Browsing by Author "Gautam, Shikha"

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The Arabidopsis F-box protein SKP1-INTERACTING PARTNER 31 modulates seed maturation and seed vigor by targeting JASMONATE ZIM DOMAIN proteins independently of jasmonic acid-isoleucine
(Oxford University Press, 2023) Varshney, Vishal; Hazra, Abhijit; Rao, Venkateswara; Ghosh, Shraboni; Kamble, Nitin Uttam; Achary, Rakesh Kumar; Gautam, Shikha; Majee, Manoj
F-box proteins have diverse functions in eukaryotic organisms, including plants, mainly targeting proteins for 26S proteasomal degradation. Here, we demonstrate the role of the F-box protein SKP1-INTERACTING PARTNER 31 (SKIP31) from Arabidopsis (Arabidopsis thaliana) in regulating late seed maturation events, seed vigor, and viability through biochemical and genetic studies using skip31 mutants and different transgenic lines. We show that SKIP31 is predominantly expressed in seeds and that SKIP31 interacts with JASMONATE ZIM DOMAIN (JAZ) proteins, key repressors in jasmonate (JA) signaling, directing their ubiquitination for proteasomal degradation independently of coronatine/jasmonic acid-isoleucine (JA-Ile), in contrast to CORONATINE INSENSITIVE 1, which sends JAZs for degradation in a coronatine/JA-Ile dependent manner. Moreover, JAZ proteins interact with the transcription factor ABSCISIC ACID-INSENSITIVE 5 (ABI5) and repress its transcriptional activity, which in turn directly or indirectly represses the expression of downstream genes involved in the accumulation of LATE EMBRYOGENESIS ABUNDANT proteins, protective metabolites, storage compounds, and abscisic acid biosynthesis. However, SKIP31 targets JAZ proteins, deregulates ABI5 activity, and positively regulates seed maturation and consequently seed vigor. Furthermore, ABI5 positively influences SKIP31 expression, while JAZ proteins repress ABI5-mediated transactivation of SKIP31 and exert feedback regulation. Taken together, our findings reveal the role of the SKIP31-JAZ-ABI5 module in seed maturation and consequently, establishment of seed vigor.
Methionine sulfoxide reductase B5 plays a key role in preserving seed vigor and longevity in rice (Oryza sativa)
(John Wiley & Sons, 2022) Hazra, Abhijit; Varshney, Vishal; Verma, Pooja; Kamble, Nitin Uttam; Ghosh, Shraboni; Achary, Rakesh Kumar; Gautam, Shikha; Majee, Manoj
Oxidation of methionine leads to the formation of methionine S-sulfoxide and methionine R-sulfoxide, which can be reverted by two types of Methionine Sulfoxide Reductase (MSR), MSRA and MSRB, respectively. Despite the role of MSR enzymes being elucidated in various physiological processes, regulation and implication of MSR in seeds remained poorly explored. In this study, through molecular, biochemical, and genetic studies using seed-specific overexpression and RNAi lines of OsMSRB5 in Oryza sativa, we demonstrate the role of OsMSRB5 in maintaining seed vigor and longevity. We show that age-induced reduced vigor and viability of seeds is correlated with reduced MSR activity and increased methionine sulfoxide (MetSO) formation. OsMSRB5 expression increases during seed maturation and predominantly localizes in the embryo. Further analyses on transgenic lines reveal the role of OsMSRB5 in modulating reactive oxygen species (ROS) homeostasis to preserve seed vigor and longevity. We show that ascorbate peroxidase (APX) and PROTEIN L-ISOASPARTYL METHYLTRANSFERASE (PIMT) undergo MetSO modification in seeds that affect their functional competence. OsMSRB5 physically interacts with these proteins and reverts this modification to facilitate their functions and preserve seed vigor and longevity of seeds. Our results thus illustrate the role of OsMSRB5 in preserving seed vigor and longevity by modulating ROS homeostasis in seeds.
Molecular intricacies of modulating seed dormancy through CRISPR/Cas9 technology
(Oxford University Press, 2026) Gautam, Shikha; Kamble, Nitin Uttam; Majee, Manoj
The processes of seed development, maturation, and dormancy acquisition are complex and tightly regulated, and play a critical role in plant survival and propagation. Over the past decades, significant advances have been made in elucidating the molecular mechanisms that govern these intricate processes. The interplay among hormone signaling, epigenetic regulation, reactive oxygen species (ROS), and environmental cues has been recognized as central to determining seed fate. Despite these advancements, many molecular components remain to be fully discovered. Recent developments in CRISPR-based gene-editing technologies have provided promising tools for the precise regulation of seed dormancy without compromising other seed traits. Although CRISPR has been effectively utilized to modify genes controlling physiological characteristics in a wide range of crops, its application in regulating dormancy remains at an early stage. This review synthesizes current knowledge on the molecular and genetic mechanisms controlling seed maturation, dormancy acquisition and germination, with particular emphasis on emerging CRISPR-based strategies. Realizing this potential, however, requires a deeper understanding of the complex regulatory networks orchestrating seed dormancy acquisition and germination. Identifying optimal gene targets and refining editing strategies will be crucial for developing reliable and sustainable dormancy-control systems. Therefore, we highlight key gene targets, summarize their functional relevance, and discuss how genome editing could be leveraged to fine-tune dormancy and germination behaviour. The studies discussed herein underscore the transformative potential of CRISPR/Cas9 and related genome-editing platforms in advancing seed biology and crop improvement, paving the way for next-generation seed technologies.
PROTEIN L-ISOASPARTYL METHYLTRANSFERASE protects enolase dysfunction by repairing isoaspartyl-induced damage and is positively implicated in agronomically important seed traits
(John Wiley & Sons, 2024) Kamble, Nitin Uttam; Ghosh, Shraboni; Petla, Bhanu Prakash; Achary, Rakesh Kumar; Gautam, Shikha; Rao, Venkateswara; Salvi, Prafull; Hazra, Abhijit; Varshney, Vishal; Majee, Manoj
The protein-repairing enzyme (PRE) PROTEIN L-ISOASPARTYL METHYLTRANSFERASE (PIMT) influences seed vigor by repairing isoaspartyl-mediated protein damage in seeds. However, PIMTs function in other seed traits, and the mechanisms by which PIMT affects such seed traits are still poorly understood. Herein, through molecular, biochemical, and genetic studies using overexpression and RNAi lines in Oryza sativa and Arabidopsis thaliana, we demonstrate that PIMT not only affects seed vigor but also affects seed size and weight by modulating enolase (ENO) activity. We have identified ENO2, a glycolytic enzyme, as a PIMT interacting protein through Y2H cDNA library screening, and this interaction was further validated by BiFC and co-immunoprecipitation assay. We show that mutation or suppression of ENO2 expression results in reduced seed vigor, seed size, and weight. We also proved that ENO2 undergoes isoAsp modification that affects its activity in both in vivo and in vitro conditions. Further, using MS/MS analyses, amino acid residues that undergo isoAsp modification in ENO2 were identified. We also demonstrate that PIMT repairs such isoAsp modification in ENO2 protein, protecting its vital cellular functions during seed maturation and storage, and plays a vital role in regulating seed size, weight, and seed vigor. Taken together, our study identified ENO2 as a novel substrate of PIMT, and both ENO2 and PIMT in turn implicate in agronomically important seed traits.
The rice heat shock transcription factor OsHSFC1b increases seed weight, size, and vigor, but its function is disrupted by isoaspartyl modification
(John Wiley & Sons, 2025) Achary, Rakesh Kumar; Kamble, Nitin Uttam; Gautam, Shikha; Hazra, Abhijit; Varshney, Vishal; Mahawar, Shivangi; Laha, Saroj; Majee, Manoj
Plant optimizes seed size, weight, vigor, and various other features during seed development, which are important not only for their successful propagation and establishment but also for effective agriculture. Despite several studies conducted, understanding how plants coordinate the regulatory mechanisms to achieve optimal seed size, weight, and vigor remains elusive. Here, our study reveals the role of rice heat shock transcription factor OsHSFC1b in modulating various seed attributes. We observe that OsHSFC1b expression increases during the later stage of seed development and is primarily localized in the embryo. We found that hsfc1b genome-edited lines exhibit compromised seed size, weight, and vigor, while overexpression lines exhibit increased seed size, weight, and vigor compared with the wild-type seeds. Our study further reveals that OsHSFC1b improves seed vigor by activating HSPs and RFO biosynthetic genes involved in protection mechanisms, while also mediating seed size and weight by modulating auxin biosynthesis, endosperm development, and seed filling. We found that upon ageing and stressful environments, OsHSFC1b undergoes isoaspartyl modification that negatively impacts its biological function in seeds. Our MS/MS analyses confirm that asparagine residues near the DNA-binding domain and nuclear localization sequence of OsHSFC1b undergo isoaspartyl modification that adversely affects OsHSFC1b's transactivation activity. However, PROTEIN L-ISOASPARTYL METHYLTRANSFERASE interacts and repairs this isoaspartate-mediated damage, and restores the function of OsHSFC1b. Taken together, our study uncovers how isoaspartyl modification affects the transactivation ability of OsHSFC1b, yet the intervention of PIMT not only repairs this damage but also elevates agronomically important seed traits.
Rice PROTEIN L-ISOASPARTYL METHYLTRANSFERASES provides tolerance against sheath blight disease and repairs ALDH and PBZ1
(Nature Publishing Group, 2026) Gautam, Shikha; Kamble, Nitin Uttam; Achary, Rakesh Kumar; Chandan, Ravindra Kumar; Varshney, Vishal; Hazra, Abhijit; Laha, Saroj; Mahawar, Shivangi; Mehandiratta, Sohela; Singh, Sarvanand; Jha, Gopaljee; Majee, Manoj
Protein L-isoaspartyl methyltransferase (PIMT) regulates key seed traits and abiotic stress tolerance in plants by repairing isoaspartyl (isoAsp) damaged proteins. However, whether PIMT-mediated repair is induced and is required during biotic stress tolerance remains unknown. Using rice lines with OsPIMT overexpression, RNAi-mediated suppression, and genome editing, we show that PIMT enhances tolerance to sheath blight (ShB) caused by Rhizoctonia solani. OsPIMT restricts fungal penetration and colonization of rice sheaths. Co-immunoprecipitation coupled with LC-MS/MS identify various proteins including antioxidant proteins, aldehyde dehydrogenases (ALDH) and pathogenesis-related protein 10 (PBZ1), that undergo isoAsp modification during R. solani infection and interact with PIMT. We show that OsALDH and OsPBZ1 exhibit intrinsic antifungal activity against R. solani, but isoAsp modification impairs their activity, making PIMT mediated repair important. Further, OsALDH enhances tolerance to R. solani by inhibiting lipid peroxidation and ROS homeostasis in rice and fungus. Overall, our study reveals that PIMT enhances ShB tolerance through the repair of isoAsp-damaged proteins important for disease tolerance.