Publications of NIPGR Scientists
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Item MPK3 mediated phosphorylation inhibits the dimerization of ABI5 to fine-tune the ABA signaling in Arabidopsis(Elsevier B.V., 2025) Bhagat, Prakash Kumar; Verma, Neetu; Pandey, Shubhangi; Verma, Deepanjali; Sinha, Alok KrishnaSeed germination is, a critical physiological process, is tightly regulated by the phytohormone abscisic acid (ABA). However, the cross talk between multiple regulatory pathways involved in seed germination remains poorly understood. Here, we show that ABA activates two MAP kinases, AtMPK3/AtMPK6, which interact with and phosphorylate AtABI5, a master regulator of ABA signaling. MAP kinase-mediated AtABI5 phosphorylation at the serine-314 position regulates its nuclear localization and dimerization. Interestingly, AtABI5 provides feedback regulation by directly binding to the promoter of AtMPK3 to modulate its transcription. Further, functional analyses revealed that overexpression of a phospho-null AtABI5S314A variant in the abi5-8 mutant background conferred increased ABA sensitivity during seed germination, heightened drought sensitivity, and delayed flowering compared to wild-type plants. Conversely, overexpression of phospho-mimic AtABI5S314D in abi5-8 mutant showed ABA insensitivity during seed germination, drought tolerance, and early floral transition similar to abi5-8 mutant. Collectively, our findings highlight that MAP kinase-mediated phosphorylation of AtABI5 fine-tunes ABA signaling by regulating its dimerization, providing new insights into the dynamic regulation of plant responses to environmental and developmental cues.Item Phosphorylation of AGO1a by MAP kinases is required for miRNA mediated resistance against Xanthomonas oryzae pv. oryzae infection in rice(Elsevier B.V., 2024) Singh, Kirti; Sharma, Deepika; Bhagat, Prakash Kumar; Tayyeba, Sumaira; Noryang, Stanzin; Sinha, Alok KrishnaBacterial leaf blight is a devastating disease caused by Xanthomonas oryzae pv. oryzae (Xoo) which causes severe crop loss in rice. The molecular mechanism that initiates defense against such pathogens remains unexplored. Reports have suggested crucial role of several miRNAs in regulating immune responses in plants. Argonaute (AGO) proteins have been implicated in imparting immunity against pathogens by using small RNAs as guide molecules. Here, we show that phosphorylation of rice AGO1a by MAP kinases is required for miRNA expression regulation during Xoo infection. AGO1a is induced in response to pathogen infection and is under the control of SA signaling pathway. The pathogen responsive MAP kinases MPK3, MPK4 and MPK6, interact with AGO1a in planta and can phosphorylate the protein in vitro. Overexpression of AGO1a extends disease resistance against Xoo in rice and leads to a higher accumulation of miRNAs. Conversely, overexpression of a non phosphorylatable mutant protein aggravates disease susceptibility and remarkably suppresses the miRNA expression levels. At a molecular level, phosphorylation of AGO1a by MAP kinase is required for increased accumulation of miRNAs during pathogen challenge. Taken together, the data suggests that OsAGO1a is a direct phosphorylation target of MAP kinases and this phosphorylation is crucial for its role in imparting disease resistance.Item Phosphorylation of PIF3 by MPK6 is required for coordinated regulation of miRNA biogenesis and hypocotyl elongation in Arabidopsis(Elsevier B.V., 2023) Sharma, Deepika; Singh, Dhanraj; Singh, Kirti; Dwivedi, Aditi; Ranjan, Aashish; Sinha, Alok KrishnaLight is one of the most important environmental factors that affect overall growth and development in plants. PHYTOCHROME INTERACTING FACTORs (PIFs) are negative regulators of photomorphogenesis. PIFs mediate light responses by interacting with downstream molecular partners and are essential regulators for hypocotyl elongation in Arabidopsis. Light induce activation of phytochromes (phys), promotes rapid phosphorylation, ubiquitination and degradation of PIFs. However the kinase responsible for the phosphorylation of PIFs and the signaling mechanism governing the adaptive changes leading to hypocotyl elongation to differential light intensities is not well understood. Here, we report interaction and phosphorylation of PIF3 by a mitogen-activated protein kinase 6 (MPK6) both in vitro and in vivo. Phosphorylation was significantly abolished when all the PIF3 putative phosphorylation sites were mutated to alanine. Further, we found that PIF3 directly binds to the bHLH binding domain in the promoter of RL responsive miRNA, miR163. Interestingly, phosphorylation status of PIF3 substantially affects the binding of PIF3 and mutated proteins to miR163 promoter. Further, overexpression of PIF3 affects the expression of red light (RL) responsive miRNAs and downstream genes involved in hypocotyl elongation and seedling development. These results suggest that MPK6-PIF3 module functions upstream of RL-responsive miRNAs and PIF3-regulated genes involved in photomorphogenesis, thus interconnecting these pathways together.Item Concurrent overexpression of rice G-protein β and γ subunits provide enhanced tolerance to sheath blight disease and abiotic stress in rice(Springer Nature Publishing AG, 2019) Swain, Durga Madhab; Sahoo, Ranjan Kumar; Chandan, Ravindra Kumar; Ghosh, Srayan; Kumar, Rahul; Jha, Gopaljee; Tuteja, NarendraThe heterotrimeric G-proteins act as signalling molecules and modulate various cellular responses including stress tolerance in eukaryotes. The gamma (γ) subunit of rice G-protein (RGG1) was earlier reported to promote salinity stress tolerance in rice. In the present study, we report that a rice gene-encoding beta (β) subunit of G-protein (RGB1) gets upregulated during both biotic (upon a necrotrophic fungal pathogen, Rhizoctonia solani infection) and drought stresses. Markerfree transgenic IR64 rice lines that simultaneously overexpress both RGB1 and RGG1 genes under CaMV35S promoter were raised. The overexpressing (OE) lines showed enhanced tolerance to R. solani infection and salinity/drought stresses. Several defense marker genes including OsMPK3 were signifcantly upregulated in the R. solani-infected OE lines. We also found the antioxidant machineries to be upregulated during salinity as well as drought stress in the OE lines. Overall, the present study provides evidence that concurrent overexpression of G-protein subunits (RGG1 and RGB1) impart multiple (both biotic and abiotic) stress tolerance in rice which could be due to the enhanced expression of stress-marker genes and better management of reactive oxygen species (ROS)-scavenging/photosynthetic machinery. The current study suggests an improved approach for simultaneous improvement of biotic and abiotic stress tolerance in rice which remains a major challenge for its sustainable cultivation.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 PiHOG1, a stress regulator MAP kinase from the root endophyte fungus Piriformospora indica, confers salinity stress tolerance in rice plants(Nature Publishing Group, 2016) Jogawat, Abhimanyu; Vadassery, Jyothilakshmi; Verma, Nidhi; Oelmüller, Ralf; Dua, Meenakshi; Nevo, Eviatar; Johri, Atul KumarIn this study, yeast HOG1 homologue from the root endophyte Piriformospora indica (PiHOG1) was isolated and functionally characterized. Functional expression of PiHOG1 in S. cerevisiae ∆hog1 mutant restored osmotolerance under high osmotic stress. Knockdown (KD) transformants of PiHOG1 generated by RNA interference in P. indica showed that genes for the HOG pathway, osmoresponse and salinity tolerance were less stimulated in KD-PiHOG1 compared to the wild-type under salinity stress. Furthermore, KD lines are impaired in the colonization of rice roots under salinity stress of 200 mM NaCl, and the biomass of the host plants, their shoot and root lengths, root number, photosynthetic pigment and proline contents were reduced as compared to rice plants colonized by WT P. indica. Therefore, PiHOG1 is critical for root colonisation, salinity tolerance and the performance of the host plant under salinity stress. Moreover, downregulation of PiHOG1 resulted not only in reduced and delayed phosphorylation of the remaining PiHOG1 protein in colonized salinity-stressed rice roots, but also in the downregulation of the upstream MAP kinase genes PiPBS2 and PiSSK2 involved in salinity tolerance signalling in the fungus. Our data demonstrate that PiHOG1 is not only involved in the salinity response of P. indica, but also helping host plant to overcome salinity stress.Item MicroRNA biogenesis factor DRB1 is a phosphorylation target of mitogen activated protein kinase, MPK3 in both rice and Arabidopsis(John Wiley & Sons, 2015) Raghuram, Badmi; Sheikh, Arsheed H.; Rustagi, Yashika; Sinha, Alok KrishnaMicroRNA (miRNA) biogenesis requires AtDRB1 (double-stranded RNA binding protein)/HYL1 (Hyponastic Leaves1) protein for processing and maturation of miRNA precursors. The AtDRB1/HYL1 protein associates with AtDCL1 (Dicer-Like1) and accurately processes primary-miRNAs (pri-mRNAs) first to precursor-miRNAs (pre-miRNAs) and finally to mature miRNAs. The dephosphorylation of AtDRB1/HYL1 protein is very important for the precise processing of miRNA precursors. The monocot model crop plant Oryza sativa encodes four orthologues of AtDRB1/HYL1 protein, the only one encoded by Arabidopsis thaliana. The present study focuses on the functionality of the O. sativa DRBs as the orthologues of AtDRB1/HYL1 by using RNA binding assays and in planta protein–protein interaction analysis. Further, mitogen-activated protein kinase MPK3 is established as the kinase phosphorylating DRB1 protein in both the model plants, O. sativa and Arabidopsis. MicroRNA microarray analysis in atmpk3 and atmpk6 mutants indicate the importance of AtMPK3 in maintaining the level of miRNAs in the plant.Item Regulation of MAP kinase signaling cascade by microRNAs in Oryza sativa(Taylor & Francis Group, 2014) Raghuram, Badmi; Sheikh, Arsheed Hussain; Sinha, Alok KrishnaMitogen activated protein kinase (MAPK) pathway is one of the most conserved signaling cascade in plants regulating a plethora of cellular processes including normal growth and development, abiotic and biotic stress responses. The perception of external cues triggers the phosphorylation of three tier MAPKKK-MAPKK-MAPK cascade which finally modifies a downstream substrate thereby regulating the cellular processes. Whereas, the transcription regulation by MAPKs, mediated through their substrates is well studied in plants, the transcription and post-transcriptional regulation of the MAPK genes are poorly understood. Previous studies from the animals systems suggested the miRNAs regulate the post-transcriptional regulation of MAPK transcripts. Here we attempt to unravel the post-transcriptional regulation of MAPKs by miRNAs in model crop plant Oryza sativa. Using in silico tools, we predict the miRNAs for 98 out of 99 MAPK transcripts. The predicted miRNAs were validated for the biological relevance of their function. The inverse correlation between relative transcript levels between the MAPKs and their predicted miRNAs validated the in silico prediction. Taken together, this report demonstrates the significance of miRNAs in regulation of the MAPK pathway in plants with a new direction to study the plant signaling molecules.Item 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 KrishnaBackground: 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.Item Differential responses of arsenic stress in two varieties of Brassica juncea(Elsevier B.V., 2009) Gupta, Meetu; Sharma, Pallavi; Sarin, Neera Bhalla; Sinha, Alok KrishnaPresent study showed the toxicity caused by Arsenite (As(III)) and its detoxification responses in two varieties (Varuna and Pusa Bold) of Brassica juncea. Comparisons were made in leaves and roots of both the varieties, which showed that the accumulation pattern in both the varieties were dose and duration dependent, being more in roots for two days and in leaves for four days. Increase/decrease of antioxidant enzymes activities (SOD, CAT, GPX) showed not much changes at the given concentrations except that the enzyme activities showed significant increase at the lower concentrations. Semi quantitative RT-PCR analysis of PCS showed more expression of its transcript in P. Bold as compared to Varuna variety due to As(III) stress. The analysis of isoenzyme pattern in leaves of P. Bold showed five and two major bands of SOD and GPX, respectively. As(III) treatment leads to the activation of MAPK activity indicating role of this important cascade in transducing As(III) mediated signals. The data presented indicates the differential responses in both the varieties and also that the increased tolerance in P. Bold may be due to the defensive role of antioxidant enzymes, induction of MAPK and up regulation of PCS transcript which is responsible for the production of metal binding peptides.