Transcriptional regulation of chickpea ferritin CaFer1 influences its role in iron homeostasis and stress response.

Ferritin, ubiquitous among all living organisms except yeast, exhibits iron-regulated expression. In plants, this regulation is applied through transcriptional control. Previous studies established the presence of two types of cis-acting elements in the promoter region: the iron regulatory element (FRE) in soybean and the iron-dependent regulatory sequence (IDRS) in maize and Arabidopsis. Adverse environmental conditions (e.g. water-deficit and oxidative stress) are known to modulate the expression of phytoferritin genes. In this study, we cloned and investigated the promoter sequence of a chickpea ferritin, designated CaFer1. Phylogenetic analysis of the CaFer1 promoter revealed its evolutionary relationship with other phytoferritins. The CaFer1 promoter exhibited several putative regulatory elements including two known transcription factor (TF) binding sites, Athb-1 and Myb.Ph. Electrophoretic mobility shift assay confirmed the sequence-specific binding of Athb-1 and Myb.Ph on the CaFer1 promoter. The TF-binding dynamics of CaFer1 showed high induction under conditions of iron-deficiency and water-deficit. We also demonstrated the possible interaction of CaFer1 with IRT1, a key component of the iron uptake system in plants, indicating its involvement in maintaining cellular iron levels. These results provide new insights into the underlying mechanisms of function of these interacting factors in CaFer1-mediated iron homeostasis and the stress response in plants.

Nuclear Proteome: Isolation of Intact Nuclei, Extraction of Nuclear Proteins, and 2-DE Analysis.

Proteome profiling aims to unravel the mystery of biological complexity encoded by the genome. The successful proteome profiling largely depends upon analytical approaches because single-step proteome characterization of eukaryotic cells is difficult due to the large number of proteins expressed and their complex physiochemical properties. Organellar proteomics helps in identifying a refined set of proteins by pinpointing certain activities to specific organelles, thereby increasing our knowledge of cellular processes. The reliability of a plant organelle proteome is intimately dependent on the purity of the organelle preparation. Methodological improvements in sample handling, organelle fractionation, and protein extraction are therefore crucial to plant subcellular proteomics. The nuclear proteins are organized into complex regulatory networks and perform varied cellular functions. Therefore, characterization of the nuclear proteome is an important step toward accumulating knowledge about regulation of gene expression and function. In this chapter, we present methods for the isolation of nuclei, purification of nuclear proteins, and proteome profiling that have been adapted for proteomic characterization of economically important crop species, such as chickpea.

Chickpea transcription factor CaTLP1 interacts with protein kinases, modulates ROS accumulation and promotes ABA-mediated stomatal closure.

Tubby and Tubby-like proteins (TLPs), in mammals, play critical roles in neural development, while its function in plants is largely unknown. We previously demonstrated that the chickpea TLP, CaTLP1, participates in osmotic stress response and might be associated with ABA-dependent network. However, how CaTLP1 is connected to ABA signaling remains unclear. The CaTLP1 was found to be engaged in ABA-mediated gene expression and stomatal closure. Complementation of the yeast yap1 mutant with CaTLP1 revealed its role in ROS scavenging. Furthermore, complementation of Arabidopsis attlp2 mutant displayed enhanced stress tolerance, indicating the functional conservation of TLPs across the species. The presence of ABA-responsive element along with other motifs in the proximal promoter regions of TLPs firmly established their involvement in stress signalling pathways. The CaTLP1 promoter driven GUS expression was restricted to the vegetative organs, especially stem and rosette leaves. Global protein expression profiling of wild-type, attlp2 and complemented Arabidopsis plants revealed 95 differentially expressed proteins, presumably involved in maintaining physiological and biological processes under dehydration. Immunoprecipitation assay revealed that protein kinases are most likely to interact with CaTLP1. This study provides the first demonstration that the TLPs act as module for ABA-mediated stomatal closure possibly via interaction with protein kinase.

Chickpea Ferritin CaFer1 Participates in Oxidative Stress Response, and Promotes Growth and Development.

Ferritins store and sequester iron, and regulate iron homeostasis. The cDNA for a stress-responsive phytoferritin, previously identified in the extracellular matrix (ECM) of chickpea (Cicer arietinum), was cloned and designated CaFer1. The CaFer1 transcript was strongly induced in chickpea exposed to dehydration, hypersalinity and ABA treatment. Additionally, it has role in the defense against Fusarium oxysporum infection. Functional complementation of the yeast frataxin-deficient mutant, Δyfh1, indicates that CaFer1 functions in oxidative stress. The presence of CaFer1 in the extracellular space besides chloroplast establishes its inimitable nature from that of other phytoferritins. Furthermore, CaFer1 expression in response to iron suggests its differential mechanism of accumulation at two different iron conditions. CaFer1-overexpressing transgenic plants conferred improved growth and development, accompanied by altered expression of iron-responsive genes. Together, these results suggest that the phytoferritin, CaFer1, might play a key role in maintenance of iron buffering and adaptation to environmental challenges.

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.

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.

Birth of plant proteomics in India: a new horizon.

In the post-genomic era, proteomics is acknowledged as the next frontier for biological research. Although India has a long and distinguished tradition in protein research, the initiation of proteomics studies was a new horizon. Protein research witnessed enormous progress in protein separation, high-resolution refinements, biochemical identification of the proteins, protein-protein interaction, and structure-function analysis. Plant proteomics research, in India, began its journey on investigation of the proteome profiling, complexity analysis, protein trafficking, and biochemical modeling. The research article by Bhushan et al. in 2006 marked the birth of the plant proteomics research in India. Since then plant proteomics studies expanded progressively and are now being carried out in various institutions spread across the country. The compilation presented here seeks to trace the history of development in the area during the past decade based on publications till date. In this review, we emphasize on outcomes of the field providing prospects on proteomic pathway analyses. Finally, we discuss the connotation of strategies and the potential that would provide the framework of plant proteome research. BIOLOGICAL SIGNIFICANCE: The past decades have seen rapidly growing number of sequenced plant genomes and associated genomic resources. To keep pace with this increasing body of data, India is in the provisional phase of proteomics research to develop a comparative hub for plant proteomes and protein families, but it requires a strong impetus from intellectuals, entrepreneurs, and government agencies. Here, we aim to provide an overview of past, present and future of Indian plant proteomics, which would serve as an evaluation platform for those seeking to incorporate proteomics into their research programs. This article is part of a Special Issue entitled: Proteomics in India.

Characterisation of the nuclear proteome of a dehydration-sensitive cultivar of chickpea and comparative proteomic analysis with a tolerant cultivar.

Water deficit or dehydration hampers plant growth and development, and shrinks harvest size of major crop species worldwide. Therefore, a better understanding of dehydration response is the key to decipher the regulatory mechanism of better adaptation. In recent years, nuclear proteomics has become an attractive area of research, particularly to study the role of nucleus in stress response. In this study, a proteome of dehydration-sensitive chickpea cultivar (ICCV-2) was generated from nuclei-enriched fractions. The LC-MS/MS analysis led to the identification of 75 differentially expressed proteins presumably associated with different metabolic and regulatory pathways. Nuclear localisation of three candidate proteins was validated by transient expression assay. The ICCV-2 proteome was then compared with that of JG-62, a tolerant cultivar. The differential proteomics and in silico analysis revealed cultivar-specific differential expression of many proteins involved in various cellular functions. The differential tolerance could be attributed to altered expression of many structural proteins and the proteins involved in stress adaptation, notably the ROS catabolising enzymes. Further, a comprehensive comparison on the abiotic stress-responsive nuclear proteome was performed using the datasets published thus far. These findings might expedite the functional determination of the dehydration-responsive proteins and their prioritisation as potential molecular targets for better adaptation.

Analysis of the grasspea proteome and identification of stress-responsive proteins upon exposure to high salinity, low temperature, and abscisic acid treatment.

Abiotic stress causes diverse biochemical and physiological changes in plants and limits crop productivity. Plants respond and adapt to such stress by altering their cellular metabolism and activating various defense machineries. To understand the molecular basis of stress tolerance in plants, we have developed differential proteomes in a hardy legume, grasspea (Lathyrus sativus L.). Five-week-old grasspea seedlings were subjected independently to high salinity, low temperature and abscisic acid treatment for duration of 36h. The physiological changes of stressed seedlings were monitored, and correlated with the temporal changes of proteome using two-dimensional gel electrophoresis. Approximately, 400 protein spots were detected in each of the stress proteome with one-fourth showing more than 2-fold differences in expression values. Eighty such proteins were subjected to LC-tandem MS/MS analyses that led to the identification of 48 stress-responsive proteins (SRPs) presumably involved in a variety of functions, including metabolism, signal transduction, protein biogenesis and degradation, and cell defense and rescue. While 33 proteins were responsive to all three treatments, 15 proteins were expressed in stress-specific manner. Further, we explored the possible role of ROS in triggering the stress-induced degradation of large subunit (LSU) of ribulose-1,5-bisphosphate carboxylase (Rubisco). These results might help in understanding the spectrum of stress-regulated proteins and the biological processes they control as well as having implications for strategies to improve stress adaptation in plants.

Identification of extracellular matrix proteins of rice (Oryza sativa L.) involved in dehydration-responsive network: a proteomic approach.

Water-deficit or dehydration impairs almost all physiological processes and greatly influences the geographical distribution of many crop species. It has been postulated that higher plants rely mostly on induction mechanisms to maintain cellular integrity during stress conditions. Plant cell wall or extracellular matrix (ECM) forms an important conduit for signal transduction between the apoplast and symplast and acts as front-line defense, thereby playing a key role in cell fate decision under various stress conditions. To better understand the molecular mechanism of dehydration response in plants, four-week-old rice seedlings were subjected to progressive dehydration by withdrawing water and the changes in the ECM proteome were examined using two-dimensional gel electrophoresis. Dehydration-responsive temporal changes revealed 192 proteins that change their intensities by more than 2.5-fold, at one or more time points during dehydration. The proteomic analysis led to the identification of about 100 differentially regulated proteins presumably involved in a variety of functions, including carbohydrate metabolism, cell defense and rescue, cell wall modification, cell signaling and molecular chaperones, among others. The differential rice proteome was compared with the dehydration-responsive proteome data of chickpea and maize. The results revealed an evolutionary divergence in the dehydration response as well as organ specificity, with few conserved proteins. The differential expression of the candidate proteins, in conjunction with previously reported results, may provide new insight into the underlying mechanisms of the dehydration response in plants. This may also facilitate the targeted alteration of metabolic routes in the cell wall for agricultural and industrial exploitation.

Proteomics approach to identify dehydration responsive nuclear proteins from chickpea (Cicer arietinum L.).

Dehydration or water-deficit is one of the most important environmental stress factors that greatly influences plant growth and development and limits crop productivity. Plants respond and adapt to such stress by altering their cellular metabolism and activating various defense machineries. Mechanisms that operate signal perception, transduction, and downstream regulatory events provide valuable information about the underlying pathways involved in environmental stress responses. The nuclear proteins constitute a highly organized, complex network that plays diverse roles during cellular development and other physiological processes. To gain a better understanding of dehydration response in plants, we have developed a comparative nuclear proteome in a food legume, chickpea (Cicer arietinum L.). Three-week-old chickpea seedlings were subjected to progressive dehydration by withdrawing water and the changes in the nuclear proteome were examined using two-dimensional gel electrophoresis. Approximately 205 protein spots were found to be differentially regulated under dehydration. Mass spectrometry analysis allowed the identification of 147 differentially expressed proteins, presumably involved in a variety of functions including gene transcription and replication, molecular chaperones, cell signaling, and chromatin remodeling. The dehydration responsive nuclear proteome of chickpea revealed a coordinated response, which involves both the regulatory as well as the functional proteins. This study, for the first time, provides an insight into the complex metabolic network operating in the nucleus during dehydration.

Comparative proteomics analysis of differentially expressed proteins in chickpea extracellular matrix during dehydration stress.

Water deficit or dehydration is the most crucial environmental factor that limits crop productivity and influences geographical distribution of many crop plants. It is suggested that dehydration-responsive changes in expression of proteins may lead to cellular adaptation against water deficit conditions. Most of the earlier understanding of dehydration-responsive cellular adaptation has evolved from transcriptome analyses. By contrast, comparative analysis of dehydration-responsive proteins, particularly proteins in the subcellular fraction, is limiting. In plants, cell wall or extracellular matrix (ECM) serves as the repository for most of the components of the cell signaling process and acts as a frontline defense. Thus, we have initiated a proteomics approach to identify dehydration-responsive ECM proteins in a food legume, chickpea. Several commercial chickpea varieties were screened for the status of dehydration tolerance using different physiological and biochemical indexes. Dehydration-responsive temporal changes of ECM proteins in JG-62, a relatively tolerant variety, revealed 186 proteins with variance at a 95% significance level statistically. The comparative proteomics analysis led to the identification of 134 differentially expressed proteins that include predicted and novel dehydration-responsive proteins. This study, for the first time, demonstrates that over a hundred ECM proteins, presumably involved in a variety of cellular functions, viz. cell wall modification, signal transduction, metabolism, and cell defense and rescue, impinge on the molecular mechanism of dehydration tolerance in plants.

The nuclear proteome of chickpea (Cicer arietinum L.) reveals predicted and unexpected proteins.

Nuclear proteins constitute a highly organized, complex network that plays diverse roles during cellular development and other physiological processes. The yeast nuclear proteome corresponds to about one-fourth of the total cellular proteins, suggesting the involvement of the nucleus in a number of diverse functions. In an attempt to understand the complexity of plant nuclear proteins, we have developed a proteome reference map of a legume, chickpea, using two-dimensional gel electrophoresis (2-DE). Approximately, 600 protein spots were detected, and LC-ESI-MS/MS analyses led to the identification of 150 proteins that have been implicated in a variety of cellular functions. The largest percentage of the identified proteins was involved in signaling and gene regulation (36%), while 17% were involved in DNA replication and transcription. The chickpea nuclear proteome indicates the presence of few new nuclear proteins of unknown functions vis-à-vis many known resident proteins. To the best of our knowledge, this is the first report of a nuclear proteome of an unsequenced genome.

Extracellular matrix proteome of chickpea (Cicer arietinum L.) illustrates pathway abundance, novel protein functions and evolutionary perspect.

The extracellular matrix (ECM) or cell wall is a dynamic system and serves as the first line mediator in cell signaling to perceive and transmit extra- and intercellular signals in many pathways. Although ECM is a conserved compartment ubiquitously present throughout evolution, a compositional variation does exist among different organisms. ECM proteins account for 10% of the ECM mass, however, comprise several hundreds of different molecules with diverse functions. To understand the function of ECM proteins, we have developed the cell wall proteome of a crop legume, chickpea (Cicer arietinum). This comprehensive overview of the proteome would provide a basis for future comparative proteomic efforts for this important crop. Proteomic analyses revealed new ECM proteins of unknown functions vis-à-vis the presence of many known cell wall proteins. In addition, we report here evidence for the presence of unexpected proteins with known biochemical activities, which have never been associated with ECM.