Institutional Publications
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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 Chickpea (Cicer arietinum) PHO1 family members function redundantly in Pi transport and root nodulation(Elsevier B.V., 2024) Mani, Balaji; Maurya, Kanika; Kohli, Pawandeep Singh; Giri, JitenderPhosphorus (P), a macronutrient, plays key roles in plant growth, development, and yield. Phosphate (Pi) transporters (PHTs) and PHOSPHATE1 (PHO1) are central to Pi acquisition and distribution. Potentially, PHO1 is also involved in signal transduction under low P. The current study was designed to identify and functionally characterize the PHO1 gene family in chickpea (CaPHO1s). Five CaPHO1 genes were identified through a comprehensive genome-wide search. Phylogenetically, CaPHO1s formed two clades, and protein sequence analyses confirmed the presence of conserved domains. CaPHO1s are expressed in different plant organs including root nodules and are induced by Pi-limiting conditions. Functional complementation of atpho1 mutant with three CaPHO1 members, CaPHO1, CaPHO1;like, and CaPHO1;H1, independently demonstrated their role in root to shoot Pi transport, and their redundant functions. To further validate this, we raised independent RNA-interference (RNAi) lines of CaPHO1, CaPHO1;like, and CaPHO1;H1 along with triple mutant line in chickpea. While single gene RNAi lines behaved just like WT, triple knock-down RNAi lines (capho1/like/h1) showed reduced shoot growth and shoot Pi content. Lastly, we showed that CaPHO1s are involved in root nodule development and Pi content. Our findings suggest that CaPHO1 members function redundantly in root to shoot Pi export and root nodule development in chickpea.Item RNA interference for improving disease resistance in plants and its relevance in this clustered regularly interspaced short palindromic repeats-dominated era in terms of dsRNA-based biopesticides(Frontiers Media S.A., 2022) Halder, Koushik; Chaudhuri, Abira; Abdin, Malik Z.; Majee, Manoj; Datta, AsisRNA interference (RNAi) has been exploited by scientists worldwide to make a significant contribution in the arena of sustainable agriculture and integrated pest management. These strategies are of an imperative need to guarantee food security for the teeming millions globally. The already established deleterious effects of chemical pesticides on human and livestock health have led researchers to exploit RNAi as a potential agri-biotechnology tool to solve the burning issue of agricultural wastage caused by pests and pathogens. On the other hand, CRISPR/Cas9, the latest genome-editing tool, also has a notable potential in this domain of biotic stress resistance, and a constant endeavor by various laboratories is in progress for making pathogen-resistant plants using this technique. Considerable outcry regarding the ill effects of genetically modified (GM) crops on the environment paved the way for the research of RNAi-induced double-stranded RNAs (dsRNA) and their application to biotic stresses. Here, we mainly focus on the application of RNAi technology to improve disease resistance in plants and its relevance in today’s CRISPR-dominated world in terms of exogenous application of dsRNAs. We also focused on the ongoing research, public awareness, and subsequent commercialization of dsRNA-based biocontrol products.Item Study of plant exclusive virus-derived small interfering RNAs(Springer Nature Publishing AG, 2019) Singh, Ajeet; Kumar, ShaileshPlants, being sessile, are vividly change with respect to gene expression profiling during stress conditions. Regulation of gene expression is controlled by many of the factors, in which ribonucleic acids interference (RNAi) mechanism has been proved to be an important regulator of both transcriptional and post-transcription controls of gene expression. RNAi mechanism provides the anti-viral resistance to plants, in which virus-derived small interfering RNAs (vsiRNAs) is a well-known component. Apart from some databases like siRNAdb, HIVsirDB and VIRsiRNAdb, which are available online pertaining to siRNAs as well as vsiRNAs generated during viral infection in humans, ‘PVsiRNAdb (http://www.nipgr.res.in/PVsiRNAdb)’, a manually curated plant-exclusive database having information related to vsiRNAs found in different virus-infected plants, collected by exhaustive data mining of published literature so far. This chapter describes the data retrieval and functioning of PVsiRNAdb. Major emphasis is also given to the tools available at this database and explanation of all the results output. The information in this plant exclusive database is very useful for the researcher to explore the complex plants and virus interaction and furthermore in the agriculture field, virus-resistant varieties of crops can be raised.Item Improvement of phosphate acquisition and utilization by a secretory purple acid phosphatase (OsPAP21b) in rice(John Wiley & Sons, 2017) Mehra, Poonam; Pandey, Bipin Kumar; Giri, JitenderPhosphate (Pi) deficiency in soil system is a limiting factor for rice growth and yield. Majority of the soil Phosphorus (P) is organic in nature; not readily available for root uptake. Low Pi inducible purple acid phosphatases (PAPs) are hypothesized to enhance the availability of Pi in soil and cellular system. However, information on molecular and physiological roles of rice PAPs is very limited. Here, we demonstrate the role of a novel rice PAP, OsPAP21b in improving plant utilization of organic-P. OsPAP21b was found to be under the transcriptional control of OsPHR2 and strictly regulated by plant Pi-status at both transcript and protein levels. Biochemically, OsPAP21b showed hydrolysis of several organophosphates at acidic pH and possessed sufficient thermostability befitting for high temperature rice ecosystems with acidic soils. Interestingly, OsPAP21b was revealed to be a secretory PAP and encodes a distinguishable major APase (acid phosphatase) isoform under low Pi in roots. Further, OsPAP21b overexpressing transgenics showed increased biomass, APase activity and P content both in hydroponics supplemented with organic-P sources and soil containing organic manure as sole P source. Additionally, overexpression lines depicted increased root length, biomass and lateral roots under low Pi while RNAi lines showed reduced root length and biomass as compared to WT. In light of these evidences, present study strongly proposes OsPAP21b as a useful candidate for improving Pi acquisition and utilization in rice.Item A calmodulin like EF hand protein positively regulates oxalate decarboxylase expression by interacting with E-box elements of the promoter(Nature Publishing Group, 2015) Kamthan, Ayushi; Kamthan, Mohan; Kumar, Avinash; Sharma, Pratima; Ansari, Sekhu; Thakur, Sarjeet Singh; Chaudhuri, Abira; Datta, AsisOxalate decarboxylase (OXDC) enzyme has immense biotechnological applications due to its ability to decompose anti-nutrient oxalic acid. Flammulina velutipes, an edible wood rotting fungus responds to oxalic acid by induction of OXDC to maintain steady levels of pH and oxalate anions outside the fungal hyphae. Here, we report that upon oxalic acid induction, a calmodulin (CaM) like protein-FvCaMLP, interacts with the OXDC promoter to regulate its expression. Electrophoretic mobility shift assay showed that FvCamlp specifically binds to two non-canonical E-box elements (AACGTG) in the OXDC promoter. Moreover, substitutions of amino acids in the EF hand motifs resulted in loss of DNA binding ability of FvCamlp. F. velutipes mycelia treated with synthetic siRNAs designed against FvCaMLP showed significant reduction in FvCaMLP as well as OXDC transcript pointing towards positive nature of the regulation. FvCaMLP is different from other known EF hand proteins. It shows sequence similarity to both CaMs and myosin regulatory light chain (Cdc4), but has properties typical of a calmodulin, like binding of 45Ca2+, heat stability and Ca2+ dependent electrophoretic shift. Hence, FvCaMLP can be considered a new addition to the category of unconventional Ca2+ binding transcriptional regulators.Item Small RNAs in plants: recent development and application for crop improvement(Frontiers Media S.A., 2015) Kamthan, Ayushi; Chaudhuri, Abira; Kamthan, Mohan; Datta, AsisThe phenomenon of RNA interference (RNAi) which involves sequence-specific gene regulation by small non-coding RNAs, i.e., small interfering RNA (siRNA) and microRNA (miRNA) has emerged as one of most powerful approaches for crop improvement. RNAi based on siRNA is one of the widely used tools of reverse genetics which aid in revealing gene functions in many species. This technology has been extensively applied to alter the gene expression in plants with an aim to achieve desirable traits. RNAi has been used for enhancing the crop yield and productivity by manipulating the gene involved in biomass, grain yield and enhanced shelf life of fruits and vegetables. It has also been applied for developing resistance against various biotic (bacteria, fungi, viruses, nematodes, insects) and abiotic stresses (drought, salinity, cold, etc.). Nutritional improvements of crops have also been achieved by enriching the crops with essential amino acids, fatty acids, antioxidants and other nutrients beneficial for human health or by reducing allergens or anti-nutrients. microRNAs are key regulators of important plant processes like growth, development, and response to various stresses. In spite of similarity in size (20–24 nt), miRNA differ from siRNA in precursor structures, pathway of biogenesis, and modes of action. This review also highlights the miRNA based genetic modification technology where various miRNAs/artificial miRNAs and their targets can be utilized for improving several desirable plant traits. microRNA based strategies are much efficient than siRNA-based RNAi strategies due to its specificity and less undesirable off target effects. As per the FDA guidelines, small RNA (sRNA) based transgenics are much safer for consumption than those over-expressing proteins. This review thereby summarizes the emerging advances and achievement in the field of sRNAs and its application for crop improvement.Item Modified expression of an auxin-responsive rice CC-type glutaredoxin gene affects multiple abiotic stress responses(Springer, 2013) Sharma, Raghvendra; Priya, Pushp; Jain, MukeshGlutaredoxins (GRXs) are the ubiquitous oxidoreductase enzymes, which play an important role in defense against various stresses. Here, we report the role of a CC-type GRX gene from rice, OsGRX8, in abiotic stress tolerance. OsGRX8 protein was found to be localized in nucleus and cytosol and its gene expression is induced by various stress conditions and plant hormone auxin. The over-expression of OsGRX8 in Arabidopsis plants conferred reduced sensitivity to auxin and stress hormone, abscisic acid. In addition, the transgenic Arabidopsis plants exhibited enhanced tolerance to various abiotic stresses, including salinity, osmotic and oxidative stress. Further, the transgenic RNAi rice plants exhibited increased susceptibility to various abiotic stresses, which further confirmed the role of OsGRX8 in abiotic stress responses. The microarray data analysis revealed that expression of a large number of auxin-responsive, known stress-associated and transcription factor encoding genes was altered in GRX transgenic Arabidopsis plants in response to exogenous auxin and stress conditions as compared to wild-type plants. Altogether, these findings suggest the role of OsGRX8 in regulating abiotic stress response and may be used to engineer stress tolerance in crop plants.Item Plant innate immunity: an updated insight into defense mechanism(Indian Academy of Sciences, 2013) Muthamilarasan, Mehanathan; Prasad, ManojPlants are invaded by an array of pathogens of which only a few succeed in causing disease. The attack by others is countered by a sophisticated immune system possessed by the plants. The plant immune system is broadly divided into two, viz. microbial-associated molecular-patterns-triggered immunity (MTI) and effector-triggered immunity (ETI). MTI confers basal resistance, while ETI confers durable resistance, often resulting in hypersensitive response. Plants also possess systemic acquired resistance (SAR), which provides long-term defense against a broad-spectrum of pathogens. Salicylic-acid-mediated systemic acquired immunity provokes the defense response throughout the plant system during pathogen infection at a particular site. Trans-generational immune priming allows the plant to heritably shield their progeny towards pathogens previously encountered. Plants circumvent the viral infection through RNA interference phenomena by utilizing small RNAs. This review summarizes the molecular mechanisms of plant immune system, and the latest breakthroughs reported in plant defense. We discuss the plant–pathogen interactions and integrated defense responses in the context of presenting an integral understanding in plant molecular immunity.Item The N-glycan processing enzymes {alpha}-mannosidase and {beta}-D-N-acetylhexosaminidase are involved in ripening-associated softening in the non-climacteric fruits of Capsicum(Oxford University Press, 2011) Ghosh, Sumit; Meli, Vijaykumar S.; Kumar, Anil; Thakur, Archana; Chakraborty, Niranjan; Chakraborty, Subhra; Datta, AsisExcessive softening of fruits during the ripening process leads to deterioration. This is of significant global importance as softening-mediated deterioration leads to huge postharvest losses. N-glycan processing enzymes are reported to play an important role during climacteric fruit softening: however, to date these enzymes have not been characterized in non-climacteric fruit. Two ripening-specific N-glycan processing enzymes, α-mannosidase (α-Man) and β-D-N-acetylhexosaminidase (β-Hex), have been identified and targeted to enhance the shelf life in non-climacteric fruits such as capsicum (Capsicum annuum). The purification, cloning, and functional characterization of α-Man and β-Hex from capsicum, which belong to glycosyl hydrolase (GH) families 38 and 20, respectively, are described here. α-Man and β-Hex are cell wall glycoproteins that are able to cleave terminal α-mannose and β-D-N-acetylglucosamine residues of N-glycans, respectively. α-Man and β-Hex transcripts as well as enzyme activity increase with the ripening and/or softening of capsicum. The function of α-Man and β-Hex in capsicum softening is investigated through RNA interference (RNAi) in fruits. α-Man and β-Hex RNAi fruits were approximately two times firmer compared with the control and fruit deterioration was delayed by approximately 7 d. It is shown that silencing of α-Man and β-Hex enhances fruit shelf life due to the reduced degradation of N-glycoproteins which resulted in delayed softening. Altogether, the results provide evidence for the involvement of N-glycan processing in non-climacteric fruit softening. In conclusion, genetic engineering of N-glycan processing can be a common strategy in both climacteric and non-climacteric species to reduce the post-harvest crop losses.