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Item OsLdh3 interacts with OsGAPC3 and OsLos2 to maintain the glycolytic continuum for tolerance to multiple abiotic stresses in rice(Oxford University Press, 2026) Chatterjee, Yajnaseni; Babuta, Priyanka; Gupta, Kapuganti Jagadis; Pareek, Ashwani; Singla-Pareek, Sneh LataLactate dehydrogenases are oxidoreductases present in almost all living organisms. They catalyze the interconversion of pyruvate and L-lactate with simultaneous oxidation of NADH and reduction of NAD+. Since their function remains largely unexplored in rice, in this study we deciphered the role of the rice lactate dehydrogenase, OsLdh3. OsLdh3 showed optimum enzyme activity at pH 6.6 for the forward reaction (pyruvate to L-lactate) and pH 9 for the reverse reaction (L-lactate to pyruvate). Protein-protein interaction studies revealed that OsLdh3 interacts with the glycolytic enzymes glyceraldehyde 3-phosphate dehydrogenaseC3 (OsGAPC3) and Enolase2 (OsLos2), suggesting its role in regulating glycolytic flux. Further, overexpression of OsLdh3 in rice showed enhanced abiotic stress tolerance by exhibiting elevated NAD+ levels and OsGAPC3 activity, thereby facilitating an improved glycolytic continuum and higher pyruvate accumulation. Consequently, these lines also showed increased mitochondrial respiration and ATP synthesis, and reduced reactive oxygen species (ROS) accumulation. Further, enhanced photosynthetic efficiency and reduced yield penalty of the stress-imposed OsLdh3 overexpression lines underscore its importance in crop productivity under adverse climatic conditions. Thus, our findings show that OsLdh3 enhances stress tolerance in rice by regulating redox homeostasis and respiration, reducing ROS levels, and maintaining energy balance. This makes OsLdh3 a promising candidate gene for developing climate-resilient rice cultivars with reduced yield gap.Item N-glycan remodeling by α-D-mannosidase and β-D-N-acetylhexosaminidase regulates fruit softening, redox balance, and post-harvest pathogen resistance(John Wiley & Sons, 2025) Irfan, Mohammad; Kumar, Pankaj; Kumar, Vinay; Ahmad, Irshad; Ansari, Sekhu; Ghosh, Sumit; Datta, AsisPost-harvest loss of fruits and vegetables poses significant challenges to food security and economic sustainability, primarily due to ripening-associated excessive softening that shortens shelf life and increases susceptibility to pathogens. N-glycans, N-glycoproteins, and their processing enzymes are integral to various plant processes, including fruit ripening. Among these, α-D-mannosidase (α-Man) and β-D-N-acetylhexosaminidase (β-Hex) are key ripening-specific enzymes that modulate fruit softening. Previously, we have shown that RNAi-mediated suppression of α-Man or β-Hex improves fruit shelf life and firmness in both climacteric and non-climacteric fruits. However, the underlying molecular and biochemical basis of fruit softening regulation by α-Man and β-Hex was not well understood. In this study, we developed transgenic tomato (Solanum lycopersicum) plants by silencing α-Man and β-Hex simultaneously using RNAi. Suppression of these enzymes reduces N-glycoprotein degradation, downregulates pectin dissolution, and inhibits ripening-related gene expression. RNAi fruits exhibited enhanced shelf life, greater firmness, reduced reactive oxygen species (ROS) accumulation and increased resistance against post-harvest pathogens without affecting plant growth, fruit development, yield, or nutritional quality. To further explore the molecular mechanism of α-Man and β-Hex function, we purified and quantified N-glycans in RNAi fruits and other ripening-impaired mutants, identifying key N-glycan species. We also carried out iTRAQ-based quantitative proteome profiling to investigate the abundance of proteins in ripened fruit affected by silencing of α-Man and β-Hex. Molecular insights revealed that N-glycan processing and degradation are key events during ripening, influencing cell wall softening, fruit redox state, and post-harvest quality attributes. This study highlights the potential of co-silencing α-Man and β-Hex as a novel approach to extending the shelf life of fruits, regardless of their climacteric behavior, without compromising quality or yield.Item Suppressing plant defence: Scavenge the ROS!(John Wiley & Sons, 2023) Prasad, Ashish; Sharma, Shambhavi; Prasad, ManojROS-mediated defence against fungal pathogens is an essential arm of plant immunity. As a counter defence, these pathogens synthesize antioxidant enzymes that scavenge the ROS produced by plants. The molecular mechanism behind the upregulation of these enzymes in fungal pathogens was unknown. A recent study by Zhang and colleagues has shed light on the mechanism, and it has been shown that deacetylation of FolSrpk1 protein on the K304 residue following oxidative stress is an important event in the signalling cascade leading to ROS detoxification in Fusarium oxysporum f. sp. lycopersici. Deacetylated FolSrpk1 moves to the nucleus where it hyperphosphorylates FolSr1, which further regulates the transcription of antioxidant enzymes (Zhang et al. 2023). This mechanism of ROS detoxification is conserved in Botrytis cinerea as well. Several other phytopathogenic fungi have a corresponding K304 site and NLS present in their Srpk1, suggesting a similar mechanism of ROS detoxification and suppression of plant defence. In this article, we have presented our views on how future studies can be synthesized based on the pathway deciphered by Zhang et al. 2023.Item Protein l-isoAspartyl Methyltransferase (PIMT) and antioxidants in plants(Elsevier B.V., 2023) Ghosh, Shraboni; Majee, ManojAll life forms, including plants, accumulate reactive oxygen species (ROS) as a byproduct of metabolism; however, environmental stresses, including abiotic stresses and pathogen attacks, cause enhanced accumulation of ROS in plants. The increased accumulation of ROS often causes oxidative damage to cells. Organisms are able to maintain levels of ROS below permissible limits by several mechanisms, including efficient antioxidant systems. In addition to antioxidant systems, recent studies suggest that protein l-isoaspartyl methyltransferase (PIMT), a highly conserved protein repair enzyme across evolutionary diverse organisms, plays a critical role in maintaining ROS homeostasis by repairing isoaspartyl-mediated damage to antioxidants in plants. Under stress conditions, antioxidant proteins undergo spontaneous isoaspartyl (isoAsp) modification which is often detrimental to protein structure and function. This reduces the catalytic action of antioxidants and disturbs the ROS homeostasis of cells. This chapter focuses on PIMT and its interaction with antioxidants in plants, where PIMT constitutes a secondary level of protection by shielding a primary level of antioxidants from dysfunction and permitting them to guard during unfavorable situations.Item 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, ManojPROTEIN 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.Item Adaptive reprogramming during early seed germination requires temporarily enhanced fermentation-A critical role for alternative oxidase regulation that concerns also microbiota effectiveness(Frontiers Media S.A., 2021) Bharadwaj, Revuru; Noceda, Carlos; Mohanapriya, Gunasekharan; Kumar, Sarma Rajeev; Thiers, Karine Leitão Lima; Costa, José Hélio; Macedo, Elisete Santos; Kumari, Aprajita; Gupta, Kapuganti Jagadis; Srivastava, Shivani; Adholeya, Alok; Oliveira, Manuela; Velada, Isabel; Sircar, Debabrata; Sathishkumar, Ramalingam; Arnholdt-Schmitt, BirgitPlants respond to environmental cues via adaptive cell reprogramming that can affect whole plant and ecosystem functionality. Microbiota constitutes part of the inner and outer environment of the plant. This Umwelt underlies steady dynamics, due to complex local and global biotic and abiotic changes. Hence, adaptive plant holobiont responses are crucial for continuous metabolic adjustment at the systems level. Plants require oxygen-dependent respiration for energy-dependent adaptive morphology, such as germination, root and shoot growth, and formation of adventitious, clonal, and reproductive organs, fruits, and seeds. Fermentative paths can help in acclimation and, to our view, the role of alternative oxidase (AOX) in coordinating complex metabolic and physiological adjustments is underestimated. Cellular levels of sucrose are an important sensor of environmental stress. We explored the role of exogenous sucrose and its interplay with AOX during early seed germination. We found that sucrose-dependent initiation of fermentation during the first 12 h after imbibition (HAI) was beneficial to germination. However, parallel upregulated AOX expression was essential to control negative effects by prolonged sucrose treatment. Early downregulated AOX activity until 12 HAI improved germination efficiency in the absence of sucrose but suppressed early germination in its presence. The results also suggest that seeds inoculated with arbuscular mycorrhizal fungi (AMF) can buffer sucrose stress during germination to restore normal respiration more efficiently. Following this approach, we propose a simple method to identify organic seeds and low-cost on-farm perspectives for early identifying disease tolerance, predicting plant holobiont behavior, and improving germination. Furthermore, the research strengthens the view that AOX can serve as a powerful functional marker source for seed hologenomes.Item 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, ManojSaline 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.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 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, ManojRaffinose 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.Item Fruit ripening mutants reveal cell metabolism and redox state during ripening(Springer, 2016) Kumar, Vinay; Irfan, Mohammad; Ghosh, Sumit; Chakraborty, Niranjan; Chakraborty, Subhra; Datta, AsisRipening which leads to fruit senescence is an inimitable process characterized by vivid changes in color, texture, flavor, and aroma of the fleshy fruits. Our understanding of the mechanisms underlying the regulation of fruit ripening and senescence is far from complete. Molecular and biochemical studies on tomato (Solanum lycopersicum) ripening mutants such as ripening inhibitor (rin), nonripening (nor), and never ripe (Nr) have been useful in our understanding of fruit development and ripening. The MADS-box transcription factor RIN, a global regulator of fruit ripening, is vital for the broad aspects of ripening, in both ethylene-dependent and independent manners. Here, we have carried out microarray analysis to study the expression profiles of tomato genes during ripening of wild type and rin mutant fruits. Analysis of the differentially expressed genes revealed the role of RIN in regulation of several molecular and biochemical events during fruit ripening including fruit specialized metabolism and cellular redox state. The role of reactive oxygen species (ROS) during fruit ripening and senescence was further examined by determining the changes in ROS level during ripening of wild type and mutant fruits and by analyzing expression profiles of the genes involved in maintaining cellular redox state. Taken together, our findings suggest an important role of ROS during fruit ripening and senescence, and therefore, modulation of ROS level during ripening could be useful in achieving desired fruit quality.