Sangram K. Lenka

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.

Comparative analysis of drought‐responsive transcriptome in Indica rice genotypes with contrasting drought tolerance

Genetic improvement in drought tolerance in rice is the key to save water for sustainable agriculture. Drought tolerance is a complex trait and involves interplay of a vast array of genes. Several genotypes of rice have evolved features that impart tolerance to drought and other abiotic stresses. Comparative analysis of drought stress-responsive transcriptome between drought-tolerant (DT) landraces/genotypes and drought-sensitive modern rice cultivars will unravel novel genetic regulatory mechanisms involved in stress tolerance. Here, we report transcriptome analysis in a highly DT rice landrace, Nagina 22 (N22), versus a high-yielding but drought-susceptible rice variety IR64. Both genotypes exhibited a diverse global transcriptional response under normal and drought conditions. Gene ontology (GO) analysis suggested that drought tolerance of N22 was attributable to the enhanced expression of several enzyme-encoding genes. Drought susceptibility of IR64 was attributable to significant down-regulation of regulatory components that confer drought tolerance. Pathway analysis unravelled significant up-regulation of several components of carbon fixation, glycolysis/gluconeogenesis and flavonoid biosynthesis and down-regulation of starch and sucrose metabolism in both the cultivars under drought. However, significant up-regulation of α-linolenic acid metabolic pathway observed in N22 under drought appears to be in good agreement with high drought tolerance of this genotype. Consensus cis-motif profiling of drought-induced co-expressed genes led to the identification of novel cis-motifs. Taken together, the results of the comparative transcriptome analysis led to the identification of specific genotype-dependent genes responsible for drought tolerance in the rice landrace N22.

Genome-wide targeted prediction of ABA responsive genes in rice based on over-represented cis-motif in co-expressed genes

Abscisic acid (ABA), the popular plant stress hormone, plays a key role in regulation of sub-set of stress responsive genes. These genes respond to ABA through specific transcription factors which bind to cis-regulatory elements present in their promoters. We discovered the ABA Responsive Element (ABRE) core (ACGT) containing CGMCACGTGB motif as over-represented motif among the promoters of ABA responsive co-expressed genes in rice. Targeted gene prediction strategy using this motif led to the identification of 402 protein coding genes potentially regulated by ABA-dependent molecular genetic network. RT-PCR analysis of arbitrarily chosen 45 genes from the predicted 402 genes confirmed 80% accuracy of our prediction. Plant Gene Ontology (GO) analysis of ABA responsive genes showed enrichment of signal transduction and stress related genes among diverse functional categories.

Identification and expression analysis of methyl jasmonate responsive ESTs in paclitaxel producing Taxus cuspidata suspension culture cells.

BackgroundTaxol® (paclitaxel) promotes microtubule assembly and stabilization and therefore is a potent chemotherapeutic agent against wide range of cancers. Methyl jasmonate (MJ) elicited Taxus cell cultures provide a sustainable option to meet the growing market demand for paclitaxel. Despite its increasing pharmaceutical importance, the molecular genetics of paclitaxel biosynthesis is not fully elucidated. This study focuses on identification of MJ responsive transcripts in cultured Taxus cells using PCR-based suppression subtractive hybridization (SSH) to identify genes involved in global pathway control.ResultsSix separate SSH cDNA libraries of paclitaxel-accumulating Taxus cuspidata P991 cell lines were constructed at three different post-elicitation time points (6h, 18h and 5 day) to identify genes that are either induced or suppressed in response to MJ. Sequencing of 576 differentially screened clones from the SSH libraries resulted in 331 unigenes. Functional annotation and Gene Ontology (GO) analysis of up-regulated EST libraries showed enrichment of several known paclitaxel biosynthetic genes and novel transcripts that may be involved in MJ-signaling, taxane transport, or taxane degradation. Macroarray analysis of these identified genes unravelled global regulatory expression of these transcripts. Semi-quantitative RT-PCR analysis of a set of 12 candidate genes further confirmed the MJ-induced gene expression in a high paclitaxel accumulating Taxus cuspidata P93AF cell line.ConclusionsThis study elucidates the global temporal expression kinetics of MJ responsive genes in Taxus suspension cell culture. Functional characterization of the novel genes identified in this study will further enhance the understanding of paclitaxel biosynthesis, taxane transport and degradation.

Jasmonate-responsive expression of paclitaxel biosynthesis genes in Taxus cuspidata cultured cells is negatively regulated by the bHLH transcription factors TcJAMYC1, TcJAMYC2, and TcJAMYC4

Taxus cell suspension culture is a sustainable technology for the industrial production of paclitaxel (Taxol®), a highly modified diterpene anti-cancer agent. The methyl jasmonate (MJ)-mediated paclitaxel biosynthetic pathway is not fully characterized, making metabolic engineering efforts difficult. Here, promoters of seven genes (TASY, T5αH, DBAT, DBBT, PAM, BAPT, and DBTNBT), encoding enzymes of the paclitaxel biosynthetic pathway were isolated and used to drive MJ-inducible expression of a GUS reporter construct in transiently transformed Taxus cells, showing that elicitation of paclitaxel production by MJ is regulated at least in part at the level of transcription. The paclitaxel biosynthetic pathway promoters contained a large number of E-box sites (CANNTG), similar to the binding sites for the key MJ-inducible transcription factor AtMYC2 from Arabidopsis thaliana. Three MJ-inducible MYC transcription factors similar to AtMYC2 (TcJAMYC1, TcJAMYC2, and TcJAMYC4) were identified in Taxus. Transcriptional regulation of paclitaxel biosynthetic pathway promoters by transient over expression of TcJAMYC transcription factors indicated a negative rather than positive regulatory role of TcJAMYCs on paclitaxel biosynthetic gene expression.

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/.

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.

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 characterization and homology modeling of thylakoid-bound ascorbate peroxidase from a drought tolerant wheat cultivar.

Ascorbate peroxidase, a haem protein (EC 1.11.1.11), efficiently scavenges hydrogen peroxide (H2O2) in cytosol and chloroplasts of plants. In this study, a full-length coding sequence of thylakoid-bound ascorbate peroxidase cDNA (TatAPX) was cloned from a drought tolerant wheat cultivar C306. Homology modeling of the TatAPX protein was performed by using the template crystal structure of chloroplastic ascorbate peroxidase from tobacco plant (PDB: 1IYN). The model structure was further refined by molecular mechanics and dynamic methods using various tools such as PROCHECK, ProSA and Verify3D. The predicted model was then tested for docking with H2O2, the substrate for TatAPX enzyme. The results revealed that Arg233 and Glu255 in the predicted active site of the enzyme are two important amino acid residues responsible for strong hydrogen bonding affinity with H2O2, which might play an important role in scavenging of H2O2 from the plant system.

Ectopic Expression of Rice PYL3 Enhances Cold and Drought Tolerance in Arabidopsis thaliana

Abscisic acid (ABA) plays an important role in plant development and adaptation to abiotic stresses. The pyrabactin resistance-like (PYL) gene family has been characterized as intracellular ABA receptors in Arabidopsis. We describe here the functional characterization of PYL3 ABA receptor from a drought-tolerant rice landrace Nagina 22 (N22). The induced expression level of the PYL3 transcript was observed in the N22 under different stress treatments, including cold, drought, high temperature, salt and ABA. In contrast, the expression of PYL3 was down-regulated in drought-susceptible rice cv. IR64 in response to above stresses. C-terminal GFP translational fusion of OsPYL3 was localized to both cytosol and nucleus explaining in part functional conservation of PYL protein as ABA receptor. Arabidopsis transgenic lines overexpressing OsPYL3 were hypersensitive to ABA suggesting ABA signaling pathway-dependent molecular response of the OsPYL3. Further, constitutive overexpression of OsPYL3 in Arabidopsis led to improved cold and drought stress tolerance. Thus, OsPYL3 identified in this study could be a good candidate for genetic improvement of cold and drought stress tolerance of rice and other crop plants.