Publications of NIPGR Scientists
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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 Fruit ripening specific expression of β-D-N-acetylhexosaminidase (β-Hex) gene in tomato is transcriptionally regulated by Ethylene Response Factor SlERF.E4(Elsevier B.V., 2022) Irfan, Mohammad; Kumar, Pankaj; Kumar, Vinay; Datta, AsisN-glycans and N-glycan processing enzymes are key players in regulating the ripening of tomato (Solanum lycopersicum) fruits, a model for fleshy fruit ripening. β-D-N-acetylhexosaminidase (β-Hex) is a N-glycan processing enzyme involved in fruit ripening. The suppression of β-Hex results in enhanced fruit shelf life and firmness in both climacteric and non-climacteric fruits. Previously, we have shown that ripening specific expression of β-Hex is regulated by RIPENING INHIBITOR (RIN), ABSCISIC ACID STRESS RIPENING 1 (SlASR1) and ethylene. However, the precise mechanism of ethylene-mediated regulation of β-Hex remains elusive. To gain insights into this, we have performed 5’ deletion mapping of tomato β-Hex promoter and a shorter promoter fragment (pD-200, 200 bp upstream to translational start site) is identified, which was found critical for spatio-temporal transcriptional regulation of β-Hex. Further, site specific mutagenesis in RIN and ASR1 binding sites in pD-200 provides key insights into ripening specific promoter activity. Furthermore, induction of GUS activity by ethylene, yeast one hybrid assay and EMSA identify Ethylene Response Factor SlERF.E4 as a positive regulator of β-Hex. Taken together, our study suggest that SlERF.E4 together with RIN and SlASR1 transcriptionally regulates β-Hex and all these three proteins are essential for fruit ripening specific expression of β-Hex in tomato.Item N-acetylglucosamine sensing and metabolic engineering for attenuating human and plant pathogens(MDPI AG, 2022) Ansari, Sekhu; Kumar, Vinay; Bhatt, Dharmendra Nath; Irfan, Mohammad; Datta, AsisDuring evolution, both human and plant pathogens have evolved to utilize a diverse range of carbon sources. N-acetylglucosamine (GlcNAc), an amino sugar, is one of the major carbon sources utilized by several human and phytopathogens. GlcNAc regulates the expression of many virulence genes of pathogens. In fact, GlcNAc catabolism is also involved in the regulation of virulence and pathogenesis of various human pathogens, including Candida albicans, Vibrio cholerae, Leishmania donovani, Mycobacterium, and phytopathogens such as Magnaporthe oryzae. Moreover, GlcNAc is also a well-known structural component of many bacterial and fungal pathogen cell walls, suggesting its possible role in cell signaling. Over the last few decades, many studies have been performed to study GlcNAc sensing, signaling, and metabolism to better understand the GlcNAc roles in pathogenesis in order to identify new drug targets. In this review, we provide recent insights into GlcNAc-mediated cell signaling and pathogenesis. Further, we describe how the GlcNAc metabolic pathway can be targeted to reduce the pathogens’ virulence in order to control the disease prevalence and crop productivity.Item Unraveling the role of tomato Bcl-2-associated athanogene (BAG) proteins during abiotic stress response and fruit ripening(Springer Nature Publishing AG, 2021) Irfan, Mohammad; Kumar, Pankaj; Ahmad, Irshad; Datta, AsisB-cell lymphoma2 (Bcl-2)-associated athanogene (BAG) family proteins are evolutionary conserved across all eukaryotes. These proteins interact with HSP70/HSC70 and function as co-chaperones during stress response and developmental pathways. Compared to the animal counterpart, the BAG proteins in plants are much less studied and primarily Arabidopsis BAG proteins have been identified and characterized for their role in programmed cell death, homeostasis, growth and development, abiotic and biotic stress response. Here, we have identified BAG protein family (SlBAGs) in tomato, an economically important and a model fruit crop using genome-wide scanning. We have performed phylogenetic analysis, genes architecture assessment, chromosomal location and in silico promoter analysis. Our data suggest that SlBAGs show differential tissue specific expression pattern during plant development particularly fruit development and ripening. Furthermore, we reported that expression of SlBAGs is modulated during abiotic stresses and is regulated by stress hormones ABA and ethylene. In planta subcellular localization reveals their diverse subcellular localization, and many members are localized in nucleus and cytoplasm. Like previous reports, our protein–protein interaction network and yeast two-hybrid analysis uncover that SlBAGs interact with HSP70. The current study provides insights into role of SlBAGs in plant development particualry fruit ripening and abiotic stress response.Item Manipulation of oxalate metabolism in plants for improving food quality and productivity(Elsevier B.V., 2019) Kumar, Vinay; Irfan, Mohammad; Datta, AsisOxalic acid is a naturally occurring metabolite in plants and a common constituent of all plant-derived human diets. Oxalic acid has diverse unrelated roles in plant metabolism, including pH regulation in association with nitrogen metabolism, metal ion homeostasis and calcium storage. In plants, oxalic acid is also a pathogenesis factor and is secreted by various fungi during host infection. Unlike those of plants, fungi and bacteria, the human genome does not contain any oxalate-degrading genes, and therefore, the consumption of large amounts of plant-derived oxalate is considered detrimental to human health. In this review, we discuss recent biotechnological approaches that have been used to reduce the oxalate content of plant tissues.Item A comprehensive analysis of Candida albicans phosphoproteome reveals dynamic changes in phosphoprotein abundance during hyphal morphogenesis(Springer Nature, 2018) Ghorai, Priyanka; Irfan, Mohammad; Narula, Alka; Datta, AsisThe morphological plasticity of Candida albicans is a virulence determinant as the hyphal form has significant roles in the infection process. Recently, phosphoregulation of proteins through phosphorylation and dephosphorylation events has gained importance in studying the regulation of pathogenicity at the molecular level. To understand the importance of phosphorylation in hyphal morphogenesis, global analysis of the phosphoproteome was performed after hyphal induction with elevated temperature, serum, and N-acetyl-glucosamine (GlcNAc) treatments. The study identified 60, 20, and 53 phosphoproteins unique to elevated temperature-, serum-, and GlcNAc-treated conditions, respectively. Distribution of unique phosphorylation sites sorted by the modified amino acids revealed that predominant phosphorylation occurs in serine, followed by threonine and tyrosine residues in all the datasets. However, the frequency distribution of phosphorylation sites in the proteins varied with treatment conditions. Further, interaction network-based functional annotation of protein kinases of C. albicans as well as identified phosphoproteins was performed, which demonstrated the interaction of kinases with phosphoproteins during filamentous growth. Altogether, the present findings will serve as a base for further functional studies in the aspects of protein kinase-target protein interaction in effectuating phosphorylation of target proteins, and delineating the downstream signaling networks linked to virulence characteristics of C. albicans.Item Improving food nutritional quality and productivity through genetic engineering(Juniper Publishers, 2017) Irfan, Mohammad; Datta, AsisGenetic engineering has provided new tools for effectively ensuring food and nutritional security to improve agriculture across the world. Conventional agricultural practices can be assisted by molecular biology and biotechnology tools to develop crops with superior traits in a relatively fast way. Genetic engineering allowed solving important problems in many crops such as susceptibility to pests, diseases, environmental stress and development of crops with higher productivity and enhanced nutritional quality. Genetically-modified (GM) crops can prove to be powerful complements to those produced by conventional methods for meeting the worldwide demand for quality foods.Item Fruit ripening regulation of α-Mannosidase expression by the MADS box transcription factor RIPENING INHIBITOR and ethylene(Frontiers Media S.A., 2016) Irfan, Mohammad; Ghosh, Sumit; Meli, Vijaykumar S.; Kumar, Anil; Kumar, Vinay; Chakraborty, Niranjan; Chakraborty, Subhra; Datta, Asisα-Mannosidase (α-Man), a fruit ripening-specific N-glycan processing enzyme, is involved in ripening-associated fruit softening process. However, the regulation of fruit-ripening specific expression of α-Man is not well understood. We have identified and functionally characterized the promoter of tomato (Solanum lycopersicum) α-Man to provide molecular insights into its transcriptional regulation during fruit ripening. Fruit ripening-specific activation of the α-Man promoter was revealed by analysing promoter driven expression of beta-glucuronidase (GUS) reporter in transgenic tomato. We found that RIPENING INHIBITOR (RIN), a MADS box family transcription factor acts as positive transcriptional regulator of α-Man during fruit ripening. RIN directly bound to the α-Man promoter sequence and promoter activation/α-Man expression was compromised in rin mutant fruit. Deletion analysis revealed that a promoter fragment (567 bp upstream of translational start site) that contained three CArG boxes (binding sites for RIN) was sufficient to drive GUS expression in fruits. In addition, α-Man expression was down-regulated in fruits of Nr mutant which is impaired in ethylene perception and promoter activation/α-Man expression was induced in wild type following treatment with a precursor of ethylene biosynthesis, 1-aminocyclopropane-1-carboxylic acid (ACC). Although, α-Man expression was induced in rin mutant after ACC treatment, the transcript level was less as compared to ACC-treated wild type. Taken together, these results suggest RIN-mediated direct transcriptional regulation of α-Man during fruit ripening and ethylene may acts in RIN-dependent and -independent ways to regulate α-Man expression.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.Item Improving nutritional quality and fungal tolerance in soya bean and grass pea by expressing an oxalate decarboxylase(John Wiley & Sons, 2016) Kumar, Vinay; Chattopadhyay, Arnab; Ghosh, Sumit; Irfan, Mohammad; Chakraborty, Niranjan; Chakraborty, Subhra; Datta, AsisSoya bean (Glycine max) and grass pea (Lathyrus sativus) seeds are important sources of dietary proteins; however, they also contain antinutritional metabolite oxalic acid (OA). Excess dietary intake of OA leads to nephrolithiasis due to the formation of calcium oxalate crystals in kidneys. Besides, OA is also a known precursor of β-N-oxalyl-L-α,β-diaminopropionic acid (β-ODAP), a neurotoxin found in grass pea. Here, we report the reduction in OA level in soya bean (up to 73%) and grass pea (up to 75%) seeds by constitutive and/or seed-specific expression of an oxalate-degrading enzyme, oxalate decarboxylase (FvOXDC) of Flammulina velutipes. In addition, β-ODAP level of grass pea seeds was also reduced up to 73%. Reduced OA content was interrelated with the associated increase in seeds micronutrients such as calcium, iron and zinc. Moreover, constitutive expression of FvOXDC led to improved tolerance to the fungal pathogen Sclerotinia sclerotiorum that requires OA during host colonization. Importantly, FvOXDC-expressing soya bean and grass pea plants were similar to the wild type with respect to the morphology and photosynthetic rates, and seed protein pool remained unaltered as revealed by the comparative proteomic analysis. Taken together, these results demonstrated improved seed quality and tolerance to the fungal pathogen in two important legume crops, by the expression of an oxalate-degrading enzyme.