Publications of NIPGR Scientists

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Author: search.filters.author.Kumar, RoshanSubject: search.filters.subject.Glucosinolates

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    An LC-MS/MS assay for enzymatic characterization of methylthioalkylmalate synthase (MAMS) involved in glucosinolate biosynthesis
    (Elsevier B.V., 2022) Kumar, Roshan; Reichelt, Michael; Bisht, Naveen C.
    Brassicaceae are blessed with specialized metabolites called glucosinolates (GSLs), which along with their degradation products, are beneficial in agriculture and human health. To date, more than 130 GSL structures have been identified, mostly derived from the amino acid methionine. The biosynthesis of methionine-derived aliphatic GSLs starts with a side-chain elongation step involving a recursive three-step cyclic process that incorporates a new methylene group into the 2-oxo acid to form a series of elongated 2-oxo acids. Methylthioalkylmalate synthase (MAMS) catalyzes the first committed step in the side-chain elongation of methionine-derived GSLs. The substrate specificity of MAMS with different 2-oxo acids determines whether reaction products of a given cycle enter for an additional round of chain elongation or enter into core GSLs structure formation. Multiple MAMS encoding genes are present in the Brassicaceae species and are known to play a central role in shaping the diverse profile of aliphatic GSLs. We recently established a highly sensitive LC-MS/MS-based methodology that quantifies the MAMS activity by estimating the amount of the next intermediate of the pathway, the 2-malate derivatives. Overall, this chapter describes the protocol for the expression, purification, and steady-state kinetic analysis of the recombinant MAMS protein.
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    Molecular basis of the evolution of methylthioalkylmalate synthase and diversity of methionine-derived glucosinolates
    (American Society of Plant Biologists, 2019) Kumar, Roshan; Lee, Soon Goo; Augustine, Rehna; Reichelt, Micheal; Vassão, Daniel G.; Palavalli, Manoj H.; Allen, Aron; Gershenzon, Jonathan; Jez, Joseph M.; Bisht, Naveen C.
    Methylthioalkylmalate synthase catalyzes the committed step in the side-chain elongation of methionine-derived aliphatic glucosinolates and likely evolved from the isopropylmalate synthases of leucine biosynthesis. The globally cultivated Brassica species possess diverse aliphatic glucosinolates important for plant defense and animal nutrition; however, the molecular basis for the evolution of methylthioalkylmalate synthase and its generation of natural product diversity in Brassica is poorly understood. Here we show that Brassica genomes encode multiple methylthioalkylmalate synthase that have differences in expression profiles and 2-oxo substrate preference that account for diversity of aliphatic glucosinolates across Brassica accessions. The 2.1 Å resolution x-ray crystal structure of B. juncea methylthioalkylmalate synthase identifies key active site residues responsible for controlling specificity for different 2-oxo substrates and the determinants of side-chain length in aliphatic glucosinolates. Overall, these results provide the evolutionary and biochemical foundation for diversification of glucosinolates profiles across globally-cultivated Brassica species, which could be used with ongoing breeding strategies towards manipulation of beneficial glucosinolates compounds for animal health and plant protection.
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    Translational genomics in Brassica crops: challenges, progress, and future prospects
    (Springer, 2014) Augustine, Rehna; Arya, Gulab C.; Nambiar, Deepti M.; Kumar, Roshan; Bisht, Naveen C.
    The last two decades have been a period of rapid advancement in our understanding of plant biology and its related developmental processes. This advancement has been facilitated by the adoption of plant models for most of the economically important plant families, as well as the development of enriched genetic, genomic, transcriptomic and metabolomic resources. In recent years, sequencing projects on major crops have further enhanced our understanding of their genomic structure, evolution, gene functions, and, most importantly, this knowledge has been utilized for crop improvement. The Brassicaceae family contains several important research and agricultural species, including the model plant Arabidopsis thaliana and economically important Brassica crops that are of great importance to human health and agriculture. Exploiting heterosis for yield enhancement, increasing tolerance against biotic and abiotic factors, and improving nutritional value remain the priorities in Brassica crop improvement. This review summarizes the potential of recently adopted genetic and genomic resources, as well as the basic knowledge obtained from studying the closest model plant A. thaliana, to accelerate the crop improvement programs in Brassica crops.