Prasanna Nori Venkatesh

A Comprehensive Transcriptional Profiling of the WRKY Gene Family in Rice Under Various Abiotic and Phytohormone Treatments

WRKY transcription factors play important roles in the regulation of various biological processes. We have analyzed the publicly available rice genome sequence databases and predicted 103 genes encoding WRKY transcription factors. Among them, the majority of rice WRKY genes (77.7%) were located in duplicated regions; 45.6% of WRKY genes were fragmentally duplicated and 35% of them were tandemly duplicated. These results suggested that genome duplications might be regarded as a major mechanism for expansion of this family in the rice genome. Subsequently, we analyzed their expression profiles under normal and abiotic stress, as well as various hormone treatments. Under normal growth conditions, 65 WRKY genes were expressed differentially either in their transcript abundance or in their expression patterns. Under abiotic (cold, drought and salinity) stresses and various phytohormone treatments, 54 WRKY genes exhibited significant differences in their transcript abundance; among them three genes were expressed only in stressed conditions. Among the stress-inducible genes, 13 genes were regulated only by abiotic stresses, another set of 13 genes were responsive to only phytohormone treatments and the remaining 28 genes were regulated by both factors, suggesting an interaction between abiotic stress and hormone signaling. On the other hand, we have also surveyed the expression divergence of duplicated genes under normal or stressed conditions, and the results showed that high expression divergence has occurred not only among fragmentally but also among tandemly duplicated genes. These results suggested that the high expression divergence could be one of the mechanisms for the retention of these duplicated WRKY genes.

Genome-wide analysis of cyclin family in rice ( Oryza Sativa L.)

The cyclins together with highly conserved cyclin-dependent kinases regulate cell cycle progression in plants. Although extensive and systematic study on cell cycle mechanisms and cyclin functions in yeasts and animals has been carried out, only a small number of plant cyclins have been characterized and classified functionally and phylogenetically. We identified several types of cyclin genes in the rice genome and characterized them by phylogenetic, tandem and segmental duplications analyses. Our results indicated that there were at least 49 predicted rice cyclin genes in the rice genome, and they were distributed on 12 chromosomes. Of these cyclins, one possessed only cyclin_C domain and no cyclin_N domain, and the remaining 48 cyclins with cyclin_N domains were classified as nine types based on evolutionary relationships. Eight of these nine types were common between rice and Arabidopsis, whereas only one, known as F-type cyclins, was unique to rice. No homologues of the F-type cyclins in plants could be retrieved from the public databases, and reverse transcription-PCR analysis supported an existence of the F-type cyclin genes. Sequence alignment suggested that the cyclin genes in the rice genome experienced a mass of gene tandem and segmental duplications occurred on seven chromosomes related to the origins of new cyclin genes. Our study provided an opportunity to facilitate assessment and classification of new members, serving as a guide for further functional elucidation of rice cyclins.

Ds insertion mutagenesis as an efficient tool to produce diverse variations for rice breeding

The availability of diversified germplasm resources is the most important for developing improved rice varieties with higher seed yield or tolerance to various biotic or abiotic stresses. Here we report an efficient tool to create increased variations in rice by maize Ac/Ds transposon (a gene trap system) insertion mutagenesis. We have generated around 20,000 Ds insertion rice lines of which majority are homozygous for Ds element. We subjected these lines to phenotypic and abiotic stress screens and evaluated these lines with respect to their seed yields and other agronomic traits as well as their tolerance to drought, salinity and cold. Based on this evaluation, we observed that random Ds insertions into rice genome have led to diverse variations including a range of morphological and conditional phenotypes. Such differences in phenotype among these lines were accompanied by differential gene expression revealed by GUS histochemical staining of gene trapped lines. Among the various phenotypes identified, some Ds lines showed significantly higher grain yield compared to wild-type plants under normal growth conditions indicating that rice could be improved in grain yield by disrupting certain endogenous genes. In addition, several 1,000s of Ds lines were subjected to abiotic stresses to identify conditional mutants. Subsequent to these screens, over 800 lines responsive to drought, salinity or cold stress were obtained, suggesting that rice has the genetic potential to survive under abiotic stresses when appropriate endogenous genes were suppressed. The mutant lines that have higher seed yielding potential or display higher tolerance to abiotic stresses may be used for rice breeding by conventional backcrossing combining with molecular marker-assisted selection. In addition, by exploiting the behavior of Ds to leave footprints upon remobilization, we have shown an alternative strategy to develop new rice varieties without foreign DNA sequences in their genome.

Over-expression of OSRIP18 increases drought and salt tolerance in transgenic rice plants

Both drought and high salinity stresses are major abiotic factors that limit the yield of agricultural crops. Transgenic techniques have been regarded as effective ways to improve crops in their tolerance to these abiotic stresses. Functional characterization of genes is the prerequisite to identify candidates for such improvement. Here, we have investigated the biological functions of an Oryza sativa Ribosome-inactivating protein gene 18 (OSRIP18) by ectopically expressing this gene under the control of CaMV 35S promoter in the rice genome. We have generated 11 independent transgenic rice plants and all of them showed significantly increased tolerance to drought and high salinity stresses. Global gene expression changes by Microarray analysis showed that more than 100 probe sets were detected with up-regulated expression abundance while signals from only three probe sets were down-regulated after over-expression of OSRIP18. Most of them were not regulated by drought or high salinity stresses. Our data suggested that the increased tolerance to these abiotic stresses in transgenic plants might be due to up-regulation of some stress-dependent/independent genes and OSRIP18 may be potentially useful in further improving plant tolerance to various abiotic stresses by over-expression.

Metabolic engineering of terpene biosynthesis in plants using a trichome-specific transcription factor MsYABBY5 from spearmint (Mentha spicata)

Summary In many aromatic plants including spearmint (Mentha spicata), the sites of secondary metabolite production are tiny specialized structures called peltate glandular trichomes (PGT). Having high commercial values, these secondary metabolites are exploited largely as flavours, fragrances and pharmaceuticals. But, knowledge about transcription factors (TFs) that regulate secondary metabolism in PGT remains elusive. Understanding the role of TFs in secondary metabolism pathway will aid in metabolic engineering for increased yield of secondary metabolites and also the development of new production techniques for valuable metabolites. Here, we isolated and functionally characterized a novel MsYABBY5 gene that is preferentially expressed in PGT of spearmint. We generated transgenic plants in which MsYABBY5 was either overexpressed or silenced using RNA interference (RNAi). Analysis of the transgenic lines showed that the reduced expression of MsYABBY5 led to increased levels of terpenes and that overexpression decreased terpene levels. Additionally, ectopic expression of MsYABBY5 in Ocimum basilicum and Nicotiana sylvestris decreased secondary metabolite production in them, suggesting that the encoded transcription factor is probably a repressor of secondary metabolism.

A terpenoid phytoalexin plays a role in basal defense of Nicotiana benthamiana against Potato virus X

Terpenoid phytoalexins function as defense compound against a broad spectrum of pathogens and pests in the plant kingdom. However, the role of phytoalexin in antiviral defense is still elusive. In this study, we identified the biosynthesis pathway of a sesquiterpenoid phytoalexin, capsidiol 3-acetate as an antiviral response against RNA virus Potato Virus X (PVX) in Nicotiana benthamiana. NbTPS1 and NbEAH genes were found strongly induced by PVX-infection. Enzymatic activity and genetic evidence indicated that both genes were involved in the PVX-induced biosynthesis of capsidiol 3-acetate. NbTPS1- or NbEAH-silenced plant was more susceptible to PVX. The accumulation of capsidiol 3-acetate in PVX-infected plant was partially regulated by jasmonic acid signaling receptor COI1. These findings provide an insight into a novel mechanism of how plant uses the basal arsenal machinery to mount a fight against virus attack even in susceptible species.

Spearmint R2R3-MYB transcription factor MsMYB negatively regulates monoterpene production and suppresses the expression of geranyl diphosphate synthase large subunit (MsGPPS.LSU)

Summary Many aromatic plants, such as spearmint, produce valuable essential oils in specialized structures called peltate glandular trichomes (PGTs). Understanding the regulatory mechanisms behind the production of these important secondary metabolites will help design new approaches to engineer them. Here, we identified a PGT‐specific R2R3‐MYB gene, MsMYB, from comparative RNA‐Seq data of spearmint and functionally characterized it. Analysis of MsMYB‐RNAi transgenic lines showed increased levels of monoterpenes, and MsMYB‐overexpressing lines exhibited decreased levels of monoterpenes. These results suggest that MsMYB is a novel negative regulator of monoterpene biosynthesis. Ectopic expression of MsMYB, in sweet basil and tobacco, perturbed sesquiterpene‐ and diterpene‐derived metabolite production. In addition, we found that MsMYB binds to cis‐elements of MsGPPS.LSU and suppresses its expression. Phylogenetic analysis placed MsMYB in subgroup 7 of R2R3‐MYBs whose members govern phenylpropanoid pathway and are regulated by miR858. Analysis of transgenic lines showed that MsMYB is more specific to terpene biosynthesis as it did not affect metabolites derived from phenylpropanoid pathway. Further, our results indicate that MsMYB is probably not regulated by miR858, like other members of subgroup 7.