Institutional Publications

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    Development of efficient protocol for rice transformation overexpressing MAP kinase and their effect on root phenotypic traits
    (Springer Nature, 2019) Singh, Pallavi; Ara, Hussain; Tayyeba, Sumaira; Pandey, Chandana; Sinha, Alok Krishna
    Exhaustive studies on mitogen-activated protein kinase (MAPK) have reported the importance in regulating a variety of responses during plant growth and development. In particular, the potential MAPK genes, MPK3 and MPK6, seem to regulate a plethora of responses, conferring tolerance to varied abiotic, biotic, and developmental stimuli. This makes both MPK3 and MPK6 potential targets for further studies. It would be an important concern to overexpress and knock out these pivotal proteins and then, in turn, to monitor the plant response which is expected to correlate action of a gene to a trait in cellular and organismal contexts. However, overexpression of MAPK genes has remained a puzzle in plants. In the present study, we report the generation of stable transgenic lines overexpressing OsMPK3 in indica and japonica cultivars and OsMPK6 in japonica cultivar under the control of an inducible promoter. We also establish the crucial steps and troubleshooting for each of the indicated rice transformation medium components. Later, we study the potential role of these MAPKs in high-throughput analysis of root system architectural (RSA) traits. It was observed that OsMPK6 overexpression lines had a more robust and spread out root architectural system while OsMPK3 overexpression lines had a typical bushy phenotype.
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    Interplay between auxin and cytokinin and its impact on mitogen activated protein kinase (MAPK)
    (Springer, 2017) Singh, Pallavi; Sinha, Alok Krishna
    Plant physiology, in particular, is governed by a repertoire of endogenous as well as environmental cues. Auxin and cytokinin constitute an indispensable phytohormonal system required for plant growth and development. Another pivotal aspect of plant physiological process that thoroughly affects various plant growth and developmental attributes is the signaling network, majorly comprising the canonical mitogen activated protein kinase (MAPK) cascade. Striking a fine balance between the phytohormonal and signaling components could be adopted as an intricate strategy by plants to counteract various stresses in question. Thus, a brief understanding of this multifaceted complex could be of use for delineating numerous plant physiological and developmental phenomena. Thus, the present section discusses the various MAPK related assays in context to auxin and cytokinin crosstalk. Briefly, this chapter outlines the discrete MAPK methods to better understand the fundamentals of MAPK signaling network in auxin and cytokinin treated rice seedlings. Further, various phenotypic, genomic as well as proteomic protocols are discussed for a better understanding of MAPK networks in the backdrop of auxin and cytokinin interplay.
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    UV‐B activates a ‘group A’ mitogen activated protein kinase in Oryza sativa
    (Springer, 2016) Wankhede, Dhammaprakash Pandhari; Singh, Pallavi; Jaggi, Monika; Rao, Kudupudi Prabhakara; Raina, Susheel Kumar; Sinha, Alok Krishna
    Increased level of Ultra violet-B radiation at earth’s surface has several deleterious consequences for plants and ecosystems. Higher UV-B level affects crop plants in several ways and also gives penalty in terms of crop yield. With changing climatic conditions it is crucial to elucidate signal transduction pathway involved in UV-B stress. Here, involvement of mitogen activated protein kinases (MAPKs) in UV-B stress was investigated in rice (Oryza sativa). Transcripts profiling of all 15 rice MAPKs have shown UV-B induced expressions of a few MAPK genes including OsMPK3, OsMPK6, OsMPK4, OsMPK17-1, OsMPK17-2, OsMPK20-1, OsMPK20-4 and OsMPK20-5. In-gel kinase assay as well as immuno-kinase assay showed activation of single MAPK of size ~45 kDa in response to UV-B treatment. Further UV-B responsive activity of rice MAPKs, OsMPK3, OsMPK4 and OsMPK6 was checked using antibodies for the respective orthologs in Arabidopsis which indicated activation of OsMPK3 in UV-B stress. Additionally, GST: OsMPK3 protein showed UV responsive phosphorylation when incubated with crude protein extract from UV exposed plants in an in-vitro phosphorylation assay. These results indicate activation of OsMPK3 in UV-B stress. Further, possible involvement of UV-B responsive OsWRKY89 downstream of MAPK cascade was studied. It was observed that staurosporin and other specific chemical inhibitors of MAPK could attenuate UV-B induced expression of OsWRKY89. OsMPK3 was also found to interact with OsWRKY89 in yeast two-hybrid assay, although the interaction was weak in nature. The present work gives an account of MAPKs in UV-B stress in rice.
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    Differential expression of Mitogen Activated Protein Kinase (MAPK) cascade components post submergence in Oryza sativa ssp indica cv Pusa Basmati 1
    (Taylor & Francis Group, 2016) Singh, Pallavi; Sinha, Alok Krishna
    Mitogen-activated protein kinase (MAPK) signaling cascade has been extensively studied in plants. Recently, this important network has been implicated in imparting submergence tolerance by facilitating the phosphorylation of the SUB1A1 protein. In this study, we try to examine the speculative role of MAPK cascade components in another rice mega-variety Pusa Basmati 1. The study reports that potential MAPK cascade components are triggered by submergence stress. Overall, the results provide a brief understanding of activation of MAPK cascade components post submergence stress in a rice variety, which does not harbor the Sub1A1, and in turn, succumbs to prolonged submergence stress.
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    A positive feedback loop governed by SUB1A1 interaction with Mitogen Activated Protein Kinase 3 imparts submergence tolerance in rice
    (American Society of Plant Biologists, 2016) Singh, Pallavi; Sinha, Alok Krishna
    Mitogen-activated protein kinase (MAPK) signal transduction networks have been extensively explored in plants; however, the connection between MAPK signalling cascades and submergence tolerance is currently unknown. The ethylene response factor-like protein SUB1A orchestrates a plethora of responses during submergence stress tolerance in rice. In the present study, we report that MPK3 is activated by submergence in a SUB1A-dependent manner. MPK3 physically interacts with and phosphorylates SUB1A in a tolerant-allele-specific manner. Furthermore, the tolerant allele SUB1A1 binds to the MPK3 promoter and regulates its expression in a positive regulatory loop during submergence stress signalling. We present molecular and physiological evidence for the key role of the MPK3-SUB1A1 module in acclimation of rice seedlings to the adverse effects of submergence. Overall, the results provide a mechanistic understanding of submergence tolerance in Oryza sativa.
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    Unraveling the intricate nexus of molecular mechanisms governing rice root development: OsMPK3/6 and auxin-cytokinin interplay
    (PLOS, 2015) Singh, Pallavi; Mohanta, Tapan Kumar; Sinha, Alok Krishna
    The root system is an imperative component of a plant, involved in water and nutrient acquisition from the soil. Any subtle change in the root system may lead to drastic changes in plant productivity. Both auxin and cytokinin are implicated in regulating various root developmental aspects. One of the major signaling cascades facilitating various hormonal and developmental allocations is the Mitogen Activated Protein Kinase (MAPK) cascade. Innumerable efforts have been made to unravel the complex nexus involved in rice root development. In spite of a plethora of studies, a comprehensive study aiming to decipher the plausible cross-talk of MAPK signaling module with auxin and cytokinin signaling components in rice is missing. In the present study, extensive phenomics analysis of different stages of rice roots; transcript profiling by qRT-PCR of entire gene family of MAPK, MAPKK and PIN genes; as well as protein level and activity of potential MAPKs was investigated using western and immuno kinase assays both on auxin and cytokinin treatment. The above study led to the identification of various novel rice root specific phenotypic traits by using GiA roots software framework. High expression profile of OsMPK3/6, OsMKK4/5 and OsPIN 1b/9 and their marked transcript level modulation in response to both auxin and cytokinin was observed. Finally, the protein levels and activity assay further substantiated our present findings. Thus, OsMPK3/6-OsMKK4/5 module is elucidated as the putative, key player in auxin-cytokinin interaction augmenting their role by differentially regulating the expression patterns of OsPIN 1b/9 in root development in rice.
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    Rice mitogen activated protein kinase kinase and mitogen activated protein kinase interaction network revealed by in-silico docking and yeast two-hybrid approaches
    (PLOS, 2013) Wankhede, Dhammaprakash Pandhari; Misra, Mohit; Singh, Pallavi; Sinha, Alok Krishna
    Protein-protein interaction is one of the crucial ways to decipher the functions of proteins and to understand their role in complex pathways at cellular level. Such a protein-protein interaction network in many crop plants remains poorly defined owing largely to the involvement of high costs, requirement for state of the art laboratory, time and labour intensive techniques. Here, we employed computational docking using ZDOCK and RDOCK programmes to identify interaction network between members of Oryza sativa mitogen activated protein kinase kinase (MAPKK) and mitogen activated protein kinase (MAPK). The 3-dimentional (3-D) structures of five MAPKKs and eleven MAPKs were determined by homology modelling and were further used as input for docking studies. With the help of the results obtained from ZDOCK and RDOCK programmes, top six possible interacting MAPK proteins were predicted for each MAPKK. In order to assess the reliability of the computational prediction, yeast two-hybrid (Y2H) analyses were performed using rice MAPKKs and MAPKs. A direct comparison of Y2H assay and computational prediction of protein interaction was made. With the exception of one, all the other MAPKK-MAPK pairs identified by Y2H screens were among the top predictions by computational dockings. Although, not all the predicted interacting partners could show interaction in Y2H, yet, the harmony between the two approaches suggests that the computational predictions in the present work are reliable. Moreover, the present Y2H analyses per se provide interaction network among MAPKKs and MAPKs which would shed more light on MAPK signalling network in rice.
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    Involvement of mitogen activated protein kinase kinase 6 in UV induced transcripts accumulation of genes in phytoalexin biosynthesis in rice
    (Springer, 2013) Wankhede, Dhammaprakash Pandhari; Kumar, Kundan; Singh, Pallavi; Sinha, Alok Krishna
    BACKGROUND: Ultra violet radiation leads to accumulation of phytoalexins (PA) in rice (Oryza sativa) which are typically accumulated when the plants are infected with rice blast pathogen Magnaporthe oryzae. Although extensive works have been done in elucidating phytoalexin biosynthesis, UV stress signal transduction leading to accumulations of rice phytoalexin is largely unknown.
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    Interaction between two rice mitogen activated protein kinases and its possible role in plant defense
    (BioMed Central Ltd, 2013) Sheikh, Arsheed H; Raghuram, Badmi; Jalmi, Siddhi K; Wankhede, Dhammaprakash P; Singh, Pallavi; Sinha, Alok Krishna
    Background: The canonical mitogen activated protein kinase (MAPK) signaling pathway plays a vital role in carrying out the normal growth and development of the plant. The pathway, connecting the upstreams signal with the downstream target is considered to be linear, mostly starting with a MAPKKK and ending in a MAPK. Results: Here we report a novel interaction between two rice MAPKs, OsMPK20-4 and OsMPK3 suggesting the complex nature of the pathway rather than a linear one at individual steps. The interaction between OsMPK20-4 and OsMPK3 found by yeast two-hybrid analysis was confirmed in planta by co-immunoprecipitation and fluorescence resonance energy transfer (FRET) assays. The interaction is specific and is phosphorylation independent. The results suggest a role of the interaction between OsMPK20-4 and OsMPK3 in basic plant defense. Conclusions: The current novel work showing the physical interaction between two plant MAPKs, OsMPK20-4 and OsMPK3 is the diversion from the dogma of a typical MAPK cascade thereby opening a new dimension to the MAPK signal transduction.
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    CrMPK3, a mitogen activated protein kinase from Catharanthus roseus and its possible role in stress induced biosynthesis of monoterpenoid indole alkaloids
    (BMC, 2012) Raina, Susheel Kumar; Wankhede, Dhammaprakash Pandhari; Jaggi, Monika; Singh, Pallavi; Jalmi, Siddhi Kashinath; Raghuram, Badmi; Sheikh, Arsheed Hussain; Sinha, Alok Krishna
    Mitogen activated protein kinase (MAPK) cascade is an important signaling cascade that operates in stress signal transduction in plants. The biologically active monoterpenoid indole alkaloids (MIA) produced in Catharanthus roseus are known to be induced under several abiotic stress conditions such as wounding, UV-B etc. However involvement of any signaling component in the accumulation of MIAs remains poorly investigated so far. Here we report isolation of a novel abiotic stress inducible Catharanthus roseus MAPK, CrMPK3 that may have role in accumulation of MIAs in response to abiotic stress.