Institutional Publications
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Item Plant isopropylmalate synthases: in and beyond leucine biosynthesis(Springer Nature Publishing AG, 2026) Varghese, Mohan; Lone, Asif; Bisht, Naveen C.Primary metabolic enzymes in amino acid biosynthesis pathways are involved in amino acid synthesis and maintenance of metabolic homeostasis through feedback regulatory mechanisms. The genes encoding these enzymes are also known to undergo functional diversification through evolutionary processes to encode specific secondary metabolic enzymes. One such enzyme is α-isopropylmalate synthase (α-IPMS), which catalyzes and regulates leucine (Leu) biosynthesis in eubacteria, archaebacteria, fungi, and plants, and has served as an evolutionary progenitor for specialized enzymes in distinct secondary metabolic pathways. However, most information on IPMS comes from the bacterial research community, particularly in the context of Leu overproduction or as a target for developing drugs against tuberculosis. In plants, only a few studies have reported on IPMS, focusing primarily on its role in regulating Leu homeostasis. Herein, we review the complex regulatory network that exists in Leu metabolism, focusing on the regulation of its biosynthesis and its key regulatory enzyme, IPMS. This review also highlights how evolution has independently recruited IPMS for specialized metabolism in several plant lineages. Finally, we describe the emerging roles of IPMS as a candidate for engineering amino acid and yield-related traits in crop plants. We also identify important open questions in this area that remain to be addressed.Item A guide to culturing, maintenance, and leaf inoculation methods for rapid screening and quantification of sclerotinia sclerotiorum infection in mustard(Springer Nature Publishing AG, 2026) Malhotra, Bhanu; Tiwari, Ruchi; Varghese, Mohan; Bisht, Naveen C.Sclerotinia rot poses a significant challenge to the cultivation of oilseed Brassica crops, causing the reduction of seed number, weight, and quality leading to >95% yield losses globally. Due to its substantial economic impact on agriculture, Sclerotinia sclerotiorum has been extensively studied at the molecular level and has now been adopted as a model to investigate the host-pathogen interactions. Despite various procedures and strategies described in different reports, there exists a considerable disparity in how the pathogen is cultured, maintained, and manipulated across studies.This chapter provides a comprehensive guide to the fundamental procedures of working with S. sclerotiorum. It describes step-by-step methods for its routine culturing and maintenance over artificial media, replenishing and storing laboratory stocks, and conducting detached leaf assays-a robust method used for screening the mustard cultivars. Additionally, we outline the quantification of pathogen load in planta through qRT-PCR analysis. The methods presented in the current chapter are reproducible and can be suitably applied when working with different isolates of the pathogen.Through the present compilation, we aim to address the research gaps in methods involving the pathogen that would not only benefit the new researchers entering the field but also future research endeavors on white mold.Item High-level production of health-beneficial glucoraphanin by multiplex editing of AOP2 gene family in mustard(John Wiley & Sons, 2025) Kumar, Pravin; Bisht, Naveen C.Intake of glucosinolates through the consumption of cruciferous vegetables has been associated with numerous health benefits. In recent decades, glucosinolate glucoraphanin has gained a lot of attention, as its hydrolysis product (sulforaphane) is known to possess numerous health-promoting benefits, including anti-cancer and chemopreventive activities. However, due to the low availability of glucoraphanin in most of the cultivated Brassica crops (except broccoli), there is an increasing interest in many laboratories around the world to manipulate the glucosinolate profile for human benefit. Here, we report the high-level production of health-beneficial glucoraphanin by CRISPR/Cas9 editing of the ALKENYL HYDROXALKYL PRODUCING 2 (BjuAOP2) gene family, displaying distinct expression profiles in the allotetraploid mustard, Brassica juncea. Multiplex editing of five BjuAOP2 homologues, using four gRNAs, provided glucoraphanin accumulation up to 41.60, 75.10, 59.21 and 27.64 μmoles/g dry weight in sprouts, microgreens, seeds and leaves, respectively, of the transgene-free BjuAOP2-edited lines, while providing a significant reduction of the anti-nutritional and goitrogenic alkenyl glucosinolates including progoitrin. The glucoraphanin enhancement in BjuAOP2-edited lines was found to be dose-dependent, wherein loss-of-function mutations in BjuAOP2.A09 and BjuAOP2.B01 homologues had a more prominent effect. The transgene-free BjuAOP2-edited lines were stable for high glucoraphanin and performed at par with the wild-type plants for various seed quality and yield parameters when tested under containment conditions in the field. The development of high-glucoraphanin mustard will help its adoption as a global superfood with health-promoting benefits and as a bioactive source of high-value sulforaphane for industrial production.Item Isopropylmalate synthase regulatory domain removal abolishes feedback regulation at the expense of leucine homeostasis in plants(Oxford University Press, 2025) Varghese, Mohan; Kumar, Roshan; Sharma, Aprajita; Lone, Asif; Gershenzon, Jonathan; Bisht, Naveen C.In the leucine (Leu) biosynthesis pathway, homeostasis is achieved through a feedback regulatory mechanism facilitated by the binding of the end-product Leu at the C-terminal regulatory domain of the first committed enzyme, isopropylmalate synthase (IPMS). In vitro studies have shown that removing the regulatory domain abolishes the feedback regulation on plant IPMS while retaining its catalytic activity. However, the physiological consequences and underlying molecular regulation on Leu flux upon removing the IPMS C-terminal domain remain to be explored in plants. Here, we removed the IPMS C-terminal regulatory domain using a CRISPR/Cas9-based gene editing system and studied the resulting impact on the Leu biosynthesis pathway under in planta conditions. Absence of the IPMS regulatory domain unexpectedly reduced the formation of the end product Leu but increased the levels of Leu pathway intermediates in mustard (Brassica juncea). Additionally, delayed growth was observed when IPMS devoid of the regulatory domain was introduced into IPMS-null mutants of Escherichia coli and Arabidopsis thaliana. Further, a detailed biochemical analysis showed that in the absence of the C-terminal regulatory domain, a Leu pathway intermediate (α-ketoisocaproate) could compete with the native IPMS substrate (2-oxoisovalerate) for the active site. Combining these metabolomic, biochemical, and in planta analyses, we demonstrate that the C-terminal regulatory domain of IPMS is critical for maintaining Leu-Val homeostasis in plants.Item MediatorWeb: a protein-protein interaction network database for the RNA polymerase II Mediator complex(John Wiley & Sons, 2024) Maji, Sourobh; Waseem, Mohd; Sharma, Manish Kumar; Singh, Maninder; Singh, Anamika; Dwivedi, Nidhi; Thakur, Pallabi; Cooper, David G.; Bisht, Naveen C.; Fassler, Jan S.; Subbarao, Naidu; Khurana, Jitendra P.; Bhavesh, Neel Sarovar; Thakur, Jitendra K.The protein-protein interaction (PPI) network of the Mediator complex is very tightly regulated and depends on different developmental and environmental cues. Here, we present an interactive platform for comparative analysis of the Mediator subunits from humans, baker's yeast Saccharomyces cerevisiae, and model plant Arabidopsis thaliana in a user-friendly web-interface database called MediatorWeb. MediatorWeb provides an interface to visualize and analyze the PPI network of Mediator subunits. The database facilitates downloading the untargeted and unweighted network of Mediator complex, its submodules, and individual Mediator subunits to better visualize the importance of individual Mediator subunits or their submodules. Further, MediatorWeb offers network visualization of the Mediator complex and interacting proteins that are functionally annotated. This feature provides clues to understand functions of Mediator subunits in different processes. In an additional tab, MediatorWeb provides quick access to secondary and tertiary structures, as well as residue-level contact information for Mediator subunits in each of the three model organisms. Another useful feature of MediatorWeb is detection of interologs based on orthologous analyses, which can provide clues to understand the functions of Mediator complex in less explored kingdoms. Thus, MediatorWeb and its features can help the user to understand the role of Mediator complex and its subunits in the transcription regulation of gene expression.Item XLG2 and CORI3 function additively to regulate plant defense against the necrotrophic pathogen Sclerotinia sclerotiorum(John Wiley & Sons, 2024) Tiwari, Ruchi; Garg, Kajal; Senthil-Kumar, Muthappa; Bisht, Naveen C.The membrane-bound heterotrimeric G-proteins in plants play a crucial role in defending against a broad range of pathogens. This study emphasizes the significance of Extra-large Gα protein 2 (XLG2), a plant-specific G-protein, in mediating the plant response to Sclerotinia sclerotiorum, which infects over 600 plant species worldwide. Our analysis of Arabidopsis G-protein mutants showed that loss of XLG2 function increased susceptibility to S. sclerotiorum, accompanied by compromised accumulation of jasmonic acid (JA) during pathogen infection. Overexpression of the XLG2 gene in xlg2 mutant plants resulted in higher resistance and increased JA accumulation during S. sclerotiorum infection. Co-immunoprecipitation (co-IP) analysis on S. sclerotiorum infected Col-0 samples, using two different approaches, identified 201 XLG2-interacting proteins. The identified JA-biosynthetic and JA-responsive proteins had compromised transcript expression in the xlg2 mutant during pathogen infection. XLG2 was found to interact physically with a JA-responsive protein, Coronatine induced 1 (CORI3) in Co-IP, and confirmed using split firefly luciferase complementation and bimolecular fluorescent complementation assays. Additionally, genetic analysis revealed an additive effect of XLG2 and CORI3 on resistance against S. sclerotiorum, JA accumulation, and expression of the defense marker genes. Overall, our study reveals two independent pathways involving XLG2 and CORI3 in contributing resistance against S. sclerotiorum.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 Targeted editing of multiple homologues of GTR1 and GTR2 genes provides the ideal low-seed, high-leaf glucosinolate oilseed mustard with uncompromised defence and yield(John Wiley & Sons, 2023) Mann, Avni; Kumari, Juhi; Kumar, Roshan; Kumar, Pawan; Pradhan, Akshay K.; Pental, Deepak; Bisht, Naveen C.Glucosinolate content in the two major oilseed Brassica crops-rapeseed and mustard has been reduced to the globally accepted Canola quality level (<30 μmoles/g of seed dry weight, DW), making the protein-rich seed meal useful as animal feed. However, the overall lower glucosinolate content in seeds as well as in the other parts of such plants renders them vulnerable to biotic challenges. We report CRISPR/Cas9-based editing of glucosinolate transporter (GTR) family genes in mustard (Brassica juncea) to develop ideal lines with the desired low seed glucosinolate content (SGC) while maintaining high glucosinolate levels in the other plant parts for uncompromised plant defence. Use of three gRNAs provided highly efficient and precise editing of four BjuGTR1 and six BjuGTR2 homologues leading to a reduction of SGC from 146.09 μmoles/g DW to as low as 6.21 μmoles/g DW. Detailed analysis of the GTR-edited lines showed higher accumulation and distributional changes of glucosinolates in the foliar parts. However, the changes did not affect the plant defence and yield parameters. When tested against the pathogen Sclerotinia sclerotiorum and generalist pest Spodoptera litura, the GTR-edited lines displayed a defence response at par or better than that of the wild-type line. The GTR-edited lines were equivalent to the wild-type line for various seed yield and seed quality traits. Our results demonstrate that simultaneous editing of multiple GTR1 and GTR2 homologues in mustard can provide the desired low-seed, high-leaf glucosinolate lines with an uncompromised defence and yield.Item The mysterious non-arbuscular mycorrhizal status of Brassicaceae species(John Wiley & Sons, 2023) Sharma, Aprajita; Sinharoy, Senjuti; Bisht, Naveen C.The Brassicaceae family is unique in not fostering functional symbiosis with Arbuscular Mycorrhiza (AM). The family is also special in possessing glucosinolates -, a class of secondary metabolites predominantly functioning for plant defence. We have reviewed what effect the glucosinolates of this non-symbiotic host have on AM or vice-versa . Isothiocyanates, the toxic degradation product of the glucosinolates, particularly the indolic and benzenic glucosinolates, are known to be involved in the inhibition of AM. Interestingly, AM colonization enhances glucosinolate production in two AM-host in the Brassicales family- Moringa oleifera and Tropaeolum spp. PHOSPHATE STARVATION RESPONSE 1 (PHR1), a central transcription factor that controls phosphate starvation response also activates the glucosinolate biosynthesis in AM non-host Arabidopsis thaliana. Recently, the advances in whole-genome sequencing, enabling extensive ecological microbiome studies have helped unravel the Brassicaceae microbiome, identifying new mutualists that compensate for the loss of AM symbiosis, and reporting cues for some influence of glucosinolates on the microbiome structure. We advocate that glucosinolate is an important candidate in determining the mycorrhizal status of Brassicaceae and has played a major role in its symbiosis-defence trade-off. We also identify key open questions in this area that remain to be addressed in the future.Item Defense versus growth trade-offs: insights from glucosinolates and their catabolites(John Wiley & Sons, 2023) Malhotra, Bhanu; Kumar, Pawan; Bisht, Naveen C.Specialized metabolites are a structurally diverse group of naturally occurring compounds that facilitate plant-environment interactions. Their synthesis and maintenance in plants is overall a resource-demanding process that occurs at the expense of growth and reproduction and typically incurs several costs. Evidence emerging on different specialized compounds suggests that they serve multiple auxiliary functions to influence and moderate primary metabolism in plants. These new functionalities enable them to mediate trade-offs from defenses to growth and also to offset their production and maintenance costs in plants. Recent research on glucosinolates (GSLs), which are specialized metabolites of Brassicales, demonstrates their emerging multifunctionalities to fine-tune plant growth and development under variable environments. Herein, we present findings from the septennium on individual GSLs and their catabolites (GHPs) per se, that work as mobile signals within plants to mediate precise regulations of their primary physiological functions. Both GSLs and GHPs calibrate growth-defense trade-off interactions either synergistically or directly when they function as storage compounds, abiotic stress alleviators, and one-to-one regulators of growth pathways in plants. We finally summarize the overall lessons learned from GSLs and GHPs as a model and raise the most pressing questions to address the molecular-genetic intricacies of specialized metabolite-based trade-offs in plants.