Meenu Kapoor

Genome-wide identification, organization and phylogenetic analysis of Dicer-like, Argonaute and RNA-dependent RNA Polymerase gene families and their expression analysis during reproductive development and stress in rice

BackgroundImportant developmental processes in both plants and animals are partly regulated by genes whose expression is modulated at the post-transcriptional level by processes such as RNA interference (RNAi). Dicers, Argonautes and RNA-dependent RNA polymerases (RDR) form the core components that facilitate gene silencing and have been implicated in the initiation and maintenance of the trigger RNA molecules, central to process of RNAi. Investigations in eukaryotes have revealed that these proteins are encoded by variable number of genes with plants showing relatively higher number in each gene family. To date, no systematic expression profiling of these genes in any of the organisms has been reported.ResultsIn this study, we provide a complete analysis of rice Dicer-like, Argonaute and RDR gene families including gene structure, genomic localization and phylogenetic relatedness among gene family members. We also present microarray-based expression profiling of these genes during 14 stages of reproductive and 5 stages of vegetative development and in response to cold, salt and dehydration stress. We have identified 8 Dicer-like (OsDCLs), 19 Argonaute (OsAGOs) and 5 RNA-dependent RNA polymerase (OsRDRs) genes in rice. Based on phylogeny, each of these genes families have been categorized into four subgroups. Although most of the genes express both in vegetative and reproductive organs, 2 OsDCLs, 14 OsAGOs and 3 OsRDRs were found to express specifically/preferentially during stages of reproductive development. Of these, 2 OsAGOs exhibited preferential up-regulation in seeds. One of the Argonautes (OsAGO2) also showed specific up-regulation in response to cold, salt and dehydration stress.ConclusionThis investigation has identified 23 rice genes belonging to DCL, Argonaute and RDR gene families that could potentially be involved in reproductive development-specific gene regulatory mechanisms. These data provide an insight into probable domains of activity of these genes and a basis for further, more detailed investigations aimed at understanding the contribution of individual components of RNA silencing machinery during reproductive phase of plant development.

Rice cytosine DNA methyltransferases - gene expression profiling during reproductive development and abiotic stress

DNA methylation affects important developmental processes in both plants and animals. The process of methylation of cytosines at C‐5 is catalysed by DNA methyltransferases (MTases), which are highly conserved, both structurally and functionally, in eukaryotes. In this study, we identified and characterized cytosine DNA MTase genes that are activated with the onset of reproductive development in rice. The rice genome (Oryza sativa L. subsp. japonica) encodes a total of 10 genes that contain the highly conserved MTase catalytic domain. These genes have been categorized into subfamilies on the basis of phylogenetic relationships. A microarray‐based gene expression profile of all 10 MTases during 22 stages/tissues that included 14 stages of reproductive development and five vegetative tissues together with three stresses, cold, salt and dehydration stress, revealed specific windows of MTase activity during panicle and seed development. The expression of six methylases was specifically/preferentially upregulated with the initiation of floral organs. Significantly, one of the MTases was also activated in young seedlings in response to cold and salt stress. The molecular studies presented here suggest a greater role for these proteins and the epigenetic process in affecting genome activity during reproductive development and stress than was previously anticipated.

Role of DNA methylation in growth and differentiation in Physcomitrella patens and characterization of cytosine DNA methyltransferases

Epigenetic mechanisms such as DNA methylation are known to regulate important developmental processes in higher eukaryotes. However, little is known about the necessity and role of this process in early land plants. Using the methyltransferase (MTase) inhibitor zebularine (1‐(β‐d‐ribofuranosyl)‐1,2‐dihydropyrimidine‐2‐one), the impact of loss of genome‐wide methylation on the overall development in Physcomitrella patens was analyzed. It is observed that various aspects of growth and differentiation during gametophyte development become aberrant. A search for the core molecular components of methylation machinery, cytosine DNA MTases, revealed the presence of seven loci in the P. patens genome. Five of the loci code for MTases that are similar to corresponding proteins in higher plants, while two MTases appear specific to P. patens and are closely related to human DNMT3a and DNMT3b, respectively. These proteins possess all the conserved catalytic motifs characteristic of MTases and a domain of unknown function, DUF3444. Association of these highly conserved motifs with a DUF has not been reported in any of the MTases known so far. All the seven genes are differentially but ubiquitously expressed in gametophytes at low levels. Subcellular localization of GFP‐fused proteins shows patterns of distribution that can be correlated with their putative cellular functions. This work bridges the knowledge of MTases in P. patens and makes this simple model plant accessible for studies on epigenetic aspects that remain intractable in higher plants.

De Novo Methyltransferase, OsDRM2, Interacts with the ATP-Dependent RNA Helicase, OseIF4A, in Rice

Domains rearranged methyltransferases (DRMs) are the de novo methyltransferases that regulate cytosine methylation in plants in a manner similar to the animal de novo methyltransferases, DNMT3a and DNMT3b. These enzymes catalyze the establishment of new methylation patterns and are guided to target sites by small RNAs through the process of RNA-directed DNA methylation (RdDM). In the current accepted view for RdDM, intricate interactions among transcription factors/chromatin modifying proteins and the large subunits of plant-specific polymerases, Pol IV and Pol V, regulate the 24-nt small interfering RNA guided de novo methylation of cytosines. The RNA-induced silencing complex assembled on Pol-V-transcribed non-coding RNA finally facilitates the recruitment of DRM2 by unknown mechanism/protein interactions to chromatin sites. In an attempt to determine the cellular proteins that specifically interact with DRM2, a yeast two-hybrid screen was performed using young rice panicles. We report that rice DRM2 interacts with the ATP-dependent RNA helicase, eIF4A. Direct interaction between the two proteins is demonstrated in vivo by bimolecular fluorescence complementation method and in vitro by histidine-pull-down assays. Deletion analysis reveals that interaction between OsDRM2 and OseIF4A is specifically mediated through ubiquitin-associated domain of OsDRM2 while, both domains 1 and 2 of OseIF4A are critical for mediating strong interaction with OsDRM2 in vivo. Interaction between Arabidopsis eIF4AI and eIF4AII with OsDRM2 and nuclear localization of these complexes suggests possible conservation of functional interaction between de novo methyltransferases and the translation initiation factor, eIF4A, in RdDM across plant species.

Post-translational regulation of rice MADS29 function: homodimerization or binary interactions with other seed-expressed MADS proteins modulate its translocation into the nucleus

Summary OsMADS29, a seed-specific transcription factor that affects grain filling and embryo development by regulating hormone homeostasis, requires homo- or heterodimerization with eleven other MADS proteins for its localization into the nucleus.

Physcomitrella patens DNA Methyltransferase 2 is Required for Recovery from Salt and Osmotic Stress

DNA methyltransferase 2 (DNMT2) unlike other members of the cytosine DNA methyltransferase gene family has dual substrate specificity and it methylates cytosines in both the DNA and transfer RNA (tRNA). Its role in plants, however, has remained obscure to date. In this study, we demonstrate that DNMT2 from Physcomitrella patens accumulates in a temporal manner under salt and osmotic stress showing maximum accumulation during recovery, i.e. 24 h after plants are transferred to normal growth medium. Therefore, to study its role in stress tolerance, we generated PpDNMT2 targeted knockout plants (ppdnmt2ko). Mutant plants show increased sensitivity to salt and osmotic stress and are unable to recover even after 21 days of growth on optimal growth media. ppdnmt2ko, however, accumulate normal levels of dehydrin‐like and small heat shock protein encoding transcripts under stress but show dramatic reduction in levels of tRNAAsp‐GUC. The levels of tRNAAsp‐GUC, in contrast, increase ~ 25–30‐fold in ppdnmt2ko under non‐stress conditions and > 1200‐fold in wild‐type plants under stress. The role of PpDNMT2 in modulating biogenesis/stability of tRNAAsp‐GUC under salt and osmotic stress is discussed in the light of these observations.