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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.

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.

Chloroplast 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 d ifferentially r egulated p roteins (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.

Environmental perturbations are persistent threats to sustainable agriculture, and thus recruitment of resilient crops, especially legumes, exhibiting agronomically important traits has become a priority for plant biologists. It is of utmost importance that the neglected and underutilized legumes (NULs) are identified and utilized as source of germane genes and gene-products, through concerted research platforms. In the present article, we analyzed the current status of NULs with specific emphasis to the potent utility of grasspea owing to its unique characters including stress adaptation, nutritional superiority and ease of cultivation. We have highlighted the landmarks in the history of grasspea, delineating the rapid progress achieved in grasspea biology during the past decades. Despite possession of a neurotoxic compound, β-N-oxalyl-L-α,β-diaminopropionic acid (β-ODAP), this neglected legume outshines most food crops with its distinct physicochemical attributes, health and agricultural benefits and resilience to environmental constraints. With the availability of genome sequence, grasspea is now established as an appropriate genetic resource for sustainable agriculture and phytoremediation rendering its genes, proteins and metabolites for targeted genetic manipulation. We conclude that grasspea would serve as a resource for plant translational genomics (TG) research, particularly resilience of legumes to environmental challenges.

The 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.

Over the past century, the average surface temperature and recurrent heatwaves have been steadily rising, affecting the yield potential of most food crops including bread wheat, the second most important caloric source, but is particularly vulnerable to the impacts of elevated temperatures. Significantly, the past decade has witnessed tremendous advancements in multiomics approaches to extract the key regulators that influence the adaptive responses to high temperature stress (HTS). With the help of genetic engineering technologies, transgenic wheat plants have been developed showing resistance to HTS without hampering productivity. In this review, we described the effect of rising temperature at a global scale and the drastic impacts on crops, particularly on wheat production. Also, this review is focused on accomplishing a deeper understanding of the genetic and molecular basis of HTS responses of crop plants, wheat in particular along with current strategies and technologies to generate thermotolerant varieties. Collective strategy and identified thresholds of HTS tolerance and susceptibility will contribute to the value-added modelling of wheat growth and yield under predictable future climate conditions.

Small heat shock proteins (sHSPs) constitute a class of molecular chaperones, which are evolutionarily conserved yet diverse group of molecules, rapidly produced in response to stress. In this study, we sought to identify plant sHSPs, especially chaperonin 10 (Cpn10) family members in major evolutionary lineages, and determine their biological significance. Multiple sequence alignment of Cpn10 domains revealed divergent amino acids as well as conserved sites. Phylogenetic tree depicted the diversification and expansion of Cpn10 gene family. During the process of evolution, the Ka/Ks ratio of orthologous and paralogous pairs was

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.

Host specific resistance and non-host resistance are two plant immune responses to counter pathogen invasion. Gene network organizing principles leading to quantitative differences in resistant and susceptible host during host specific resistance are poorly understood. Vascular wilt caused by root pathogen Fusarium species is complex and governed by host specific resistance in crop plants, including chickpea. Here, we temporally profiled two contrasting chickpea genotypes in disease and immune state to better understand gene expression switches in host specific resistance. Integrative gene-regulatory network elucidated tangible insight into interaction coordinators leading to pathway determination governing distinct (disease or immune) phenotypes. Global network analysis identified five major hubs with 389 co-regulated genes. Functional enrichment revealed immunome containing three subnetworks involving CTI, PTI and ETI and wilt diseasome encompassing four subnetworks highlighting pathogen perception, penetration, colonization and disease establishment. These subnetworks likely represent key components that coordinate various biological processes favouring defence or disease. Furthermore, we identified core 76 disease/immunity related genes through subcellular analysis. Our regularized network with robust statistical assessment captured known and unexpected gene interaction, candidate novel regulators as future biomarkers and first time showed system-wide quantitative architecture corresponding to genotypic characteristics in wilt landscape.

Architectural proteins play key roles in genome construction and regulate the expression of many genes, albeit the modulation of genome plasticity by these proteins is largely unknown. A critical screening of the architectural proteins in five crop species, viz., Oryza sativa, Zea mays, Sorghum bicolor, Cicer arietinum, and Vitis vinifera, and in the model plant Arabidopsis thaliana along with evolutionary relevant species such as Chlamydomonas reinhardtii, Physcomitrella patens, and Amborella trichopoda, revealed 9, 20, 10, 7, 7, 6, 1, 4, and 4 Alba (acetylation lowers binding affinity) genes, respectively. A phylogenetic analysis of the genes and of their counterparts in other plant species indicated evolutionary conservation and diversification. In each group, the structural components of the genes and motifs showed significant conservation. The chromosomal location of the Alba genes of rice (OsAlba), showed an unequal distribution on 8 of its 12 chromosomes. The expression profiles of the OsAlba genes indicated a distinct tissue-specific expression in the seedling, vegetative, and reproductive stages. The quantitative real-time PCR (qRT-PCR) analysis of the OsAlba genes confirmed their stress-inducible expression under multivariate environmental conditions and phytohormone treatments. The evaluation of the regulatory elements in 68 Alba genes from the 9 species studied led to the identification of conserved motifs and overlapping microRNA (miRNA) target sites, suggesting the conservation of their function in related proteins and a divergence in their biological roles across species. The 3D structure and the prediction of putative ligands and their binding sites for OsAlba proteins offered a key insight into the structure–function relationship. These results provide a comprehensive overview of the subtle genetic diversification of the OsAlba genes, which will help in elucidating their functional role in plants.

Soya bean (Glycine max) and grass pea (Lathyrus sativus) seeds are important sources of dietary proteins; however, they also contain antinutritional metabolite oxalic acid (OA). Excess dietary intake of OA leads to nephrolithiasis due to the formation of calcium oxalate crystals in kidneys. Besides, OA is also a known precursor of β-N-oxalyl-L-α,β-diaminopropionic acid (β-ODAP), a neurotoxin found in grass pea. Here, we report the reduction in OA level in soya bean (up to 73%) and grass pea (up to 75%) seeds by constitutive and/or seed-specific expression of an oxalate-degrading enzyme, oxalate decarboxylase (FvOXDC) of Flammulina velutipes. In addition, β-ODAP level of grass pea seeds was also reduced up to 73%. Reduced OA content was interrelated with the associated increase in seeds micronutrients such as calcium, iron and zinc. Moreover, constitutive expression of FvOXDC led to improved tolerance to the fungal pathogen Sclerotinia sclerotiorum that requires OA during host colonization. Importantly, FvOXDC-expressing soya bean and grass pea plants were similar to the wild type with respect to the morphology and photosynthetic rates, and seed protein pool remained unaltered as revealed by the comparative proteomic analysis. Taken together, these results demonstrated improved seed quality and tolerance to the fungal pathogen in two important legume crops, by the expression of an oxalate-degrading enzyme.

Orphan legumes are defined as those which are grown as food, animal feed and/or other legumes of agriculture importance, but which have received very little research attention. Grasspea is one of the best examples of such legume which is cultivated worldwide, as it is the cheapest source of dietary protein particularly for the developing world. It has remained outside the realm of largescale functional genomics studies. Many grasspea cultivars are capable to withstand a myriad of constraints, not only the common abiotic stresses, but pests and pathogen attack making it one of the potential systems to study stress tolerance. In recent years, most of its traits that interest biologists worldwide, such as stress tolerance, have rated so high that a number of new initiatives have been taken by different research groups for better and safer use of grasspea. In this review, we discuss the progress made in the field of grasspea proteomics to date and dwell upon the future direction/problems/approaches towards defining the grasspea proteome.Plant Tissue Cult. & Biotech. 25(1): 117-141, 2015 (June)

Additional file 2. Fig. S1. Wild-type and transformed roots of chickpea cultivar Annigeri grown in selection medium. Fig. S2. Green fluorescent protein (GFP) visualization by confocal microscopy in transformed chickpea (cultivar Annigeri) roots. Fig. S3. Characterization of transformed roots in chickpea cultivar Annigeri. Fig. S4. Green fluorescent protein (GFP) expression in different chickpea cultivars. Fig. S5. PCR analysis of transgenic chickpea roots expressing GFP. Fig. S6. Characterization of roots of chickpea cultivar JG-62 expressing AtTT2:GFP.

Background Chickpea (Cicer arietinum L.), an important legume crop is one of the major source of dietary protein. Developing an efficient 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 fluorescent protein further confirmed the success of transformation. We demonstrate that our method adequately transforms chickpea roots at early developmental stage with high efficiency. In addition, root transformation was found to be genotype-independent and the efficacy 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 affect 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 sufficient 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. Electronic supplementary material The online version of this article (10.1186/s13007-018-0315-6) contains supplementary material, which is available to authorized users.

Although precisely controlled innate immune response is governed by conserved cellular events in phylogenetically diverse hosts, the underlying molecular mechanisms by which this process is regulated against a multi-host pathogen remain unknown. Fusarium oxysporum is a model multi-host pathogen, known to be associated with neuronal stress in humans and vascular wilt in plants. The interaction between innate immune and neuronal pathways is the basis of many diverse biological responses. How these processes are coordinated in response to fungal disease is not well understood. Here, we show that F. oxysporum f. sp. ciceri causes neuronal stress and intestinal disintegration, ultimately leading to the death of Caenorhabditis elegans. To explore the regulatory framework of Fusarium-associated disease, we analysed the gene expression during infection, integrated temporal gene expression, and network analysis with genetic inactivation data in Caenorhabditis elegans. We identified 1024 genes showing significant changes in expression (corrected P-values

The plant metabolite oxalic acid is increasingly recognized as a food toxin with negative effects on human nutrition. Decarboxylative degradation of oxalic acid is catalyzed, in a substrate-specific reaction, by oxalate decarboxylase (OXDC), forming formic acid and carbon dioxide. Attempts to date to reduce oxalic acid levels and to understand the biological significance of OXDC in crop plants have met with little success. To investigate the role of OXDC and the metabolic consequences of oxalate down-regulation in a heterotrophic, oxalic acid-accumulating fruit, we generated transgenic tomato (Solanum lycopersicum) plants expressing an OXDC (FvOXDC) from the fungus Flammulina velutipes specifically in the fruit. These E8.2-OXDC fruit showed up to a 90% reduction in oxalate content, which correlated with concomitant increases in calcium, iron, and citrate. Expression of OXDC affected neither carbon dioxide assimilation rates nor resulted in any detectable morphological differences in the transgenic plants. Comparative proteomic analysis suggested that metabolic remodeling was associated with the decrease in oxalate content in transgenic fruit. Examination of the E8.2-OXDC fruit proteome revealed that OXDC-responsive proteins involved in metabolism and stress responses represented the most substantially up- and down-regulated categories, respectively, in the transgenic fruit, compared with those of wild-type plants. Collectively, our study provides insights into OXDC-regulated metabolic networks and may provide a widely applicable strategy for enhancing crop nutritional value.

Sad1/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 N„ and N± 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.

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.

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.

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 Electronic supplementary material The online version of this article (doi:10.1186/s12870-016-0860-y) contains supplementary material, which is available to authorized users.

Storage proteins in plants, because of high nutrient value, have been a subject of intensive investigation. These proteins are synthesized de novo in the cytoplasm and transported to the storage organelles where they serve as reservoir of energy and supplement of nitrogen during rapid growth and development. Sweetpotato is the seventh most important food crop worldwide and has a significant contribution to the source of nutrition, albeit with low protein content. To determine the behaviour of seed storage proteins in non-native system, a seed albumin, AmA1, was overexpressed in sweetpotato with an additional aim of improving nutritional quality of tuber proteins. Introduction of AmA1 imparted an increase in protein and amino acid contents as well as the phytophenols. The proteometabolomics analysis revealed a rebalancing of the proteome, with no significant effects on the global metabolome profile of the transgenic tubers. Additionally, the slower degradation of starch and cellulose in transgenic tubers, led to increased post-harvest durability. Present study provides a new insight into the role of a seed storage protein in the modulation of photoassimilate movement and nutrient acquisition.

Stress adaptation or tolerance in plants is a complex phenomenon involving changes in physiological and metabolic processes. Plants must develop elaborate networks of defense mechanisms, and adapt to and survive for sustainable agriculture. Water-deficit or dehydration is the most critical environmental factor that plants are exposed to during their life cycle, which influences geographical distribution and productivity of many crop species. The cellular responses to dehydration are orchestrated by a series of multidirectional relays of biochemical events at organelle level. The new challenge is to dissect the underlying mechanisms controlling the perception of stress signals and their transmission to cellular machinery for activation of adaptive responses. The completeness of current descriptions of spatial distribution of proteins, the relevance of subcellular locations in diverse functional processes, and the changes of protein abundance in response to dehydration hold the key to understanding how plants cope with such stress conditions. During past decades, organellar proteomics has proved to be useful not only for deciphering reprograming of plant responses to dehydration, but also to dissect stress-responsive pathways. This review summarizes a range of organellar proteomics investigations under dehydration to gain a holistic view of plant responses to water-deficit conditions, which may facilitate future efforts to develop genetically engineered crops for better adaptation.

Excessive softening of fruits during the ripening process leads to deterioration. This is of significant global importance as softening-mediated deterioration leads to huge postharvest losses. N-glycan processing enzymes are reported to play an important role during climacteric fruit softening: however, to date these enzymes have not been characterized in non-climacteric fruit. Two ripening-specific N-glycan processing enzymes, α-mannosidase (α-Man) and β-D-N-acetylhexosaminidase (β-Hex), have been identified and targeted to enhance the shelf life in non-climacteric fruits such as capsicum (Capsicum annuum). The purification, cloning, and functional characterization of α-Man and β-Hex from capsicum, which belong to glycosyl hydrolase (GH) families 38 and 20, respectively, are described here. α-Man and β-Hex are cell wall glycoproteins that are able to cleave terminal α-mannose and β-D-N-acetylglucosamine residues of N-glycans, respectively. α-Man and β-Hex transcripts as well as enzyme activity increase with the ripening and/or softening of capsicum. The function of α-Man and β-Hex in capsicum softening is investigated through RNA interference (RNAi) in fruits. α-Man and β-Hex RNAi fruits were approximately two times firmer compared with the control and fruit deterioration was delayed by approximately 7 d. It is shown that silencing of α-Man and β-Hex enhances fruit shelf life due to the reduced degradation of N-glycoproteins which resulted in delayed softening. Altogether, the results provide evidence for the involvement of N-glycan processing in non-climacteric fruit softening. In conclusion, genetic engineering of N-glycan processing can be a common strategy in both climacteric and non-climacteric species to reduce the post-harvest crop losses.

Protein deficiency is the most crucial factor that affects physical growth and development and that increases morbidity and mortality especially in developing countries. Efforts have been made to improve protein quality and quantity in crop plants but with limited success. Here, we report the development of transgenic potatoes with enhanced nutritive value by tuber-specific expression of a seed protein, AmA1 (Amaranth Albumin 1), in seven genotypic backgrounds suitable for cultivation in different agro-climatic regions. Analyses of the transgenic tubers revealed up to 60% increase in total protein content. In addition, the concentrations of several essential amino acids were increased significantly in transgenic tubers, which are otherwise limited in potato. Moreover, the transgenics also exhibited enhanced photosynthetic activity with a concomitant increase in total biomass. These results are striking because this genetic manipulation also resulted in a moderate increase in tuber yield. The comparative protein profiling suggests that the proteome rebalancing might cause increased protein content in transgenic tubers. Furthermore, the data on field performance and safety evaluation indicate that the transgenic potatoes are suitable for commercial cultivation. In vitro and in vivo studies on experimental animals demonstrate that the transgenic tubers are also safe for human consumption. Altogether, these results emphasize that the expression of AmA1 is a potential strategy for the nutritional improvement of food crops.

In a globalized economy, the control of fruit ripening is of strategic importance because excessive softening limits shelf life. Efforts have been made to reduce fruit softening in transgenic tomato through the suppression of genes encoding cell wall–degrading proteins. However, these have met with very limited success.N-glycans are reported to play an important role during fruit ripening, although the role of any particular enzyme is yet unknown. We have identified and targeted two ripening-specificN-glycoprotein modifying enzymes, α-mannosidase (α-Man) and β-D-N-acetylhexosaminidase (β-Hex). We show that their suppression enhances fruit shelf life, owing to the reduced rate of softening. Analysis of transgenic tomatoes revealed ≈2.5- and ≈2-fold firmer fruits in the α-Man and β-Hex RNAi lines, respectively, and ≈30 days of enhanced shelf life. Overexpression of α-Man or β-Hex resulted in excessive fruit softening. Expression of α-Man and β-Hex is induced by the ripening hormone ethylene and is modulated by a regulator of ripening,rin(ripening inhibitor). Furthermore, transcriptomic comparative studies demonstrate the down-regulation of cell wall degradation- and ripening-related genes in RNAi fruits. It is evident from these results thatN-glycan processing is involved in ripening-associated fruit softening. Genetic manipulation ofN-glycan processing can be of strategic importance to enhance fruit shelf life, without any negative effect on phenotype, including yield.

Water deficit or dehydration is the most crucial environmental constraint on plant growth and development and crop productivity. It has been postulated that plants respond and adapt to dehydration by altering their cellular metabolism and by activating various defense machineries. The nucleus, the regulatory hub of the eukaryotic cell, is a dynamic system and a repository of various macromolecules that serve as modulators of cell signaling dictating the cell fate decision. To better understand the molecular mechanisms of dehydration-responsive adaptation in plants, we developed a comprehensive nuclear proteome of rice. The proteome was determined using a sequential method of organellar enrichment followed by two-dimensional electrophoresis-based protein identification by LC-ESI-MS/MS. We initially screened several commercial rice varieties and parental lines and established their relative dehydration tolerance. The differential display of nuclear proteins in the tolerant variety under study revealed 150 spots that showed changes in their intensities by more than 2.5-fold. The proteomics analysis led to the identification of 109 differentially regulated proteins presumably involved in a variety of functions, including transcriptional regulation and chromatin remodeling, signaling and gene regulation, cell defense and rescue, and protein degradation. The dehydration-responsive nuclear proteome revealed a coordinated response involving both regulatory and functional proteins, impinging upon the molecular mechanism of dehydration adaptation. Furthermore a comparison between the dehydration-responsive nuclear proteome of rice and that of a legume, the chickpea, showed an evolutionary divergence in dehydration response comprising a few conserved proteins, whereas most of the proteins may be involved in crop-specific adaptation. These results might help in understanding the spectrum of nuclear proteins and the biological processes they control under dehydration as well as having implications for strategies to improve dehydration tolerance in plants.

Oxalate decarboxylase (OXDC) from the wood-rotting fungus Flammulina velutipes, which catalyzes the conversion of oxalate to formic acid and CO(2) in a single-step reaction, is a duplicated double-domain germin family enzyme. It has agricultural as well as therapeutic importance. We reported earlier the purification and molecular cloning of OXDC. Knowledge-based modeling of the enzyme reveals a beta-barrel core in each of the two domains organized in the hexameric state. A cluster of three histidines suitably juxtaposed to coordinate a divalent metal ion exists in both the domains. Involvement of the two histidine clusters in the catalytic mechanism of the enzyme, possibly through coordination of a metal cofactor, has been hypothesized because all histidine knockout mutants showed total loss of decarboxylase activity. The atomic absorption spectroscopy analysis showed that OXDC contains Mn(2+) at up to 2.5 atoms per subunit. Docking of the oxalate in the active site indicates a similar electrostatic environment around the substrate-binding site in the two domains. We suggest that the histidine coordinated manganese is critical for substrate recognition and is directly involved in the catalysis of the enzyme.

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.

α-Mannosidase (α-Man), a fruit ripening-specific N-glycan processing enzyme, is involved in ripening-associated fruit softening process. However, the regulation of fruit-ripening specific expression of α-Man is not well understood. We have identified and functionally characterized the promoter of tomato (Solanum lycopersicum) α-Man to provide molecular insights into its transcriptional regulation during fruit ripening. Fruit ripening-specific activation of the α-Man promoter was revealed by analysing promoter driven expression of beta-glucuronidase (GUS) reporter in transgenic tomato. We found that RIPENING INHIBITOR (RIN), a MADS box family transcription factor acts as positive transcriptional regulator of α-Man during fruit ripening. RIN directly bound to the α-Man promoter sequence and promoter activation/α-Man expression was compromised in rin mutant fruit. Deletion analysis revealed that a promoter fragment (567 bp upstream of translational start site) that contained three CArG boxes (binding sites for RIN) was sufficient to drive GUS expression in fruits. In addition, α-Man expression was down-regulated in fruits of Nr mutant which is impaired in ethylene perception and promoter activation/α-Man expression was induced in wild type following treatment with a precursor of ethylene biosynthesis, 1-aminocyclopropane-1-carboxylic acid (ACC). Although, α-Man expression was induced in rin mutant after ACC treatment, the transcript level was less as compared to ACC-treated wild type. Taken together, these results suggest RIN-mediated direct transcriptional regulation of α-Man during fruit ripening and ethylene may acts in RIN-dependent and -independent ways to regulate α-Man expression.

A bar chart showing distribution of transcription factors in chickpea genome on individual chromosomes and line chart showing average density of transcription factors in each chromosome. The data shows count of all TF types. Individual family distribution graph has been included in the database website. (PDF 26Â kb)

Diagram showing CicerTransDB webserver workflow. Bold red lines show user accessible pipeline. Blue and green dotted lines show workflow running through website PHP without userâ s interaction. Blue lines are normal PHP data, while green lines are secured ones. (PDF 39Â kb)

Manual curation of the primary list of transcription factors generated through domain search. Tubby* domain proteins are putative transcription factors (Wardhan et al. [13]), but not included in PlantTFDB. (PDF 1721Â kb)

Distribution of TF family genes across the chromosomes. (PDF 42Â kb)

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.

Rice is the most widely consumed staple food for both developed as well as developing world, more so for Asia. According to the data of FAOSTAT (2012), rice has the third-highest worldwide production after sugarcane and maize, among all agricultural crops [1]. Developing countries account for 95% of the total rice production, with China and India contributing for nearly half of the world output [1].

The poly(A) signal and downstream elements with transcriptional pausing activity play an important role in termination of RNA polymerase II transcription. We show that an intronic sequence derived from the plant seed protein gene (AmA1) specifically acts as a transcriptional terminator in the fission yeast, Schizosaccharomyces pombe. The 3′-end points of mRNA encoded by the AmA1 gene were mapped at different positions in S.pombe and in native cells of Amaranthus hypochondriacus. Deletion analyses of the AmA1 intronic sequence revealed that multiple elements essential for proper transcriptional termination in S.pombe include two site-determining elements (SDEs) and three downstream sequence elements. RT–PCR analyses detected transcripts up to the second SDE. This is the first report showing that the highly conserved mammalian poly(A) signal, AAUAAA, is also functional in S.pombe. The poly(A) site was determined as Y(A) both in native and heterologous systems but at different positions. Deletion of these cis-elements abolished 3′-end processing in S.pombe and a single point mutation in this motif reduced the activity by 70% while enhancing activity at downstream SDE. These results indicate that the bipartite sequence elements as signals for 3′-end processing in fission yeast act in tandem with other cis-acting elements. A comparison of these elements in the AmA1 intron that function as a transcriptional terminator in fission yeast with that of its native genes showed that both require an AT-rich distal and proximal upstream element. However, these sequences are not identical. Transcription run-on analysis indicates that elongating RNA polymerase II molecules accumulate over these pause signals, maximal at 611–949 nt. Furthermore, we demonstrate that the AmA1 intronic terminator sequence acts in a position-independent manner when placed within another gene.

Highlight text Ripening-specific expression of β-Hex, a gene involved in the fruit-softening process, is transcriptionally regulated by the MADS-box transcription factor RIN in SlASR1-dependent and -independent manners., Tomato (Solanum lycopersicum) fruit ripening-specific N-glycan processing enzyme, β-D-N-acetylhexosaminidase (β-Hex), plays an important role in the ripening-associated fruit-softening process. However, the regulation of fruit ripening-specific expression of β-Hex is not well understood. We have identified and functionally characterized the fruit ripening-specific promoter of β-Hex and provided insights into its transcriptional regulation during fruit ripening. Our results demonstrate that RIPENING INHIBITOR (RIN), a global fruit ripening regulator, and ABSCISIC ACID STRESS RIPENING 1 (SlASR1), a poorly characterized ripening-related protein, are the transcriptional regulators of β-Hex. Both RIN and SlASR1 directly bound to the β-Hex promoter fragments containing CArG and C2-3(C/G)A cis-acting elements, the binding sites for RIN and SlASR1, respectively. Moreover, β-Hex expression/promoter activity in tomato fruits was downregulated once expression of either RIN or SlASR1 was suppressed; indicating that RIN and SlASR1 positively regulate the transcription of β-Hex during fruit ripening. Interestingly, RIN could also bind to the SlASR1 promoter, which contains several CArG cis-acting elements, and SlASR1 expression was suppressed in rin mutant fruits, indicating that RIN also acts as a positive regulator of SlASR1 expression during fruit ripening. Taken together, these results suggest that RIN, both directly and indirectly, through SlASR1, regulates the transcription of β-Hex during fruit ripening. The fruit ripening-specific promoter of β-Hex could be a useful tool in regulating gene expression during fruit ripening.

Improvement of nutritive value of crop plants, in particular the amino acid composition, has been a major long-term goal of plant breeding programs. Toward this end, we reported earlier the cloning of the seed albumin gene AmA1 from Amaranthus hypochondriacus . The AmA1 protein is nonallergenic in nature and is rich in all essential amino acids, and the composition corresponds well with the World Health Organization standards for optimal human nutrition. In an attempt to improve the nutritional value of potato, the AmA1 coding sequence was successfully introduced and expressed in tuber-specific and constitutive manner. There was a striking increase in the growth and production of tubers in transgenic populations and also of the total protein content with an increase in most essential amino acids. The expressed protein was localized in the cytoplasm as well as in the vacuole of transgenic tubers. Thus we have been able to use a seed albumin gene with a well-balanced amino acid composition as a donor protein to develop a transgenic crop plant. The results document, in addition to successful nutritional improvement of potato tubers, the feasibility of genetically modifying other crop plants with novel seed protein composition.

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.

Plant proteomics has made tremendous contributions in understanding the complex processes of plant biology. Here, its current status in India and Nepal is discussed. Gel-based proteomics is predominantly utilized on crops and non-crops to analyze majorly abiotic (49 %) and biotic (18 %) stress, development (11 %) and post-translational modifications (7 %). Rice is the most explored system (36 %) with major focus on abiotic mainly dehydration (36 %) stress. In spite of expensive proteomics setup and scarcity of trained workforce, output in form of publications is encouraging. To boost plant proteomics in India and Nepal, researchers have discussed ground level issues among themselves and with the International Plant Proteomics Organization (INPPO) to act in priority on concerns like food security. Active collaboration may help in translating this knowledge to fruitful applications.

Monoterpenes, which are among the major components of plant essential oils, are known for their ecological roles as well for pharmaceutical properties. Geraniol, an acyclic monoterpene induces cell cycle arrest and apoptosis/senescence in various cancer cells and plants; however, the genes involved in the process and the underlying molecular mechanisms are not well understood. In this study, we demonstrate that treatment of tomato plants with geraniol results in induction of senescence due to a substantial alteration in transcriptome. We have identified several geraniol-responsive protein encoding genes in tomato using suppression subtractive hybridization (SSH) approach. These genes comprise of various components of signal transduction, cellular metabolism, reactive oxygen species (ROS), ethylene signalling, apoptosis and DNA damage response. Upregulation of NADPH oxidase and antioxidant genes, and increase in ROS level after geraniol treatment point towards the involvement of ROS in geraniol-mediated senescence. The delayed onset of seedling death and induced expression of geraniol-responsive genes in geraniol-treated ethylene receptor mutant (Nr) suggest that geraniol-mediated senescence involves both ethylene dependent and independent pathways. Moreover, expression analysis during tomato ripening revealed that geraniol-responsive genes are also associated with the natural organ senescence process.

Development of an efficient genotype independent regeneration and genetic transformation system in sweet potato continues to be of great interest. Agrobacterium ‐ mediated genetic transformation protocol was established in two different cultivars of sweet potato using Agrobacterium strain EHA105 harbouring binary plasmid pBI121 containing GUS and nptII genes. The internodal stem segments from 30 ‐ day ‐ old micropropogated plants were used as explant with different combinations of media and hormones. MS and LS media with various concentrations of growth regulators proved to be non ‐ responsive and the infecundity was severe with the addition of cytokinins. Nonetheless, MS with 2,4 ‐ D and TDZ gave a good percentage of callusing but with low differentiation. In different concentrations of NAA, significant amount of callusing was observed but percentage of rooting remained low in both the genotypes. Gamborg’s B5 supplemented with NAA proved to be the most suitable media and hormone combination, which yielded shoot formation after 8 ‐ 10 weeks with a regenera ‐ tion efficiency of 40 ‐ 70%. Stable integration of transgene was confirmed by PCR analysis. Furthermore, qRT ‐ PCR analysis was performed to assess the transcript accumulation in addition to the GUS enzymatic assay in the transgenic lines. Plant Tissue Cult. & Biotech. 23 (1): 87 ‐ 100, 2013 (June) DOI: http://dx.doi.org/10.3329/ptcb.v23i1.15565 D.O.I. 10.3329/ptcb.v23i1.15565

Organeller proteomics is an emerging technology that is critical in determining the cellular signal transduction pathways. Nucleus, the regulatory hub of the eukaryotic cell is a dynamic system and a repository of various macromolecules that serve as modulators of such signaling that dictate cell fate decisions. Nuclear proteins are predicted to comprise about 10-20% of the total cellular proteins, suggesting the involvement of the nucleus in a number of diverse functions. Indeed, nuclear proteins constitute a highly organized but complex network that plays diverse roles during development and physiological processes. In plants, relatively little is known about the nature of the molecular components and mechanisms involved in coordinating nuclear protein synthesis, their action and function. Proteomic study hold promise to understand the molecular basis of nuclear function using an unbiased comparative and differential approach. We identified a few hundred proteins that include classical and non-canonical nuclear components presumably associated with variety of cellular functions impinge on the complexity of nuclear proteome. Here, we review the nuclear proteome based on our own findings, available literature, and databases focusing on detailed comparative analysis of nuclear proteins and their functions in order to understand how plant nucleus works. The review also shed light on the current status of plant nuclear proteome and discusses the future prospect.