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Item Regulation of photosynthesis by mitogen-activated protein kinase in rice: antagonistic adjustment by OsMPK3 and OsMPK6(Springer Nature Publishing AG, 2023) Jonwal, Sarvesh; Rengasamy, Balakrishnan; Sinha, Alok KrishnaPhotosynthesis is the basis of almost all life on earth and is the main component of crop yield that contributes to the carbohydrate partitioning to the grains. Maintaining the photosynthetic efficiency of plants in challenging environmental conditions by regulating the associated factors is a potential research arena which will help in the improvement of crop yield. Phosphorylation is known to play a pivotal role in the regulation of photosynthesis. Mitogen Activated Protein Kinases (MAPKs) cascade although known to regulate a diverse range of processes does not have any exact reported function in the regulation of photosynthesis. To elucidate the regulatory role of MAPKs in photosynthesis we investigated the changes in net photosynthesis rate and related parameters in DEX inducible over-expressing (OE) lines of two members of MAPK gene family namely, OsMPK3 and OsMPK6 in rice. Interestingly, significant changes were found in net photosynthesis rate and related physiological parameters in OsMPK3 and OsMPK6-OE lines compared to its wild-type relatives. OsMPK3 and OsMPK6 have regulatory effects on nuclear-encoded photosynthetic genes. Untargeted metabolite profiling reveals a higher accumulation of sugars and their derivatives in MPK6 overexpressing plants and a lower accumulation of sugars and organic acids in MPK3 overexpressing plants. The accumulation of amino acids was found in abundance in both MPK3 and MPK6 overexpressing plants. Understanding the effects of MPK3 and MPK6 on the CO2 assimilation of rice plants under normal growth conditions, will help in devising strategies that can be extended for crop improvement.Item Unraveling the molecular aspects of iron-mediated OsWRKY76 signaling under arsenic stress in rice(Elsevier B.V., 2023) Mirza, Zainab; Jonwal, Sarvesh; Saini, Himanshu; Sinha, Alok Krishna; Gupta, MeetuArsenic (As) is a significant environmental element that restricts the growth and production of rice plants. Although the role of iron (Fe) to sequester As in rice is widely known, the molecular mechanism regarding As–Fe interaction remains opaque. Here, we show the differential response of two rice varieties (Ratna and Lalat) in terms of their morphological and biochemical changes in the presence of As and Fe. These results together with in-silico screening, gene expression analysis, and protein-protein interaction studies suggest the role of OsWRKY76 in Fe-mediated As stress alleviation. When OsWRKY76 is activated by MAPK signaling, it inhibits the gene expression of Fe transporters OsIRT1 and OsYSL2, which reduces the amount of Fe accumulated. However, MAPK signaling and OsWRKY76 remain down-regulated during Fe supplementation with As, which subsequently encourages the up-regulation of OsIRT1 and OsYSL2. This results in greater Fe content and decreased As accumulation and toxicity. The lower H2O2 and SOD, CAT, and APX activities were likewise seen under the As + Fe condition. Overall, results revealed the molecular aspects of Fe-mediated control of OsWRKY76 signaling and showed that Ratna is a more As tolerant variety than Lalat. Lalat, however, performs better in As stress due to the presence of Fe.Item Rice Mitogen-Activated Protein Kinase regulates serotonin accumulation and interacts with cell cycle regulators under prolonged UV-B exposure(Elsevier B.V., 2023) Banerjee, Gopal; Singh, Dhanraj; Pandey, Chandana; Jonwal, Sarvesh; Basu, Udita; Parida, Swarup K.; Pandey, Ashutosh; Sinha, Alok KrishnaStress conditions such as UV-B exposure activates MAPKs in Arabidopsis and rice. UV-B radiation is hazardous to plant as it causes photosystem disruption, DNA damage and ROS generation. Here we report its effect on biological pathways by studying the global changes in transcript profile in rice seedling exposed to UV-B radiation for 1 h and 16 h. Short UV-B exposure (1 h) led to moderate changes, while a drastic change in transcript landscape was observed after long term UV-B exposure (16 h) in rice seedlings. Prolonged UV-B exposure negatively impacts the expression of cell cycle regulating genes and several other metabolic pathways in developing seedlings. MAP kinase signaling cascade gets activated upon UV-B exposure similar to reports in Arabidopsis indicating conservation of its function in both dicot and monocot. Expression analysis in inducible overexpression transgenic lines of MPK3 and MPK6 shows higher transcript abundance of phytoalexin biosynthesis gene like Oryzalexin D synthase and Momilactone A synthase, along with serotonin biosynthesis genes. An accumulation of serotonin was observed upon UV-B exposure and its abundance positively correlates with the MPK3 and MPK6 transcript level in the respective over-expression lines. Interestingly, multiple cell cycle inhibitor proteins including WEE1 and SMR1 interact with MPK3 and MPK6 thus, implying a major role of this pathway in cell cycle regulation under stress condition. Overall overexpression of MPK3 and MPK6 found to be detrimental for rice as overexpression lines shows higher cell death and compromised tolerance to UV-B.Item Possible role of WRKY transcription factors in regulating immunity in Oryza sativa ssp. indica(Elsevier B.V., 2021) Sheikh, Arsheed H.; Hussain, Rana M. Fraz; Tabassum, Naheed; Badmi, Raghuram; Marillonnet, Sylvestre; Scheel, Dierk; Lee, Justin; Sinha, Alok KrishnaPlants have developed a robust transcription machinery to combat potential pathogenic organisms. One of the hallmarks of early immune responses is the activation of the WRKY transcription factors post infection. Specific WRKYs proteins from Arabidopsis are known substrates of MAPK pathway to mediate the flg22 elicited early immunity. In the current study, using the Golden Gate cloning strategy, we aim to clone the entire WRKY transcription factor family from Oryza sativa ssp. indica consisting of more than 100 members and study their MAPK interaction and subsequent role in PTI. Using a reporter LUC assay in protoplasts we investigated the early defense responses in a few interesting OsWRKY candidates. Interestingly, we observed stringent regulation of WRKY expression in cells and their transcriptional expression only under specific stress responses. The phenomenon of gene expression regulation by intron retention (IR) was prevalently observed in rice WRKY transcripts. We could show the role of WRKY8, 24, and 77 in early defense responses. It was observed that WRKY24 enhanced the expression of early defense response marker genes like NHL10 while WRKY8 and WRKY77 supressed their expression. This study highlights the complicated mechanism by which OsWRKYs expression is possibly regulated and the distinctive roles of some individual members in plant immunity. At the same time this study serves as a cautionary warning for plant researchers to be mindful of the intron retention mechanism while cloning OsWRKYs.Item 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 KrishnaExhaustive 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.Item Possible role of plant MAP kinases in the biogenesis and transcription regulation of rice microRNA pathway factors(Elsevier B.V., 2018) Badmi, Raghuram; Sheikh, Arsheed Hussain; Bhagat, Prakash Kumar; Verma, Deepanjali; Noryang, Stanzin; Sinha, Alok KrishnaSignalling pathways play vital roles as determinants of almost all the molecular processes inside a eukaryotic cell. They are more often considered to be the link between extracellular and intracellular environmental cues. Gene silencing pathways have emerged to be involved in regulation of stress responses and developmental processes. However, very little is known about the crosstalk between signalling and silencing pathways and their influence on each other. The present work describes the effects of general protein kinase inhibitors and specific mitogen activated protein kinase (MAPK) pathway inhibitors on the components of microRNA pathway in rice. The kinase inhibitors significantly reduced the activities of miRNA biogenesis complex and changed the transcript expression of miRNA pathway factors. This study suggests a possible regulation of microRNA machinery by plant kinases and MAP kinases in particular.Item Functional involvement of a mitogen activated protein kinase module, OsMKK3-OsMPK7-OsWRK30 in mediating resistance against Xanthomonas oryzae in rice(Nature Publishing Group, 2016) Jalmi, Siddhi Kashinath; Sinha, Alok KrishnaMitogen-activated protein kinases (MAPKs) are highly conserved signaling modules in eukaryotes, transmitting signals from upstream receptor to downstream target by phosphorelay mechanism. Here we report involvement of a poorly characterized group C MAPK of rice namely, OsMPK7 along with its upstream MAPK kinase, OsMKK3 and downstream target, OsWRKY30 during Xanthomonas oryzae infection, a causal agent of leaf blight disease in rice. X. oryzae infection resulted in induction of OsMPK7 and OsMKK3. OsMKK3 was found to physically interact and phosphorylate OsMPK7. Overexpression of OsMPK7 and OsMKK3, individually and in combinations resulted in inhibition of disease symptoms caused by X. oryzae, however silencing of OsMPK7 resulted in disease susceptibility. Furthermore, OsWRKY30 was identified as downstream target of OsMPK7 through protein-protein interaction techniques and was found to be a positive regulator of defence response against X. oryzae pathogen. The overexpression of OsMKK3-OsMPK7 upregulated genes involved in pathogenesis, cell wall structure maintenance and cell metabolism indicating possible mechanism of disease resistance. These leaves also showed restricted movement of the pathogen from the point of infection to uninfected area. Taken together, this work suggests a positive involvement of OsMKK3-OsMPK7-OsWRKY30 module in imparting disease resistance against X. oryzae infection in rice.Item 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 KrishnaIncreased 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.Item 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 KrishnaMitogen-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.Item 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 KrishnaMitogen-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.