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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 MKKK20 works as an upstream triple-kinase of MKK3-MPK6-MYC2 module in Arabidopsis seedling development(Cell Press, 2023) Ojha, Madhusmita; Verma, Deepanjali; Chakraborty, Nibedita; Pal, Abhideep; Bhagat, Prakash Kumar; Singh, Anshuman; Verma, Neetu; Sinha, Alok Krishna; Chattopadhyay, SudipThe mitogen-activated protein kinase (MAPK) cascade is involved in several signal transduction processes in eukaryotes. Here, we report a mechanistic function of MAP kinase kinase kinase 20 (MKKK20) in light signal transduction pathways. We show that MKKK20 acts as a negative regulator of photomorphogenic growth at various wavelengths of light. MKKK20 not only regulates the expression of light signaling pathway regulatory genes but also gets regulated by the same pathway genes. The atmyc2 mkkk20 double mutant analysis shows that MYC2 works downstream to MKKK20 in the regulation of photomorphogenic growth. MYC2 directly binds to the promoter of MKKK20 to modulate its expression. The protein-protein interaction study indicates that MKKK20 physically interacts with MYC2, and this interaction likely suppresses the MYC2-mediated promotion of MKKK20 expression. Further, the protein phosphorylation studies demonstrate that MKKK20 works as the upstream kinase of MKK3-MPK6-MYC2 module in photomorphogenesis.Item Arabidopsis MPK3 and MPK6 regulates D-Glucose signaling and interacts with G-Protein, RGS1(Elsevier B.V., 2022) Bhagat, Prakash Kumar; Sharma, Deepika; Verma, Deepanjali; Singh, Kirti; Sinha, Alok KrishnaSugar as a signaling molecule has attracted lots of attention. Even though several kinases have been shown to play a crucial role in the sugar signaling and response to exogenous D-glucose (Glc), the information on the involvement of MAP kinase cascade in sugar signaling has remain largely unexplored. In this report we demonstrate that MAP kinase signaling is essential for sensitivity to higher concentrations of D-Glc in Arabidopsis. We found that D-Glc activates MAP kinases, MPK3 and MPK6 in a concentration and time-dependent manner. The mutants of mpk3 and mpk6 display hyposensitivity to 6% D-Glc during seed germination, cotyledon greening and root growth. Interestingly, the altered sensitivity to increased D-Glc is severely enhanced by addition of 1% Sucrose in the media. Our study also deciphered the role of one of the Glc sensor proteins, RGS1 that interacts and gets phosphorylated at its C-terminal domain by MPK3 and MPK6. Overall our study provides a new insight on the involvement of MAP kinases in association with G-proteins that might regulate sugar signaling and sugar responsive growth and development in Arabidopsis.Item Dynamic phosphorylation of miRNA biogenesis factor HYL1 by MPK3 involving nuclear–cytoplasmic shuttling and protein stability in Arabidopsis(MDPI AG, 2022) Bhagat, Prakash Kumar; Verma, Deepanjali; Singh, Kirti; Badmi, Raghuram; Sharma, Deepika; Sinha, Alok KrishnaMicroRNAs (miRNAs) are one of the prime regulators of gene expression. The recruitment of hyponastic leaves 1 (HYL1), a double-stranded RNA binding protein also termed as DRB1, to the microprocessor complex is crucial for accurate primary-miRNA (pri-miRNA) processing and the accumulation of mature miRNA in Arabidopsis thaliana. In the present study, we investigated the role of the MAP kinase-mediated phosphorylation of AtHYL1 and its sub-cellular activity. AtMPK3 specifically phosphorylates AtHYL1 at the evolutionarily conserved serine-42 present at the Nterminal regions and plays an important role in its nuclear–cytosolic shuttling. Additionally, we identified that AtHYL1 is cleaved by trypsin-like proteases into an N-terminal fragment, which renders its subcellular activities. We, for the first time, report that the dimerization of AtHYL1 not only takes place in the nucleus, but also in the cytosol, and the C-terminal of AtHYL1 has a role in regulating its stability, as well as its subcellular localization. AtHYL1 is hyper-phosphorylated in mpk3 mutants, leading to higher stability and reduced degradation. Our data show that AtMPK3 is a negative regulator of AtHYL1 protein stability and that the AtMPK3-induced phosphorylation of AtHYL1 leads to its protein degradation.Item MAP kinase as regulators for stress responses in plants: An overview(John Wiley & Sons, 2021) Sharma, Deepika; Verma, Neetu; Pandey, Chandana; Verma, Deepanjali; Bhagat, Prakash Kumar; Noryang, Stanzin; Singh, Kirti; Tayyeba, Sumaira; Banerjee, Gopal; Sinha, Alok KrishnaLiving cells adapt to various kind of stresses by perceiving changes in the extracellular environment and communicating these changes from outside of the cell to the inside and ultimately to the nucleus where expression of a specific set of genes occurs in order to provide appropriate response to the stress. Plant cells have evolved several signaling cascades to accomplish this signal transduction. One of the major mechanisms is the cascade of protein phosphorylation by specific Mitogen Activated Protein Kinase (MAPK) cascade. This cascade comprises of three kinase modules, a MAPK kinase kinase (MAPKKK/MAKKK/MEKK/MAP3K), a MAPK kinase (MAPKK/MKK/MEK/MAP2K) and a MAPK, which are sequentially activated by an event of protein phosphorylation. In plants, MAPKs are large signaling families of proteins, which have been shown to be involved in cross-talk with various abiotic and biotic stress responses forming complex networks in cells. Abiotic stresses such as heat, cold, changing light intensities, heavy metals and salinity, and biotic stress such as pathogen attack are key factors, which affect growth and development of plants. In response to these stresses, MAPK signaling cascade regulate growth of plants by transcriptional and post-transcriptional regulation such as protein–protein interactions. In this chapter, we discuss the latest findings and insights in relation to the role of all the modules, MAPKKK, MAPKK, and MAPKs of different MAPK signaling cascades in various abiotic and biotic stresses in plants.Item HY5 and ABI5 transcription factors physically interact to fine tune light and ABA signaling in Arabidopsis(Springer Nature Publishing AG, 2021) Bhagat, Prakash Kumar; Verma, Deepanjali; Sharma, Deepika; Sinha, Alok KrishnaPlants undergo numerous transitions during their life-cycle and have developed a very complex network of signaling to integrate information from their surroundings to effectively survive in the ever-changing environment. Light signaling is one of the crucial factors that govern the plant growth and development from the very first step of that is from seedling germination to the flowering. Similarly, Abscisic acid (ABA) signaling transduces the signals from external unfavorable condition to the internal developmental pathways and is crucial for regulation of seed maturation, dormancy germination and early seedling development. These two fundamental factors coordinately regulate plant wellbeing, but the underlying molecular mechanisms that drive this regulation are poorly understood. Here, we identified that two bZIP transcription factors, ELONGATED HYPOCOTYLE 5 (HY5), a positive regulator of light signaling and ABA-INSENSITIVE 5 (ABI5), a positive regulator of ABA signaling interacts and integrates the two pathways together. Our phenotypic data suggest that ABI5 may act as a negative regulator during photomorphogenesis in contrast, HY5 acts as a positive regulator of ABA signaling in an ABA dependent manner. We further showed that over-expression of HY5 leads to ABA-hypersensitive phenotype and late flowering phenotype. Taken together, our data provides key insights regarding the mechanism of interaction between ABI5-HY5 that fine tunes the stress and developmental response in Arabidopsis.Item A dual-specificity phosphatase, MAP kinase phosphatase 1, positively regulates blue light-mediated seedling development in Arabidopsis(Springer Nature Publishing AG, 2021) Verma, Deepanjali; Bhagat, Prakash Kumar; Sinha, Alok KrishnaReversible phosphorylation of proteins is one of the major post-translational modifications in nearly all signaling pathways in plants. MAP kinase phosphatases are very crucial in the regulation of MAPKs as they dephosphorylate both threonine (Thr) and tyrosine (Tyr) residues within the T-X-Y motif of active MAPKs. Therefore, to gain insight of involvement of MAP kinase phosphatases in the regulation of light signaling, we searched for the potential phosphatase which may regulate the function of MPK6, a negative regulator of blue light (BL)-mediated photomorphogenic development. We report here the identification of a dual-specificity phosphatase, MAP kinase phosphatase 1 (MKP1) as a positive regulator of BL-mediated seedling development. Overexpression of MKP1 enhances the BL-induced inhibition of hypocotyl elongation and displays more opened cotyledons. We also show that MKP1OE accumulates more pigments and positively affects the expression of downstream light-related genes in response to BL. In vitro and in vivo evidences also demonstrate that MKP1 not only interacts with but also dephosphorylates MPK6 in BL. In addition, MKP1 regulates stability as well as activity of MPK6 upon BL. Taken together our study highlights the important role of phosphatases in the regulation of a signaling pathway and identifies the role of MKP1 in the negative regulation of MPK6 activity leading to a change in BL-induced photomorphogenic responses.Item MKK3-MPK6-MYC2 module positively regulates ABA biosynthesis and signalling in Arabidopsis(Springer Nature Publishing AG, 2020) Verma, Deepanjali; Bhagat, Prakash Kumar; Sinha, Alok KrishnaInvolvement of MAPK cascades is well studied in the regulation of ABA mediated responses such as stomatal opening, seed germination and stress tolerance. However most of the studies have focused on ABA signalling and involvement of MAPKs in ABA biosynthesis remains elusive. Hence, in the current study, we have identified the new role of MKK3- MPK6-MYC2 module in positive regulation of ABA biosynthesis as well as ABA responsive genes. Seed germination studies disclosed the ABA insensitive phenotype of myc2, mpk6 and mkk3 mutants. Reduced expression of ABA biosynthesis genes, ABA2 and NCED3 in myc2 and MAPK cascade mutants was confirmed by witnessing the direct binding of MYC2 to the promoter of ABA2 in EMSA assays. Additionally, ABA responsive genes also displayed altered expression patterns in these mutants. Further, transcript and biochemical studies validated the role of MPK6 upstream to MYC2 and downstream to MKK3 in response to ABA. Altogether, we have shown that MKK3-MPK6-MYC2 module induces the expression of several ABA related genes and regulate ABA mediated inhibition of seed germination.Item A bHLH transcription factor, MYC2, imparts salt intolerance by regulating proline biosynthesis in Arabidopsis(John Wiley & Sons, 2020) Verma, Deepanjali; Jalmi, Siddhi Kashinath; Bhagat, Prakash Kumar; Verma, Neetu; Sinha, Alok KrishnaMYC2, a bHLH TF, acts as regulatory hub within several signaling pathways by integration of various endogenous and exogenous signals which shape plant growth and development. However, its involvement in salt stress regulation is still elusive. This study has deciphered a novel role of MYC2 in imparting salt stress intolerance by regulating P5CS1 gene and hence proline synthesis. P5CS1 is a rate limiting enzyme in the biosynthesis of proline. Y‐1‐H and EMSA studies confirmed the binding of MYC2 with the 5’ UTR region of P5CS1. Transcript and biochemical studies have revealed MYC2 as a negative regulator of proline biosynthesis. Proline is necessary for imparting tolerance towards abiotic stress however, its overaccumulation is toxic for the plants. Hence studying the regulation of proline biosynthesis is requisite to understand the mechanism of stress tolerance. We have also studied that MYC2 is regulated by MAPK cascade MKK3‐MPK6 and vice versa. Altogether, this study demonstrates salt stress mediated activation of MYC2 by MAPK cascade, regulating proline biosynthesis and thus salt stress.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.