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    Ubiquitination: a tool for plant adaptation to changing environments
    (Springer Nature, 2018) Mandal, Arunava; Sharma, Namisha; Muthamilarasan, Mehanathan; Prasad, Manoj
    Post-translational modifcations namely ubiquitination, phosphorylation, methylation and acetylation play distinct roles in regulating the growth and development of plants. Among these, the ubiquitination regulates the abundance, activities, subcellular compartmentalization and trafcking of regulatory proteins involved in diverse developmental as well as stress-responsive processes. The ubiquitin–proteasome system (UPS) involves fve essential components namely ubiquitin, ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), ubiquitin ligase (E3) and the intact 26S proteasome. The E3 ubiquitin ligase is the major component of UPS that recognizes and tethers poly-ubiquitins on the target proteins. Owing to its specifcity of substrate recognition, the E3 ubiquitin ligase contributes not only to the proteome plasticity of the cell but also regulates the plant’s response to environmental cues. In this context, the review summarizes the components involved in UPS and elaborates the role of E3 ubiquitin ligase in biotic and abiotic stress responses.
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    Characterization of DEAD-box family of RNA helicases in tomato provides insights into their roles in biotic and abiotic stresses
    (Elsevier B.V., 2019) Pandey, Saurabh; Muthamilarasan, Mehanathan; Sharma, Namisha; Chaudhry, Vaishali; Dulani, Priya; Shweta, Shweta; Jha, Sarita; Mathur, Saloni; Prasad, Manoj
    In plants, RNA helicases play significant roles in growth, development and stress response. In a previous study, a three-fold upregulation of a DEAD-box RNA helicase in a tomato cultivar tolerant to Tomato leaf curl New Delhi virus (ToLCNDV) as compared to susceptible cultivar during virus infection was shown. Given this, a comprehensive study was performed to identify the members of RNA helicase family in tomato and analyze their functional properties in response to abiotic stresses, hormone treatments and ToLCNDV infection. A total of 131 genes were identified and classified into DEAD- (42), DEAH- (38), and DExD/H-box (51) RNA helicases. Expression profiling of candidate genes in response to abiotic stresses and ToLCNDV infection in contrasting tomato cultivars suggested the putative roles of SlDEAD23 and SlDEAD35 in biotic and abiotic stresses. Heterologous overexpression of these genes in yeast enhanced the tolerance of transgenic cells to salt and cold stresses. Further, virus-induced silencing of SlDEAD35 in ToLCNDV tolerant cultivar resulted in susceptibility to virus infection, thus suggesting its involvement in tolerance mechanism. Altogether, this study provides novel insights into the structure, organization and involvement of DEAD-box RNA helicase genes in biotic and abiotic stress responses in tomato.
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    Salinity induced differential methylation patterns in contrasting cultivars of foxtail millet (Setaria italica L.)
    (Springer, 2017) Pandey, Garima; Yadav, Chandra Bhan; Sahu, Pranav Pankaj; Muthamilarasan, Mehanathan; Prasad, Manoj
    Reduced productivity and significant yield loss are the adverse effects of environmental conditions on physiological and biochemical pathways in crop plants. In this context, understanding the epigenetic machinery underlying the tolerance traits in a naturally stress tolerant crop is imperative. Foxtail millet (Setaria italica) is known for its better tolerance to abiotic stresses compared to other cereal crops. In the present study, methylation-sensitive amplified polymorphism (MSAP) technique was used to quantify the salt-induced methylation changes in two foxtail millet cultivars contrastingly differing in their tolerance levels to salt stress. The study highlighted that the DNA methylation level was significantly reduced in tolerant cultivar compared to sensitive cultivar. A total of 86 polymorphic MSAP fragments were identified, sequenced and functionally annotated. These fragments showed sequence similarity to several genes including ABC transporter, WRKY transcription factor, serine threonine-protein phosphatase, disease resistance, oxidoreductases, cell wall-related enzymes and retrotransposon and transposase like proteins, suggesting salt stress-induced methylation in these genes. Among these, four genes were chosen for expression profiling which showed differential expression pattern between both cultivars of foxtail millet. Altogether, the study infers that salinity stress induces genome-wide DNA demethylation, which in turn, modulates expression of corresponding genes.
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    Comprehensive analysis of SET domain gene family in foxtail millet identifies the putative role of SiSET14 in abiotic stress tolerance
    (Nature Publishing Group, 2016) Yadav, Chandra Bhan; Muthamilarasan, Mehanathan; Dangi, Anand; Shweta, Shweta; Prasad, Manoj
    SET domain-containing genes catalyse histone lysine methylation, which alters chromatin structure and regulates the transcription of genes that are involved in various developmental and physiological processes. The present study identified 53 SET domain-containing genes in C4 panicoid model, foxtail millet (Setaria italica) and the genes were physically mapped onto nine chromosomes. Phylogenetic and structural analyses classified SiSET proteins into five classes (I–V). RNA-seq derived expression profiling showed that SiSET genes were differentially expressed in four tissues namely, leaf, root, stem and spica. Expression analyses using qRT-PCR was performed for 21 SiSET genes under different abiotic stress and hormonal treatments, which showed differential expression of these genes during late phase of stress and hormonal treatments. Significant upregulation of SiSET gene was observed during cold stress, which has been confirmed by over-expressing a candidate gene, SiSET14 in yeast. Interestingly, hypermethylation was observed in gene body of highly differentially expressed genes, whereas methylation event was completely absent in their transcription start sites. This suggested the occurrence of demethylation events during various abiotic stresses, which enhance the gene expression. Altogether, the present study would serve as a base for further functional characterization of SiSET genes towards understanding their molecular roles in conferring stress tolerance.
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    Genome-wide analysis of heat shock proteins in C4 model, foxtail millet identifies potential candidates for crop improvement under abiotic stress
    (Nature Publishing Group, 2016) Singh, Roshan Kumar; Jaishankar, Jananee; Muthamilarasan, Mehanathan; Shweta, Shweta; Dangi, Anand; Prasad, Manoj
    Heat shock proteins (HSPs) perform significant roles in conferring abiotic stress tolerance to crop plants. In view of this, HSPs and their encoding genes were extensively characterized in several plant species; however, understanding their structure, organization, evolution and expression profiling in a naturally stress tolerant crop is necessary to delineate their precise roles in stress-responsive molecular machinery. In this context, the present study has been performed in C4 panicoid model, foxtail millet, which resulted in identification of 20, 9, 27, 20 and 37 genes belonging to SiHSP100, SiHSP90, SiHSP70, SiHSP60 and SisHSP families, respectively. Comprehensive in silico characterization of these genes followed by their expression profiling in response to dehydration, heat, salinity and cold stresses in foxtail millet cultivars contrastingly differing in stress tolerance revealed significant upregulation of several genes in tolerant cultivar. SisHSP-27 showed substantial higher expression in response to heat stress in tolerant cultivar, and its over-expression in yeast system conferred tolerance to several abiotic stresses. Methylation analysis of SiHSP genes suggested that, in susceptible cultivar, higher levels of methylation might be the reason for reduced expression of these genes during stress. Altogether, the study provides novel clues on the role of HSPs in conferring stress tolerance.
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    Comprehensive genome-wide identification and expression profiling of foxtail millet [Setaria italica (L.)] miRNAs in response to abiotic stress and development of miRNA database
    (Springer, 2014) Khan, Yusuf; Yadav, Amita; Bonthala, Venkata Suresh; Muthamilarasan, Mehanathan; Yadav, Chandra Bhan; Prasad, Manoj
    MicroRNA (miRNA)-guided post-transcriptional regulation is an important mechanism of gene regulation during multiple biological processes including response to abiotic stresses. Foxtail millet is a model crop, which is genetically closely related to several bioenergy grasses and also known for its potential abiotic stress tolerance. Hence deciphering the role of miRNAs in regulating stress-responsive mechanism would enable imparting durable stress tolerance in both millets and bioenergy grasses. Considering this, a comprehensive genome-wide in silico analysis was performed in foxtail millet which identified 355 mature miRNAs along with their secondary structure as well as corresponding targets. Predicted miRNA targets were found to encode various DNA binding proteins, transcription factors or important functional enzymes, which could be the crucial regulators in plant abiotic stress responses. All the 355 miRNAs were physically mapped onto the foxtail millet genome and in silico tissue-specific expression for these miRNAs were studied. Comparative mapping of the 355 miRNAs between foxtail millet and other related grass species would assist miRNA studies in these genetically closely-related plants. Expression profiling was performed for eight candidate miRNAs under diverse abiotic stresses in foxtail millet, which unravelled the putative involvement of these miRNAs in stress tolerance. With an aim of providing the generated miRNA marker information to the global scientific community, a foxtail millet MiRNA Database (FmMiRNADb: http://​59.​163.​192.​91/​FmMiRNADb/​index.​html) has also been constructed. Overall, the present study provides novel insights onto the role of miRNAs in abiotic stress tolerance and would promisingly expedite research on post-transcriptional regulation of stress-related genes in millets and bioenergy grasses.
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    Reference genes for quantitative real-time PCR analysis in the model plant foxtail millet (Setaria italica L.) subjected to abiotic stress conditions
    (Springer, 2013) Kumar, Karunesh; Muthamilarasan, Mehanathan; Prasad, Manoj
    Reference genes are standards for quantifying gene expression through quantitative real-time PCR (qRT-PCR); however, the variation observed in their expression levels is the major hindrance towards realising their effective use. Hence, a systematic validation of reference genes is required to ensure proper normalization. However, no such study has been conducted in foxtail millet [Setaria italica (L.)], which has recently emerged as a model crop for genetic and genomic studies. In the present study, 8 commonly used reference genes were evaluated, including 18S ribosomal RNA, elongation factor-1α, Actin2, alpha tubulin, beta tubulin, translation factor, RNA polymerase II and adenine phosphoribosyl transferase. Expression stability of candidate internal control genes was investigated under salinity and dehydration treatments. The results obtained suggested a wide range of Ct values and variable expression of all reference genes. geNorm and NormFinder analysis had revealed that Act2 and RNA POL II are suitable reference genes for salinity stress-related studies and EF-1α and RNA POL II are appropriate internal controls for dehydration stress-related expression analyses. These qualified reference genes has also been validated for relative quantification of 14-3-3 expression analysis which demonstrated their applicability. Thus, this is the first report on selection and validation of superior reference genes for qRT-PCR in foxtail millet under different abiotic stress conditions.