Browsing by Author "Kamble, Nitin Uttam"

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ABI transcription factors and PROTEIN L-ISOASPARTYL METHYLTRANSFERASE module mediate seed desiccation tolerance and longevity in Oryza sativa
(The Company of Biologists, 2022) Kamble, Nitin Uttam; Majee, Manoj
In contrast to desiccation-tolerant orthodox seeds, recalcitrant seeds are desiccation sensitive and are unable to survive for a prolonged time. Here, our analyses of Oryza species with contrasting seed desiccation tolerance reveals that PROTEIN L-ISOASPARTYL METHYLTRANSFERASE (PIMT), an enzyme that repairs abnormal isoaspartyl (isoAsp) residues in proteins, acts as a key player that governs seed desiccation tolerance to orthodox seeds but is ineffective in recalcitrant seeds. We observe that, unlike the orthodox seed of Oryza sativa, desiccation intolerance of the recalcitrant seeds of Oryza coarctata are linked to reduced PIMT activity and increased isoAsp accumulation due to the lack of coordinated action of ABA and ABI transcription factors to upregulate PIMT during maturation. We show that suppression of PIMT reduces, and its overexpression increases, seed desiccation tolerance and seed longevity in O. sativa. Our analyses further reveal that the ABI transcription factors undergo isoAsp formation that affect their functional competence; however, PIMT interacts with and repairs isoAsp residues and facilitates their functions. Our results thus illustrate a new insight into the mechanisms of acquisition of seed desiccation tolerance and longevity by ABI transcription factors and the PIMT module.
Arabidopsis ABSCISIC ACID INSENSITIVE4 targets PROTEIN L-ISOASPARTYL METHYLTRANSFERASE1 in seed
(Springer Nature Publishing AG, 2022) Kamble, Nitin Uttam; Ghosh, Shraboni; Achary, Rakesh Kumar; Majee, Manoj
PROTEIN L-ISOASPARTYL METHYLTRANSFERASE (PIMT) is a protein repairing enzyme (PRE) and is implicated in seed vigor and longevity. PIMT has been shown to be induced by ABA, however, its detailed regulation by ABA signaling components is unknown. Herein, we report that ABSCISIC ACID INSENSITIVE4 (ABI4) directly binds to the PIMT1 promoter and regulates its expression in Arabidopsis seeds. AtPIMT1 promoter analysis demonstrated the presence of putative ABI4 binding sites. Our Y1H analysis revealed that AtABI4 transcription factor binds to the AtPIMT1 promoter. Dual luciferase assay also demonstrated the binding of the AtABI4 transcription factor to the AtPIMT1 promoter. Subsequently, we have generated AtPIMT1 promoter GUS lines and revealed that ABA induced expression of GUS in Arabidopsis thaliana. Expression analyses exhibited reduced accumulation of PIMT1 protein and transcript with significant reduction in total PIMT activity in abi4-1 mutants as compared to that of the wild type. The AtPIMT1 promoter GUS expression in abi4-1 mutants was also found to be severely affected in both the control and ABA treatment. Hence, through molecular and genetic evidences we show that the AtABI4 plays a central role in regulating the expression of AtPIMT1 to impart seed vigor and longevity to orthodox seeds.
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.
Arabidopsis Protein L-Isoaspartyl Methyltransferase repairs isoaspartyl damage to antioxidant enzymes and increases heat and oxidative stress tolerance
(American Society for Biochemistry and Molecular Biology, 2020) Ghosh, Shraboni; Kamble, Nitin Uttam; Verma, Pooja; Salvi, Prafull; Petla, Bhanu Prakash; Roy, Shweta; Rao, Venkateswara; Hazra, Abhijit; Varshney, Vishal; Kaur, Harmeet; Majee, Manoj
Stressful environments accelerate the formation of isoaspartyl (isoAsp) residues in proteins, which detrimentally affect protein structure and function. The enzyme Protein L-Isoaspartyl Methyltransferase (PIMT) repairs other proteins by reverting deleterious isoAsp residues to functional aspartyl residues. PIMT function previously has been elucidated in seeds, but its role in plant survival under stress conditions remains undefined. Herein, we used molecular, biochemical, and genetic approaches, including protein overexpression and knockdown experiments, in Arabidopsis to investigate the role of PIMTs in plant growth and survival during heat and oxidative stresses. We demonstrate that these stresses increase isoAsp accumulation in plant proteins, that PIMT activity is essential for restricting isoAsp accumulation, and that both PIMT1 and PIMT2 play an important role in this restriction and Arabidopsis growth and survival. Moreover, we show that PIMT improves stress tolerance by facilitating efficient reactive oxygen species (ROS) scavenging and thereby protecting the functionality of antioxidant enzymes from isoAsp-mediated damage during stress. Specifically, biochemical and MS/MS analyses revealed that antioxidant enzymes acquire deleterious isoAsp residues during stress, which adversely affect their catalytic activities, and that PIMT repairs the isoAsp residues and thereby restores antioxidant enzyme function. Collectively, our results suggest that the PIMT-mediated protein repair system is an integral part of the stress tolerance mechanism in plants, in which PIMTs protect antioxidant enzymes that maintain proper ROS homeostasis against isoAsp-mediated damage in stressful environments.
Arabidopsis SKP1-like protein 13 (ASK13) positively regulates seed germination and seedling growth under abiotic stresses
(Oxford University Press, 2018) Rao, Venkateswara; Petla, Bhanu Prakash; Verma, Pooja; Salvi, Prafull; Kamble, Nitin Uttam; Ghosh, Sharboni; Kaur, Harmeet; Saxena, Saurabh C; Majee, Manoj
SKP1 (S-Phase Kinase Associated Protein1) proteins are key members of the SCF (SKP-Cullin-F-box protein) E3 ligase complexes that ubiquitinate the target proteins and play diverse roles in plant biology. However, as compared to other members of the SCF complex, study of SKP1-like proteins is very limited in plants. In the present work, we report that Arabidopsis SKP1-like protein13 (ASK13) is differentially regulated in different organs, during seed development and germination, and is upregulated in response to abiotic stresses. Y2H library screening and subsequent in vivo interaction through BiFC analysis revealed that ASK13 not only interacts with F-box proteins but also with other proteins which are not components of SCF complexes. Biochemical analysis revealed that ASK13 not only exists as a monomer but also as a homo-oligomer or heteromer with other ASK proteins. Functional analysis using ASK13 overexpression and knockdown lines revealed that ASK13 positively influences seed germination and seedling growth particularly under abiotic stresses. Taken together, our data strongly suggests that apart from participation to form SCF complexes, ASK13 interacts with several other proteins and is implicated in different cellular processes distinct from protein degradation. Overall, ASK13 positively regulates seed germination and seedling growth particularly under abiotic stress conditions.
A conserved NAG motif is critical to the catalytic activity of galactinol synthase, a key regulatory enzyme of RFO biosynthesis
(Portland Press, 2021) Salvi, Prafull; Kumar, Bhavnesh; Kamble, Nitin Uttam; Hazra, Abhijit; Majee, Manoj
Galactinol synthase (GolS) catalyzes the key regulatory step in the biosynthesis of Raffinose Family Oligosaccharides (RFOs). Even though the physiological role and regulation of this enzyme has been well studied, little is known about active site amino acids and the structure-function relationship with substrates of this enzyme. In the present study, we investigate the active site amino acid and structure function relationship for this enzyme. Using a combination of three-dimensional homology modelling, molecular docking along with a series of deletion, site directed mutagenesis followed by in vitro biochemical and in vivo functional analysis; we have studied active site amino acids and their interaction with the substrate of chickpea and Arabidopsis GolS enzyme. Our study reveals that the GolS protein possesses GT8 family-specific several conserved motifs in which NAG motif plays a crucial role in substrate binding and catalytic activity of this enzyme. Deletion of entire NAG motif or deletion or the substitution (with alanine) of any residues of this motif results in complete loss of catalytic activity in in vitro condition. Furthermore, disruption of NAG motif of CaGolS1 enzyme disrupts it’s in vivo cellular function in yeast as well as in planta. Together, our study offers a new insight into the active site amino acids and their substrate interaction for the catalytic activity of GolS enzyme. We demonstrate that NAG motif plays a vital role in substrate binding for the catalytic activity of galactinol synthase that affects overall RFO synthesis.
Deciphering the structural basis of the broad substrate specificity of myo-inositol monophosphatase (IMP) from Cicer arietinum
(Elsevier B.V., 2020) Yadav, Prakarsh K.; Salvi, Prafull; Kamble, Nitin Uttam; Petla, Bhanu Prakash; Majee, Manoj; Saxena, Saurabh C.
Myo-inositol monophosphatase (IMP) is a crucial enzyme in the inositol biosynthetic pathway that dephosphorylates myo-inositol 1-phosphate and other inositol phosphate derivative compounds to maintain the homeostasis of cellular inositol pool. In our previous research, we have biochemically and functionally characterized IMP enzyme from chickpea (CaIMP), which was able to catalyze diverse substrates. We cloned, overexpressed recombinant CaIMP protein and purified it and further characterized the CaIMP with its three main substrates viz. galactose 1-P, inositol 6-P and fructose 1,6-bisP. Homology model of CaIMP was generated to elucidate the factors contributing to the broad substrate specificity of the protein. The active site of the CaIMP protein was analysed with respect to its interactions with the proposed substrates. Structural features such as, high B-factor and flexible loop regions in the active site, inspired further investigation into the static and dynamic behaviour of the active site of CaIMP protein. The electrostatic biding of each of the key substrates was assessed through molecular docking. Furthermore, molecular dynamics simulations showed that these interactions indeed were stable for extended periods of time under physiological conditions. These experiments conclusively allowed us to establish the primary factors contributing to the promiscuity in substrate binding by CaIMP protein.
Differentially expressed galactinol synthase(s) in chickpea are implicated in seed vigor and longevity by limiting the age induced ROS accumulation
(Nature Publishing Group, 2016) Salvi, Prafull; Saxena, Saurabh Chandra; Petla, Bhanu Prakash; Kamble, Nitin Uttam; Kaur, Harmeet; Verma, Pooja; Rao, Venkateswara; Ghosh, Shraboni; Majee, Manoj
Galactinol synthase (GolS) catalyzes the first and rate limiting step of Raffinose Family Oligosaccharide (RFO) biosynthetic pathway, which is a highly specialized metabolic event in plants. Increased accumulation of galactinol and RFOs in seeds have been reported in few plant species, however their precise role in seed vigor and longevity remain elusive. In present study, we have shown that galactinol synthase activity as well as galactinol and raffinose content progressively increase as seed development proceeds and become highly abundant in pod and mature dry seeds, which gradually decline as seed germination progresses in chickpea (Cicer arietinum). Furthermore, artificial aging also stimulates galactinol synthase activity and consequent galactinol and raffinose accumulation in seed. Molecular analysis revealed that GolS in chickpea are encoded by two divergent genes (CaGolS1 and CaGolS2) which potentially encode five CaGolS isoforms through alternative splicing. Biochemical analysis showed that only two isoforms (CaGolS1 and CaGolS2) are biochemically active with similar yet distinct biochemical properties. CaGolS1 and CaGolS2 are differentially regulated in different organs, during seed development and germination however exhibit similar subcellular localization. Furthermore, seed-specific overexpression of CaGolS1 and CaGolS2 in Arabidopsis results improved seed vigor and longevity through limiting the age induced excess ROS and consequent lipid peroxidation.
Ectopic over-expression of ABA-responsive Chickpea galactinol synthase (CaGolS) gene results in improved tolerance to dehydration stress by modulating ROS scavenging
(Elsevier B.V., 2020) Salvi, Prafull; Kamble, Nitin Uttam; Majee, Manoj
Galactinol synthase (EC: 2.4.1.123) is a crucial enzyme of raffinose family oligosaccharides (RFO’s) biosynthesis, essentially catalyzes the first crucial step in the raffinose biosynthetic pathway. Galactinol and raffinose accumulation along with its higher homologs such as stachyose and verbascose has been considered to participate in plant abiotic stress tolerance. Previously, we reported the function of chickpea galactinol synthase (CaGolS) genes in seed vigor and heat and oxidative stress tolerance. Here, we demonstrate the role and regulation of CaGolS in response to dehydration stress in chickpea. Through transcript accumulation and promoter-gus analyses, we demonstrate that the expression of CaGolS1 and CaGolS2 are positively influenced by dehydration stress and ABA treatment. Further, we have shown that the level of galactinol and raffinose are remarkably enhanced in response to dehydration stress and ABA. Utilizing CaGolS1 and CaGolS2 over-expression Arabidopsis lines, we demonstrate the role of CaGolS in dehydration stress tolerance. The biochemical and physiological analysis revealed that CaGolS over-expressing transgenic lines exhibited improved phenotype with respect to higher number of siliques, plant height, and rosette diameter under dehydration stress. The improved dehydration stress tolerance was corelated with higher chlorophyll retention and relative water content of transgenic lines. Further, lower H2O2, MDA content, and ion-leakage in transgenic lines suggest that CaGolS mediates dehydration stress tolerance by protecting the membrane damage from ROS attack. Collectively, our data highlight the prospect of CaGolS genes in improving dehydration stress tolerance in plants.
Ectopic overexpression of cytosolic ascorbate peroxidase gene (Apx1) improves salinity stress tolerance in Brassica juncea by strengthening antioxidative defense mechanism
(Springer Nature, 2020) Saxena, Saurabh C.; Salvi, Prafull; Kamble, Nitin Uttam; Joshi, Pankaj K.; Majee, Manoj; Arora, Sandeep
Salinity stress is considered to be a key constrain that reduces the crop productivity by impairing plant growth and development. During salt stress condition, an underlying mechanism for reduction in crop yield is increase in ROS level that can potentially harm cellular macromolecules, leading to disruption of essential physiological and biochemical processes. Plants possess a complex antioxidative defense machinery for scavenging these ROS. Ascorbate peroxidase (APX, E.C. 1.11.1.11), is a crucial antioxidant enzyme involved in Ascorbate–Glutathione pathway that primarily detoxifies the negative impact of H2O2 in cell. The efficient scavenging of H2O2 is a prerequisite for enhanced tolerance to salinity stress. Here, we have inspected whether over-expression of APX could provide protection against salinity stress. Cytosolic ascorbate peroxidase (Apx1) gene, isolated from Arabidopsis thaliana, was chosen as the candidate gene for strengthening the antioxidative defense system of Brassica juncea. Physiological parameters were employed to analyze the growth status of transgenic plants. Leaf disc assay was done to evaluate the salinity stress tolerance potential of transgenic plants, using several physiological and biochemical parameters. Under salinity stress, the transgenic plants performed well as compared to their non-transgenic counterparts; as revealed through greater proline accumulation, increased chlorophyll stability index, lower chlorophyll a/b ratio, and higher antioxidative enzyme activities. Further, the lower H2O2 levels were well correlated with lesser membrane damage as measured through MDA content. Collectively, our results clearly depicted that ectopic overexpression of AtApx1 gene was able to confer salinity stress tolerance by strengthening the antioxidative defense system in B. juncea.
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.
Oryza coarctata PROTEIN L-ISOASPARTYL METHYLTRANSFERASE (PIMT) repairs isoaspartyl modification to antioxidative enzymes and is implicated in seed traits in rice
(Elsevier B.V., 2022) Kamble, Nitin Uttam; Petla, Bhanu Prakash; Ghosh, Shraboni; Achary, Rakesh Kumar; Majee, Manoj
PROTEIN L-ISOASPARTYL METHYLTRANSFERASE (PIMT) is a protein repairing enzyme, which is highly abundant in orthodox seeds, and plays an important role in seed vigor and longevity. PIMT essentially repairs isoaspartyl modification in proteins. Despite PIMT has been characterized from several orthodox seed producing plant species, role and regulation of PIMTs in recalcitrant seed producing plants are still limited. In the present study, PIMT from Oryza coarctata, which produces recalcitrant seeds and possess both enzymatically active (OcPIMT1–1 and OcPIMT2–1) and inactive (OcPIMT1–2 and OcPIMT2–2) PIMT isoforms, are functionally characterized through biochemical and genetic approach. We show that PIMT isoforms are differentially localized in Oryza sativa and Oryza coarctata. We also report that enzymatically active OcPIMTs isoforms, but not enzymatically inactive OcPIMTs isoforms, could impart seed vigor, viability and longevity in A. thaliana. Likewise, rice transgenic lines were also generated, and ectopic overexpression of enzymatically active OcPIMT isoforms resulted in increased seed length and weight with improved seed vigor and longevity. Subsequent analysis revealed that antioxidant enzymes (OsAPX and OsCAT) are susceptible to isoAsp modification, which negatively influences their biological functions; however, OcPIMTs physically interact, repairs and protect their function from harmful isoAsp modification, and thereby modulate ROS homeostasis in seeds during aging. Collectively, our results highlight the mechanisms and importance of ectopic expression of OcPIMT isoforms in seed desiccation tolerance and subsequent vigor, viability and longevity in rice.
PROTEIN L-ISOASPARTYL METHYLTRANSFERASE (PIMT) in plants: regulations and functions
(Portland Press, 2020) Kamble, Nitin Uttam; Majee, Manoj
Proteins are essential molecules that carry out key functions in a cell. However, as a result of aging or stressful environments, the protein undergoes a range of spontaneous covalent modifications, including the formation of abnormal l-isoaspartyl residues from aspartyl or asparaginyl residues, which can disrupt the protein's inherent structure and function. PROTEIN l-ISOASPARTYL METHYLTRANSFERASE (PIMT: EC 2.1.1.77), an evolutionarily conserved ancient protein repairing enzyme (PRE), converts such abnormal l-isoaspartyl residues to normal l-aspartyl residues and re-establishes the protein's native structure and function. Although originally discovered in animals as a PRE, PIMT emerged as a key PRE in plants, particularly in seeds, in which PIMT plays a predominant role in preserving seed vigor and viability for prolonged periods of time. Interestingly, higher plants encode a second PIMT (PIMT2) protein which possesses a unique N-terminal extension, and exhibits several distinct features and far more complexity than non-plant PIMTs. Recent studies indicate that the role of PIMT is not restricted to preserving seed vigor and longevity but is also implicated in enhancing the growth and survivability of plants under stressful environments. Furthermore, expression studies indicate the tantalizing possibility that PIMT is involved in various physiological processes apart from its role in seed vigor, longevity and plant's survivability under abiotic stress. This review article particularly describes new insights and emerging interest in all facets of this enzyme in plants along with a concise comparative overview on isoAsp formation, and the role and regulation of PIMTs across evolutionary diverse species. Additionally, recent methods and their challenges in identifying isoaspartyl containing proteins (PIMT substrates) are highlighted.
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.
A protein repairing enzyme, PROTEIN L- ISOASPARTYL METHYLTRANSFERASE is involved in salinity stress tolerance by increasing efficiency of ROS-scavenging enzymes
(Elsevier B.V., 2020) Ghosh, Shraboni; Kamble, Nitin Uttam; Majee, Manoj
Saline conditions can significantly affect plant growth and development, leading to massive reduction in crop yield. Herein, we show that a protein repairing enzyme PROTEIN L-ISOASPARTYL METHYLTRANSFERASE imparts salinity stress tolerance in Arabidopsis thaliana by repairing deleterious isoAsp accumulation during salinity stress. We demonstrate that salinity stress accelerates isoAsp accumulation in proteins and also induces PIMT activity in Arabidopsis. Transcript analysis indicates that both PIMT1 and PIMT2 are upregulated in response to salinity stress. Subsequent functional analysis reveals that PIMT1 and PIMT2 overexpression lines are tolerant, while RNAi lines are hyper sensitive to salinity stress in comparison to wild type (WT). Biochemical analyses of thesePIMT transgenic lines also reveals that compromised salinity tolerance of RNAi lines are linked to increased isoAsp accumulation, while improved tolerance of overexpression lines is associated with reduced isoAsp accumulation in proteins. Histochemical and biochemical studies further confirm lower accumulation of ROS and reduced lipid peroxidation in PIMT overexpression lines, while increased ROS accumulation and increased lipid peroxidation in RNAi lines as compared to WT under salinity stress. Interestingly, PIMToverexpression lines exhibit improved antioxidant enzyme efficiency, while RNAi lines display compromised antioxidant enzyme efficacy as compared to WT type plants. Our study suggests that PIMT improves salinity stress tolerance by restricting salt induced-excess ROS accumulation possibly by repairing isoAsp mediated protein damage of antioxidant enzymes. Our study can be utilized for enhancing salinity stress tolerance of economically important crops.
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 METHYLTRANSFERASE isoforms differentially accumulate during seed maturation to restrict deleterious isoAsp and reactive oxygen species accumulation and are implicated in seed vigor and longevity
(John Wiley & Sons, 2016) Petla, Bhanu Prakash; Kamble, Nitin Uttam; Kumar, Meenu; Verma, Pooja; Ghosh, Shraboni; Singh, Ajeet; Rao, Venkateswara; Salvi, Prafull; Kaur, Harmeet; Saxena, Saurabh Chandra; Majee, Manoj
PROTEIN l-ISOASPARTYL O-METHYLTRANSFERASE (PIMT) is a protein-repairing enzyme involved in seed vigor and longevity. However, the regulation of PIMT isoforms during seed development and the mechanism of PIMT-mediated improvement of seed vigor and longevity are largely unknown. In this study in rice (Oryza sativa), we demonstrate the dynamics and correlation of isoaspartyl (isoAsp)-repairing demands and PIMT activity, and their implications, during seed development, germination and aging, through biochemical, molecular and genetic studies. Molecular and biochemical analyses revealed that rice possesses various biochemically active and inactive PIMT isoforms. Transcript and western blot analyses clearly showed the seed development stage and tissue-specific accumulation of active isoforms. Immunolocalization studies revealed distinct isoform expression in embryo and aleurone layers. Further analyses of transgenic lines for each OsPIMT isoform revealed a clear role in the restriction of deleterious isoAsp and age-induced reactive oxygen species (ROS) accumulation to improve seed vigor and longevity. Collectively, our data suggest that a PIMT-mediated, protein repair mechanism is initiated during seed development in rice, with each isoform playing a distinct, yet coordinated, role. Our results also raise the intriguing possibility that PIMT repairs antioxidative enzymes and proteins which restrict ROS accumulation, lipid peroxidation, etc. in seed, particularly during aging, thus contributing to seed vigor and longevity.
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.
Stress inducible galactinol synthase of chickpea (CaGolS) implicates in heat and oxidative stress tolerance through reducing stress induced excessive reactive oxygen species accumulation
(Oxford University Press, 2018) Salvi, Prafull; Kamble, Nitin Uttam; Majee, Manoj
Raffinose Family Oligosaccharides (RFOs) participates in various aspects of plant physiology and galactinol synthase (GolS, EC 2.4.1.123) catalyzes the key step of RFO biosynthesis. Stress induced accumulation of RFOs particularly galactinol and raffinose has been reported in few plants; however their precise role and mechanistic insight in stress adaptation remain elusive. In present study, we have shown that the GolS activity as well as galactinol and raffinose content are significantly increased in response to various abiotic stresses in chickpea. Transcriptional analysis indicated that the CaGolS1 and CaGolS2 genes are induced in response to different abiotic stresses. Interestingly, heat and oxidative stress preferentially induce CaGolS1 over CaGolS2. Insilco analysis revealed several common yet distinct cis-acting regulatory elements in their 5' upstream regulatory sequences. Further, in vitro biochemical analysis revealed that CaGolS1 enzyme functions better in stressful conditions than CaGolS2 enzyme. Finally, Arabidopsis transgenic plants constitutively overexpressing CaGolS1 or CaGolS2 not only exhibits significantly increased galactinol but also raffinose content and display better growth responses than wild type or vector control plants when exposed to heat and oxidative stress. Further, improved tolerance of transgenic lines are associated with reduced accumulation of reactive oxygen species (ROS) and consequent lipid peroxidation as compared to control plants.Collectively, our data implies that GolS enzyme activity and consequent galactinol and raffinose content are significantly increased in response to stresses to mitigate stress induced growth inhibition by restricting excessive ROS accumulation and consequent lipid peroxidation in plants.