Publications of NIPGR Scientists
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Item Heat-responsive MaHSF11 transcriptional activator positively regulates flavonol biosynthesis and flavonoid B-ring hydroxylation in banana (Musa acuminata)(John Wiley & Sons, 2025) Naik, Jogindra; Rajput, Ruchika; Singh, Samar; Stracke, Ralf; Pandey, AshutoshPlant flavonols act primarily as ultraviolet radiation absorbers, reactive oxygen species scavengers, and phytoalexins, and they contribute to biotic and abiotic stress tolerance in plants. Banana (Musa acuminata), an herbaceous monocot and important fruit crop, accumulates flavonol derivatives in different organs, including the edible fruit pulp. Although flavonol content varies greatly in different organs, the molecular mechanisms involving transcriptional regulation of flavonol synthesis in banana are not known. Here, we characterized three SG7-R2R3 MYB transcription factors (MaMYBFA1, MaMYBFA2, and MaMYBFA3) and heat shock transcription factor (MaHSF11), to elucidate the molecular mechanism involved in transcriptional regulation of flavonol biosynthesis in banana. MaMYBFA positively regulates flavonol synthase 2 (MaFLS2) and downregulates MaFLS1. We show these transcription factors to be weak regulators of flavonol synthesis. Overexpression of MaHSF11 enhances flavonol contents, particularly that of myricetin, and promotes flavonol B-ring hydroxylation, which contributes to the diversity of flavonol derivatives. MaHSF11 directly interacts with the MaFLS1 and flavonoid 3',5'-hydroxylase1 (MaF3'5'H1) promoters, both in vitro and in vivo. MaHSF11 activates the expression of MaDREB1 directly, which is known to promote cold and chilling tolerance in banana fruit. Overall, our study elucidates a regulatory mechanism for flavonol synthesis in banana and suggests possible targets for genetic optimization to enhance nutritional value and stress responses in this globally important fruit crop.Item Broadening the epigenetic horizon of abiotic stress response in plants(Springer Nature Publishing AG, 2024) Chhatwal, Himani; Naik, Jogindra; Pandey, Ashutosh; Trivedi, Prabodh KumarPlants, unlike animals, cannot move from one place to another and have to face different climatic disturbances wherever they are growing. So, they have innumerable built-in mechanisms to adapt to various abiotic stressful conditions like drought, heat, cold, and salinity. The changing environmental conditions influence the expression patterns of genes. Epigenetics involves heritable changes in DNA bases or histone proteins, which ultimately create different conformational states of chromatin. The regulatory enzymes of epigenetic modifications are grouped as writers, readers and erasers, which add, recognize and remove the epigenetic marks, respectively. Here, we provide a comprehensive overview of the mechanism of DNA methylation by the RdDM pathway, its maintenance and removal, and different histone modification categories like acetylation, methylation, phosphorylation and ubiquitination. This review further discusses in detail the crucial role these modifications play in adapting to major abiotic stresses and how plants preserve these experiences as stress memory to respond to recurring stresses. It emphasizes the role of epigenetic modifications as a crucial mechanism for building plant’s tolerance and how it can be an important research priority to improve plant growth and development under abiotic stress conditions.Item Flavonols affect the interrelated glucosinolate and camalexin biosynthetic pathways in Arabidopsis thaliana(Oxford University Press, 2024) Naik, Jogindra; Tyagi, Shivi; Rajput, Ruchika; Kumar, Pawan; Pucker, Boas; Bisht, Naveen C.; Misra, Prashant; Stracke, Ralf; Pandey, AshutoshFlavonols are structurally and functionally diverse biomolecules involved in plant biotic and abiotic stress tolerance, pollen development, and inhibition of auxin transport. Despite the ubiquitous nature and multifunctionality of flavonols in land plants, their effects on global gene expression and signaling pathways are unclear. To explore the roles of flavonol metabolites in signaling, we performed comparative transcriptome and targeted metabolite profiling of seedlings from the flavonol-deficient Arabidopsis (Arabidopsis thaliana) loss-of-function mutant flavonol synthase1 (fls1) with and without exogenous supplementation of flavonol derivatives (kaempferol, quercetin, and rutin). Our RNA-seq results indicated that flavanols modulate various biological and metabolic pathways, with significant alteration in camalexin and aliphatic glucosinolate synthesis. Flavonols negatively regulated camalexin biosynthesis but appeared to promote the accumulation of aliphatic glucosinolates via transcription factor–mediated upregulation of biosynthesis genes. Interestingly, upstream amino acid biosynthesis genes involved in methionine and tryptophan synthesis were altered under flavonol deficiency and exogenous supplementation. Quercetin treatment significantly upregulated aliphatic glucosinolate biosynthesis genes compared to kaempferol and rutin. In addition, expression and metabolite analysis of the transparent testa7 mutant, which lacks hydroxylated flavonol derivatives, clarified the role of quercetin in the glucosinolate biosynthesis pathway. This study elucidates the molecular mechanisms by which flavonols interfere with signaling pathways, their molecular targets, and the multiple biological activities of flavonols in plants.Item The R2R3-MYB-SG7 transcription factor CaMYB39 orchestrates surface phenylpropanoid metabolism and pathogen resistance in chickpea(John Wiley & Sons, 2023) Saxena, Samiksha; Pal, Lalita; Naik, Jogindra; Singh, Yeshveer; Verma, Praveen K.; Chattopadhyay, Debasis; Pandey, AshutoshFlavonoids are important plant pigments and defense compounds; understanding the transcriptional regulation of flavonoid biosynthesis may enable engineering crops with improved nutrition and stress tolerance. Here, we characterize R2R3-MYB domain subgroup 7 transcription factor CaMYB39, which regulates flavonol biosynthesis primarily in chickpea trichomes. CaMYB39 overexpression in chickpea was accompanied by a change in flux availability for the phenylpropanoid pathway, particularly flavonol biosynthesis. Lines overexpressing CaMYB39 showed higher isoflavonoid levels, suggesting its role in regulating isoflavonoid pathway. CaMYB39 transactivates the transcription of early flavonoid biosynthetic genes (EBG). FLAVONOL SYNTHASE2, an EBG, encodes an enzyme with higher substrate specificity for dihydrokaempferol than other dihydroflavonols explaining the preferential accumulation of kaempferol derivatives as prominent flavonols in chickpea. Interestingly, CaMYB39 overexpression increased trichome density and enhanced accumulation of diverse flavonol derivatives in trichome-rich tissues. Moreover, CaMYB39 overexpression reduced ROS levels and induced defense gene expression which aids in partially blocking the penetration efficiency of the fungal pathogen, Ascochyta rabiei, resulting in lesser symptoms, thus establishing its role against deadly Ascochyta blight(AB) disease. Overall, our study reports an instance where R2R3-MYB-SG7 member, CaMYB39, besides regulating flavonol biosynthesis, modulates diverse pathways like general phenylpropanoid, isoflavonoid, trichome density and defense against necrotrophic fungal infection in chickpea.Item Gene pyramiding in transgenic plant development: Approaches and challenges(Springer Nature Publishing AG, 2023) Rajput, Ruchika; Naik, Jogindra; Misra, Prashant; Trivedi, Prabodh Kumar; Pandey, AshutoshClimate change leads to frequent alterations in environmental factors with a reciprocal impact on crop productivity. Over the last few decades, various approaches have been used for producing more stress-tolerant and climate-flexible crops. Genetic engineering is one of the approaches used to modify multiple characters or to improve more than one agronomic trait in plants. These instances simultaneously demand simultaneous genetic manipulation of multiple genes, necessitating stacking or pyramiding of multiple genes as compared to single-gene manipulations, and the genetic engineering of plants using multiple genes is technically challenging. In the last two decades, considerable progress has been made with respect to the development and application of the methods for gene pyramiding in transgenic context. The conventional methods of gene stacking include the crossing of individual transgenic plants, co-transformation using multiple plant expression constructs, transformation with single constructs carrying multiple transgenes as well as with the constructs carrying polycistronic transgenes. These methods have been instrumental for gene stacking in several commercialized crops. The tools of targeted genome editing (ZFN, TALEN, and CRISPR) that carry out precise genetic modifications, have opened new avenues in the area of crop biotechnology for defending plants against various stresses. The present review covers the current status of biotechnological techniques used to combat biotic and abiotic stresses in crop plants and describes multiple associated challenges.Item The R2R3-MYB gene family in Cicer arietinum: genome-wide identification and expression analysis leads to functional characterization of proanthocyanidin biosynthesis regulators in the seed coat(Springer Nature Publishing AG, 2022) Rajput, Ruchika; Tyagi, Shivi; Naik, Jogindra; Pucker, Boas; Stracke, Ralf; Pandey, AshutoshChickpea (Cicer arietinum) is among the eight oldest crops and has two main types, i.e., desi and kabuli, whose most obvious difference is the color of their seeds. We show that this color difference is due to differences in proanthocyanidin content of seed coats. Using a targeted approach, we performed in silico analysis, metabolite profiling, molecular, genetic, and biochemical studies to decipher the transcriptional regulatory network involved in proanthocyanidin biosynthesis in the seed coat of C. arietinum. Based on the annotated C. arietinum reference genome sequence, we identified 119 typical CaMYB encoding genes, grouped in 32 distinct clades. Two CaR2R3-MYB transcription factors, named CaPAR1 and CaPAR2, clustering with known proanthocyanidin regulators (PARs) were identified and further analyzed. The expression of CaPAR genes correlated well with the expression of the key structural proanthocyanidin biosynthesis genes CaANR and CaLAR and with proanthocyanidin levels. Protein–protein interaction studies suggest the in vivo interaction of CaPAR1 and CaPAR2 with the bHLH-type transcription factor CaTT8. Co-transfection analyses using Arabidopsis thaliana protoplasts showed that the CaPAR proteins form a MBW complex with CaTT8 and CaTTG1, able to activate the promoters of CaANR and CaLAR in planta. Finally, transgenic expression of CaPARs in the proanthocyanidin-deficient A. thaliana mutant tt2-1 leads to complementation of the transparent testa phenotype. Taken together, our results reveal main components of the proanthocyanidin regulatory network in C. arietinum and suggest that CaPARs are relevant targets of genetic engineering toward improved agronomic traits.Item Interplay between R2R3 MYB-type activators and repressors regulates proanthocyanidin biosynthesis in banana (Musa acuminata)(John Wiley & Sons, 2022) Rajput, Ruchika; Naik, Jogindra; Stracke, Ralf; Pandey, AshutoshProanthocyanidins are oligomeric flavonoids that promote plant disease resistance and benefit human health. Banana is one of the world's most extensively farmed crops and its fruit pulp contain proanthocyanidins. However, the transcriptional regulatory network that fine-tunes proanthocyanidin biosynthesis in banana remains poorly understood. We characterized two proanthocyanidin-specific R2R3 MYB activators (MaMYBPA1-MaMYBPA2) and four repressors (MaMYBPR1-MaMYBPR4) to elucidate the mechanisms underlying the transcriptional regulation of proanthocyanidin biosynthesis in banana. Heterologous expression of MaMYBPA1 and MaMYBPA2 partially complemented the Arabidopsis thaliana proanthocyanidin-deficient transparent testa2 mutant. MaMYBPA1 and MaMYBPA2 interacted physically with MaMYCs to transactivate anthocyanin synthase, leucoanthocyanidin reductase, and anthocyanidin reductase genes in vitro and form functional MBW complexes with MaTTG1 to transactivate these promoters in vivo. Overexpression of MaMYBPAs alone or with MaMYC in banana fruits induced proanthocyanidin accumulation and transcription of proanthocyanidin biosynthesis-related genes. MaMYBPR repressors are also shown to interact with MaMYCs forming repressing MBW complexes, and diminished proanthocyanidin accumulation. Interestingly overexpression of MaMYBPA induces the expression of MaMYBPR, indicating an agile regulation of proanthocyanidin biosynthesis via the formation of competitive MBW complex. Our results reveal regulatory modules of R2R3 MYB- that fine-tune proanthocyanidin biosynthesis and offer possible targets for genetic manipulation for nutritional improvement of banana.Item Genetics of plant organelles: Plastid and mitochondrial genomes(Springer Nature Publishing AG, 2022) Singh, Samar; Naik, Jogindra; Pandey, AshutoshPlant organelles like chloroplasts and mitochondria are essential organelles serving critical functions like photosynthesis and respiration, respectively, in plants. While most of the processes and the components required by the functioning of these organelles are contributed by nuclear DNA, they have few of their own components encoded by their respective genome. Mitochondrial and chloroplast genomes give a real insight into the evolution of land plants, as evident by several studies. Few studies have successfully conducted gene transfer technology into these organelles’ genomes. Although extensive research on plant organelle genome is yet to be done, recent research has shown the probability of these organelles as a target of genome engineering. From targeting individual genes of their genome to incorporating new genes from other species, they hold promises to produce improved traits. Packaging of their genome, which varies significantly in various hierarchies of land and primitive plants, has also been studied in few plant species. This chapter summarizes the current studies and findings in the study of the organellar genome concerning their structure, organization, distribution, regulatory mechanism, and gene transfer technologies. This chapter provides an updated account of the evolution of these organelle genomes.Item Molecular components associated with the regulation of flavonoid biosynthesis(Elsevier B.V., 2022) Naik, Jogindra; Misra, Prashant; Trivedi, Prabodh Kumar; Pandey, AshutoshFlavonoids exhibit amazing structural diversity and play different roles in plants. Besides, these compounds have been associated with several health benefits in humans. Several exogenous and endogenous cues, for example, light, temperature, nutrient status, and phytohormones have been reported as modulators of biosynthesis and accumulation of flavonoids. Thus, multiple hormones and stress-related signaling pathways are involved in the regulation of gene expression associated with this pathway. The transcriptional regulators belonging to the MYB and bHLH family transcription factors are well documented as the direct regulators of the structural genes associated with flavonoid biosynthesis. Recent studies also suggest that some of these factors are regulated by molecular components involved in stress and hormone signaling pathways. Adapter proteins for transcriptional activation or repression via recruitment of co-activators and co-repressors, respectively, E2 ubiquitin ligases, miRNA processing complex, and DNA methylation/demethylation factors have been recently discovered in various plants to play key roles in fine-tuning flavonoids synthesis. In the present review, we aim to provide comprehensive information about the role of different factors in the regulation of flavonoid biosynthesis. Besides, we describe the potential upstream regulators involved in the regulation of flavonoid biosynthesis within the context of available information. To sum up, the present review furnishes an updated account of signal transduction pathways modulating the biosynthesis of flavonoids.Item The R2R3‑MYB transcription factor MtMYB134 orchestrates favonol biosynthesis in Medicago truncatula(Springer Nature Publishing AG, 2021) Naik, Jogindra; Rajput, Ruchika; Pucker, Boas; Stracke, Ralf; Pandey, AshutoshFlavonols are plant specialized metabolites with vital roles in plant development and defense and are known as diet compound beneficial to human health. In leguminous plants, the regulatory proteins involved in flavonol biosynthesis are not well characterized. Using a homology-based approach, three R2R3-MYB transcription factor encoding genes have been identified in the Medicago truncatula reference genome sequence. The gene encoding a protein with highest similarity to known flavonol regulators, MtMYB134, was chosen for further experiments and was characterized as a functional flavonol regulator from M. truncatula. MtMYB134 expression levels are correlated with the expression of MtFLS2, encoding a key enzyme of flavonol biosynthesis, and with flavonol metabolite content. MtMYB134 was shown to activate the promoters of the A. thaliana flavonol biosynthesis genes AtCHS and AtFLS1 in Arabidopsis protoplasts in a transactivation assay and to interact with the Medicago promoters of MtCHS2 and MtFLS2 in yeast 1-hybrid assays. To ascertain the functional aspect of the identified transcription factor, we developed a sextuple mutant, which is defective in anthocyanin and flavonol biosynthesis. Ectopic expression of MtMYB134 in a multiple myb A. thaliana mutant restored flavonol biosynthesis. Furthermore, overexpression of MtMYB134 in hairy roots of M. truncatula enhanced the biosynthesis of various flavonol derivatives. Taken together, our results provide insight into the understanding of flavonol biosynthesis regulation in M. truncatula and provides MtMYB134 as tool for genetic manipulation to improve flavonol synthesis.