Drought Stress Tolerance in Plants.

The current climate change scenario is accelerating degradation, desertification, and salinisation: all destructive processes that are negatively impacting arable lands and food production [...].

Strategies to Apply Water-Deficit Stress: Similarities and Disparities at the Whole Plant Metabolism Level in Medicago truncatula.

Water-deficit stresses such as drought and salinity are the most important factors limiting crop productivity. Hence, understanding the plant responses to these stresses is key for the improvement of their tolerance and yield. In this study M. truncatula plants were subjected to 250 mM NaCl as well as reduced irrigation (No-W) and 250 g/L polyethylene glycol (PEG)-6000 to induce salinity and drought stress, respectively, provoking a drop to -1.7 MPa in leaf water potential. The whole plant physiology and metabolism was explored by characterizing the stress responses at root, phloem sap and leaf organ level. PEG treatment led to some typical responses of plants to drought stress, but in addition to PEG uptake, an important impairment of nutrient uptake and a different regulation of carbon metabolism could be observed compared to No-W plants. No-W plants showed an important redistribution of antioxidants and assimilates to the root tissue, with a distinctive increase in root proline degradation and alkaline invertase activity. On the contrary, salinity provoked an increase in leaf starch and isocitrate dehydrogenase activity, suggesting key roles in the plant response to this stress. Overall, results suggest higher protection of salt-stressed shoots and non-irrigated roots through different mechanisms, including the regulation of proline and carbon metabolism, while discarding PEG as safe mimicker of drought. This raises the need to understand the effect at the whole plant level of the different strategies employed to apply water-deficit stress.

Medicago truncatula and Glycine max: Different Drought Tolerance and Similar Local Response of the Root Nodule Proteome.

Legume crops present important agronomical and environmental advantages mainly due to their capacity to reduce atmospheric N2 to ammonium via symbiotic nitrogen fixation (SNF). This process is very sensitive to abiotic stresses such as drought, but the mechanism underlying this response is not fully understood. The goal of the current work is to compare the drought response of two legumes with high economic impact and research importance, Medicago truncatula and Glycine max, by characterizing their root nodule proteomes. Our results show that, although M. truncatula exhibits lower water potential values under drought conditions compared to G. max, SNF declined analogously in the two legumes. Both of their nodule proteomes are very similar, and comparable down-regulation responses in the diverse protein functional groups were identified (mainly proteins related to the metabolism of carbon, nitrogen, and sulfur). We suggest lipoxygenases and protein turnover as newly recognized players in SNF regulation. Partial drought conditions applied to a split-root system resulted in the local down-regulation of the entire proteome of drought-stressed nodules in both legumes. The high degree of similarity between both legume proteomes suggests that the vast amount of research conducted on M. truncatula could be applied to economically important legume crops, such as soybean.

Drought stress provokes the down-regulation of methionine and ethylene biosynthesis pathways in Medicago truncatula roots and nodules.

Symbiotic nitrogen fixation is one of the first physiological processes inhibited in legume plants under water-deficit conditions. Despite the progress made in the last decades, the molecular mechanisms behind this regulation are not fully understood yet. Recent proteomic work carried out in the model legume Medicago truncatula provided the first indications of a possible involvement of nodule methionine (Met) biosynthesis and related pathways in response to water-deficit conditions. To better understand this involvement, the drought-induced changes in expression and content of enzymes involved in the biosynthesis of Met, S-adenosyl-L-methionine (SAM) and ethylene in M. truncatula root and nodules were analyzed using targeted approaches. Nitrogen-fixing plants were subjected to a progressive water deficit and a subsequent recovery period. Besides the physiological characterization of the plants, the content of total sulphur, sulphate and main S-containing metabolites was measured. Results presented here show that S availability is not a limiting factor in the drought-induced decline of nitrogen fixation rates in M. truncatula plants and provide evidences for a down-regulation of the Met and ethylene biosynthesis pathways in roots and nodules in response to water-deficit conditions.

A proteomic approach reveals new actors of nodule response to drought in split-root grown pea plants.

Drought is considered the more harmful abiotic stress resulting in crops yield loss. Legumes in symbiosis with rhizobia are able to fix atmospheric nitrogen. Biological nitrogen fixation (SNF) is a very sensitive process to drought and limits legumes agricultural productivity. Several factors are known to regulate SNF including oxygen availability to bacteroids, carbon and nitrogen metabolisms; but the signaling pathways leading to SNF inhibition are largely unknown. In this work, we have performed a proteomic approach of pea plants grown in split-root system where one half of the root was well-irrigated and the other was subjected to drought. Water stress locally provoked nodule water potential decrease that led to SNF local inhibition. The proteomic approach revealed 11 and 7 nodule proteins regulated by drought encoded by Pisum sativum and Rhizobium leguminosarum genomes respectively. Among these 18 proteins, 3 proteins related to flavonoid metabolism, 2 to sulfur metabolism and 3 RNA-binding proteins were identified. These proteins could be molecular targets for future studies focused on the improvement of legumes tolerance to drought. Moreover, this work also provides new hints for the deciphering of SNF regulation machinery in nodules.

Split-root systems applied to the study of the legume-rhizobial symbiosis: what have we learned?

Split-root system (SRS) approaches allow the differential treatment of separate and independent root systems, while sharing a common aerial part. As such, SRS is a useful tool for the discrimination of systemic (shoot origin) versus local (root/nodule origin) regulation mechanisms. This type of approach is particularly useful when studying the complex regulatory mechanisms governing the symbiosis established between legumes and Rhizobium bacteria. The current work provides an overview of the main insights gained from the application of SRS approaches to understand how nodule number (nodulation autoregulation) and nitrogen fixation are controlled both under non-stressful conditions and in response to a variety of stresses. Nodule number appears to be mainly controlled at the systemic level through a signal which is produced by nodule/root tissue, translocated to the shoot, and transmitted back to the root system, involving shoot Leu-rich repeat receptor-like kinases. In contrast, both local and systemic mechanisms have been shown to operate for the regulation of nitrogenase activity in nodules. Under drought and heavy metal stress, the regulation is mostly local, whereas the application of exogenous nitrogen seems to exert a regulation of nitrogen fixation both at the local and systemic levels.

Development of tools for the biochemical characterization of the symbiotic receptor-like kinase DMI2.

The Medicago truncatula DMI2 gene encodes a leucine-rich repeat receptor-like kinase that is essential for symbiosis with nitrogen-fixing rhizobia. While phenotypic analyses have provided a description for the host's responses mediated by DMI2, a lack of tools for in vivo biochemical analysis has hampered efforts to elucidate the mechanisms by which DMI2 mediates symbiotic signal transduction. Here, we report stably transformed M. truncatula lines that express a genomic DMI2 construct that is fused to a dual-affinity tag containing three copies of the hemagglutinin epitope and a single StrepII tag (gDMI2:HAST). gDMI2: HAST complements the dmi2-1 mutation, and transgenic plants expressing this construct behave similarly to wild-type plants. We show that the expression patterns of gDMI2:HAST recapitulate those of endogenous DMI2 and that we can detect and purify DMI2:HAST from microsomal root and nodule extracts. Using this line, we show that DMI2 resides in a high-molecular weight complex, which is consistent with our observation that DMI2:GFP localizes to plasma membrane-associated puncta and cytoplasmic vesicles. We further demonstrate that Nod factor (NF) perception increases the abundance of DMI2 vesicles. These tools should be a valuable resource for the Medicago community to dissect the biochemical function of DMI2.

Is N-feedback involved in the inhibition of nitrogen fixation in drought-stressed Medicago truncatula?

Drought stress is a major factor limiting nitrogen fixation (NF) in crop production. However, the regulatory mechanism involved and the origin of the inhibition, whether local or systemic, is still controversial and so far scarcely studied in temperate forage legumes. Medicago truncatula plants were symbiotically grown with a split-root system and exposed to gradual water deprivation. Physiological parameters, NF activity, and amino acid content were measured. The partial drought treatment inhibited NF in the nodules directly exposed to drought stress. Concomitantly, in the droughted below-ground organs, amino acids accumulated prior to any drop in evapotranspiration (ET). It is concluded that drought exerts a local inhibition of NF and drives an overall accumulation of amino acids in diverse plant organs which is independent of the decrease in ET. The general increase in the majority of single amino acids in the whole plant questions the commonly accepted concept of a single amino acid acting as an N-feedback signal.

Local inhibition of nitrogen fixation and nodule metabolism in drought-stressed soybean.

Drought stress is a major factor limiting symbiotic nitrogen fixation (NF) in soybean crop production. However, the regulatory mechanisms involved in this inhibition are still controversial. Soybean plants were symbiotically grown in a split-root system (SRS), which allowed for half of the root system to be irrigated at field capacity while the other half remained water deprived. NF declined in the water-deprived root system while nitrogenase activity was maintained at control values in the well-watered half. Concomitantly, amino acids and ureides accumulated in the water-deprived belowground organs regardless of transpiration rates. Ureide accumulation was found to be related to the decline in their degradation activities rather than increased biosynthesis. Finally, proteomic analysis suggests that plant carbon metabolism, protein synthesis, amino acid metabolism, and cell growth are among the processes most altered in soybean nodules under drought stress. Results presented here support the hypothesis of a local regulation of NF taking place in soybean and downplay the role of ureides in the inhibition of NF.

Phloem Sap Proteins Are Part of a Core Stress Responsive Proteome Involved in Drought Stress Adjustment.

During moderate drought stress, plants can adjust by changes in the protein profiles of the different organs. Plants transport and modulate extracellular stimuli local and systemically through commonly induced inter- and intracellular reactions. However, most proteins are frequently considered, cell and organelle specific. Hence, while signaling molecules and peptides can travel systemically throughout the whole plant, it is not clear, whether protein isoforms may exist ubiquitously across organs, and what function those may have during drought regulation. By applying shotgun proteomics, we extracted a core proteome of 92 identical protein isoforms, shared ubiquitously amongst several Medicago truncatula tissues, including roots, phloem sap, petioles, and leaves. We investigated their relative distribution across the different tissues and their response to moderate drought stress. In addition, we functionally compared this plant core stress responsive proteome with the organ-specific proteomes. Our study revealed plant ubiquitous protein isoforms, mainly related to redox homeostasis and signaling and involved in protein interaction networks across the whole plant. Furthermore, about 90% of these identified core protein isoforms were significantly involved in drought stress response, indicating a crucial role of the core stress responsive proteome (CSRP) in the plant organ cross-communication, important for a long-distance stress-responsive network. Besides, the data allowed for a comprehensive characterization of the phloem proteome, revealing new insights into its function. For instance, CSRP protein levels involved in stress and redox are relatively more abundant in the phloem compared to the other tissues already under control conditions. This suggests a major role of the phloem in stress protection and antioxidant activity enabling the plants metabolic maintenance and rapid response upon moderate stress. We anticipate our study to be a starting point for future investigations of the role of the core plant proteome. Under an evolutionary perspective, CSRP would enable communication of different cells with each other and the environment being crucial for coordinated stress response of multicellular organisms.

Medicago sativa and Medicago truncatula Show Contrasting Root Metabolic Responses to Drought.

Drought is an environmental stressor that affects crop yield worldwide. Understanding plant physiological responses to stress conditions is needed to secure food in future climate conditions. In this study, we applied a combination of plant physiology and metabolomic techniques to understand plant responses to progressive water deficit focusing on the root system. We chose two legume plants with contrasting tolerance to drought, the widely cultivated alfalfa Medicago sativa (Ms) and the model legume Medicago truncatula (Mt) for comparative analysis. Ms taproot (tapR) and Mt fibrous root (fibR) biomass increased during drought, while a progressive decline in water content was observed in both species. Metabolomic analysis allowed the identification of key metabolites in the different tissues tested. Under drought, carbohydrates, abscisic acid, and proline predominantly accumulated in leaves and tapRs, whereas flavonoids increased in fibRs in both species. Raffinose-family related metabolites accumulated during drought. Along with an accumulation of root sucrose in plants subjected to drought, both species showed a decrease in sucrose synthase (SUS) activity related to a reduction in the transcript level of SUS1, the main SUS gene. This study highlights the relevance of root carbon metabolism during drought conditions and provides evidence on the specific accumulation of metabolites throughout the root system.

Carbon metabolism and bacteroid functioning are involved in the regulation of nitrogen fixation in Medicago truncatula under drought and recovery.

Regulation of symbiotic nitrogen fixation (SNF) during drought stress is complex and not yet fully understood. In the present work, the involvement of nodule C and N metabolism in the regulation of SNF in Medicago truncatula under drought and a subsequent rewatering treatment was analyzed using a combination of metabolomic and proteomic approaches. Drought induced a reduction of SNF rates and major changes in the metabolic profile of nodules, mostly an accumulation of amino acids (Pro, His, and Trp) and carbohydrates (sucrose, galactinol, raffinose, and trehalose). This accumulation was coincidental with a decline in the levels of bacteroid proteins involved in SNF and C metabolism, along with a partial reduction of the levels of plant sucrose synthase 1 (SuSy1). In contrast, the variations in enzymes related to N assimilation were found not to correlate with the reduction in SNF, suggesting that these enzymes do not have a role in the regulation of SNF. Unlike the situation in other legumes such as pea and soybean, the drought-induced inhibition of SNF in M. truncatula appears to be caused by impairment of bacteroid metabolism and N(2)-fixing capacity rather than a limitation of respiratory substrate.

Evidence for transcriptional and post-translational regulation of sucrose synthase in pea nodules by the cellular redox state.

Nitrogen fixation (NF) in legume nodules is very sensitive to environmental constraints. Nodule sucrose synthase (SS; EC 2.4.1.13) has been suggested to play a crucial role in those circumstances because its downregulation leads to an impaired glycolytic carbon flux and, therefore, a depletion of carbon substrates for bacteroids. In the present study, the likelihood of SS being regulated by oxidative signaling has been addressed by the in vivo supply of paraquat (PQ) to nodulated pea plants and the in vitro effects of oxidizing and reducing agents on nodule SS. PQ produced cellular redox imbalance leading to an inhibition of NF. This was preceded by the downregulation of SS gene expression, protein content, and activity. In vitro, oxidizing agents were able to inhibit SS activity and this inhibition was completely reversed by the addition of dithiothreitol. The overall results are consistent with a regulation model of nodule SS exerted by the cellular redox state at both the transcriptional and post-translational levels. The importance of such mechanisms for the regulation of NF in response to environmental stresses are discussed.

Use of recombinant iron-superoxide dismutase as a marker of nitrative stress.

Superoxide dismutases (SODs; EC 1.15.1.1) are a group of metalloenzymes which are essential to protect cells under aerobic conditions. In biological systems, it has been reported that SODs and other proteins are susceptible to be attacked by peroxynitrite (ONOO(-)) which can be originated from the reaction of nitric oxide with superoxide radical. ONOO(-) is a strong oxidant molecule capable of nitrating peptides and proteins at the phenyl side chain of the tyrosine residues. In the present work, bovine serum albumin (BSA) and recombinant iron-superoxide dismutase from the plant cowpea (Vu_FeSOD) are used as target molecules to estimate ONOO(-) production. The method employs the compound SIN-1, which simultaneously generates *NO and O(2)(-) in aerobic aqueous solutions. First, assay conditions were optimized incubating BSA with different concentrations of SIN-1, and at a later stage, the effect on the tyrosine nitration and catalytic activity of Vu_FeSOD was examined by in-gel activity and spectrophotometric assays. Both BSA and Vu_FeSOD are nitrated in a dose-dependent manner, and, at least in BSA nitration, the reaction seems to be metal catalyzed.

Absolute quantification of Medicago truncatula sucrose synthase isoforms and N-metabolism enzymes in symbiotic root nodules and the detection of novel nodule phosphoproteins by mass spectrometry.

Mass spectrometry (MS) has become increasingly important for tissue specific protein quantification at the isoform level, as well as for the analysis of protein post-translational regulation mechanisms and turnover rates. Thanks to the development of high accuracy mass spectrometers, peptide sequencing without prior knowledge of the amino acid sequence--de novo sequencing--can be performed. In this work, absolute quantification of a set of key enzymes involved in carbon and nitrogen metabolism in Medicago truncatula 'Jemalong A17' root nodules is presented. Among them, sucrose synthase (SuSy; EC 2.4.1.13), one of the central enzymes in sucrose cleavage in root nodules, has been further characterized and the relative phosphorylation state of the three most abundant isoforms has been quantified. De novo sequencing provided sequence information of a so far unidentified peptide, most probably belonging to SuSy2, the second most abundant isoform in M. truncatula root nodules. TiO(2)-phosphopeptide enrichment led to the identification of not only a phosphorylation site at Ser11 in SuSy1, but also of several novel phosphorylation sites present in other root nodule proteins such as alkaline invertase (AI; EC 3.2.1.26) and an RNA-binding protein.

Drought Stress Causes a Reduction in the Biosynthesis of Ascorbic Acid in Soybean Plants.

Drought provokes a number of physiological changes in plants including oxidative damage. Ascorbic acid (AsA), also known as vitamin C, is one of the most abundant water-soluble antioxidant compound present in plant tissues. However, little is known on the regulation of AsA biosynthesis under drought stress conditions. In the current work we analyze the effects of water deficit on the biosynthesis of AsA by measuring its content, in vivo biosynthesis and the expression level of genes in the Smirnoff-Wheeler pathway in one of the major legume crop, soybean (Glycine max L. Merr). Since the pathway has not been described in legumes, we first searched for the putative orthologous genes in the soybean genome. We observed a significant genetic redundancy, with multiple genes encoding each step in the pathway. Based on RNA-seq analysis, expression of the complete pathway was detected not only in leaves but also in root tissue. Putative paralogous genes presented differential expression patterns in response to drought, suggesting the existence of functional specialization mechanisms. We found a correlation between the levels of AsA and GalLDH biosynthetic rates in leaves of drought-stressed soybean plants. However, the levels of GalLDH transcripts did not show significant differences under water deficit conditions. Among the other known regulators of the pathway, only the expression of VTC1 genes correlated with the observed decline in AsA in leaves.

Evidence for a rhizobia-induced drought stress response strategy in Medicago truncatula.

Drought stress hampers plant energy and biomass production; however it is still unknown how internal C:N balance and rhizobial symbiosis impact on plant response to water limitation. Here, the effect of differential optimal nitrogen nutrition and root nodule symbiosis on drought stress and rehydration responses of Medicago truncatula was assessed. Two groups of plants were nodulated with Sinorhizobium medicae or Sinorhizobium meliloti--differing in the performance of N fixation; the third group grew in a rhizobia-free medium and received mineral nitrogen fertilizer. In addition to growth analyses, physiological and molecular responses of the two systems were studied using ionomic, metabolomic and proteomic techniques. We found a significant delay in drought-induced leaf senescence in nodulated relative to non-nodulated plants, independent of rhizobial strain and uncoupled from initial leaf N content. The major mechanisms involved are increased concentrations of potassium and shifts in the carbon partitioning between starch and sugars under well-watered conditions, as well as the enhanced allocation of reserves to osmolytes during drought. Consequently, nodulated plants recovered more effectively from drought, relative to non-nodulated M. truncatula. Proteomic data suggest that phytohormone interactions and enhanced translational regulation play a role in increased leaf maintenance in nodulated plants during drought.

Fermentative metabolism is induced by inhibiting different enzymes of the branched-chain amino acid biosynthesis pathway in pea plants.

The inhibition of branched-chain amino acid (BCAA) biosynthesis was evaluated in pea plants in relation to the ability for induction of fermentative metabolism under aerobic conditions. Chlorsulfuron and imazethapyr (inhibitors of acetolactate synthase, ALS, EC 4.1.3.18) produced a strong induction of pyruvate decarboxylase (PDC, EC 4.1.1.1) and alcohol dehydrogenase (ADH, EC 1.1.1.1) activities and a lesser induction of lactate dehydrogenase (LDH, EC 1.1.1.27) and alanine aminotransferase (AlaAT, EC 2.6.1.2) activities in roots. Inhibition of the second enzyme of the BCAA biosynthesis (ketol-acid reductoisomerase, KARI, EC 1.1.1.86) by Hoe 704 (2-dimethylphosphinoyl-2-hydroxyacetic acid) and CPCA (1,1-cyclopropanedicarboxylic acid) enhanced fermentative enzyme activities including PDC, ADH, and AlaAT. Fermentative metabolism induction occurring with ALS- and KARI-inhibitors was related to a higher expression of PDC. In the case of KARI inhibition, it is proposed that fermentation induction is due to an inhibition of ALS activity resulted from an increase in acetolactate concentration. Fermentative metabolism induction in roots, or at least ethanolic fermentation, appeared to be a general physiological response to the BCAA biosynthesis inhibition.

Imazethapyr, an inhibitor of the branched-chain amino acid biosynthesis, induces aerobic fermentation in pea plants.

Acetolactate synthase (ALS; EC 4.1.3.18) inhibition is the primary mechanism of action of imazethapyr (IM). However, the precise mechanisms that links ALS inhibition with plant death have not been elucidated. Supply of IM to pea (Pisum sativum L) plants produced an immediate cessation of growth, caused a 50% inhibition of the in vivo ALS activity within 1 day of treatment, and a remarkable accumulation (2.7-times) of free amino acids after 3 days. Carbohydrates (soluble and starch) were accumulated in both leaves and roots. Accumulation of soluble sugars in roots preceded that of starch in leaves, suggesting that the accumulation of carbohydrates in leaves is not the reason for the arrested root growth. A transient pyruvate accumulation was observed in roots, 1 day after the onset of IM supply. This was coincident with an increase in pyruvate decarboxylase (EC 4.1.1.1), and later increases in alcohol dehydrogenase (EC 1.1.1.1), lactate dehydrogenase (EC 1.1.1.27), and alanine amino transferase (EC 2.6.1.2) activities. This enhancement of fermentative activities was coincident with a slight decrease in aerobic respiration. The overall data suggest that the impairment of ALS activity may lead to a fermentative metabolism that may be involved in growth inhibition and plant death.