Browsing by Author "Singh, Dhanraj"

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KRP3 stability controls rice plant architecture and productivity via MPK3-mediated phosphorylation
(John Wiley & Sons, 2026) Banerjee, Gopal; Jonwal, Sarvesh; Rengasamy, Balakrishnan; Pal, Uttam; Singh, Dhanraj; Mohit, Mohit; Sinha, Alok Krishna
Yield is a critical agronomic trait in cereal crops, shaped by factors like tiller and seed number, and seed weight. Understanding the factors governing these traits will help in improving the yield of plants. In this study, we identified an orphan gene, KRP3, belonging to cereal crops as a key regulator of rice plant architecture. Altered KRP3 protein homeostasis affected plant height, tiller number, and seed production, highlighting its role in maintaining rice plant vigor and productivity. The stability of the KRP3 protein is positively regulated by MPK3-mediated phosphorylation, as unphosphorylated KRP3 is targeted for degradation via the ubiquitin-proteasome pathway. Our findings reveal that the identified MPK3-KRP3 module operates as an S-phase checkpoint, modulating the pace of cell division in the actively dividing zones and maintaining a balance between cell division and elongation. These findings provide valuable insights for improving plant growth and grain yield in rice.
MAP kinases may mediate regulation of the cell cycle in rice by E2F2 phosphorylation
(John Wiley & Sons, 2023) Singh, Dhanraj; Banerjee, Gopal; Verma, Neetu; Sinha, Alok Krishna
E2F is the key transcription factor that determines the proliferative status of cells by regulating the G1/S phase of the cell cycle. In this study, we show that in rice (Oryza sativa), OsE2F2 is a phosphorylation target of MAP kinases. The MAP kinases OsMPK3, OsMPK4, and OsMPK6 interact with and phosphorylate OsE2F2. Next, we determined the serine and threonine residues that could play a role in the phosphorylation of OsE2F2. Subsequently, our study suggests a possible link between MAP kinase-mediated OsE2F2 phosphorylation and its impact on DNA proliferation in the roots of rice seedlings. Finally, we found positive feedback regulation of OsMPK4 by OsE2F2. Therefore, our study hints at the potential impact of MAP kinase signaling on the cell cycle of rice plants.
MPK4-mediated phosphorylation of PHYTOCHROME INTERACTING FACTOR4 controls thermosensing by regulating histone variant H2A.Z deposition
(Oxford University Press, 2024) Verma, Neetu; Singh, Dhanraj; Mittal, Lavanya; Banerjee, Gopal; Noryang, Stanzin; Sinha, Alok Krishna
Plants can perceive a slight upsurge in ambient temperature and respond by undergoing morphological changes, such as elongated hypocotyls and early flowering. The dynamic functioning of PHYTOCHROME INTERACTING FACTOR4 (PIF4) in thermomorphogenesis is well established, although the complete regulatory pathway involved in thermosensing remains elusive. We establish that an increase in temperature from 22˚C to 28˚C induces upregulation and activation of MITOGEN-ACTIVATED PROTEIN KINASE 4 (MPK4) in Arabidopsis (Arabidopsis thaliana), subsequently leading to the phosphorylation of PIF4. Phosphorylated PIF4 represses the expression of ACTIN-RELATED PROTEIN 6 (ARP6), which is required for mediating the deposition of histone variant H2A.Z at its target loci. Furthermore, we demonstrate that variations in ARP6 expression in PIF4 phosphor-null and phosphor-mimetic seedlings affect hypocotyl growth at 22˚C and 28˚C by modulating the regulation of ARP6-mediated H2A.Z deposition at the loci of genes involved in elongating hypocotyl cells. Interestingly, the expression of MPK4 is also controlled by H2A.Z deposition in a temperature-dependent manner. Taken together, these findings highlight the regulatory mechanism of thermosensing by which MPK4-mediated phosphorylation of PIF4 affects ARP6-mediated H2A.Z deposition at the genes involved in hypocotyl cell elongation.
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 Krishna
Light 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.
Plant cell cycle regulators: Mitogen-activated protein kinase, a new regulating switch?
(Elsevier B.V., 2020) Banerjee, Gopal; Singh, Dhanraj; Sinha, Alok Krishna
Cell cycle is essential for the maintenance of genetic material and continuity of a species. Its regulation involves a complex interplay between multiple proteins with diverse molecular functions such as the kinases, transcription factors, proteases and phosphatases. Every step of this cycle requires a certain combination of these protein regulators which paves the way for the next stage. It is now evident that plants have their own unique features in the context of cell cycle regulation. Cell cycle in plants is not only necessary for maintenance of its physiomorphological parameter but it also regulates traits important for mankind like grain or fruit size. This makes it even more important to understand how plants regulate its cell cycle amidst various a/biotic stresses it is subjected to during its lifetime. The association of MAPK signaling pathways with every major developmental and stress response pathways in plants raises the question of its potential role in cell cycle regulation. There are number of cell cycle regulating proteins with putative sites for MAPK phosphorylation. The MAPK signaling pathway may directly or in a parallel pathway regulate the plant cell cycle. Unraveling the role of MAPK in cell cycle will open up new arenas to explore.
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 Krishna
Stress 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.
The small RNA biogenesis in rice is regulated by MAP kinase-mediated OsCDKD phosphorylation
(John Wiley & Sons, 2024) Singh, Dhanraj; Verma, Neetu; Rengasamy, Balakrishnan; Banerjee, Gopal; Sinha, Alok Krishna
CDKs are the master regulator of cell division and their activity is controlled by the regulatory subunit cyclins and phosphorylation by the CAKs. However, the role of MAP kinases in regulating plant cell cycle or CDKs have not been explored. Here, we report that the MAP kinases OsMPK3, OsMPK4, and OsMPK6 physically interact and phosphorylate OsCDKD and its regulatory subunit OsCYCH in rice. MAP kinases phosphorylate CDKD at Ser-168 and Thr-235 residues in OsCDKD. The MAP kinase-mediated phosphorylation of OsCDKD is required for its activation to control the small RNA biogenesis. The phosphodead version of OsCDKD fails to activate the C-terminal domain of RNA Polymerase II, thereby negatively impacting small RNA transcription. Further, the overexpression lines of wild-type (WT) OsCDKD and phosphomimic OsCDKD show increased root growth, plant height, tiller number, panicle number, and seed number in comparison to WT, phosphodead OsCDKD-OE, and kinase-dead OsCDKD-OE plants. In a nutshell, our study establishes a novel regulation of OsCDKD by MAPK-mediated phosphorylation in rice. The phosphorylation of OsCDKD by MAPKs imparts a positive effect on rice growth and development by regulating miRNAs transcription.
Unveiling the molecular mechanism underlying PSKR-mediated amplification of the ABA signaling in Arabidopsis thaliana
(Springer Nature Publishing AG, 2025) Yadav, Nikita; Nagar, Preeti ; Rawat, Abhilasha ; Rakhi, R. ; Singh, Dhanraj; Habibzai, Hedayaturahman ; Sinha, Alok Krishna; Mustafiz, Ananda
Abscisic acid (ABA) serves as a vital signaling molecule that help plants respond to various environmental stresses, ensuring their survival and adaptability. The ABA signaling pathway begins when ABA is recognized by receptors known as PYR/PYL/RCAR. Upon ABA binding, these receptors undergo structural changes, but the precise modifications occurring during post-translational stages and their impact on ABA signaling are not fully understood. In this study, we have identified and characterized the ABA receptor family as target of PSKRs in both Arabidopsis and rice. In addition, we pinpointed the critical active sites in AtPSKR1 (N865) and OsPSKR15 (N892) that are responsible for kinase activity of the respective receptors and also important for direct interaction with ABA receptors. In vitro kinase experiments demonstrated phosphorylation of ABA receptors at S99 in AtPYL4, and S79 in AtPYL9. In addition, our genetic analysis demonstrated that PSKR plays a positive role in regulating ABA-mediated physiological responses, and promotes ABA-dependent leaf senescence in Arabidopsis. Phenotypic studies and expression analysis of ABA-related genes in complementation lines (AtPSKR1:pyl9 and OsPSKR15:pyl9) suggested that the overexpression of PSKR can partially restore the insensitivity of pyl9 mutant plants to ABA. These findings underscore the critical role of PSKR in enhancing ABA signaling via phosphorylation of PYL4/PYL9 in Arabidopsis.