Publications of NIPGR Scientists

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Subject: search.filters.subject.Plant molecular biology

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    Duplicated RGS (Regulator of G-protein signaling) proteins exhibit conserved biochemical but differential transcriptional regulation of heterotrimeric G-protein signaling in Brassica species
    (Nature Publishing Group, 2018) Kumar, Roshan; Bisht, Naveen C.
    G-alpha (Gα) and 'Regulator of G-protein Signaling (RGS)' proteins are the two key components primarily involved in regulation of heterotrimeric G-proteins signaling across phyla. Unlike Arabidopsis thaliana, our knowledge about G-protein regulation in polyploid Brassica species is sparse. In this study, we identified one Gα and two RGS genes each from three species of Brassica 'U' triangle and assessed the effects of whole genome triplication on the divergence of gene sequence and structure, protein-protein interaction, biochemical activities, and gene expression. Sequence and phylogenetic analysis revealed that the deduced Gα and RGS proteins are evolutionarily conserved across Brassica species. The duplicated RGS proteins of each Brassica species interacted with their cognate Gα but displayed varying levels of interaction strength. The Gα and the duplicated RGS proteins of Brassica species exhibited highly conserved G-protein activities when tested under in-vitro conditions. Expression analysis of the B. rapa RGS genes revealed a high degree of transcriptional differentiation across the tested tissue types and in response to various elicitors, particularly under D-glucose, salt and phytohormone treatments. Taken together, our results suggest that the RGS-mediated regulation of G-protein signaling in Brassica species is predominantly governed by stage and condition-specific expression differentiation of the duplicated RGS genes.
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    Identification of miRNA-mediated drought responsive multi-tiered regulatory network in drought tolerant rice, Nagina 22
    (Nature Publishing Group, 2017) Balyan, Sonia; Kumar, Mukesh; Mutum, Roseeta Devi; Raghuvanshi, Utkarsh; Agarwal, Priyanka; Mathur, Saloni; Raghuvanshi, Saurabh
    Comparative characterization of microRNA-mediated stress regulatory networks in contrasting rice cultivars is critical to decipher plant stress response. Consequently, a multi-level comparative analysis, using sRNA sequencing, degradome analysis, enzymatic and metabolite assays and metal ion analysis, in drought tolerant and sensitive rice cultivars was conducted. The study identified a group of miRNAs "Cultivar-specific drought responsive" (CSDR)-miRNAs (osa-miR159f, osa-miR1871, osa-miR398b, osa-miR408-3p, osa-miR2878-5p, osa-miR528-5p and osa-miR397a) that were up-regulated in the flag-leaves of tolerant cultivar, Nagina 22 (N22) and Vandana, but down-regulated in the sensitive cultivar, Pusa Basmati 1 (PB1) and IR64, during drought. Interestingly, CSDR-miRNAs target several copper-protein coding transcripts like plantacyanins, laccases and Copper/Zinc superoxide dismutases (Cu/Zn SODs) and are themselves found to be similarly induced under simulated copper-starvation in both N22 and PB1. Transcription factor OsSPL9, implicated in Cu-homeostasis also interacted with osa-miR408-3p and osa-miR528-5p promoters. Further, N22 flag leaves showed lower SOD activity, accumulated ROS and had a higher stomata closure. Interestingly, compared to PB1, internal Cu levels significantly decreased in the N22 flag-leaves, during drought. Thus, the study identifies the unique drought mediated dynamism and interplay of Cu and ROS homeostasis, in the flag leaves of drought tolerant rice, wherein CSDR-miRNAs play a pivotal role.
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    Genome-wide identification and co-expression network analysis provide insights into the roles of auxin response factor gene family in chickpea
    (Nature Publishing Group, 2017) Singh, Vikash K.; Rajkumar, Mohan Singh; Garg, Rohini; Jain, Mukesh
    Auxin response factors (ARFs) are the transcription factors that regulate auxin responses in various aspects of plant growth and development. Although genome-wide analysis of ARF gene family has been done in some species, no information is available regarding ARF genes in chickpea. In this study, we identified 28 ARF genes (CaARF) in the chickpea genome. Phylogenetic analysis revealed that CaARFs can be divided into four different groups. Duplication analysis revealed that 50% of CaARF genes arose from duplication events. We analyzed expression pattern of CaARFs in various developmental stages. CaARF16.3, CaARF17.1 and CaARF17.2 showed highest expression at initial stages of flower bud development, while CaARF6.2 had higher expression at later stages of flower development. Further, CaARF4.2, CaARF9.2, CaARF16.2 and CaARF7.1 exhibited differential expression under different abiotic stress conditions, suggesting their role in abiotic stress responses. Co-expression network analysis among CaARF, CaIAA and CaGH3 genes enabled us to recognize components involved in the regulatory network associated with CaARFs. Further, we identified microRNAs that target CaARFs and TAS3 locus that trigger production of trans-acting siRNAs targeting CaARFs. The analyses presented here provide comprehensive information on ARF family members and will help in elucidating their exact function in chickpea.
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    Phylogenomic analysis of MKKs and MAPKs from 16 legumes and detection of interacting pairs in chickpea divulge MAPK signalling modules
    (Nature Publishing Group, 2017) Purayannur, Savithri; Kumar, Kamal; Kaladhar, Vemula Chandra; Verma, Praveen K.
    The mitogen-activated protein kinase (MAPK)-mediated phosphorylation cascade is a vital component of plant cellular signalling. Despite this, MAPK signalling cascade is less characterized in crop legumes. To fill this void, we present here a comprehensive phylogeny of MAPK kinases (MKKs) and MAPKs identified from 16 legume species belonging to genistoid (Lupinus angustifolius), dalbergioid (Arachis spp.), phaseoloid (Glycine max, Cajanus cajan, Phaseolus vulgaris, and Vigna spp.), and galegoid (Cicer arietinum, Lotus japonicus, Medicago truncatula, Pisum sativum, Trifolium spp., and Vicia faba) clades. Using the genes of the diploid crop chickpea (C. arietinum), an exhaustive interaction analysis was performed between MKKs and MAPKs by split-ubiquitin based yeast two-hybrid (Y2H). Twenty seven interactions of varying strengths were identified between chickpea MKKs and MAPKs. These interactions were verified in planta by bimolecular fluorescence complementation (BiFC). As a first report in plants, four intra-molecular interactions of weak strength were identified within chickpea MKKs. Additionally; two TEOSINTE-BRANCHED1/CYCLOIDEA/PCF (TCP) transcription factors of class I were identified as novel down-stream interacting partners of seven MAPKs. We propose that this highly reliable MAPK interaction network, presented here for chickpea, can be utilized as a reference for legumes and thus will help in deciphering their role in legume-specific events.
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    MicroRNA profiling provides insights into post-transcriptional regulation of gene expression in chickpea root apex under salinity and water deficiency
    (Nature Publishing Group, 2017) Khandal, Hitaishi; Parween, Sabiha; Roy, Riti; Meena, Mukesh Kumar; Chattopadhyay, Debasis
    Activity of root apical meristem (RAM) at the root apex is critical for stress-mediated modulation of root-architecture. Chickpea, like other legumes, possesses a basic open root meristem. Deep sequencing was used to perform microRNA expression profiling in root apex of chickpea (Cicer arietinum L.) in order to investigate post-transcriptional regulation of gene expression in this tissue in response to salinity and water deficit. Five small RNA libraries prepared from chickpea root apices at different stages of stress treatments were sequenced to obtain 284 unique miRNA sequences including 60 novel miRNAs belonging to total 255 families. Two hundred and fiftynine miRNAs were differentially expressed in stress. Six hundred and nine mRNA targets involved in diverse cellular processes were predicted for 244 miRNAs. Stress-responsive expression patterns of selected miRNAs, inverse expression patterns of their target genes and the target-cleavage sites were validated. Three candidate miRNA-target gene relationships were validated in transient expression system in chickpea. The miRNA expression profiling under salinity and water deficiency in a legume root apex and the reported function of their target genes suggested important roles of miRNA-mediated post-transcriptional regulation of gene expression involved in re-patterning of root hair cells, lateral root formation and high-affinity K+-uptake under these stresses.
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    Phytohormonal crosstalk modulates the expression of miR166/165s, target Class III HD-ZIPs, and KANADI genes during root growth in Arabidopsis thaliana
    (Nature Publishing Group, 2017) Singh, Archita; Roy, Shradha; Singh, Sharmila; Das, Shabari Sarkar; Gautam, Vibhav; Yadav, Sandeep; Kumar, Ashutosh; Singh, Alka; Samantha, Sukanya; Sarkar, Ananda K.
    Both phytohormones and non-coding microRNAs (miRNAs) play important role in root development in Arabidopsis thaliana. Mature miR166/165 s, which are derived from precursor transcripts of concerned genes, regulate developmental processes, including leaf and root patterning, by targeting Class III HOMEODOMAIN LEUCINE-ZIPPER (HD-ZIP III) transcription factors (TFs). However, their regulation through hormones remained poorly understood. Here, we show that several phytohormones dynamically regulate the spatio-temporal expression pattern of miR166/165 and target HD-ZIP IIIs in developing roots. Hormone signaling pathway mutants show differential expression pattern of miR166/165, providing further genetic evidence for multilayered regulation of these genes through phytohormones. We further show that a crosstalk of at least six different phytohormones regulate the miR166/165, their target HD-ZIP IIIs, and KANADI (KANs). Our results suggest that HD-ZIP IIIs mediated root development is modulated both transcriptionally through phytohormones and KANs, and post-transcriptionally by miR166/165 that in turn are also regulated by the phytohormonal crosstalk.
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    Chickpea transcription factor CaTLP1 interacts with protein kinases, modulates ROS accumulation and promotes ABA-mediated stomatal closure
    (Nature Publishing Group, 2016) Wardhan, Vijay; Pandey, Aarti; Chakraborty, Subhra; Chakraborty, Niranjan
    Tubby and Tubby-like proteins (TLPs), in mammals, play critical roles in neural development, while its function in plants is largely unknown. We previously demonstrated that the chickpea TLP, CaTLP1, participates in osmotic stress response and might be associated with ABA-dependent network. However, how CaTLP1 is connected to ABA signaling remains unclear. The CaTLP1 was found to be engaged in ABA-mediated gene expression and stomatal closure. Complementation of the yeast yap1 mutant with CaTLP1 revealed its role in ROS scavenging. Furthermore, complementation of Arabidopsis attlp2 mutant displayed enhanced stress tolerance, indicating the functional conservation of TLPs across the species. The presence of ABA-responsive element along with other motifs in the proximal promoter regions of TLPs firmly established their involvement in stress signalling pathways. The CaTLP1 promoter driven GUS expression was restricted to the vegetative organs, especially stem and rosette leaves. Global protein expression profiling of wild-type, attlp2 and complemented Arabidopsis plants revealed 95 differentially expressed proteins, presumably involved in maintaining physiological and biological processes under dehydration. Immunoprecipitation assay revealed that protein kinases are most likely to interact with CaTLP1. This study provides the first demonstration that the TLPs act as module for ABA-mediated stomatal closure possibly via interaction with protein kinase.
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    bHLH142 regulates various metabolic pathway-related genes to affect pollen development and anther dehiscence in rice
    (Nature Publishing Group, 2017) Ranjan, Rajeev; Khurana, Reema; Malik, Naveen; Badoni, Saurabh; Parida, Swarup K.; Kapoor, Sanjay; Tyagi, Akhilesh K.
    Apposite development of anther and its dehiscence are important for the reproductive success of the flowering plants. Recently, bHLH142, a bHLH transcription factor encoding gene of rice has been found to show anther-specific expression and mutant analyses suggest its functions in regulating tapetum differentiation and degeneration during anther development. However, our study on protein level expression and gain-of-function phenotype revealed novel aspects of its regulation and function during anther development. Temporally dissimilar pattern of bHLH142 transcript and polypeptide accumulation suggested regulation of its expression beyond transcriptional level. Overexpression of bHLH142 in transgenic rice resulted in indehiscent anthers and aborted pollen grains. Defects in septum and stomium rupture caused anther indehiscence while pollen abortion phenotype attributed to abnormal degeneration of the tapetum. Furthermore, RNA-Seq-based transcriptome analysis of tetrad and mature pollen stage anthers of wild type and bHLH142OEplants suggested that it might regulate carbohydrate and lipid metabolism, cell wall modification, reactive oxygen species (ROS) homeostasis and cell death-related genes during rice anther development. Thus, bHLH142 is an anther-specific gene whose expression is regulated at transcriptional and post-transcriptional/translational levels. It plays a role in pollen maturation and anther dehiscence by regulating expression of various metabolic pathways-related genes.
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    Sirtinol, a Sir2 protein inhibitor, affects stem cell maintenance and root development in Arabidopsis thaliana by modulating auxin-cytokinin signaling components
    (Nature Publishing Group, 2017) Singh, Sharmila; Singh, Alka; Yadav, Sandeep; Gautam, Vibhav; Singh, Archita; Sarkar, Ananda K.
    In Arabidopsis thaliana, besides several key transcription factors and chromatin modifiers, phytohormones auxin and cytokinin play pivotal role in shoot and root meristem maintenance, and lateral root (LR) development. Sirtinol, a chemical inhibitor of Sir2 proteins, is known to promote some auxin induced phenotypes in Arabidopsis. However, its effect on plant stem cell maintenance or organ formation remained unaddressed. Here we show that sirtinol affects meristem maintenance by altering the expression of key stem cell regulators, cell division and differentiation by modulating both auxin and cytokinin signaling in Arabidopsis thaliana. The expression of shoot stem cell niche related genes WUSCHEL (WUS) and CLAVATA3 (CLV3) was upregulated, whereas SHOOT MERISTEMLESS (STM) was downregulated in sirtinol treated seedlings. The expression level and domain of key root stem cell regulators PLETHORA (PLTs) and WUS-Related Homeobox 5 (WOX5) were altered in sirtinol treated roots. Sirtinol affects LR development by disturbing proper auxin transport and maxima formation, similar to 2,4-dichlorophenoxyacetic acid (2,4-D). Sirtinol also affects LR formation by altering cytokinin biosynthesis and signaling genes in roots. Therefore, sirtinol affects shoot and root growth, meristem maintenance and LR development by altering the expression of cytokinin-auxin signaling components, and regulators of stem cells, meristems, and LRs.
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    Root transcriptome of two contrasting indica rice cultivars uncovers regulators of root development and physiological responses
    (Nature Publishing Group, 2016) Singh, Alka; Kumar, Pramod; Gautam, Vibhav; Rengasamy, Balakrishnan; Adhikari, Bijan; Udayakumar, Makarla; Sarkar, Ananda K.
    The huge variation in root system architecture (RSA) among different rice (Oryza sativa) cultivars is conferred by their genetic makeup and different growth or climatic conditions. Unlike model plant Arabidopsis, the molecular basis of such variation in RSA is very poorly understood in rice. Cultivars with stable variation are valuable resources for identification of genes involved in RSA and related physiological traits. We have screened for RSA and identified two such indica rice cultivars, IR-64 (OsAS83) and IET-16348 (OsAS84), with stable contrasting RSA. OsAS84 produces robust RSA with more crown roots, lateral roots and root hairs than OsAS83. Using comparative root transcriptome analysis of these cultivars, we identified genes related to root development and different physiological responses like abiotic stress responses, hormone signaling, and nutrient acquisition or transport. The two cultivars differ in their response to salinity/dehydration stresses, phosphate/nitrogen deficiency, and different phytohormones. Differential expression of genes involved in salinity or dehydration response, nitrogen (N) transport, phosphate (Pi) starvation signaling, hormone signaling and root development underlies more resistance of OsAS84 towards abiotic stresses, Pi or N deficiency and its robust RSA. Thus our study uncovers gene-network involved in root development and abiotic stress responses in rice.