Shuchi Smita

Genome-wide classification and expression analysis of MYB transcription factor families in rice and Arabidopsis.

BackgroundThe MYB gene family comprises one of the richest groups of transcription factors in plants. Plant MYB proteins are characterized by a highly conserved MYB DNA-binding domain. MYB proteins are classified into four major groups namely, 1R-MYB, 2R-MYB, 3R-MYB and 4R-MYB based on the number and position of MYB repeats. MYB transcription factors are involved in plant development, secondary metabolism, hormone signal transduction, disease resistance and abiotic stress tolerance. A comparative analysis of MYB family genes in rice and Arabidopsis will help reveal the evolution and function of MYB genes in plants.ResultsA genome-wide analysis identified at least 155 and 197 MYB genes in rice and Arabidopsis, respectively. Gene structure analysis revealed that MYB family genes possess relatively more number of introns in the middle as compared with C- and N-terminal regions of the predicted genes. Intronless MYB-genes are highly conserved both in rice and Arabidopsis. MYB genes encoding R2R3 repeat MYB proteins retained conserved gene structure with three exons and two introns, whereas genes encoding R1R2R3 repeat containing proteins consist of six exons and five introns. The splicing pattern is similar among R1R2R3 MYB genes in Arabidopsis. In contrast, variation in splicing pattern was observed among R1R2R3 MYB members of rice. Consensus motif analysis of 1kb upstream region (5′ to translation initiation codon) of MYB gene ORFs led to the identification of conserved and over-represented cis-motifs in both rice and Arabidopsis. Real-time quantitative RT-PCR analysis showed that several members of MYBs are up-regulated by various abiotic stresses both in rice and Arabidopsis.ConclusionA comprehensive genome-wide analysis of chromosomal distribution, tandem repeats and phylogenetic relationship of MYB family genes in rice and Arabidopsis suggested their evolution via duplication. Genome-wide comparative analysis of MYB genes and their expression analysis identified several MYBs with potential role in development and stress response of plants.

Identification of miRNAs in sorghum by using bioinformatics approach

MicroRNAs (miRNAs) regulate gene expression mainly by post-transcriptional gene silencing (PTGS) and in some cases by transcriptional genes silencing (TGS). miRNAs play critical roles in developmental processes, nutrient homeostasis, abiotic stress and pathogen responses of plants. In contrast to the large number of miRNAs predicted in cereal model plant rice, only 148 miRNAs were predicted in sorghum till date (miRBase release 17). This suggested that miRNAs identified in sorghum is far from saturation. Hence, we developed a bioinformatics pipeline using an in-house PERL script and publicly available structure prediction tools to identify miRNAs and their target genes from publically available Expressed Sequence Tags (EST) and Genomic Survey Sequence (GSS). About 1,379 known and unique plant miRNAs from 33 different crops were used to predict new miRNAs in sorghum. We identified 31 new miRNAs belonging to 10 different miRNA families. We predicted 72 potential target genes for 31 miRNAs, and most of these target genes are predicted to be involved in plant growth and development. These newly identified miRNAs add to the growing database of miRNA and lay the foundation for further understanding of miRNA function in sorghum plant development.

Identification of novel drought-responsive microRNAs and trans-acting siRNAs from Sorghum bicolor (L.) Moench by high-throughput sequencing analysis

Small non-coding RNAs (sRNAs) namely microRNAs (miRNAs) and trans-acting small interfering RNAs (tasi-RNAs) play a crucial role in post-transcriptional regulation of gene expression and thus the control plant development and stress responses. In order to identify drought-responsive miRNAs and tasi-RNAs in sorghum, we constructed small RNA libraries from a drought tolerant (M35-1) and susceptible (C43) sorghum genotypes grown under control and drought stress conditions, and sequenced by Illumina Genome Analyzer IIx. Ninety seven conserved and 526 novel miRNAs representing 472 unique miRNA families were identified from sorghum. Ninety-six unique miRNAs were found to be regulated by drought stress, of which 32 were up- and 49 were down-regulated (fold change ≥ 2 or ≤ −2) at least in one genotype, while the remaining 15 miRNAs showed contrasting drought-regulated expression pattern between genotypes. A maximum of 17 and 18 miRNAs was differentially regulated under drought stress condition in the sensitive and tolerant genotypes, respectively. These results suggest that genotype dependent stress responsive regulation of miRNAs may contribute, at least in part, to the differential drought tolerance of sorghum genotypes. We also identified two miR390-directed TAS3 gene homologs and the auxin response factors as tasi-RNA targets. We predicted more than 1300 unique target genes for the novel and conserved miRNAs. These target genes were predicted to be involved in different cellular, metabolic, response to stimulus, biological regulation, and developmental processes. Genome-wide identification of stress-responsive miRNAs, tasi-RNAs and their targets identified in this study will be useful in unraveling the molecular mechanisms underlying drought stress responses and genetic improvement of biomass production and stress tolerance in sorghum.

Synergistic regulatory networks mediated by microRNAs and transcription factors under drought, heat and salt stresses in Oryza Sativa spp.

BACKGROUND Transcription factors (TFs) and microRNAs (miRNAs) are primary gene regulators within the cell. Regulatory mechanisms of these two main regulators are of great interest to biologists and may provide insights into the abiotic and biotic stresses. However, the interaction between miRNAs and TFs in a gene regulatory network (GRN) still remains uncovered. Previous research has been mostly directed at inferring either miRNA or TF regulatory networks from data. However, networks involving a single type of regulator may not fully reveal the complex gene regulatory mechanisms, therefore study of interplay among these two regulators in gene regulation is important towards explaining the mechanism of different abiotic stresses. RESULT Oligonucleotide microarrays containing 51,279 transcripts were used to identify total 133 salt responsive target genes regulated by 11 TFs that are also differentially regulated by miRNA under salinity, heat and drought stresses in Oryza sativa. TFs-target interactions which are most enriched in their downstream regulation were also identified. Many genes whose encoded proteins are implicated in response to light and radiation stimulus, hormone stimuli, oxidative stress, copper ion binding and electron transport were found to be enriched. However the majority were novel for the combined abiotic stress, which indicates that there are a great number of genes induced after the exposure these abiotic stresses and regulated by miRNA. CONCLUSION Analysis of the expression profile data of Oryza provides clues regarding some putative cellular and molecular processes that are undertaken in response to these stresses. The study also identified a large number of candidate functional genes that appear to be constitutively involved in salt, drought and heat stresses tolerance. Further examination of these genes may enable the molecular basis of abiotic stress tolerance in Oryza, to be elucidated.

QlicRice: a web interface for abiotic stress responsive QTL and loci interaction channels in rice

The QlicRice database is designed to host publicly accessible, abiotic stress responsive quantitative trait loci (QTLs) in rice (Oryza sativa) and their corresponding sequenced gene loci. It provides a platform for the data mining of abiotic stress responsive QTLs, as well as browsing and annotating associated traits, their location on a sequenced genome, mapped expressed sequence tags (ESTs) and tissue and growth stage-specific expressions on the whole genome. Information on QTLs related to abiotic stresses and their corresponding loci from a genomic perspective has not yet been integrated on an accessible, user-friendly platform. QlicRice offers client-responsive architecture to retrieve meaningful biological information—integrated and named ‘Qlic Search’—embedded in a query phrase autocomplete feature, coupled with multiple search options that include trait names, genes and QTL IDs. A comprehensive physical and genetic map and vital statistics have been provided in a graphical manner for deciphering the position of QTLs on different chromosomes. A convenient and intuitive user interface have been designed to help users retrieve associations to agronomically important QTLs on abiotic stress response in rice. Database URL: http://nabg.iasri.res.in:8080/qlic-rice/.

Transcriptional Regulatory Network Analysis of MYB Transcription Factor Family Genes in Rice.

MYB transcription factor (TF) is one of the largest TF families and regulates defense responses to various stresses, hormone signaling as well as many metabolic and developmental processes in plants. Understanding these regulatory hierarchies of gene expression networks in response to developmental and environmental cues is a major challenge due to the complex interactions between the genetic elements. Correlation analyses are useful to unravel co-regulated gene pairs governing biological process as well as identification of new candidate hub genes in response to these complex processes. High throughput expression profiling data are highly useful for construction of co-expression networks. In the present study, we utilized transcriptome data for comprehensive regulatory network studies of MYB TFs by “top-down” and “guide-gene” approaches. More than 50% of OsMYBs were strongly correlated under 50 experimental conditions with 51 hub genes via “top-down” approach. Further, clusters were identified using Markov Clustering (MCL). To maximize the clustering performance, parameter evaluation of the MCL inflation score (I) was performed in terms of enriched GO categories by measuring F-score. Comparison of co-expressed cluster and clads analyzed from phylogenetic analysis signifies their evolutionarily conserved co-regulatory role. We utilized compendium of known interaction and biological role with Gene Ontology enrichment analysis to hypothesize function of coexpressed OsMYBs. In the other part, the transcriptional regulatory network analysis by “guide-gene” approach revealed 40 putative targets of 26 OsMYB TF hubs with high correlation value utilizing 815 microarray data. The putative targets with MYB-binding cis-elements enrichment in their promoter region, functional co-occurrence as well as nuclear localization supports our finding. Specially, enrichment of MYB binding regions involved in drought-inducibility implying their regulatory role in drought response in rice. Thus, the co-regulatory network analysis facilitated the identification of complex OsMYB regulatory networks, and candidate target regulon genes of selected guide MYB genes. The results contribute to the candidate gene screening, and experimentally testable hypotheses for potential regulatory MYB TFs, and their targets under stress conditions.

Expression profile of genes coding for carotenoid biosynthetic pathway during ripening and their association with accumulation of lycopene in tomato fruits

Fruit ripening process is associated with change in carotenoid profile and accumulation of lycopene in tomato (Solanum lycopersicum L.). In this study, we quantified the β-carotene and lycopene content at green, breaker and red-ripe stages of fruit ripening in eight tomato genotypes by using high-performance liquid chromatography. Among the genotypes, lycopene content was found highest in Pusa Rohini and lowest in VRT-32-1. To gain further insight into the regulation of lycopene biosynthesis and accumulation during fruit ripening, expression analysis of nine carotenoid pathway-related genes was carried out in the fruits of high lycopene genotype—Pusa Rohini. We found that expression of phytoene synthase and β-carotene hydroxylase-1 was four and thirty-fold higher, respectively, at breaker stage as compared to red-ripe stage of fruit ripening. Changes in the expression level of these genes were associated with a 40% increase in lycopene content at red-ripe stage as compared with breaker stage. Thus, the results from our study suggest the role of specific carotenoid pathway-related genes in accumulation of high lycopene during the fruit ripening processes.

Identification of conserved drought stress responsive gene-network across tissues and developmental stages in rice

Identification of genes that are coexpressed across various tissues and environmental stresses is biologically interesting, since they may play coordinated role in similar biological processes. Genes with correlated expression patterns can be best identified by using coexpression network analysis of transcriptome data. In the present study, we analyzed the temporal-spatial coordination of gene expression in root, leaf and panicle of rice under drought stress and constructed network using WGCNA and Cytoscape. Total of 2199 differentially expressed genes (DEGs) were identified in at least three or more tissues, wherein 88 genes have coordinated expression profile among all the six tissues under drought stress. These 88 highly coordinated genes were further subjected to module identification in the coexpression network. Based on chief topological properties we identified 18 hub genes such as ABC transporter, ATP-binding protein, dehydrin, protein phosphatase 2C, LTPL153 - Protease inhibitor, phosphatidylethanolaminebinding protein, lactose permease-related, NADP-dependent malic enzyme, etc. Motif enrichment analysis showed the presence of ABRE cis-elements in the promoters of > 62% of the coordinately expressed genes. Our results suggest that drought stress mediated upregulated gene expression was coordinated through an ABA-dependent signaling pathway across tissues, at least for the subset of genes identified in this study, while down regulation appears to be regulated by tissue specific pathways in rice.

Comparative Analysis of Fruit Transcriptome in Tomato (Solanum lycopersicum) Genotypes with Contrasting Lycopene Contents

Carotenoid metabolism is regulated by several genes encoding carotenoid biosynthetic pathway enzymes. In the present study, a fruit transcriptome in tomato (Solanum lycopersicum) was compared between high lycopene accumulating genotype EC-521086 and low lycopene accumulating genotype VRT-32-1 at three different stages (green, breaker and red) of fruit ripening. This analysis led to the identification of 2,558 differentially expressed genes at three stages of fruit ripening. Among these genes, 123 were carotenoid-correlated genes. Quantitative RT-PCR analysis revealed high expression of genes encoding enzymes involved in lycopene biosynthesis like IPP isomerase, phytoene synthase, phytoene desaturase, z-carotene desaturase; and comparatively lower expression of genes encoding enzymes involved in lycopene catabolism like lycopene cyclase, carotenoid e-ring hydroxylase, zeaxanthin epoxidase, violaxanthin de-epoxidase and neoxanthin synthase in EC-521086, thereby possibly explaining the high lycopene content in EC-521086 as compared with the low lycopene genotype VRT-32-1. Further, the EC-521086 genotype exhibited high expression of the TOMATO AGAMOUS-LIKE 1 (TAGL1) MADS box gene—a positive regulator of lycopene accumulation—at breaker stage, and low expression of the ethylene receptor LeETR4 gene—a negative regulator of trans-lycopene and β-carotene accumulation, at the red stage of fruit ripening. Our results clearly demonstrate the role of specific genes in accumulation of high lycopene in the EC-521086 tomato genotype during the fruit ripening processes.

In silico analysis of motifs in promoters of Differentially Expressed Genes in rice (Oryza sativa L.) under anoxia

The aim of this study was to characterise the molecular mechanisms of transcriptional regulation of Differentially Expressed Genes (DEGs) in rice coleoptiles under anoxia by identifying motifs that are common in the promoter region of co-regulated genes. Un-changed DEGs (<2 fold and >-2), up-regulated DEGs (>or=2 fold) and down-regulated DEGs (<or=-2 fold) were separated in three different data sets. Their gene promoters were extracted from eukaryotic promoter database. Statistically significant consensus promoter motifs were detected by in silico method. A significant variation in the number of promoter motifs, consensus promoter motif and their sequences between UR-DEGs and DR-DEGs were detected that might be responsible for their related expression.