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.

Dehydration-responsive nuclear proteome landscape of chickpea (Cicer arietinum L.) reveals phosphorylation-mediated regulation of stress response.

Nonavailability of water or dehydration remains recurring climatic disorder affecting yield of major food crops, legumes in particular. Nuclear proteins (NPs) and phosphoproteins (NPPs) execute crucial cellular functions that form the regulatory hub for coordinated stress response. Phosphoproteins hold enormous influence over cellular signalling. Four-week-old seedlings of a grain legume, chickpea, were subjected to gradual dehydration, and NPs were extracted from unstressed control and from 72- and 144-hr stressed tissues. We identified 4,832 NPs and 478 phosphosites, corresponding to 299 unique NPPs involved in multivariate cellular processes including protein modification and gene expression regulation, among others. The identified proteins included several novel kinases, phosphatases, and transcription factors, besides 660 uncharacterized proteins. Spliceosome complex and splicing related proteins were dominant among differentially regulated NPPs, indicating their dehydration modulated regulation. Phospho-motif analysis revealed stress-induced enrichment of proline-directed serine phosphorylation. Association mapping of NPPs revealed predominance of differential phosphorylation of spliceosome and splicing associated proteins. Also, regulatory proteins of key processes viz., protein degradation, regulation of flowering time, and circadian clock were observed to undergo dehydration-induced dephosphorylation. The characterization of novel regulatory proteins would provide new insights into stress adaptation and enable directed genetic manipulations for developing climate-resilient crops.

CicerTransDB 1.0: a resource for expression and functional study of chickpea transcription factors.

BACKGROUND: Transcription factor (TF) databases are major resource for systematic studies of TFs in specific species as well as related family members. Even though there are several publicly available multi-species databases, the information on the amount and diversity of TFs within individual species is fragmented, especially for newly sequenced genomes of non-model species of agricultural significance. DESCRIPTION: We constructed CicerTransDB (Cicer Transcription Factor Database), the first database of its kind, which would provide a centralized putatively complete list of TFs in a food legume, chickpea. CicerTransDB, available at www.cicertransdb.esy.es , is based on chickpea (Cicer arietinum L.) annotation v 1.0. The database is an outcome of genome-wide domain study and manual classification of TF families. This database not only provides information of the gene, but also gene ontology, domain and motif architecture. CONCLUSION: CicerTransDB v 1.0 comprises information of 1124 genes of chickpea and enables the user to not only search, browse and download sequences but also retrieve sequence features. CicerTransDB also provides several single click interfaces, transconnecting to various other databases to ease further analysis. Several webAPI(s) integrated in the database allow end-users direct access of data. A critical comparison of CicerTransDB with PlantTFDB (Plant Transcription Factor Database) revealed 68 novel TFs in the chickpea genome, hitherto unexplored. Database URL: http://www.cicertransdb.esy.es.

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.

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.

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.

Dehydration-induced proteomic landscape of mitochondria in chickpea reveals large-scale coordination of key biological processes.

Mitochondria 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.

Global Proteomic Profiling and Identification of Stress-Responsive Proteins Using Two-Dimensional Gel Electrophoresis.

Global proteome profiling is a direct representation of the protein set in an organism, organ, tissues, or an organelle. One of the main objectives of proteomic analysis is the comparison and relative quantitation of proteins under a defined set of conditions. Two-dimensional gel electrophoresis (2-DE) has gained prominence over the last 4 decades for successfully aiding differential proteomics, providing visual confirmation of changes in protein abundance, which otherwise cannot be predicted from genome analysis. Each protein spot on 2-DE gel can be analyzed by its abundance, location, or even its presence or absence. This versatile gel-based method combines and utilizes the finest principle for separation of protein complexes by virtue of their charge and mass, visual mapping coupled with successful mass spectrometric identification of individual proteins.

Dissecting the chloroplast proteome of chickpea (Cicer arietinum L.) provides new insights into classical and non-classical functions.

Chloroplast, the energy organelle unique to plant cells, is a dynamic entity which integrates an array of metabolic pathways and serves as first level for energy conversion for the entire ecological hierarchy. Increasing amount of sequence data and evolution of mass spectrometric approaches has opened up new avenues for opportune exploration of the global proteome of this organelle. In our study, we aimed at generation of a comprehensive catalogue of chloroplast proteins in a grain legume, chickpea and provided a reference proteome map. To accurately assign the identified proteins, purity of chloroplast-enriched fraction was stringently monitored by multiple chemical and immunological indexes, besides pigment and enzyme analyses. The proteome analysis led to the identification of 2451 proteins, including 27 isoforms, which include predicted and novel chloroplast constituents. The identified proteins were validated through their sequence analysis. Extensive sequence based localization prediction revealed more than 50% proteins to be chloroplast resident by at least two different algorithms. Chromosomal distribution of identified proteins across nuclear and chloroplast genome unveiled the presence of 55 chloroplast encoded gene. In depth comparison of our dataset with the non-redundant set of chloroplast proteins identified so far across other species revealed novel as well as overlapping candidates. BIOLOGICAL SIGNIFICANCE: Pulses add large amount of nitrogen to the soil and has very low water footprint and therefore, contributes to fortification of sustainable agriculture. Chickpea is one of the earliest cultivated legumes and serves as an energy and protein source for humans and animals. Chloroplasts are the unique organelles which conduct photosynthesis. Investigation on chloroplast proteome is of particular significance, especially to plant biologists, as it would allow a better understanding of chloroplast function in plants. Generation of a saturated proteome map would not only validate the proteome inventory from its genome sequencing, but also serve as a comprehensive catalogue for future studies. We identified 2451 proteins, encoded by both the nuclear as well as chloroplast genomes, presumably involved in multivariate metabolic processes. The chloroplast deduced proteome and putative chloroplast proteins identified in this study would provide a foundation for future investigation of the expression and function of the chloroplast proteins of chickpea in specific and other crops species in general.

Gel-based and gel-free search for plasma membrane proteins in chickpea (Cicer arietinum L.) augments the comprehensive data sets of membrane protein repertoire.

UNLABELLED: Plasma membrane (PM) encompasses total cellular contents, serving as semi-porous barrier to cell exterior. This living barrier regulates all cellular exchanges in a spatio-temporal fashion. Most of the essential tasks of PMs including molecular transport, cell-cell interaction and signal transduction are carried out by their proteinaceous components, which make the PM protein repertoire to be diverse and dynamic. Here, we report the systematic analysis of PM proteome of a food legume, chickpea and develop a PM proteome reference map. Proteins were extracted from highly enriched PM fraction of four-week-old seedlings using aqueous two-phase partitioning. To address a population of PM proteins that is as comprehensive as possible, both gel-based and gel-free approaches were employed, which led to the identification of a set of 2732 non-redundant proteins. These included both integral proteins having bilayer spanning domains as well as peripheral proteins associated with PMs through posttranslational modifications or protein-protein interactions. Further, the proteins were subjected to various in-silico analyses and functionally classified based on their gene ontology. Finally an inventory of the complete set of PM proteins, identified in several monocot and dicot species, was created for comparative study with the generated PM protein dataset of chickpea. BIOLOGICAL SIGNIFICANCE: Chickpea, a rich source of dietary proteins, is the second most cultivated legume, which is grown over 10 million hectares of land worldwide. The annual global production of chickpea hovers around 8.5 million metric tons. Recent chickpea genome sequencing effort has provided a broad genetic basis for highlighting the important traits that may fortify other crop legumes. Improvement in chickpea varieties can further strengthen the world food security, which includes food availability, access and utilization. It is known that the phenotypic trait of a cultivar is the manifestation of the orchestrated functions of its proteins. Study of the PM proteome offers insights into the mechanism of communication between the cell and its environment by identification of receptors, signalling proteins and membrane transporters. Knowledge of the PM protein repertoire of a relatively dehydration tolerant chickpea variety, JG-62, can contribute in development of strategies for metabolic reprograming of crop species and breeding applications.