Physiological, transcriptomic and metabolomic insights of three extremophyte woody species living in the multi-stress environment of the Atacama Desert.

MAIN CONCLUSIONS: In contrast to Neltuma species, S. tamarugo exhibited higher stress tolerance, maintaining photosynthetic performance through enhanced gene expression and metabolites. Differentially accumulated metabolites include chlorophyll and carotenoids and accumulation of non-nitrogen osmoprotectants. Plant species have developed different adaptive strategies to live under extreme environmental conditions. Hypothetically, extremophyte species present a unique configuration of physiological functions that prioritize stress-tolerance mechanisms while carefully managing resource allocation for photosynthesis. This could be particularly challenging under a multi-stress environment, where the synthesis of multiple and sequential molecular mechanisms is induced. We explored this hypothesis in three phylogenetically related woody species co-occurring in the Atacama Desert, Strombocarpa tamarugo, Neltuma alba, and Neltuma chilensis, by analyzing their leaf dehydration and freezing tolerance and by characterizing their photosynthetic performance under natural growth conditions. Besides, the transcriptomic profiling, biochemical analyses of leaf pigments, and metabolite analysis by untargeted metabolomics were conducted to study gene expression and metabolomic landscape within this challenging multi-stress environment. S. tamarugo showed a higher photosynthetic capacity and leaf stress tolerance than the other species. In this species, a multifactorial response was observed, which involves high photochemical activity associated with a higher content of chlorophylls and ß-carotene. The oxidative damage of the photosynthetic apparatus is probably attenuated by the synthesis of complex antioxidant molecules in the three species, but S. tamarugo showed the highest antioxidant capacity. Comparative transcriptomic and metabolomic analyses among the species showed the differential expression of genes involved in the biosynthetic pathways of key stress-related metabolites. Moreover, the synthesis of non-nitrogen osmoprotectant molecules, such as ciceritol and mannitol in S. tamarugo, would allow the nitrogen allocation to support its high photosynthetic capacity without compromising leaf dehydration tolerance and freezing stress avoidance.

Pyrroline-5-carboxylate metabolism protein complex detected in Arabidopsis thaliana leaf mitochondria.

Proline dehydrogenase (ProDH) and pyrroline-5-carboxylate (P5C) dehydrogenase (P5CDH) catalyse the oxidation of proline into glutamate via the intermediates P5C and glutamate-semialdehyde (GSA), which spontaneously interconvert. P5C and GSA are also intermediates in the production of glutamate from ornithine and α-ketoglutarate catalysed by ornithine δ-aminotransferase (OAT). ProDH and P5CDH form a fused bifunctional PutA enzyme in Gram-negative bacteria and are associated in a bifunctional substrate-channelling complex in Thermus thermophilus; however, the physical proximity of ProDH and P5CDH in eukaryotes has not been described. Here, we report evidence of physical proximity and interactions between Arabidopsis ProDH, P5CDH, and OAT in the mitochondria of plants during dark-induced leaf senescence when all three enzymes are expressed. Pairwise interactions and localization of the three enzymes were investigated using bimolecular fluorescence complementation with confocal microscopy in tobacco and sub-mitochondrial fractionation in Arabidopsis. Evidence for a complex composed of ProDH, P5CDH, and OAT was revealed by co-migration of the proteins in native conditions upon gel electrophoresis. Co-immunoprecipitation coupled with mass spectrometry analysis confirmed the presence of the P5C metabolism complex in Arabidopsis. Pull-down assays further demonstrated a direct interaction between ProDH1 and P5CDH. P5C metabolism complexes might channel P5C among the constituent enzymes and directly provide electrons to the respiratory electron chain via ProDH.

Pyrroline-5-carboxylate dehydrogenase is an essential enzyme for proline dehydrogenase function during dark-induced senescence in Arabidopsis thaliana.

During leaf senescence, nitrogen is remobilized and carbon backbones are replenished by amino acid catabolism, with many of the key reactions occurring in mitochondria. The intermediate Δ1 -pyrroline-5-carboxylate (P5C) is common to some catabolic pathways, thus linking the metabolism of several amino acids, including proline and arginine. Specifically, mitochondrial proline catabolism involves sequential action of proline dehydrogenase (ProDH) and P5C dehydrogenase (P5CDH) to produce P5C and then glutamate. Arginine catabolism produces urea and ornithine, the latter in the presence of α-ketoglutarate being converted by ornithine δ-aminotransferase (OAT) into P5C and glutamate. Metabolic changes during dark-induced leaf senescence (DIS) were studied in Arabidopsis thaliana leaves of Col-0 and in prodh1prodh2, p5cdh and oat mutants. Progression of DIS was followed by measuring chlorophyll and proline contents for 5 days. Metabolomic profiling of 116 compounds revealed similar profiles of Col-0 and oat metabolism, distinct from prodh1prodh2 and p5cdh metabolism. Metabolic dynamics were accelerated in p5cdh by 1 day. Notably, more P5C and proline accumulated in p5cdh than in prodh1prodh2. ProDH1 enzymatic activity and protein amount were significantly down-regulated in p5cdh mutant at Day 4 of DIS. Mitochondrial P5C levels appeared critical in determining the flow through interconnected amino acid remobilization pathways to sustain senescence.

Carbon and nitrogen remobilization during seed filling in Arabidopsis is strongly impaired in the pyrroline-5-carboxylate dehydrogenase mutant.

Proline is an amino acid that is degraded in the mitochondria by the sequential action of proline dehydrogenase (ProDH) and pyrroline-5-carboxylate dehydrogenase (P5CDH) to form glutamate. We investigated the phenotypes of Arabidopsis wild-type plants, the knockout prodh1 prodh2 double-mutant, and knockout p5cdh allelic mutants grown at low and high nitrate supplies. Surprisingly, only p5cdh presented lower seed yield and produced lighter seeds. Analyses of elements in above-ground organs revealed lower C concentrations in the p5cdh seeds. Determination of C, N, and dry matter partitioning among the above-ground organs revealed a major defect in stem-to-seed resource allocations in this mutant. Again surprisingly, defects in C, N, and biomass allocation to seeds dramatically increased in high-N conditions. 15N-labelling consistently confirmed the defect in N remobilization from the rosette and stem to seeds in p5cdh. Consequently, the p5cdh mutants produced morphologically abnormal, C-depleted seeds that displayed very low germination rates. The most striking result was the strong amplification of the N-remobilization defects in p5cdh under high nitrate supply, and interestingly this phenotype was not observed in the prodh1 prodh2 double-mutant irrespective of nitrate supply. This study reveals an essential role of P5CDH in carbon and nitrogen remobilization for reserve accumulation during seed development in Arabidopsis.

The proline cycle as an eukaryotic redox valve.

The amino acid proline has been known for many years to be a component of proteins as well as an osmolyte. Many recent studies have demonstrated that proline has other roles such as regulating redox balance and energy status. In animals and plants, the well-described proline cycle is concomitantly responsible for the preferential accumulation of proline and shuttling of redox equivalents from the cytosol to mitochondria. The impact of the proline cycle goes beyond regulating proline levels. In this review, we focus on recent evidence of how the proline cycle regulates redox status in relation to other redox shuttles. We discuss how the interconversion of proline and glutamate shuttles reducing power between cellular compartments. Spatial aspects of the proline cycle in the entire plant are considered in terms of proline transport between organs with different metabolic regimes (photosynthesis versus respiration). Furthermore, we highlight the importance of this shuttle in the regulation of energy and redox power in plants, through a particularly intricate coordination, notably between mitochondria and cytosol.

Proline oxidation fuels mitochondrial respiration during dark-induced leaf senescence in Arabidopsis thaliana.

Leaf senescence is a form of developmentally programmed cell death that allows the remobilization of nutrients and cellular materials from leaves to sink tissues and organs. Among the catabolic reactions that occur upon senescence, little is known about the role of proline catabolism. In this study, the involvement in dark-induced senescence of proline dehydrogenases (ProDHs), which catalyse the first and rate-limiting step of proline oxidation in mitochondria, was investigated using prodh single- and double-mutants with the help of biochemical, proteomic, and metabolomic approaches. The presence of ProDH2 in mitochondria was confirmed by mass spectrometry and immunogold labelling in dark-induced leaves of Arabidopsis. The prodh1 prodh2 mutant exhibited enhanced levels of most tricarboxylic acid cycle intermediates and free amino acids, demonstrating a role of ProDH in mitochondrial metabolism. We also found evidence of the involvement and the importance of ProDH in respiration, with proline as an alternative substrate, and in remobilization of proline during senescence to generate glutamate and energy that can then be exported to sink tissues and organs.

Phospholipases Dζ1 and Dζ2 have distinct roles in growth and antioxidant systems in Arabidopsis thaliana responding to salt stress.

MAIN CONCLUSION: Phospholipases Dζ play different roles in Arabidopsis salt tolerance affecting the regulation of ion transport and antioxidant responses. Lipid signalling mediated by phospholipase D (PLD) plays essential roles in plant growth including stress and hormonal responses. Here we show that PLDζ1 and PLDζ2 have distinct effects on Arabidopsis responses to salinity. A transcriptome analysis of a double pldζ1pldζ2 mutant revealed a cluster of genes involved in abiotic and biotic stresses, such as the high salt-stress responsive genes DDF1 and RD29A. Another cluster of genes with a common expression pattern included ROS detoxification genes involved in electron transport and biotic and abiotic stress responses. Total superoxide dismutase (SOD) activity was induced early in the shoots and roots of all pldζ mutants exposed to mild or severe salinity with the highest SOD activity measured in pldζ2 at 14 days. Lipid peroxidation in shoots and roots was higher in the pldζ1 mutant upon salt treatment and pldζ1 accumulated H2O2 earlier than other genotypes in response to salt. Salinity caused less deleterious effects on K+ accumulation in shoots and roots of the pldζ2 mutant than of wild type, causing only a slight variation in Na+/K+ ratio. Relative growth rates of wild-type plants, pldζ1, pldζ2 and pldζ1pldζ2 mutants were similar in control conditions, but strongly affected by salt in WT and pldζ1. The efficiency of photosystem II, estimated by measuring the ratio of chlorophyll fluorescence (F v/F m ratio), was strongly decreased in pldζ1 under salt stress. In conclusion, PLDζ2 plays a key role in determining Arabidopsis sensitivity to salt stress allowing ion transport and antioxidant responses to be finely regulated.

Proteomic and functional analysis of proline dehydrogenase 1 link proline catabolism to mitochondrial electron transport in Arabidopsis thaliana.

Proline accumulates in many plant species in response to environmental stresses. Upon relief from stress, proline is rapidly oxidized in mitochondria by proline dehydrogenase (ProDH) and then by pyrroline-5-carboxylate dehydrogenase (P5CDH). Two ProDH genes have been identified in the genome of the model plant Arabidopsis thaliana To gain a better understanding of ProDH1 functions in mitochondria, proteomic analysis was performed. ProDH1 polypeptides were identified in Arabidopsis mitochondria by immunoblotting gels after 2D blue native (BN)-SDS/PAGE, probing them with an anti-ProDH antibody and analysing protein spots by MS. The 2D gels showed that ProDH1 forms part of a low-molecular-mass (70-140 kDa) complex in the mitochondrial membrane. To evaluate the contribution of each isoform to proline oxidation, mitochondria were isolated from wild-type (WT) and prodh1, prodh2, prodh1prodh2 and p5cdh mutants. ProDH activity was high for genotypes in which ProDH, most likely ProDH1, was strongly induced by proline. Respiratory measurements indicate that ProDH1 has a role in oxidizing excess proline and transferring electrons to the respiratory chain.

Hydrogen peroxide produced by NADPH oxidases increases proline accumulation during salt or mannitol stress in Arabidopsis thaliana.

Many plants accumulate proline, a compatible osmolyte, in response to various environmental stresses such as water deficit and salinity. In some stress responses, plants generate hydrogen peroxide (H2 O2 ) that mediates numerous physiological and biochemical processes. The aim was to study the relationship between stress-induced proline accumulation and H2 O2 production. Using pharmacological and reverse genetic approaches in Arabidopsis thaliana, we investigated the role of NADPH oxidases, Respiratory burst oxidase homologues (Rboh), in the induction of proline accumulation was investigated in response to stress induced by either 200 mM NaCl or 400 mM mannitol. Stress from NaCl or mannitol resulted in a transient increase in H2 O2 content accompanied by accumulation of proline. Dimethylthiourea, a scavenger of H2 O2 , and diphenylene iodonium (DPI), an inhibitor of H2 O2 production by NADPH oxidase, were found to significantly inhibit proline accumulation in these stress conditions. DPI also reduced the expression level of Δ(1) -pyrroline-5-carboxylate synthetase, the key enzyme involved in the biosynthesis of proline. Similarly, less proline accumulated in knockout mutants lacking either AtRbohD or AtRbohF than in wild-type plants in response to the same stresses. Our data demonstrate that AtRbohs (A. thaliana Rbohs) contribute to H2 O2 production in response to NaCl or mannitol stress to increase proline accumulation in this plant.

Diversity, distribution and roles of osmoprotective compounds accumulated in halophytes under abiotic stress.

BACKGROUND AND AIMS: Osmolytes are low-molecular-weight organic solutes, a broad group that encompasses a variety of compounds such as amino acids, tertiary sulphonium and quaternary ammonium compounds, sugars and polyhydric alcohols. Osmolytes are accumulated in the cytoplasm of halophytic species in order to balance the osmotic potential of the Na(+) and Cl(-) accumulated in the vacuole. The advantages of the accumulation of osmolytes are that they keep the main physiological functions of the cell active, the induction of their biosynthesis is controlled by environmental cues, and they can be synthesized at all developmental stages. In addition to their role in osmoregulation, osmolytes have crucial functions in protecting subcellular structures and in scavenging reactive oxygen species. SCOPE: This review discusses the diversity of osmolytes among halophytes and their distribution within taxonomic groups, the intrinsic and extrinsic factors that influence their accumulation, and their role in osmoregulation and osmoprotection. Increasing the osmolyte content in plants is an interesting strategy to improve the growth and yield of crops upon exposure to salinity. Examples of transgenic plants as well as exogenous applications of some osmolytes are also discussed. Finally, the potential use of osmolytes in protein stabilization and solvation in biotechnology, including the pharmaceutical industry and medicine, are considered.

Effects of Foliar Boron Application on Physiological and Antioxidants Responses in Highbush Blueberry (Vaccinium corymbosum L.) Cultivars.

Boron (B) is a micronutrient crucial for the growth, development, productivity, and quality of crops. However, in areas characterized by acid soil (pHwater < 5.0) and high rainfall, soil B concentration tends to decrease, leading to insufficient supply to crops. This study was aimed at determining the optimal rate of B fertilization to enhance Vaccinium corymbosum L. performance in acid conditions. One-year-old cultivars with contrasting Al resistance (Al-sensitive Star and Al-resistant Cargo) were used. Plants were conditioned in plastic pots containing 18 L of half-ionic-strength Hoagland solution (pH 4.5) for 2 weeks. Thereafter, the following B treatments were applied foliarly: control, without B application (distilled water), 200, 400, and 800 mg L-1 of B as Solubor® for up to 72 h. Photosynthetic performance, root and shoot B levels, antioxidants, and oxidative stress were evaluated. Root and shoot B concentrations increased with the increasing B application, being higher in leaves than in roots of both cultivars. Net photosynthesis decreased at 800 mg L-1 B supply and effective quantum yield of PSII at 72 h in all B treatments. Lipid peroxidation increased in both cultivars at 800 mg L-1 B treatment. Antioxidant activity increased in all B treatments in both cultivars; while, at 400 and 800 mg L-1 B, total phenols increased in leaves of cultivar Star and decreased in cultivar Cargo. In conclusion, optimal B foliar application for highbush blueberry appears to be around 400 mg L-1 B. The appropriate B foliar application could help mitigate potential stress-induced problems in highbush blueberry cultivation. However, the optimal foliar B application should be confirmed in field experiments to help the farmers manage B nutrition.

The older, the better: Ageing improves the efficiency of biochar-compost mixture to alleviate drought stress in plant and soil.

Due to increased drought frequency following climate change, practices improving water use efficiency and reducing water-stress are needed. The efficiency of organic amendments to improve plant growth conditions under drought is poorly known. Our aim was to investigate if organic amendments can attenuate plant water-stress due to their effect on the plant-soil system and if this effect may increase upon ageing. To this end we determined plant and soil responses to water shortage and organic amendments added to soil. We compared fresh biochar/compost mixtures to similar amendments after ageing in soil. Results indicated that amendment application induced few plant physiological responses under water-stress. The reduction of leaf gas exchange under watershortage was alleviated when plants were grown with biochar and compost amendments: stomatal conductance was least reduced with aged mixture aged mixture (-79 % compared to -87 % in control), similarly to transpiration (-69 % in control and not affected with aged mixture). Belowground biomass production (0.25 times) and nodules formation (6.5 times) were enhanced under water-stress by amendment addition. This effect was improved when grown on soil containing the aged as compared to fresh amendments. Plants grown with aged mixtures also showed reduced leaf proline concentrations (two to five times) compared to fresh mixtures indicating stress reduction. Soil enzyme activities were less affected by water-stress in soil with aged amendments. We conclude that the application of biochar-compost mixtures may be a solution to reduce the effect of water-stress to plants. Our findings revealed that this beneficial effect is expected to increase with aged mixtures, leading to a better water-stress resistance over time. However, while being beneficial for plant growth under water-stress, the use of amendments may not be suited to increase water use efficiency.

Phospholipases C and D modulate proline accumulation in Thellungiella halophila/salsuginea differently according to the severity of salt or hyperosmotic stress.

Proline accumulation is one of the most common responses of plants to environmental constraints. Thellungiella halophila/salsuginea, a model halophyte, accumulates high levels of proline in response to abiotic stress and in the absence of stress. Recently, lipid signaling pathways have been shown to be involved in the regulation of proline metabolism in Arabidopsis thaliana. Here we investigated the relationship between lipid signaling enzymes and the level of proline in T. salsuginea. Inhibition of phospholipase C (PLC) enzymes by the specific inhibitor U73122 demonstrated that proline accumulation is negatively controlled by PLCs in the absence of stress and under moderate salt stress (200 mM NaCl). The use of 1-butanol to divert some of the phospholipase D (PLD)-derived phosphatidic acid by transphosphatidylation revealed that PLDs exert a positive control on proline accumulation under severe stress (400 mM NaCl or 400 mM mannitol) but have no effect on its accumulation in non-stress conditions. This experimental evidence shows that positive and negative lipid regulatory components are involved in the fine regulation of proline metabolism. These signaling pathways in T. salsuginea are regulated in the opposite sense to those previously described in A. thaliana, revealing that common signaling components affect the physiology of closely related glycophyte and salt-tolerant plants differently.

Proline metabolism as regulatory hub.

Proline is a multifunctional amino acid that is accumulated in high concentrations in plants under various stress conditions. Proline accumulation is intimately connected to many cellular processes, such as osmotic pressure, energy status, nutrient availability, changes in redox balance, and defenses against pathogens. Proline biosynthesis and catabolism is linked to photosynthesis and mitochondrial respiration, respectively. Proline can function as a signal, modulating gene expression and certain metabolic processes. We review important findings on proline metabolism and function of the last decade, giving a more informative picture about the function of this unusual amino acid in maintaining cellular homeostasis, modulating plant development, and promoting stress acclimation.

Metabolome and water homeostasis analysis of Thellungiella salsuginea suggests that dehydration tolerance is a key response to osmotic stress in this halophyte.

Thellungiella salsuginea, a Brassicaceae species closely related to Arabidopsis thaliana, is tolerant to high salinity. The two species were compared under conditions of osmotic stress to assess the relationships between stress tolerance, the metabolome, water homeostasis and growth performance. A broad range of metabolites were analysed by metabolic fingerprinting and profiling, and the results showed that, despite a few notable differences in raffinose and secondary metabolites, the same metabolic pathways were regulated by salt stress in both species. The main difference was quantitative: Thellungiella had much higher levels of most metabolites than Arabidopsis whatever the treatment. Comprehensive quantification of organic and mineral solutes showed a relative stability of the total solute content regardless of the species or treatment, meaning that little or no osmotic adjustment occurred under stress. The reduction in osmotic potential observed in plants under stress was found to result from a passive loss of water. Thellungiella shoots contain less water than Arabidopsis shoots, and have the ability to lose more water, which could contribute to maintain a water potential gradient between soil and plant. Significant differences between Thellungiella and Arabidopsis were also observed in terms of the physicochemical properties of their metabolomes, such as water solubility and polarity. On the whole, the Thellungiella metabolome appears to be more compatible with dehydration. Osmotic stress was also found to impact the metabolome properties in both species, increasing the overall polarity. Together, the results suggest that Thellungiella copes with osmotic stress by tolerating dehydration, with its metabolic configuration lending itself to osmoprotective strategies rather than osmo-adjustment.

Mutations in the hyperosmotic stress-responsive mitochondrial BASIC AMINO ACID CARRIER2 enhance proline accumulation in Arabidopsis.

Mitochondrial carrier family proteins are diverse in their substrate specificity, organellar location, and gene expression. In Arabidopsis (Arabidopsis thaliana), 58 genes encode these six-transmembrane-domain proteins. We investigated the biological role of the basic amino acid carrier Basic Amino Acid Carrier2 (BAC2) from Arabidopsis that is structurally and functionally similar to ARG11, a yeast ornithine and arginine carrier, and to Arabidopsis BAC1. By studying the expression of BAC2 and the consequences of its mutation in Arabidopsis, we showed that BAC2 is a genuine mitochondrial protein and that Arabidopsis requires expression of the BAC2 gene in order to use arginine. The BAC2 gene is induced by hyperosmotic stress (with either 0.2 m NaCl or 0.4 m mannitol) and dark-induced senescence. The BAC2 promoter contains numerous stress-related cis-regulatory elements, and the transcriptional activity of BAC2:beta-glucuronidase is up-regulated by stress and senescence. Under hyperosmotic stress, bac2 mutants express the P5CS1 proline biosynthetic gene more strongly than the wild type, and this correlates with a greater accumulation of Pro. Our data suggest that BAC2 is a hyperosmotic stress-inducible transporter of basic amino acids that contributes to proline accumulation in response to hyperosmotic stress in Arabidopsis.

How Does Proline Treatment Promote Salt Stress Tolerance During Crop Plant Development?

Soil salinity is one of the major abiotic stresses restricting the use of land for agriculture because it limits the growth and development of most crop plants. Improving productivity under these physiologically stressful conditions is a major scientific challenge because salinity has different effects at different developmental stages in different crops. When supplied exogenously, proline has improved salt stress tolerance in various plant species. Under high-salt conditions, proline application enhances plant growth with increases in seed germination, biomass, photosynthesis, gas exchange, and grain yield. These positive effects are mainly driven by better nutrient acquisition, water uptake, and biological nitrogen fixation. Exogenous proline also alleviates salt stress by improving antioxidant activities and reducing Na+ and Cl- uptake and translocation while enhancing K+ assimilation by plants. However, which of these mechanisms operate at any one time varies according to the proline concentration, how it is applied, the plant species, and the specific stress conditions as well as the developmental stage. To position salt stress tolerance studies in the context of a crop plant growing in the field, here we discuss the beneficial effects of exogenous proline on plants exposed to salt stress through well-known and more recently described examples in more than twenty crop species in order to appreciate both the diversity and commonality of the responses. Proposed mechanisms by which exogenous proline mitigates the detrimental effects of salt stress during crop plant growth are thus highlighted and critically assessed.

Appropriate Activity Assays Are Crucial for the Specific Determination of Proline Dehydrogenase and Pyrroline-5-Carboxylate Reductase Activities.

Accumulation of proline is a widespread plant response to a broad range of environmental stress conditions including salt and osmotic stress. Proline accumulation is achieved mainly by upregulation of proline biosynthesis in the cytosol and by inhibition of proline degradation in mitochondria. Changes in gene expression or activity levels of the two enzymes catalyzing the first reactions in these two pathways, namely pyrroline-5-carboxylate (P5C) synthetase and proline dehydrogenase (ProDH), are often used to assess the stress response of plants. The difficulty to isolate ProDH in active form has led several researchers to erroneously report proline-dependent NAD+ reduction at pH 10 as ProDH activity. We demonstrate that this activity is due to P5C reductase (P5CR), the second and last enzyme in proline biosynthesis, which works in the reverse direction at unphysiologically high pH. ProDH does not use NAD+ as electron acceptor but can be assayed with the artificial electron acceptor 2,6-dichlorophenolindophenol (DCPIP) after detergent-mediated solubilization or enrichment of mitochondria. Seemingly counter-intuitive results from previous publications can be explained in this way and our data highlight the importance of appropriate and specific assays for the detection of ProDH and P5CR activities in crude plant extracts.

Combined effects of long-term salinity and soil drying on growth, water relations, nutrient status and proline accumulation of Sesuvium portulacastrum.

The interaction between soil drying and salinity was studied in the perennial halophyte, Sesuvium portulacastrum. Rooted cuttings were individually cultivated for three months in silty-sandy soil under two irrigation modes: 100 and 25% of field capacity (FC). The amount of the evapotranspirated water was replaced by a nutrient solution containing either 0 or 100 mM NaCl. Whole-plant growth, leaf water content, leaf water potential (Psi(w)), and Na+, K+, and proline concentrations in the tissues were measured. When individually applied, both drought and salinity significantly restricted whole-plant growth, with a more marked effect of the former stress. However, the effects of the two stresses were not additive on whole-plant biomass or on leaf expansion. Root growth was more sensitive to salt than to soil drying, the latter being even magnified by the adverse impact of salinity. Leaf water content was significantly reduced following exposure to water-deficit stress, but was less affected in salt-treated plants. When simultaneously submitted to water-deficit stress and salinity, plants displayed higher values of water and potassium use efficiencies, leaf proline and Na+ concentrations, associated with lower leaf water potential (-1.87 MPa), suggesting the ability of S. portulacastrum to use Na+ and proline for osmotic adjustment.

Effects of exogenous nitric oxide on growth, proline accumulation and antioxidant capacity in Cakile maritima seedlings subjected to water deficit stress.

Nitric oxide (NO) - an endogenous signalling molecule in plants and animals - mediates responses to biotic and abiotic stresses. In the present study, we examined the role of exogenous application of NO in mediating stress responses in Cakile maritima Scop. seedlings under water deficit stress using sodium nitroprusside (SNP) as NO donor and as a pre-treatment before the application of stress. Water deficit stress was applied by withholding water for 14 days. Growth, leaf water content (LWC), osmotic potential (ψs), chlorophyll, malondialdehyde (MDA), electrolyte leakage (EL), proline and Δ1-pyrroline-5-carboxylate synthetase (P5CS) and proline dehydrogenase (ProDH) protein levels were determined. Enzyme activities involved in antioxidant activities (superoxide dismutase (SOD) and catalase (CAT)) were measured upon withholding water. The results showed that shoot biomass production was significantly decreased in plants subjected to water deficit stress alone. However, in water deficit stressed plants pre-treated with SNP, growth activity was improved and proline accumulation was significantly increased. Proline accumulation was concomitant with the stimulation of its biosynthesis as shown by the accumulation of P5CS proteins. Nevertheless, no significant change in ProDH protein levels was observed. Besides plants showed lower water deficit-induced lipid membrane degradation and oxidative stress after the pretreatment with 100µM SNP. This behaviour was related to the increased activity of SOD and CAT. Thus, we concluded that NO increased C. maritima drought tolerance and mitigated damage associated with water deficit stress by the regulation of proline metabolism and the reduction of oxidative damage.