Hemant R. Kushwaha

Whole-Genome Analysis of Oryza sativa Reveals Similar Architecture of Two-Component Signaling Machinery with Arabidopsis

The two-component system (TCS), which works on the principle of histidine-aspartate phosphorelay signaling, is known to play an important role in diverse physiological processes in lower organisms and has recently emerged as an important signaling system in plants. Employing the tools of bioinformatics, we have characterized TCS signaling candidate genes in the genome of Oryza sativa L. subsp. japonica. We present a complete overview of TCS gene families in O. sativa, including gene structures, conserved motifs, chromosome locations, and phylogeny. Our analysis indicates a total of 51 genes encoding 73 putative TCS proteins. Fourteen genes encode 22 putative histidine kinases with a conserved histidine and other typical histidine kinase signature sequences, five phosphotransfer genes encoding seven phosphotransfer proteins, and 32 response regulator genes encoding 44 proteins. The variations seen between gene and protein numbers are assumed to result from alternative splicing. These putative proteins have high homology with TCS members that have been shown experimentally to participate in several important physiological phenomena in plants, such as ethylene and cytokinin signaling and phytochrome-mediated responses to light. We conclude that the overall architecture of the TCS machinery in O. sativa and Arabidopsis thaliana is similar, and our analysis provides insights into the conservation and divergence of this important signaling machinery in higher plants.

Transcriptome map for seedling stage specific salinity stress response indicates a specific set of genes as candidate for saline tolerance in Oryza sativa L.

Oryza sativa L. cv IR64 is a widely cultivated, salt-sensitive indica rice, while Pokkali is a well-known, naturally salt-tolerant relative. To understand the molecular basis of differences in their salinity tolerance, three subtractive cDNA libraries were constructed. A total of 1,194 salinity-regulated cDNAs are reported here that may serve as repositories for future individual gene-based functional genomics studies. Gene expression data using macroarrays and Northern blots gives support to our hypothesis that salinity tolerance of Pokkali may be due to constitutive overexpression of many genes that function in salinity tolerance and are stress inducible in IR64. Analysis of genome architecture revealed the presence of these genes on all the chromosomes with several distinct clusters. Notably, a few mapped on one of the major quantitative trait loci – Saltol – on chromosome 1 and were found to be differentially regulated in the two contrasting genotypes. The present study also defines a set of known abiotic stress inducible genes, including CaMBP, GST, LEA, V-ATPase, OSAP1 zinc finger protein, and transcription factor HBP1B, that were expressed at high levels in Pokkali even in the absence of stress. These proposed genes may prove useful as “candidates” in improving salinity tolerance in crop plants using transgenic approach.

Genome wide expression analysis of CBS domain containing proteins in Arabidopsis thaliana (L.) Heynh and Oryza sativa L. reveals their developmental and stress regulation

BackgroundIn Arabidopsis thaliana (L.) Heynh and Oryza sativa L., a large number of genes encode proteins of unknown functions, whose characterization still remains one of the major challenges. With an aim to characterize these unknown proteins having defined features (PDFs) in plants, we have chosen to work on proteins having a cystathionine β-synthase (CBS) domain. CBS domain as such has no defined function(s) but plays a regulatory role for many enzymes and thus helps in maintaining the intracellular redox balance. Its function as sensor of cellular energy has also been widely suggested.ResultsOur analysis has identified 34 CBS domain containing proteins (CDCPs) in Arabidopsis and 59 in Oryza. In most of these proteins, CBS domain coexists with other functional domain(s), which may indicate towards their probable functions. In order to investigate the role(s) of these CDCPs, we have carried out their detailed analysis in whole genomes of Arabidopsis and Oryza, including their classification, nomenclature, sequence analysis, domain analysis, chromosomal locations, phylogenetic relationships and their expression patterns using public databases (MPSS database and microarray data). We have found that the transcript levels of some members of this family are altered in response to various stresses such as salinity, drought, cold, high temperature, UV, wounding and genotoxic stress, in both root and shoot tissues. This data would be helpful in exploring the so far obscure functions of CBS domain and CBS domain-containing proteins in plant stress responses.ConclusionWe have identified, classified and suggested the nomenclature of CDCPs in Arabidopsis and Oryza. A comprehensive analysis of expression patterns for CDCPs using the already existing transcriptome profiles and MPSS database reveals that a few CDCPs may have an important role in stress response/tolerance and development in plants, which needs to be validated further through functional genomics.

Clustered metallothionein genes are co-regulated in rice and ectopic expression of OsMT1e-P confers multiple abiotic stress tolerance in tobacco via ROS scavenging

BackgroundMetallothioneins (MT) are low molecular weight, cysteine rich metal binding proteins, found across genera and species, but their function(s) in abiotic stress tolerance are not well documented.ResultsWe have characterized a rice MT gene, OsMT1e-P, isolated from a subtractive library generated from a stressed salinity tolerant rice genotype, Pokkali. Bioinformatics analysis of the rice genome sequence revealed that this gene belongs to a multigenic family, which consists of 13 genes with 15 protein products. OsMT1e-P is located on chromosome XI, away from the majority of other type I genes that are clustered on chromosome XII. Various members of this MT gene cluster showed a tight co-regulation pattern under several abiotic stresses. Sequence analysis revealed the presence of conserved cysteine residues in OsMT1e-P protein. Salinity stress was found to regulate the transcript abundance of OsMT1e-P in a developmental and organ specific manner. Using transgenic approach, we found a positive correlation between ectopic expression of OsMT1e-P and stress tolerance. Our experiments further suggest ROS scavenging to be the possible mechanism for multiple stress tolerance conferred by OsMT1e-P.ConclusionWe present an overview of MTs, describing their gene structure, genome localization and expression patterns under salinity and development in rice. We have found that ectopic expression of OsMT1e-P enhances tolerance towards multiple abiotic stresses in transgenic tobacco and the resultant plants could survive and set viable seeds under saline conditions. Taken together, the experiments presented here have indicated that ectopic expression of OsMT1e-P protects against oxidative stress primarily through efficient scavenging of reactive oxygen species.

Presence of unique glyoxalase III proteins in plants indicates the existence of shorter route for methylglyoxal detoxification

Glyoxalase pathway, comprising glyoxalase I (GLY I) and glyoxalase II (GLY II) enzymes, is the major pathway for detoxification of methylglyoxal (MG) into D-lactate involving reduced glutathione (GSH). However, in bacteria, glyoxalase III (GLY III) with DJ-1/PfpI domain(s) can do the same conversion in a single step without GSH. Our investigations for the presence of DJ-1/PfpI domain containing proteins in plants have indicated the existence of GLY III-like proteins in monocots, dicots, lycopods, gymnosperm and bryophytes. A deeper in silico analysis of rice genome identified twelve DJ-1 proteins encoded by six genes. Detailed analysis has been carried out including their chromosomal distribution, genomic architecture and localization. Transcript profiling under multiple stress conditions indicated strong induction of OsDJ-1 in response to exogenous MG. A member of OsDJ-1 family, OsDJ-1C, showed high constitutive expression at all developmental stages and tissues of rice. MG depletion study complemented by simultaneous formation of D-lactate proved OsDJ-1C to be a GLY III enzyme that converts MG directly into D-lactate in a GSH-independent manner. Site directed mutagenesis of Cys-119 to Alanine significantly reduces its GLY III activity indicating towards the existence of functional GLY III enzyme in rice—a shorter route for MG detoxification.

Analysis of global gene expression profile of rice in response to methylglyoxal indicates its possible role as a stress signal molecule.

Methylglyoxal (MG) is a toxic metabolite produced primarily as a byproduct of glycolysis. Being a potent glycating agent, it can readily bind macromolecules like DNA, RNA, or proteins, modulating their expression and activity. In plants, despite the known inhibitory effects of MG on growth and development, still limited information is available about the molecular mechanisms and response pathways elicited upon elevation in MG levels. To gain insight into the molecular basis of MG response, we have investigated changes in global gene expression profiles in rice upon exposure to exogenous MG using GeneChip microarrays. Initially, growth of rice seedlings was monitored in response to increasing MG concentrations which could retard plant growth in a dose-dependent manner. Upon exposure to 10 mM concentration of MG, a total of 1685 probe sets were up- or down-regulated by more than 1.5-fold in shoot tissues within 16 h. These were classified into 10 functional categories. The genes involved in signal transduction such as, protein kinases and transcription factors, were significantly over-represented in the perturbed transcriptome, of which several are known to be involved in abiotic and biotic stress response indicating a cross-talk between MG-responsive and stress-responsive signal transduction pathways. Through in silico studies, we could predict 7–8 bp long conserved motif as a possible MG-responsive element (MGRE) in the 1 kb upstream region of genes that were more than 10-fold up- or down-regulated in the analysis. Since several perturbations were found in signaling cascades in response to MG, we hereby suggest that it plays an important role in signal transduction probably acting as a stress signal molecule.

Tissue specific and abiotic stress regulated transcription of histidine kinases in plants is also influenced by diurnal rhythm.

Two-component system (TCS) is one of the key signal sensing machinery which enables species to sense environmental stimuli. It essentially comprises of three major components, sensory histidine kinase proteins (HKs), histidine phosphotransfer proteins (Hpts), and response regulator proteins (RRs). The members of the TCS family have already been identified in Arabidopsis and rice but the knowledge about their functional indulgence during various abiotic stress conditions remains meager. Current study is an attempt to carry out comprehensive analysis of the expression of TCS members in response to various abiotic stress conditions and in various plant tissues in Arabidopsis and rice using MPSS and publicly available microarray data. The analysis suggests that despite having almost similar number of genes, rice expresses higher number of TCS members during various abiotic stress conditions than Arabidopsis. We found that the TCS machinery is regulated by not only various abiotic stresses, but also by the tissue specificity. Analysis of expression of some representative members of TCS gene family showed their regulation by the diurnal cycle in rice seedlings, thus bringing-in another level of their transcriptional control. Thus, we report a highly complex and tight regulatory network of TCS members, as influenced by the tissue, abiotic stress signal, and diurnal rhythm. The insights on the comparative expression analysis presented in this study may provide crucial leads toward dissection of diverse role(s) of the various TCS family members in Arabidopsis and rice.

MATH-Domain Family Shows Response toward Abiotic Stress in Arabidopsis and Rice.

Response to stress represents a highly complex mechanism in plants involving a plethora of genes and gene families. It has been established that plants use some common set of genes and gene families for both biotic and abiotic stress responses leading to cross-talk phenomena. One such family, Meprin And TRAF Homology (MATH) domain containing protein (MDCP), has been known to be involved in biotic stress response. In this study, we present genome-wide identification of various members of MDCP family from both Arabidopsis and rice. A large number of members identified in Arabidopsis and rice indicate toward an expansion and diversification of MDCP family in both the species. Chromosomal localization of MDCP genes in Arabidopsis and rice reveals their presence in a few specific clusters on various chromosomes such as, chromosome III in Arabidopsis and chromosome X in rice. For the functional analysis of MDCP genes, we used information from publicly available data for plant growth and development as well as biotic stresses and found differential expression of various members of the family. Further, we narrowed down 11 potential candidate genes in rice which showed high expression in various tissues and development stages as well as biotic stress conditions. The expression analysis of these 11 genes in rice using qRT-PCR under drought and salinity stress identified OsM4 and OsMB11 to be highly expressed in both the stress conditions. Taken together, our data indicates that OsM4 and OsMB11 can be used as potential candidates for generating stress resilient crops.

Putative osmosensor – OsHK3b – a histidine kinase protein from rice shows high structural conservation with its ortholog AtHK1 from Arabidopsis

Prokaryotes and eukaryotes respond to various environmental stimuli using the two-component system (TCS). Essentially, it consists of membrane-bound histidine kinase (HK) which senses the stimuli and further transfers the signal to the response regulator, which in turn, regulates expression of various target genes. Recently, sequence-based genome wide analysis has been carried out in Arabidopsis and rice to identify all the putative members of TCS family. One of the members of this family i.e. AtHK1, (a putative osmosensor, hybrid-type sensory histidine kinase) is known to interact with AtHPt1 (phosphotransfer proteins) in Arabidopsis. Based on predicted rice interactome network (PRIN), the ortholog of AtHK1 in rice, OsHK3b, was found to be interacting with OsHPt2. The analysis of amino acid sequence of AtHK1 showed the presence of transmitter domain (TD) and receiver domain (RD), while OsHK3b showed presence of three conserved domains namely CHASE (signaling domain), TD, and RD. In order to elaborate on structural details of functional domains of hybrid-type HK and phosphotransfer proteins in both these genera, we have modeled them using homology modeling approach. The structural motifs present in various functional domains of the orthologous proteins were found to be highly conserved. Binding analysis of the RD domain of these sensory proteins in Arabidopsis and rice revealed the role of various residues such as histidine in HPt protein which are essential for their interaction.

Draft Genome Sequence of the Paenibacillus sp. Strain ICGEB2008 (MTCC 5639) Isolated from the Gut of Helicoverpa armigera

ABSTRACT Paenibacillus sp. strain ICGEB2008 (MTCC 5639) is a Gram-positive cellulolytic bacterium, isolated from the gut of Helicoverpa armigera. Here, we report the draft genome sequence of Paenibacillus sp. ICGEB2008. The annotation of the ~5.7-Mb sequence indicated a cluster of genes related to the glycosyl hydrolase family and the butanediol biosynthesis pathway.