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.

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.

Optimization of Protein Isolation and Label-Free Quantitative Proteomic Analysis in Four Different Tissues of Korean Ginseng.

Korean ginseng is one of the most valuable medicinal plants worldwide. However, our understanding of ginseng proteomics is largely limited due to difficulties in the extraction and resolution of ginseng proteins because of the presence of natural contaminants such as polysaccharides, phenols, and glycosides. Here, we compared four different protein extraction methods, namely, TCA/acetone, TCA/acetone-MeOH/chloroform, phenol-TCA/acetone, and phenol-MeOH/chloroform methods. The TCA/acetone-MeOH/chloroform method displayed the highest extraction efficiency, and thus it was used for the comparative proteome profiling of leaf, root, shoot, and fruit by a label-free quantitative proteomics approach. This approach led to the identification of 2604 significantly modulated proteins among four tissues. We could pinpoint differential pathways and proteins associated with ginsenoside biosynthesis, including the methylerythritol 4-phosphate (MEP) pathway, the mevalonate (MVA) pathway, UDP-glycosyltransferases (UGTs), and oxidoreductases (CYP450s). The current study reports an efficient and reproducible method for the isolation of proteins from a wide range of ginseng tissues and provides a detailed organ-based proteome map and a more comprehensive view of enzymatic alterations in ginsenoside biosynthesis.

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.

Variety-specific nutrient acquisition and dehydration-induced proteomic landscape of grasspea (Lathyrus sativus L.).

Grasspea, a stress-resilient pulse crop, has largely remained outside the realm of phytochemical and functional genomics analyses despite its high nutritional significance. To unravel the intervarietal variability in nutrient acquisition of grasspea, we conducted a series of physicochemical experiments using two cultivated varieties, LP-24 and Prateek. The analyses revealed high percentage of starch, cellulose, peroxides, carotenoids, phytic acid and minerals in cv. LP-24, whereas large amounts of protein, soluble carbohydrates and antioxidants in Prateek. To dissect the mechanism of stress tolerance, 3-week-old seedlings of cv. LP-24 and Prateek were afflicted with dehydration for a period of 144 h. The physicochemical indices indicated better adaptation in cv. LP-24, with high abundance of proline, phenolics and flavonoids. Dehydration-responsive proteome landscape of cv. LP-24 revealed 152 proteins with variance at a statistically 94% significance level. The comparative proteomics analysis led to the identification of 120 dehydration-responsive proteins (DRPs), most of which were associated with carbohydrate metabolism, amino acid synthesis, antioxidant reactions and cell defense. We report, for the first time, the dehydration-induced proteome landscape of grasspea, whose genome is yet to be sequenced. The results provide unique insights into variety-specific nutrient acquisition attributes and dehydration-tolerance of grasspea. BIOLOGICAL SIGNIFICANCE: Grasspea is a great source of protein and antioxidants with nitrogen fixing ability, besides its tolerance to multivariate environmental stress as compared to major legume species. This represents the first report on nutrient profile and health-promoting attributes of grasspea. The cultivars under study are nutritionally enriched that possess high protein, amino acids and health-promoting factors and may therefore be projected as a vital part of a healthy diet. Grasspea is known for its hardy nature, water-use efficiency and efficacy as a stress-tolerant pulse. Further, this study portrays the dehydration-responsive proteomic landscape of grasspea. The proteomics analyses provide crucial insights into the dehydration response, presumably orchestrated by proteins belonging to an array of functional classes including photosynthesis, protein and RNA metabolism, protein folding, antioxidant enzymes and defense. The interplay of the differentially regulated proteins might aid in reinforcing the mechanisms of dehydration avoidance and/or tolerance.

Legume proteomics: Progress, prospects, and challenges.

Legumes are the major sources of food and fodder with strong commercial relevance, and are essential components of agricultural ecosystems owing to their ability to carry out endosymbiotic nitrogen fixation. In recent years, legumes have become one of the major choices of plant research. The legume proteomics is currently represented by more than 100 reference maps and an equal number of stress-responsive proteomes. Among the 48 legumes in the protein databases, most proteomic studies have been accomplished in two model legumes, soybean, and barrel medic. This review highlights recent contributions in the field of legume proteomics to comprehend the defence and regulatory mechanisms during development and adaptation to climatic changes. Here, we attempted to provide a concise overview of the progress in legume proteomics and discuss future developments in three broad perspectives: (i) proteome of organs/tissues; (ii) subcellular compartments; and (iii) spatiotemporal changes in response to stress. Such data mining may aid in discovering potential biomarkers for plant growth, in general, apart from essential components involved in stress tolerance. The prospect of integrating proteome data with genome information from legumes will provide exciting opportunities for plant biologists to achieve long-term goals of crop improvement and sustainable agriculture.

Secretome analysis of chickpea reveals dynamic extracellular remodeling and identifies a Bet v1-like protein, CaRRP1 that participates in stress response.

Secreted proteins maintain cell structure and biogenesis besides acting in signaling events crucial for cellular homeostasis during stress adaptation. To understand the underlying mechanism of stress-responsive secretion, the dehydration-responsive secretome was developed from suspension-cultured cells of chickpea. Cell viability of the suspension culture remained unaltered until 96 h, which gradually declined at later stages of dehydration. Proteomic analysis led to the identification of 215 differentially regulated proteins, involved in a variety of cellular functions that include metabolism, cell defence, and signal transduction suggesting their concerted role in stress adaptation. One-third of the secreted proteins were devoid of N-terminal secretion signals suggesting a non-classical secretory route. Screening of the secretome identified a leaderless Bet v 1-like protein, designated CaRRP1, the export of which was inhibited by brefeldin A. We investigated the gene structure and genomic organization and demonstrated that CaRRP1 may be involved in stress response. Its expression was positively associated with abiotic and biotic stresses. CaRRP1 could complement the aberrant growth phenotype of yeast mutant, deficient in vesicular transport, indicating a partial overlap of protein secretion and stress response. Our study provides the most comprehensive analysis of dehydration-responsive secretome and the complex metabolic network operating in plant extracellular space.

Membrane-associated proteomics of chickpea identifies Sad1/UNC-84 protein (CaSUN1), a novel component of dehydration signaling.

Dehydration affects almost all the physiological processes including those that result in the accumulation of misfolded proteins in the endoplasmic reticulum (ER), which in turn elicits a highly conserved signaling, the unfolded protein response (UPR). We investigated the dehydration-responsive membrane-associated proteome of a legume, chickpea, by 2-DE coupled with mass spectrometry. A total of 184 protein spots were significantly altered over a dehydration treatment of 120 h. Among the differentially expressed proteins, a non-canonical SUN domain protein, designated CaSUN1 (Cicer arietinum Sad1/UNC-84), was identified. CaSUN1 localized to the nuclear membrane and ER, besides small vacuolar vesicles. The transcripts were downregulated by both abiotic and biotic stresses, but not by abscisic acid treatment. Overexpression of CaSUN1 conferred stress tolerance in transgenic Arabidopsis. Furthermore, functional complementation of the yeast mutant, slp1, could rescue its growth defects. We propose that the function of CaSUN1 in stress response might be regulated via UPR signaling.