Proteomic signatures uncover phenotypic plasticity of susceptible and resistant genotypes by wall remodelers in rice blast.

Molecular communication between macromolecules dictates extracellular matrix (ECM) dynamics during pathogen recognition and disease development. Extensive research has shed light on how plant immune components are activated, regulated and function in response to pathogen attack. However, two key questions remain largely unresolved: (i) how does ECM dynamics govern susceptibility and disease resistance, (ii) what are the components that underpin these phenomena? Rice blast, caused by Magnaporthe oryzae adversely affects rice productivity. To understand ECM regulated genotype-phenotype plasticity in blast disease, we temporally profiled two contrasting rice genotypes in disease and immune state. Morpho-histological, biochemical and electron microscopy analyses revealed that increased necrotic lesions accompanied by electrolyte leakage governs disease state. Wall carbohydrate quantification showed changes in pectin level was more significant in blast susceptible compared to blast resistant cultivar. Temporally resolved quantitative disease- and immune-responsive ECM proteomes identified 308 and 334 proteins, respectively involved in wall remodelling and integrity, signalling and disease/immune response. Pairwise comparisons between time and treatment, messenger ribonucleic acid expression, diseasome and immunome networks revealed novel blast-related functional modules. Data demonstrated accumulation of α-galactosidase and phosphatase were associated with disease state, while reactive oxygen species, induction of Lysin motif proteins, CAZymes and extracellular Ca-receptor protein govern immune state.

Proteo-metabolomic Dissection of Extracellular Matrix Reveals Alterations in Cell Wall Integrity and Calcium Signaling Governs Wall-Associated Susceptibility during Stem Rot Disease in Jute.

The plant surveillance system confers specificity to disease and immune states by activating distinct molecular pathways linked to cellular functionality. The extracellular matrix (ECM), a preformed passive barrier, is dynamically remodeled at sites of interaction with pathogenic microbes. Stem rot, caused by Macrophomina phaseolina, adversely affects fiber production in jute. However, how wall related susceptibility affects the ECM proteome and metabolome remains undetermined in bast fiber crops. Here, stem rot responsive quantitative temporal ECM proteome and metabolome were developed in jute upon M. phaseolina infection. Morpho-histological examination revealed that leaf shredding was accompanied by reactive oxygen species production in patho-stressed jute. Electron microscopy showed disease progression and ECM architecture remodeling due to necrosis in the later phase of fungal attack. Using isobaric tags for relative and absolute quantitative proteomics and liquid chromatography-tandem mass spectrometry, we identified 415 disease-responsive proteins involved in wall integrity, acidification, proteostasis, hydration, and redox homeostasis. The disease-related correlation network identified functional hubs centered on α-galactosidase, pectinesterase, and thaumatin. Gas chromatography-mass spectrometry analysis pointed toward enrichment of disease-responsive metabolites associated with the glutathione pathway, TCA cycle, and cutin, suberin, and wax metabolism. Data demonstrated that wall-degrading enzymes, structural carbohydrates, and calcium signaling govern rot responsive wall-susceptibility. Proteomics data were deposited in Pride (PXD046937; PXD046939).

Combining extracellular matrix proteome and phosphoproteome of chickpea and meta-analysis reveal novel proteoforms and evolutionary significance of clade-specific wall-associated events in plant.

Extracellular matrix (ECM) plays central roles in cell architecture, innate defense and cell wall integrity (CWI) signaling. During transition to multicellularity, modular domain structures of ECM proteins and proteoforms have evolved due to continuous adaptation across taxonomic clades under different ecological niche. Although this incredible diversity has to some extent been investigated at protein level, extracellular phosphorylation events and molecular evolution of ECM proteoform families remains unexplored. We developed matrisome proteoform atlas in a grain legume, chickpea and performed meta-analyses of 74 plant matrisomes. MS/MS analysis identified 1,424 proteins and 315 phosphoproteins involved in diverse functions. Cross-species ECM protein network identified proteoforms associated with CWI maintenance system. Phylogenetic characterization of eighteen matrix protein families highlighted the role of taxon-specific paralogs and orthologs. Novel information was acquired on gene expansion and loss, co-divergence, sub functionalization and neofunctionalization during evolution. Modular networks of matrix protein families and hub proteins showed higher diversity across taxonomic clades than among organs. Furthermore, protein families differ in nonsynonymous to synonymous substitution rates. Our study pointed towards the matrix proteoform functionality, sequence divergence variation, interactions between wall remodelers and molecular evolution using a phylogenetic framework. This is the first report on comprehensive matrisome proteoform network illustrating presence of CWI signaling proteins in land plants.

Dehydration-responsive cytoskeleton proteome of rice reveals reprograming of key molecular pathways to mediate metabolic adaptation and cell survival.

The plant cytoskeletal proteins play a key role that control cytoskeleton dynamics, contributing to crucial biological processes such as cell wall morphogenesis, stomatal conductance and abscisic acid accumulation in repercussion to water-deficit stress or dehydration. Yet, it is still completely unknown which specific biochemical processes and regulatory mechanisms the cytoskeleton uses to drive dehydration tolerance. To better understand the role of cytoskeleton, we developed the dehydration-responsive cytoskeletal proteome map of a resilient rice cultivar. Initially, four-week-old rice plants were exposed to progressive dehydration, and the magnitude of dehydration-induced compensatory physiological responses was monitored in terms of physicochemical indices. The organelle fractionation in conjunction with label-free quantitative proteome analysis led to the identification of 955 dehydration-responsive cytoskeletal proteins (DRCPs). To our knowledge, this is the first report of a stress-responsive plant cytoskeletal proteome, representing the largest inventory of cytoskeleton and cytoskeleton-associated proteins. The DRCPs were apparently involved in a wide array of intra-cellular molecules transportation, organelles positioning, cytoskeleton organization followed by different metabolic processes including amino acid metabolism. These findings presented open a unique view on global regulation of plant cytoskeletal proteome is intimately linked to cellular metabolic rewiring of adaptive responses, and potentially confer dehydration tolerance, especially in rice, and other crop species, in general.

The plant cytoskeleton takes center stage in abiotic stress responses and resilience.

Stress resilience behaviours in plants are defensive mechanisms that develop under adverse environmental conditions to promote growth, development and yield. Over the past decades, improving stress resilience, especially in crop species, has been a focus of intense research for global food security and economic growth. Plants have evolved specific mechanisms to sense external stress and transmit information to the cell interior and generate appropriate responses. Plant cytoskeleton, comprising microtubules and actin filaments, takes a center stage in stress-induced signalling pathways, either as a direct target or as a signal transducer. In the past few years, it has become apparent that the function of the plant cytoskeleton and other associated proteins are not merely limited to elementary processes of cell growth and proliferation, but they also function in stress response and resilience. This review summarizes recent advances in the role of plant cytoskeleton and associated proteins in abiotic stress management. We provide a thorough overview of the mechanisms that plant cells employ to withstand different abiotic stimuli such as hypersalinity, dehydration, high temperature and cold, among others. We also discuss the crucial role of the plant cytoskeleton in organellar positioning under the influence of high light intensity.

Suspension cell secretome of the grain legume Lathyrus sativus (grasspea) reveals roles in plant development and defense responses.

Plant secretomics has been especially important in understanding the molecular basis of plant development, stress resistance and biomarker discovery. In addition to sharing a similar role in maintaining cell metabolism and biogenesis with the animal secretome, plant-secreted proteins actively participate in signaling events crucial for cellular homeostasis during stress adaptation. However, investigation of the plant secretome remains largely overlooked, particularly in pulse crops, demanding urgent attention. To better understand the complexity of the secretome, we developed a reference map of a stress-resilient orphan legume, Lathyrus sativus (grasspea), which can be utilized as a potential proteomic resource. Secretome analysis of L. sativus led to the identification of 741 nonredundant proteins belonging to a myriad of functional classes, including antimicrobial, antioxidative and redox potential. Computational prediction of the secretome revealed that ∼29% of constituents are predicted to follow unconventional protein secretion (UPS) routes. We conducted additional in planta analysis to determine the localization of two secreted proteins, recognized as cell surface residents. Sequence-based homology comparison revealed that L. sativus shares ∼40% of the constituents reported thus far from in vitro and in planta secretome analysis in model and crop species. Significantly, we identified 571 unique proteins secreted from L. sativus involved in cell-to-cell communication, organ development, kinase-mediated signaling, and stress perception, among other critical roles. Conclusively, the grasspea secretome participates in putative crosstalk between genetic circuits that regulate developmental processes and stress resilience.

Dehydration-responsive chickpea chloroplast protein, CaPDZ1, confers dehydration tolerance by improving photosynthesis.

The screening of a dehydration-responsive chloroplast proteome of chickpea led us to identify and investigate the functional importance of an uncharacterized protein, designated CaPDZ1. In all, we identified 14 CaPDZs, and phylogenetic analysis revealed that these belong to photosynthetic eukaryotes. Sequence analyses of CaPDZs indicated that CaPDZ1 is a unique member, which harbours a TPR domain besides a PDZ domain. The global expression analysis showed that CaPDZs are intimately associated with various stresses such as dehydration and oxidative stress along with certain phytohormone responses. The CaPDZ1-overexpressing chickpea seedlings exhibited distinct phenotypic and molecular responses, particularly increased photosystem (PS) efficiency, ETR and qP that validated its participation in PSII complex assembly and/or repair. The investigation of CaPDZ1 interacting proteins through Y2H library screening and co-IP analysis revealed the interacting partners to be PSII associated CP43, CP47, D1, D2 and STN8. These findings supported the earlier hypothesis regarding the role of direct or indirect involvement of PDZ proteins in PS assembly or repair. Moreover, the GUS-promoter analysis demonstrated the preferential expression of CaPDZ1 specifically in photosynthetic tissues. We classified CaPDZ1 as a dehydration-responsive chloroplast intrinsic protein with multi-fold abundance under dehydration stress, which may participate synergistically with other chloroplast proteins in the maintenance of the photosystem.

Dissection of grasspea (Lathyrus sativus L.) root exoproteome reveals critical insights and novel proteins.

The plant exoproteome is crucial because its constituents greatly influence plant phenotype by regulating physiological characteristics to adapt to environmental stresses. The root exudates constitute a dynamic aspect of plant exoproteome, as its molecular composition ensures a beneficial rhizosphere in a species-specific manner. We investigated the root exoproteome of grasspea, a stress-resilient pulse and identified 2861 non-redundant proteins, belonging to a myriad of functional classes, including root development, rhizosphere augmentation as well as defense functions against soil-borne pathogens. Significantly, we identified 1986 novel exoproteome constituents of grasspea, potentially involved in cell-to-cell communication and root meristem maintenance, among other critical roles. Sequence-based comparison revealed that grasspea shares less than 30 % of its exoproteome with the reports so far from model plants as well as crop species. Further, the exoproteome revealed 65 % proteins to be extracellular in nature and of these, 37 % constituents were predicted to follow unconventional protein secretion (UPS) mode. We validated the UPS for four stress-responsive proteins, which were otherwise predicted to follow classical protein secretion (CPS). Conclusively, we recognized not only the highest number of root exudate proteins, but also pinpointed novel signatures of dicot root exoproteome.

Proteomic dissection of rice cytoskeleton reveals the dominance of microtubule and microfilament proteins, and novel components in the cytoskeleton-bound polysome.

The plant cytoskeleton persistently undergoes remodeling to achieve its roles in supporting cell division, differentiation, cell expansion and organelle transport. However, the links between cell metabolism and cytoskeletal networks, particularly how the proteinaceous components execute such processes remain poorly understood. We investigated the cytoskeletal proteome landscape of rice to gain better understanding of such events. Proteins were extracted from highly enriched cytoskeletal fraction of four-week-old rice seedlings, and the purity of the fraction was stringently monitored. A total of 2577 non-redundant proteins were identified using both gel-based and gel-free approaches, which constitutes the most comprehensive dataset, thus far, for plant cytoskeleton. The data set includes both microtubule and microfilament-associated proteins and their binding proteins comprising hypothetical as well as novel cytoskeletal proteins. Further, various in-silico analyses were performed, and the proteins were functionally classified on the basis of their gene ontology. The catalogued proteins were validated through their sequence analysis. Extensive comparative analysis of our dataset with the non-redundant set of cytoskeletal proteins across plant species affirms unique as well as overlapping candidates. Together, these findings unveil new insights of how cytoskeletons undergo dynamic remodeling in rice to drive seedling development processes in rapidly changing in planta environment.

Wheat 2-Cys peroxiredoxin plays a dual role in chlorophyll biosynthesis and adaptation to high temperature.

The molecular mechanism of high-temperature stress (HTS) response, in plants, has so far been investigated using transcriptomics, while the dynamics of HTS-responsive proteome remain unexplored. We examined the adaptive responses of the resilient wheat cultivar 'Unnat Halna' and dissected the HTS-responsive proteome landscape. This led to the identification of 55 HTS-responsive proteins (HRPs), which are predominantly involved in metabolism and defense pathways. Interestingly, HRPs included a 2-cysteine peroxiredoxin (2CP), designated Ta2CP, presumably involved in stress perception and adaptation. Complementation of Ta2CP in yeast and heterologous expression in Arabidopsis demonstrated its role in thermotolerance. Both Ta2CP silencing and overexpression inferred the involvement of Ta2CP in plant growth and chlorophyll biosynthesis. We demonstrated that Ta2CP interacts with protochlorophyllide reductase b, TaPORB. Reduced TaPORB expression was found in Ta2cp-silenced plants, while upregulation was observed in Ta2CP-overexpressed plants. Furthermore, the downregulation of Ta2CP in Taporb-silenced plants and reduction of protochlorophyllide in Ta2cp-silenced plants suggested the key role of Ta2CP in chlorophyll metabolism. Additionally, the transcript levels of AGPase1 and starch were increased in Ta2cp-silenced plants. More significantly, HTS-treated Ta2cp-silenced plants showed adaptive responses despite increased reactive oxygen species and peroxide concentrations, which might help in rapid induction of high-temperature acclimation.

Systemic acquired resistance specific proteome of Arabidopsis thaliana.

KEY MESSAGE: A comparative proteomic study between WT and SAR-compromised rsi1/fld mutant reveals a set of proteins having possible roles in the SAR development. A partly infected plant shows enhanced resistance during subsequent infection through the development of systemic acquired resistance (SAR). Mobile signals generated at the site of primary infection travel across the plant for the activation of SAR. These mobile signals are likely to cause changes in the expression of a set of proteins in the distal tissue, which contributes to the SAR development. However, SAR-specific proteome is not revealed for any plant. The reduced systemic immunity 1 (rsi1)/(allelic to flowering locus D; fld) mutant of Arabidopsis is compromised for SAR but shows normal local resistance. Here we report the SAR-specific proteome of Arabidopsis by comparing differentially abundant proteins (DAPs) between WT and fld mutant. Plants were either mock-treated or SAR-induced by primary pathogen inoculation. For proteomic analysis, samples were collected from the systemic tissues before and after the secondary inoculation. Protein identification was carried out by using two-dimensional gel electrophoresis (2-DE) followed by tandem mass spectrometry. Our work identified a total of 94 DAPs between mock and pathogen treatment in WT and fld mutant. The DAPs were categorized into different functional groups along with their subcellular localization. The majority of DAPs are involved in metabolic processes and stress response. Among the subcellular compartments, plastids contained the highest number of DAPs, suggesting the importance of plastidic proteins in SAR activation. The findings of this study would provide resources to engineer efficient SAR activation traits in Arabidopsis and other plants.

Integrated Seed Proteome and Phosphoproteome Analyses Reveal Interplay of Nutrient Dynamics, Carbon-Nitrogen Partitioning, and Oxidative Signaling in Chickpea.

Nutrient dynamics in storage organs is a complex developmental process that requires coordinated interactions of environmental, biochemical, and genetic factors. Although sink organ developmental events have been identified, understanding of translational and post-translational regulation of reserve synthesis, accumulation, and utilization in legumes is limited. To understand nutrient dynamics during embryonic and cotyledonary photoheterotrophic transition to mature and germinating autotrophic seeds, an integrated proteomics and phosphoproteomics study in six sequential seed developmental stages in chickpea is performed. MS/MS analyses identify 109 unique nutrient-associated proteins (NAPs) involved in metabolism, storage and biogenesis, and protein turnover. Differences and similarities in 60 nutrient-associated phosphoproteins (NAPPs) containing 93 phosphosites are compared with NAPs. Data reveal accumulation of carbon-nitrogen metabolic and photosynthetic proteoforms during seed filling. Furthermore, enrichment of storage proteoforms and protease inhibitors is associated with cell expansion and seed maturation. Finally, combined proteoforms network analysis identifies three significant modules, centered around malate dehydrogenase, HSP70, triose phosphate isomerase, and vicilin. Novel clues suggest that ubiquitin-proteasome pathway regulates nutrient reallocation. Second, increased abundance of NAPs/NAPPs related to oxidative and serine/threonine signaling indicates direct interface between redox sensing and signaling during seed development. Taken together, nutrient signals act as metabolic and differentiation determinant governing storage organ reprogramming.

Chitosan-triggered immunity to Fusarium in chickpea is associated with changes in the plant extracellular matrix architecture, stomatal closure and remodeling of the plant metabolome and proteome.

Pathogen-/microbe-associated molecular patterns (PAMPs/MAMPs) initiate complex defense responses by reorganizing the biomolecular dynamics of the host cellular machinery. The extracellular matrix (ECM) acts as a physical scaffold that prevents recognition and entry of phytopathogens, while guard cells perceive and integrate signals metabolically. Although chitosan is a known MAMP implicated in plant defense, the precise mechanism of chitosan-triggered immunity (CTI) remains unknown. Here, we show how chitosan imparts immunity against fungal disease. Morpho-histological examination revealed stomatal closure accompanied by reductions in stomatal conductance and transpiration rate as early responses in chitosan-treated seedlings upon vascular fusariosis. Electron microscopy and Raman spectroscopy showed ECM fortification leading to oligosaccharide signaling, as documented by increased galactose, pectin and associated secondary metabolites. Multiomics approach using quantitative ECM proteomics and metabolomics identified 325 chitosan-triggered immune-responsive proteins (CTIRPs), notably novel ECM structural proteins, LYM2 and receptor-like kinases, and 65 chitosan-triggered immune-responsive metabolites (CTIRMs), including sugars, sugar alcohols, fatty alcohols, organic and amino acids. Identified proteins and metabolites are linked to reactive oxygen species (ROS) production, stomatal movement, root nodule development and root architecture coupled with oligosaccharide signaling that leads to Fusarium resistance. The cumulative data demonstrate that ROS, NO and eATP govern CTI, in addition to induction of PR proteins, CAZymes and PAL activities, besides accumulation of phenolic compounds downstream of CTI. The immune-related correlation network identified functional hubs in the CTI pathway. Altogether, these shifts led to the discovery of chitosan-responsive networks that cause significant ECM and guard cell remodeling, and translate ECM cues into cell fate decisions during fusariosis.

Quantitative Phosphoproteomic Analysis of Legume Using TiO2-Based Enrichment Coupled with Isobaric Labeling.

Phosphorylation of proteins is the most dynamic protein modification, and its analysis aids in determining the functional and regulatory principles of important cellular pathways. The legumes constitute the third largest family of higher plants, Fabaceae, comprising about 20,000 species and are second to cereals in agricultural importance on the basis of global production. Therefore, an understanding of the developmental and adaptive processes of legumes demands identification of their regulatory components. The most crucial signature of the legume family is the symbiotic nitrogen fixation, which makes this fascinating and interesting to investigate phosphorylation events. The research on protein phosphorylation in legumes has been focused primarily on two model species, Medicago truncatula and Lotus japonicus. The development of reciprocal research in other species, particularly the crops, is lagging behind which has limited its beneficial uses in agricultural productivity. In this chapter, we outline the titanium dioxide-based enrichment of phosphopeptides for nuclear proteome analysis of a grain legume, chickpea.

Extracellular Matrix Proteome: Isolation of ECM Proteins for Proteomics Studies.

Understanding molecular mechanisms and cellular metabolism in varied plant processes necessitates knowledge of the expressed proteins and their subcellular distribution. Spatial partitioning of organelles generates an enclosed milieu for physiochemical reactions designed and tightly linked to a specific organelle function. Of which, extracellular matrix (ECM)/cell wall (CW) is a dynamic and chemically active compartment. The ECM proteins are organized into complex structural and functional networks involved in several metabolic processes, including carbon and nitrogen metabolism. Organellar proteomics aim for comprehensive identification of resident proteins that rely on the isolation of highly purified organelle free from contamination by other intracellular components. Extraction and isolation of plant ECM proteins features key caveats due to the lack of adjoining membrane, the presence of a polysaccharide-protein network that traps contaminants, and the existence of high phenolic content. Furthermore, due to diverse biochemical forces, including labile, weakly bound and strongly bound protein in the protein-polysaccharide matrix different elution procedures are required to enrich ECM proteins. Here, we describe a method that allows efficient fractionation of plant ECM, extraction of ECM proteins and protein profiling from variety of crop plants, including rice, chickpea and potato. This method can easily be adapted to other plant species for varied experimental conditions.

Dehydration-induced alterations in chloroplast proteome and reprogramming of cellular metabolism in developing chickpea delineate interrelated adaptive responses.

Chloroplast, the energy organelle unique to photosynthetic eukaryotes, executes several crucial functions including photosynthesis. While chloroplast development and function are controlled by the nucleus, environmental stress modulated alterations perceived by the chloroplasts are communicated to the nucleus via retrograde signaling. Notably, coordination of chloroplast and nuclear gene expression is synchronized by anterograde and retrograde signaling. The chloroplast proteome holds significance for stress responses and adaptation. We unraveled dehydration-induced alterations in the chloroplast proteome of a grain legume, chickpea and identified an array of dehydration-responsive proteins (DRPs) primarily involved in photosynthesis, carbohydrate metabolism and stress response. Notably, 12 DRPs were encoded by chloroplast genome, while the rest were nuclear-encoded. We observed a coordinated expression of different multi-subunit protein complexes viz., RuBisCo, photosystem II and cytochrome b6f, encoded by both chloroplast and nuclear genome. Comparison with previously reported stress-responsive chloroplast proteomes showed unique and overlapping components. Transcript abundance of several previously reported markers of retrograde signaling revealed relay of dehydration-elicited signaling events between chloroplasts and nucleus. Additionally, dehydration-triggered metabolic adjustments demonstrated alterations in carbohydrate and amino acid metabolism. This study offers a panoramic catalogue of dehydration-responsive signatures of chloroplast proteome and associated retrograde signaling events, and cellular metabolic reprograming.

Proteomic dissection of the chloroplast: Moving beyond photosynthesis.

Chloroplast, the photosynthetic machinery, converts photoenergy to ATP and NADPH, which powers the production of carbohydrates from atmospheric CO2 and H2O. It also serves as a major production site of multivariate pro-defense molecules, and coordinate with other organelles for cell defense. Chloroplast harbors 30-50% of total cellular proteins, out of which 80% are membrane residents and are difficult to solubilize. While proteome profiling has illuminated vast areas of biological protein space, a great deal of effort must be invested to understand the proteomic landscape of the chloroplast, which plays central role in photosynthesis, energy metabolism and stress-adaptation. Therefore, characterization of chloroplast proteome would not only provide the foundation for future investigation of expression and function of chloroplast proteins, but would open up new avenues for modulation of plant productivity through synchronizing chloroplastic key components. In this review, we summarize the progress that has been made to build new understanding of the chloroplast proteome and implications of chloroplast dynamicsing generate metabolic energy and modulating stress adaptation.

Physiological plasticity to high temperature stress in chickpea: Adaptive responses and variable tolerance.

High temperature stress (HTS) is one of the most crucial factors that limits plant growth and development, and reduces crop yields worldwide. Cool-season crops, particularly the legumes, are severely affected by increasing ambient temperature associated with global climate change. We characterized the HTS-induced modulations of morpho-physicochemical traits and gene expression of several chickpea genotypes and the metabolic profile of the tolerant cultivar. Higher water use efficiency and photosynthetic capacity, minimal membrane lipid peroxidation in conjunction with increased abundance of osmolytes and secondary metabolites depicted thermotolerance of ICC 1205. The adaptive responses were accompanied by high transcript abundance of heat shock proteins and antioxidant enzymes. To integrate stress-responsive signalling and metabolic networks, the HTS-induced physicochemical analysis was further extended to metabolite profiling of the thermotolerant cultivar. The screening of the metabolome landscape led to the identification of 49 HTS-responsive metabolites that include polycarboxylic acid, sugar acids, sugar alcohols and amino acids which might confer thermotolerance in chickpea. The present study, to our knowledge, is the most comprehensive of its kind in dissecting cultivar-specific differential adaptive responses to HTS in chickpea, which might potentiate the identification of genetic traits extendible to improvement of thermotolerance of crops.

Metabolite signatures of grasspea suspension-cultured cells illustrate the complexity of dehydration response.

MAIN CONCLUSION: This represents the first report deciphering the dehydration response of suspension-cultured cells of a crop species, highlighting unique and shared pathways, and adaptive mechanisms via profiling of 330 metabolites. Grasspea, being a hardy legume, is an ideal model system to study stress tolerance mechanisms in plants. In this study, we investigated the dehydration-responsive metabolome in grasspea suspension-cultured cells (SCCs) to identify the unique and shared metabolites crucial in imparting dehydration tolerance. To reveal the dehydration-induced metabolite signatures, SCCs of grasspea were exposed to 10% PEG, followed by metabolomic profiling. Chromatographic separation by HPLC coupled with MRM-MS led to the identification of 330 metabolites, designated dehydration-responsive metabolites (DRMs), which belonged to 28 varied functional classes. The metabolome was found to be constituted by carboxylic acids (17%), amino acids (13.5%), flavonoids (10.9%) and plant growth regulators (10%), among others. Pathway enrichment analysis revealed predominance of metabolites involved in phytohormone biosynthesis, secondary metabolism and osmotic adjustment. Exogenous application of DRMs, arbutin and acetylcholine, displayed improved physiological status in stress-resilient grasspea as well as hypersensitive pea, while administration of lauric acid imparted detrimental effects. This represents the first report on stress-induced metabolomic landscape of a crop species via a suspension culture system, which would provide new insights into the molecular mechanism of stress responses and adaptation in crop species.

Transcriptome profiling illustrates expression signatures of dehydration tolerance in developing grasspea seedlings.

MAIN CONCLUSION: This study highlights dehydration-mediated temporal changes in physicochemical, transcriptome and metabolome profiles indicating altered gene expression and metabolic shifts, underlying endurance and adaptation to stress tolerance in the marginalized crop, grasspea. Grasspea, often regarded as an orphan legume, is recognized to be fairly tolerant to water-deficit stress. In the present study, 3-week-old grasspea seedlings were subjected to dehydration by withholding water over a period of 144 h. While there were no detectable phenotypic changes in the seedlings till 48 h, the symptoms appeared during 72 h and aggravated upon prolonged dehydration. The physiological responses to water-deficit stress during 72-96 h displayed a decrease in pigments, disruption in membrane integrity and osmotic imbalance. We evaluated the temporal effects of dehydration at the transcriptome and metabolome levels. In total, 5201 genes of various functional classes including transcription factors, cytoplasmic enzymes and structural cell wall proteins, among others, were found to be dehydration-responsive. Further, metabolome profiling revealed 59 dehydration-responsive metabolites including sugar alcohols and amino acids. Despite the lack of genome information of grasspea, the time course of physicochemical and molecular responses suggest a synchronized dehydration response. The cross-species comparison of the transcriptomes and metabolomes with other legumes provides evidence for marked molecular diversity. We propose a hypothetical model that highlights novel biomarkers and explain their relevance in dehydration-response, which would facilitate targeted breeding and aid in commencing crop improvement efforts.