis of the truncated CDeT11-24 protein identified this region to be responsible for both activities: enzyme protection and phosphatidic acid interaction. Possible functions of the CDeT11-24 protein are discussed in the context of desiccation tolerance. Abbreviations: ABA abscisic acid; CD circular dichroism; CS citrate synthase; IDP intrinsically disordered protein; LDH lactate dehydrogenase; LEA late embryogenesis abundant; PA phosphatidic acid; PC phosphatidylcholine; TFE trifluoroethanol.

In the present study, three closely related Linderniaceae species which differ in their sensitivity to desiccation are compared in response to light and oxidative stress defence. Lindernia brevidens, a desiccation-tolerant plant, displayed intense purple pigmentation in leaves under long-day conditions in contrast to Craterostigma plantagineum (desiccation tolerant) and Lindernia subracemosa (desiccation sensitive). The intense pigmentation in leaves does not affect the desiccation tolerance behaviour but seems to be related to oxidative stress protection. Green leaves of short-day and purple leaves of long-day plants provided suitable material for comparing basic photosynthetic parameters. An increase in non-photochemical quenching in purple leaves appears to prevent photoinhibition. Treatment with methyl viologen decreased the photochemical activities in both long-day and short-day plants but long-day plants which accumulate anthocyanins maintained a higher non-photochemical quenching than short-day plants. No differences were seen in the expression of desiccation-induced proteins and proteins involved in carbohydrate metabolism in short-day and long-day grown plants, whereas differences were observed in the expression of transcripts encoding chloroplast-localised stress proteins and transcripts encoding antioxidant enzymes. While the expression of genes encoding antioxidant enzymes were either constitutive or up-regulated during desiccation in C. plantagineum, the expression was down-regulated in L. subracemosa. RNA expression analysis indicated degradation of mRNA during desiccation in L. subracemosa but not in desiccation tolerant species. These results indicate that a better oxidative stress management and mRNA stability are correlated with desiccation tolerance.

Resurrection plants are regarded as excellent models to study the mechanisms associated with desiccation tolerance. During the past years tremendous progress has been made in understanding the phenomenon of desiccation tolerance in resurrection plants, but many questions are open concerning the mechanisms enabling these plants to survive desiccation. The photosynthetic apparatus is very sensitive to reactive oxygen species mediated injury during desiccation and must be maintained or quickly repaired upon rehydration. The photosynthetic apparatus is a primary source of generating reactive oxygen species. The unique ability of plants to withstand the oxidative stress imposed by reactive oxygen species during desiccation depends on the production of antioxidants. The present review considers the overall strategies and the mechanisms involved in the desiccation tolerance in the first part and will focus on the effects on photosynthesis, energy metabolism and antioxidative stress defenses in the second part.

Summary • Craterostigma plantagineum can lose up to 96% of its water content but fully recover within hours after rehydration. The callus tissue of the plant becomes desiccation tolerant upon pre-incubation with abscisic acid (ABA). In callus and vegetative organs, ABA addition and water depletion induce a set of dehydrationresponsive genes.  Previously, activation tagging led to the isolation of Craterostigma desiccation tolerant (CDT-1), a dehydration-related ABA-inducible gene which renders callus desiccation tolerant without ABA pre-treatment. This gene belongs to a family of retroelements, members of which are inducible by dehydration.  Craterostigma plantagineum transformation with mutated versions of CDT-1 indicated that protein is not required for the induction of callus desiccation tolerance. Northern analysis and protoplast transfection indicated that CDT-1 directs the synthesis of a double-stranded 21-bp short interfering RNA (siRNA), which opens the metabolic pathway for desiccation tolerance.  Via transposition, these retroelements have progressively increased the capacity of the species to synthesize siRNA and thus recover after dehydration. This may be a case of evolution towards the acquisition of a new trait, stimulated by the environment acting directly on intra-genomic DNA replication.

International audience; Betaine aldehyde dehydrogenases oxidize betaine aldehyde to glycine betaine in species that accumulate glycine betaine as a compatible solute under stress conditions. In contrast, the physiological function of betaine aldehyde dehydrogenase genes is at present unclear in species that do not accumulate glycine betaine, such as Arabidopsis thaliana. To address this question, we overexpressed the Arabidopsis ALDH10A8 and ALDH10A9 genes, which were identified to code for betaine aldehyde dehydrogenases, in wild-type A. thaliana. We analysed changes in metabolite contents of transgenic plants in comparison with the wild type. Using exogenous or endogenous choline, our results indicated that ALDH10A8 and ALDH10A9 are involved in the synthesis of glycine betaine in Arabidopsis. Choline availability seems to be a factor limiting glycine betaine synthesis. Moreover, the contents of diverse metabolites including sugars (glucose and fructose) and amino acids were altered in fully developed transgenic plants compared with the wild type. The plant metabolic response to salt and the salt stress tolerance were impaired only in young transgenic plants, which exhibited a delayed growth of the seedlings early after germination. Our results suggest that a balanced expression of the betaine aldehyde dehydrogenase genes is important for early growth of A. thaliana seedlings and for salt stress mitigation in young seedlings

Aldehyde dehydrogenases (ALDHs) play a major role in the detoxification processes of aldehydes generated in plants when exposed to abiotic stress. In previous studies, we have shown that the Arabidopsis thaliana ALDH3I1 gene is transcriptionally activated by abiotic stress, and over-expression of the ALDH3I1 gene confers stress tolerance in transgenic plants. The A. thaliana genome contains 14 ALDH genes expressed in different sub-cellular compartments and are presumably involved in different reactions. The purpose of this study was to compare the potential of a cytoplasmic and a chloroplastic stress-inducible ALDH in conferring stress tolerance under different conditions. We demonstrated that constitutive or stress-inducible expression of both the chloroplastic ALDH3I1 and the cytoplasmic ALDH7B4 confers tolerance to osmotic and oxidative stress. Stress tolerance in transgenic plants is accompanied by a reduction of H2O2 and malondialdehyde (MDA) derived from cellular lipid peroxidation. Involvement of ALDHs in stress tolerance was corroborated by the analysis of ALDH3I1 and ALDH7B4 T-DNA knockout (KO) mutants. Both mutant lines exhibited higher sensitivity to dehydration and salt than wild-type (WT) plants. The results indicate that ALDH3I1 and ALDH7B4 not only function as aldehyde-detoxifying enzymes, but also as efficient reactive oxygen species (ROS) scavengers and lipid peroxidation-inhibiting enzymes. The potential of ALDHs to interfere with H2O2 was also shown for recombinant bacterial proteins.

The resurrection plant Craterostigma plantagineum has the ability to survive complete dehydration. In an attempt to further understand desiccation tolerance in this plant, the CpMYB10 transcription factor gene was functionally characterized. CpMYB10 is rapidly induced by dehydration and abscisic acid (ABA) treatments in leaves and roots, but no expression was detected in fully hydrated tissues. Electrophoretic mobility shift assay experiments showed binding of rCpMYB10 to specific mybRE elements within the LEA Cp11-24 and CpMYB10 promoters. Localization of CpMYB10 transcript by in situ reverse transcription-PCR reactions showed expression in vascular tissues, parenchyma, and epidermis both in leaves and roots in response to ABA. Transgenic Arabidopsis plants transformed with CpMYB10 promoter fused to GUS gene showed reporter expression under ABA and stress conditions in several organs. Overexpression of CpMYB10 cDNA in Arabidopsis led to desiccation and salt tolerance of transgenics lines. Interestingly, it was found that plants overexpressing CpMYB10 exhibited Glc-insensitive and ABA hypersensitive phenotypes. Therefore, our results indicate that CpMYB10 in Arabidopsis is mediating stress tolerance and altering ABA and Glc signaling responses.

Although desiccation tolerant, the resurrection plant Craterostigma plantagineum is sensitive to relatively low levels of sodium chloride. Exposure to sodium chloride, but not dehydration, led to accumulation of sodium ions in leaves and roots and caused irreversible wilting. The effects of salt and dehydration on transcript accumulation patterns were studied by using selected cDNA clones that were related to water stress. Most of the clones represented genes that were up-regulated in response to both treatments. Among the transcripts specifically up-regulated by dehydration were RNAs encoding transcripts with homology to aquaporins. Expression analysis revealed dehydration-specific profiles of late embryogenesis abundant (LEA) genes, which differed from the patterns observed for the same genes under sodium chloride stress. The interconversion of octulose and sucrose, which is characteristic for the desiccation/rehydration cycle in C. plantagineum leaves, was not activated by sodium chloride. The present results suggest that dehydration-specific responses involve the synchronized expression of specific genes and the presence of a determined concentration of sucrose. These dehydration responses were not detected in response to sodium chloride treatment.

Oxidation of methionine residues during periods of oxidative stress can lead to loss of protein function. Organisms have developed defense strategies to minimize such damage. The PilB protein, which is involved in pilus formation in the pathogen Neisseria gonorrhoeae, is composed of three functional protein domains (I-III) with putative roles in oxidative stress defense. These domains are evolutionarily conserved and homologs have been discovered in diverse prokaryotes and eukaryotes. Domain III shows similarities to selenoproteins which contain selenium instead of sulfur in a conserved cysteine residue. The substitution of selenium for sulfur alters the redox properties of such proteins. Knock-out mutants were used to elucidate the function of these novel selenoprotein-like domains in yeast and in Arabidopsis thaliana. We show that organisms with non-functional genes for selenoprotein-like polypeptides accumulate higher levels of oxidized methionine residues on exposure to oxidative stress. The behavior of the mutants suggests that these novel selenoprotein-like gene products are part of a ubiquitous detoxification system that interacts with other redox-related proteins such as the thioredoxin-related protein and methionine sulfoxide reductase which are encoded by domains I and II of PilB. These proteins may be encoded by one gene as in the case of several prokaryotes, or by separate genes as in the eukaryotes examined here.

Summary In order to identify genes that are critical for the ABA-dependent stress response in the resurrection plant Craterostigma plantagineum, a gene was isolated with homology to class 3 variable substrate aldehyde dehydrogenases (ALDH). The C. plantagineum gene Cp-ALDH constitutes a novel class of plant ALDHs. In a search for corresponding genes from Arabidopsis thaliana, Ath-ALDH3 and Ath-ALDH4 were isolated, showing 70% and 80% similarity to Cp-ALDH. Phylogenetically, the Cp- and Ath-ALDH3 and -ALDH4 proteins are closely related to aldehyde dehydrogenases from bacteria and mammalian species and are separated from known plant ALDHs and betaine-aldehyde dehydrogenases (BADH). Cp-ALDH transcript and polypeptide are up-regulated in vegetative tissues and callus in response to dehydration or ABA-treatment. Ath-ALDH3 expression was induced in response to dehydration and ABA treatment, while Ath-ALDH4 is constitutively expressed at a low level. Recombinant Cp-ALDH protein oxidizes nonanal, propionaldehyde and acetaldehyde, with Km values of 2.2 µm, 0.27 mm and 3.23 mm, respectively, in an NAD-dependent manner. Immunogold electron microscopy shows that Cp-ALDH is localized in plastids.

Desiccation and abscisic acid treatment lead to major changes in thylakoid membranes of the desiccation-tolerant plant Craterostigma plantagineum. The chlorophyll contents and proteins of the light harvesting complexes decrease during desiccation, although some chlorophyll is retained in the dehydrated state. The xanthophyll cycle pigment zeaxanthin, however, increased. Under these conditions, a 22 kDa ELIP-like desiccation-induced protein (dsp 22) accumulated in the thylakoid membranes. Fractionation of pigment-protein complexes of stressed plants revealed that the dsp 22 protein co-localized with the carotenoid zeaxanthin. Inhibition of zeaxanthin production had a negative effect on the accumulation of the dsp 22 protein. It is suggested that dsp 22 contributes to the protection against photoinhibition caused by dehydration.

The enzyme sucrose synthase (UDP-glucose: D-fructose 2alpha-glucosyltransferase, EC 2.4.1.13) is a key enzyme in carbohydrate metabolism, catalyzing the reversible conversion of sucrose uridine-diphosphate into fructose and UDP-glucose. We report the molecular characterization of two classes of cDNA and genomic clones encoding sucrose synthase from Craterostigma plantagineum Hochst., a resurrection plant in which the turnover of sucrose is considered to have an important role in the unique phenomenon of surviving desiccation. Sucrose-synthase transcript and protein levels are modulated by dehydration and rehydration. In-situ hybridization revealed that transcripts preferentially accumulate in phloem tissues. Promoter analysis underlined a role for class-I sucrose-synthase genes in dehydration stress and in response to cis-abscisic acid. A DNA sequence motif common to class-I sucrose-synthase and sucrose-phosphate-synthase genes was discovered.

The molecular dissection of desiccation tolerance in the resurrection plant Craterostigma plantagineum led to the isolation of two dehydration-stress inducible homeo-domain-leucine zipper genes (CPHB-1 and -2). When the coding region of CPHB-1 was used as bait in the yeast two-hybrid system, the ability of CPHB-1 to form homodimers was demonstrated. The two-hybrid system was also used to isolate CPHB-2, which heterodimerises with CPHB-1. Both transcripts are inducible by dehydration in leaves and roots, but steady state levels vary in response to exogenously applied ABA. Although expression of CPHB-1 is not inducible by ABA, the transcript level of CPHB-2 increases during ABA-treatment. Both genes are expressed at very early stages of dehydration and thus may be involved in the regulation of gene expression during dehydration. CPHB-1 and -2 differential expression in response to ABA suggests that they act in different branches of the dehydration-induced signalling network. In vitro binding studies revealed that CPHB-1 specifically binds to the pseudopalindromic sequence CAAT(C/G)ATTG. Using this element for in vitro binding studies with nuclear proteins from dehydrated leaves, an inducible DNA-protein complex was identified.

Major intrinsic proteins (MIPs) are a family of channel proteins that are mainly represented by aquaporins in plants. These are divided into TIPs (tonoplast intrinsic proteins) and PIPs (plasma membrane intrinsic proteins) according to their subcellular localization. Homologues to PIPs and TIPs were isolated from the desiccation-tolerant resurrection plant Craterostigma plantagineum by two approaches: firstly, a cDNA library constructed from RNA of dehydrated C. plantagineum leaves was screened with an Arabidopsis thaliana Ath-PIP1b cDNA probe and, secondly, a cDNA library was screened differentially to isolate early drought-induced transcripts. According to sequence homologies the isolated cDNA clones were grouped as follows: Cp-PIPa, Cp-PIPb, Cp-PIPc and Cp-TIP. Cp-PIPa, Cp-PIPc and Cp-TIP transcript accumulation was regulated by dehydration and abscisic acid (ABA). Within the Cp-PIPa group transcripts were regulated either by drought only or by drought and ABA, indicating that ABA-dependent and -independent signal transduction pathways lead to Cp-PIPa expression. Comparison of Cp-PIPa expression in detached leaves and in whole plants suggested the involvement of a signal transmitted in the whole plant in response to drought. Cp-PIPb transcript levels were constitutive in all organs tested. Antibodies raised against a Cp-PIPA protein recognized a polypeptide with an apparent molecular mass of 28 kDa. Using these antibodies it was shown that both Cp-PIPA and Cp-PIPB proteins were localized to the plasma membrane. The role of different members of the MIP group in the dehydration response is discussed.

Sucrose-phosphate synthase (SPS) is a key enzyme in the regulation of sucrose metabolism, being responsible for the synthesis of sucrose 6-phosphate from fructose 6-phosphate and uridine 5[prime]-diphosphate-glucose. We report on the isolation and characterization of cDNA clones encoding SPS from Craterostigma plantagineum Hochst., a resurrection plant in which the accumulation of sucrose is considered to play an important role in tolerance to severe protoplastic dehydration. Two distinct classes of cDNAs encoding SPS were isolated from C. plantagineum, and are represented by the clones Cpsps1 and Cpsps2. The transcripts corresponding to both cDNAs decrease to very low levels in dehydrating leaves of C. plantagineum. Only the Cpsps1 transcript occurs in the roots, where it is present at a higher level than in leaves and increases upon dehydration of the plant. Higher enzymatic activities have been determined in protein extracts of dehydrated tissues compared with untreated tissues, which correlates with an increase in protein levels. It is suggested that the overall regulation of SPS is strongly influenced by the changing composition of the cytoplasm in C. plantagineum leaves during the dehydration-rehydration cycle.

We have used callus tissue of the desiccation-tolerant plant, Craterostigma plantagineum Hochst to probe the stereochemical requirements of ABA in the desiccation tolerance response, using ABA analogs. Callus was treated with (S)-(+)-ABA and (R)-(-)-ABA and two pure isomer derivatives of each, and viability after a drying treatment and the expression of four desiccation-induced late embryogenesis abundant (LEA)-type transcripts and sucrose synthase from Craterostigma were measured. Both stereoisomers alone caused transcript expression and highest viability. The two derivatives of (R)-(-)-ABA gave poor or no transcript expression and no viability, suggesting that alteration of a ring double bond to a single bond in this series of compounds is significant in ABA perception for both responses. One (S)-(+)-ABA derivative was effective in inducing all transcripts and causing callus viability. The other analog derivative induced all protein transcripts without concomitant tissue viability after drying. This suggests that some other ABA-inducible factor(s) are vital for desiccation tolerance of callus.

Proteins inducible by dehydration and abscisic acid (ABA), have been identified in a number of species and have been suggested to play a role in desiccation tolerance. Recently, we identified a novel boiling-stable protein (BspA) which accumulated in shoots of aspen (Populus tremula L.) cultured in vitro, in response to gradual water stress and ABA application (Pelah et al. 1995. Tree Physiol. 15: 673–678.). Accumulation of BspA, and of the water stress-related protein dehydrin dsp- 16 and sucrose synthase from the resurrection plant. Craterostigma plantagineum, was examined in two greenhouse-grown Populus species to investigate the relationship between the presence of the proteins and water stress tolerance. Detached leaves of Populus tomentosa lost more water than Populus popularis, resulting in a significant decrease in leaf water potential. Using electrolyte leakage analysis, it was found that detached leaves of Populus popularis are more tolerant to water stress than those of Populus tomentosa. Using western blots with the corresponding antibodies, we have found in Populus popularis accumulation of BspA and sucrose synthase due to water stress, and the constitutive presence of a dehydrin-like protein. In contrast, a low expression of BspA was found in Populus tomentosa, but not of sucrose synthase and dehydrin-like proteins. Desiccation tolerance in many tissues can be partly attributed to soluble sugars. Analysis of the amount of soluble sugars did not reveal clear-cut differences between the two species, except for significant sucrose accumulation and glucose reduction in water-stressed Populus tomentosa and increase in glucose in water-stressed Populus popularis. The data obtained points to a positive correlation between increased water stress tolerance of one poplar species as compared with another and accumulation of water stress-related proteins and sucrose synthase.

Summary In a previous study we identified a novel 66-kD boiling-stable protein (BspA) that accumulates in cultured shoots of aspen ( Populus tremula L.) in response to gradual water stress and ABA application, as well as to osmotic and cold stresses (Altman et al., 1996; Pelah et al., 1995). BspA was found to be the major heat-stable water stress-responsive protein in aspen. Here, the expression of other water-stress responsive proteins — DSP 16 (dehydrin), cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and sucrose synthase — was studied in aspen roots and shoots. These proteins were identified immunologically using polyclonal antibodies isolated from the resurrection plant Craterostigma plantagineum . A 43-kD homologue and a 31-kD homologue of dehydrin were found to accumulate in roots and shoots of aspen after water stress in a tissue specific manner. Shoots and roots of intact plantlets subjected to ABA application accumulated a 33-kD dehydrin homologue. A sucrose synthase homologue (90 kD) accumulated in water-stressed shoots and was constitutively present at high levels in control and water-stressed roots harvested from intact plandets. GAPDH, a key enzyme in the glycolysis and gluconeogenesis pathways, exhibited some accumulation in shoots in response to water stress. Sugar analysis revealed that water stress results in increased sucrose and decreased glucose levels in the aspen leaves. The data presented here suggest that the water-stress-response mechanisms present in herbaceous plants are also present in woody plants. Woody plants subjected to water stress accumulate dehydrin-like proteins and specific enzymes such as sucrose synthase and GAPDH, which may play a dominant role in sugar metabolism under these conditions.

The cofactor-binding site of the NAD + -dependent Arabidopsis thaliana aldehyde dehydrogenase ALDH3H1 was analyzed to understand structural features determining cofactor-specificity. Homology modeling and mutant analysis elucidated important amino acid residues. Glu149 occupies a central position in the cofactor-binding cleft, and its carboxylate group coordinates the 2′- and 3′-hydroxyl groups of the adenosyl ribose ring of NAD + and repels the 2′-phosphate moiety of NADP + . If Glu149 is mutated to Gln, Asp, Asn or Thr the binding of NAD + is altered and rendered the enzyme capable of using NADP + . This change is attributed to a weaker steric hindrance and elimination of the electrostatic repulsion force of the 2′-phosphate of NADP + . Simultaneous mutations of Glu149 and Ile200, which is situated opposite of the cofactor binding cleft, improved the enzyme efficiency with NADP + . The double mutant ALDH3H1 Glu149Thr/Ile200Val showed a good catalysis with NADP + . Subsequently a triple mutation was generated by replacing Val178 by Arg in order to create a “closed” cofactor binding site. The cofactor specificity was shifted even further in favor of NADP + , as the mutant ALDH3H1 E149T/V178R/I200V uses NADP + with almost 7-fold higher catalytic efficiency compared to NAD + . Our experiments suggest that residues occupying positions equivalent to 149, 178 and 200 constitute a group of amino acids in the ALDH3H1 protein determining cofactor affinity.

Craterostigma plantagineum is a representative of the resurrection plants, which are able to withstand complete dryness. During the dehydration process, many characteristic transcripts and proteins are induced; these have been isolated by molecular cloning. The expression of most of these gene products can also be triggered by ABA. Five representative desiccation-related cDNA clones were selected. The effect of light and developmental stage on the expression of the transcripts and corresponding proteins was analysed during dehydration and ABA treatment. Desiccation and ABA treatment in the presence of light induced a marked increase in several of the transcripts, whereas light had the reverse effect on the levels of some proteins (relative to mRNA levels) localized in the chloroplast. Although very young plants have the capacity to resume full physiological activity after dehydration, some of the desiccation-related gene products are still expressed at low levels.