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Item Physiological plasticity to high temperature stress in chickpea: Adaptive responses and variable tolerance(Elsevier B.V., 2019) Pareek, Akanksha; Rathi, Divya; Mishra, Divya; Chakraborty, Subhra; Chakraborty, NiranjanHigh 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.Item Metabolite signatures of grasspea suspension-cultured cells illustrate the complexity of dehydration response(Springer Nature Publishing AG, 2019) Rathi, Divya; Pareek, Akanksha; Zhang, Tong; Pang, Qiuying; Chen, Sixue; Chakraborty, Subhra; Chakraborty, NiranjanGrasspea, 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 profling. Chromatographic separation by HPLC coupled with MRM-MS led to the identifcation 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%), favonoids (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 efects. This represents the frst 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.Item Dehydration-responsive alterations in the chloroplast proteome and cell metabolomic profile of rice reveals key stress adaptation responses(Elsevier B.V., 2019) Gayen, Dipak; Barua, Pragya; Lande, Nilesh Vikram; Varshney, Swati; Sengupta, Shantanu; Chakraborty, Subhra; Chakraborty, NiranjanChloroplast is a semi-autonomous organelle in plants and other photosynthetic eukaryotes, playing a fundamental role of regulating photosynthesis. It is also responsible for sustaining essential biosynthetic reactions including synthesis of amino acids, fatty acids and terpenes. Photosynthesis, the conversion of light energy into chemical energy, serves as the sensor of environmental changes and augments different cellular functions to initiate adaptive responses. However, the molecular processes and regulatory mechanisms of dehydration tolerance adopted by chloroplast remain largely unknown. To gain a better understanding of dehydration response, a chloroplast proteome map of rice was developed. Four-week-old rice seedlings were subjected to dehydration by withholding water for 9 d, and the magnitude of dehydration-induced damage to the chloroplast was monitored. The iTRAQ-based quantitative proteome analysis led to the identification of 40 differentially regulated proteins (DRPs). The DRPs were presumably involved in a wide array of metabolic processes including chloroplast energy metabolism, photosynthesis and defense response. Furthermore, dehydration-induced changes in the metabolite profile and network analysis revealed a high abundance of branched chain amino acids and sugar that might reduce osmotic potential, thereby protecting cellular integrity. The proteomics approach revealed altered status of major photosynthesis related proteins, while cell metabolite profile demonstrated alteration of tricarboxylic acid cycle intermediates, indicating dehydration-triggered alterations in ATP production and energy metabolism. Altogether, these results demonstrated that the global regulation of chloroplast proteome is intimately linked to cellular metabolic rewiring of adaptive responses, which may favor genetic manipulation of crop species for better adaptation.Item Transcriptome profiling illustrates expression signatures of dehydration tolerance in developing grasspea seedlings(Springer Nature, 2019) Rathi, Divya; Gayali, Saurabh; Pareek, Akanksha; Chakraborty, Subhra; Chakraborty, NiranjanMain conclusion This study highlights dehydration-mediated temporal changes in physicochemical, transcriptome and metabolome profles 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-defcit 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-defcit stress during 72–96 h displayed a decrease in pigments, disruption in membrane integrity and osmotic imbalance. We evaluated the temporal efects 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 profling 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 eforts.Item Comparative nuclear proteomics analysis provides insight into the mechanism of signaling and immune response to blast disease caused by Magnoporthe oryzae in rice(John Wiley & Sons, 2019) Narula, Kanika; Choudhary, Pooja; Ghosh, Sudip; Elagamey, Eman; Chakraborty, Niranjan; Chakraborty, SubhraModulation of plant immune system by extrinsic/intrinsic factors and host-specific determinants fine-tunes cellular components involving multiple organelles, particularly nucleus to mount resistance against pathogen attack. Rice blast, caused by hemibiotrophic fungus Magnaporthe oryzae, is one of the most devastating diseases that adversely affect rice productivity. However, the role of nuclear proteins and their regulation in response to M. oryzae remains unknown. Here, we elucidate the nucleus-associated immune pathways in blast resistant rice genotype. Temporal analysis of nuclear proteome was carried out using 2-DE coupled MS/MS analysis. A total of 140 immune responsive proteins (IRPs) were identified associated with nuclear reorganization, cell division, energy production/deprivation, signaling and gene regulation. We interrogated the proteome data using correlation network analysis that identified significant functional modules pointing towards immune related coinciding processes through a common mechanism of remodelling and homeostasis. Novel clues regarding blast resistance include nucleus associated redox homeostasis and glycolytic enzyme mediated chromatin organization which manipulates cell division and immunity. Taken together, our study provides evidence that coordination of nuclear function and reprogramming of host translational machinery regulate resistance mechanism against blast disease.Item Heat shock proteins and abiotic stress tolerance in plants(Springer Nature, 2018) Mishra, Divya; Shekhar, Shubhendu; Singh, Deepika; Chakraborty, Subhra; Chakraborty, NiranjanAbiotic stresses restrict plant growth and development, and reduce harvest index of many crop species worldwide. Maintenance of native conformation of proteins and reducing the accumulation of non-native proteins are imperative for survival under stress conditions as such stresses frequently lead to protein aggregation causing metabolic dysfunction. Heat shock proteins (HSP) play a key role in conferring abiotic stress tolerance. Plants protect themselves from numerous stresses by inducing HSP, besides some stress-responsive proteins, suggesting analogous response mechanisms. A close association between the HSP and ROS also co-exists, indicating that plants have evolved to gain a higher degree of regulation over ROS toxicity and can use ROS as elicitor to induce HSP for better adaptations through activating an array of molecules. Therefore, unraveling the mechanisms of plant response against various stress and the role of HSP in acquired stress tolerance is utmost important to delineate their specific function as a part of stress-responsive module. The HSP have been well characterized in different crop species, albeit the knowledge about their correlation with genome sequence information as well as their functional plasticity is limited.Item Dehydration-induced proteomic landscape of mitochondria in chickpea reveals large-scale coordination of key biological processes(Elsevier B.V, 2019) Gayen, Dipak; Gayali, Saurabh; Barua, Pragya; Lande, Nilesh Vikram; Varshney, Swati; Sengupta, Shantanu; Chakraborty, Subhra; Chakraborty, NiranjanMitochondria play crucial roles in regulating multiple biological processes particularly electron transfer and energy metabolism in eukaryotic cells. Exposure to water-deficit or dehydration may affect mitochondrial function, and dehydration response may dictate cell fate decisions. iTRAQ-based quantitative proteome of a winter legume, chickpea, demonstrated the central metabolic alterations in mitochondria, presumably involved in dehydration adaptation. Three-week-old chickpea seedlings were subjected to progressive dehydration and the magnitude of dehydration-induced compensatory physiological responses was monitored in terms of physicochemical characteristics and mitochondrial architecture. The proteomics analysis led to the identification of 40 dehydration-responsive proteins whose expressions were significantly modulated by dehydration. The differentially expressed proteins were implicated in different metabolic processes, with obvious functional tendencies toward purine-thiamine metabolic network, pathways of carbon fixation and oxidative phosphorylation. The linearity of dehydration-induced proteome alteration was examined with transcript abundance of randomly selected candidates under multivariate stress conditions. The differentially regulated proteins were validated through sequence analysis. An extensive sequence based localization prediction revealed >62.5% proteins to be mitochondrial resident by, at least, one prediction algorithm. The results altogether provide intriguing insights into the dehydration-responsive metabolic pathways and useful clues to identify crucial proteins linked to stress tolerance. BIOLOGICAL SIGNIFICANCE: Investigation on plant mitochondrial proteome is of significance because it would allow a better understanding of mitochondrial function in plant adaptation to stress. Mitochondria are the unique organelles, which play a crucial role in energy metabolism and cellular homeostasis, particularly when exposed to stress conditions. Chickpea is one of the cultivated winter legumes, which enriches soil nitrogen and has very low water footprint and thus contributes to fortification of sustainable agriculture. We therefore examined the dehydration-responsive mitochondrial proteome landscape of chickpea and queried whether molecular interplay of mitochondrial proteins modulate dehydration tolerance. A total of 40 dehydration-induced mitochondrial proteins were identified, predicted to be involved in key metabolic processes. Our future efforts would focus on understanding both posttranslational modification and processing for comprehensive characterization of mitochondrial protein function. This approach will facilitate mining of more biomarkers linked to the tolerance trait and contribute to crop adaptation to climate change.Item Genotype-independent Agrobacterium rhizogenes-mediated root transformation of chickpea: a rapid and efficient method for reverse genetics studies(BioMed Central Ltd, 2018) Aggarwal, Pooja Rani; Nag, Papri; Choudhary, Pooja; Chakraborty, Niranjan; Chakraborty, SubhraBackground: Chickpea (Cicer arietinum L.), an important legume crop is one of the major source of dietary protein. Developing an efcient and reproducible transformation method is imperative to expedite functional genomics studies in this crop. Here, we present an optimized and detailed procedure for Agrobacterium rhizogenes-mediated root transformation of chickpea. Results: Transformation positive roots were obtained on selection medium after two weeks of A. rhizogenes inoculation. Expression of green fuorescent protein further confrmed the success of transformation. We demonstrate that our method adequately transforms chickpea roots at early developmental stage with high efciency. In addition, root transformation was found to be genotype-independent and the efcacy of our protocol was highest in two (Annigiri and JG-62) of the seven tested chickpea genotypes. Next, we present the functional analysis of chickpea hairy roots by expressing Arabidopsis TRANSPARENT TESTA 2 (AtTT2) gene involved in proanthocyanidins biosynthesis. Overexpression of AtTT2 enhanced the level of proanthocyanidins in hairy roots that led to the decreased colonization of fungal pathogen, Fusarium oxysporum. Furthermore, the induction of transgenic roots does not afect functional studies involving infection of roots by fungal pathogen. Conclusions: Transgenic roots expressing genes of interest will be useful in downstream functional characterization using reverse genetics studies. It requires 1 day to perform the root transformation protocol described in this study and the roots expressing transgene can be maintained for 3–4 weeks, providing sufcient time for further functional studies. Overall, the current methodology will greatly facilitate the functional genomics analyses of candidate genes in root-rhizosphere interaction in this recalcitrant but economically important legume crop.Item Carboxylate clamp tetratricopeptide repeat (TPR) domain containing Hsp90 cochaperones in Triticeace: an insight into structural and functional diversification(Elsevier B.V., 2018) Mishra, Divya; Shekhar, Shubhendu; Chakraborty, Subhra; Chakraborty, NiranjanThe molecular chaperones serve as surveillance molecules that mediates regulatory crosstalk between protein folding and degradation pathways under natural and stress conditions. In present study, we focused on the diversification and role of tetratricopeptide repeat (TPR) domain containing Hsp90 cochaperones. These cochaperone were recognized by the presence of three motifs of TPR with the basic conserved residues often referred to as carboxylate clamp (CC). A total of 213 putative CC-TPRs were found in Triticeace, clustered into 16 groups, amongst which few CC-TPR families such as TPR-RPAP3 and TPR-SMYD were documented. Domain architecture and genomic organization revealed that CC-TPRs are very diverse in nature. Evolutionary analyses showed that CC-TPRs are conserved, stable and ubiquitous in nature. Analysis of available RNA-seq data revealed a high degree of tissue-specific expression of 1-TPR and TaTPR-FKBP family members at various developmental stages. The transcripts of TaCC-TPRs displayed differential expression in two contrasting wheat cultivars under abiotic stress conditions. Complementation and heterologous expression of TaTPR-FKBP5 in yeast conferred abiotic stress tolerance. Together, these results provide a glimpse into the genetic diversity and evolution of CC-TPRs in Triticeace, which would help to better understand of how TPR-domain cochaperones function in plants.Item Comparative analysis of sequence-structure function relationship of the SUN-domain protein CaSUN1(OMICS International, 2017) Mishra, Poonam; Wardhan, Vijay; Pandey, Aarti; Chakraborty, Subhra; Garg, Gunjan; Chakraborty, NiranjanSad1/UNC-84 (SUN)-domain proteins are residents of inner nuclear membrane (INM), and share structural features across species. We previously reported a highly conserved C-terminal SUN-domain family protein, designated CaSUN1, in the stress-responsive proteomic landscape of a grain legume, chickpea. In this study, we identified two other chickpea SUN proteins, CaSUN2 and CaSUN3, and performed a comparative analysis of the sequence-structure-function relationship to better understand the diversification of SUN-domain superfamily proteins. Sequence similarity across the species was investigated using multiple sequence alignment, which showed conserved patterns between CaSUN1 and the homologs. Phylogenetic analysis showed that plant SUN-domain proteins are clustered in a unique and distinct group. Using ab-initio approach, a 3D protein structure was generated and further validated using various tools including the Ramachandran plot. The results displayed 90.1% of the à  and à ± residues angles in the most favoured regions, suggesting a high-quality structural model for CaSUN1. Model deviation and fluctuation analysis were performed using molecular dynamics (MD) simulation of CaSUN1. The secondary structure analysis of CaSUN revealed a similarity between the structural components shared among them. CaSUN1 revealed two functional domains viz., SUN and muskelin, and the presence of kelch-repeat domain pointed out its putative role in oligomerization, while its binding affinity with different ligands indicates diverse functions. These results would not only give deeper insights into the structure-function relationships within the SUNsuperfamily proteins, but also their putative physiological roles.