Publications of NIPGR Scientists

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Author: search.filters.author.Varghese, MohanStart date: 2025

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    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.
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    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.
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    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.