Institutional Publications
Permanent URI for this collectionhttps://ndkr-library.nipgr.ac.in/handle/123456789/11
Browse
10 results
Search Results
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 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.Item A comprehensive Vis-NIRS equation for rapid quantification of seed glucosinolate content and composition across diverse Brassica oilseed chemotypes(Elsevier B.V., 2021) Gohain, Bornali; Kumar, Pawan; Malhotra, Bhanu; Augustine, Rehna; Pradhan, Akshay K.; Bisht, Naveen C.The globally cultivated Brassica crops contain high deliverable concentrations of health-promoting glucosinolates. Development of a Visible-Near InfraRed Spectroscopy (Vis-NIRS) calibration to profile different glucosinolate components from 641 diverse Brassica juncea chemotypes was attempted in this study. Principal component analysis of HPLC-determined glucosinolates established the distinctiveness of four B. juncea populations used. Subsequently, modified partial least square regression based population-specific and combined Vis-NIRS models were developed, wherein the combined model exhibited higher coefficient of determination (R2; 0.81–0.97) for eight glucosinolates and higher ratio of prediction determination (RPD; 2.42–5.35) for seven glucosinolates in B. juncea populations. Furthermore, range error ratio (RER > 4) for twelve and RER > 10 for eight glucosinolates make the combined model acceptable for screening and quality control. The model also provided excellent prediction for aliphatic glucosinolates in four oilseed Brassica species. Overall, our work highlights the potential of Vis-NIR spectroscopy in estimating glucosinolate content in the economically important Brassica oilseeds.Item Editorial: Glucosinolates: Regulation of biosynthesis and hydrolysis(Frontiers Media S.A., 2020) Malhotra, Bhanu; Bisht, Naveen C.The members of the Brassicaceae family possess a class of sulfur and nitrogen containing specialized metabolites glucosinolates (GSLs), that are important for plant defense and animal nutrition. Glucosinolates are both constitutive and inducible, and their accumulation is controlled by environmental, molecular, and genetic levels. Much of the information about the glucosinolate biosynthesis, transport, hydrolysis, turnover, crosstalk amongst biosynthesis and hormonal pathways, and mechanisms controlling glucosinolate accumulation and metabolism during abiotic and biotic stresses has been derived from the model plant Arabidopsis thaliana (Figure 1). However, there are still lacunae in fully understanding these regulatory processes in crop species. Thus, the purpose of this Research Topic is to decipher several aspects under the glucosinolate regulatory mechanisms, in view of which a total of eight research articles have been published.Item A cell suspension based uptake method to study high affinity glucosinolate transporters(BioMed Central Ltd, 2020) Nambiar, Deepti M.; Kumari, Juhi; Arya, Gulab C.; Singh, Amarjeet K.; Bisht, Naveen C.Background: Glucosinolates are an important class of secondary metabolites characteristic to the order Brassicales. They are known to play a major role in plant defense and from the human perspective, can be anticarcinogenic or antinutritive. GTRs are plasma-membrane localized high afnity glucosinolate transporters, which are important components of the source (leaf ) to sink (seed) translocation of intact glucosinolates in members of Brassicaceae family. GTRs are identifed as major candidates for Brassica crop improvement, thus dictating a need for their functional characterization. However, currently there are limitations in availability of heterologous assay systems for functional characterization of plant secondary metabolite transporters. To date, the animal-based Xenopus oocyte system is the best established heterologous system for functional characterization of these transporters. Inherent biochemical and physiological attributes unique to the plant membranes necessitate the need for developing plant-based transporters assay systems as well. Methods: In this study, Agrobacterium mediated transformation was used to develop GTR expressing cotton cell lines (CCL-1) for functional characterization of the Arabidopsis high afnity glucosinolate transporters, AtGTR1 and AtGTR2. Following sub-cellular localization of AtGTRs, we standardized the glucosinolate uptake assays using cell suspension cultures of AtGTR expressing CCL-1 its requirement of pH, salt, and time based glucosinolate uptake. Using the GTR expressing CCL-1, we subsequently performed kinetic analysis of AtGTR1 and AtGTR2 for diferent glucosinolate substrates, sinigrin, gluconapin and sinalbin. Results: Several clones expressing each of AtGTR1 and AtGTR2 were obtained showing high level of GTR expression and were maintained through regular sub-culturing. Both AtGTR1 and AtGTR2 are predominantly plasma-localized proteins when overexpressed in CCL-1 cells. Uptake assays were standardized, suggesting that glucosinolate uptake of GTR expressing CCL-1 is robust within the physiological pH range 5–6, and at lower concentration of nitrate salts. GTR expressing CCL-1 cells show increasing glucosinolate accumulation in time course experiment. Kinetic studies over a wide glucosinolate concentrations (10–800 µM) revealed that our novel assay system displayed robust GTR-mediated uptake of diferent glucosinolates and unambiguously helps elucidate the saturable kinetics of GTRs. Our system confrms the high afnity of AtGTRs for both aliphatic and aromatic glucosinolates. Conclusion: The transporter assay system described in this study holds potential for studying sub-functionalization amongst GTR homologs present across Brassicaceae family. The fast growing CCL-1 cells, confer the benefts of an in vitro system for quick assays and is plant based thus enabling optimal expression without sequence modifcations. The efcient functioning of the GTR transporters in the heterologous CCL-1 opens the possibility of using this plant cell suspension system for functional characterization of other metabolite transporters.Item 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.Item Targeted silencing of genes in polyploids: lessons learned from Brassica juncea-glucosinolate system(Springer Nature, 2019) Augustine, Rehna; Bisht, Naveen C.Key message Intron-spliced hairpin RNAi construct targeting the exonic region of BjuMYB28 driven by the native promoter is the best suited strategy for developing viable low glucosinolate lines in polyploid Brassica juncea. Abstract Targeted silencing of specific homolog(s) of a multigene family in polyploids through RNA interference (RNAi) is a challenging effort. Indian oilseed mustard (Brassica juncea), a natural allotetraploid, is expected to have 4–6 copies of every Arabidopsis gene ortholog. In the current study, we have attempted to establish the best gene silencing system suitable for BjuMYB28, a transcription factor gene, with the objective of developing low seed glucosinolate lines in B. juncea. After comparing multiple combinations of BjuMYB28 gene homologs, promoters, target regions (exon and 3′ UTR) and silencing strategies (RNAi and antisense), we suggest that the intron-spliced hairpin RNAi construct targeting the specific exonic region of the BjuMYB28 gene under the control of native promoter, whose peak activity synchronises with the highest glucosinolate accumulation phase of the plant, is the best suited strategy for developing viable low glucosinolate lines in polyploid B. juncea.Item Regulation of glucosinolate metabolism: From model plant Arabidopsis thaliana to Brassica crops(Springer, 2017) Augustine, Rehna; Bisht, Naveen C.Brassicaceae are blessed with secondary metabolites called glucosinolates which form the defense arsenal of these plants. Glucosinolates and its degradation products are also proved to be beneficial in agriculture and human health even though some are known to be detrimental. The type of glucosinolates and its content displays huge diversity across different species. The glucosinolate diversity is primarily genetically controlled. The profile of glucosinolates also varies depending on the growth stages and external environment of the plant. The environmental factors include type of pest/pathogen attack, nutrient status of the plant, and other abiotic stress factors. The glucosinolate pathway is also linked to other major metabolic and signaling pathways resulting in a complex mechanism of regulation. Even though the regulatory mechanism is not completely understood, the current chapter integrates the knowledge available from the model plant Arabidopsis and related Brassica crops.Item Biotic elicitors and mechanical damage modulate glucosinolate accumulation by co-ordinated interplay of glucosinolate biosynthesis regulators in polyploid Brassica juncea(Elsevier B.V., 2015) Augustine, Rehna; Bisht, Naveen C.Glucosinolates are nitrogen and sulfur containing secondary metabolites found mainly in the Brassicaceae. They function as plant defense compounds against a broad spectrum of pathogens and pests. Since these molecules form part of the plant defense mechanism, glucosinolate biosynthesis may be modulated by environmental signals leading to activation of a biological stress response. In the current study, we have mimicked such conditions by exogenously applying biotic elicitors such as methyl jasmonate, salicylic acid, glucose and mechanical injury in Brassica juncea seedling over a time course experiment. We found that total glucosinolates over-accumulated under these stress conditions with maximum accumulation observed 24h post treatment. Indole glucosinolates like 1-methoxy-indol-3-ylmethyl and its precursor indol-3-methyl glucosinolates showed a more significant induction compared to aliphatic glucosinolates thereby suggesting a prominent role of indole glucosinolates during plant defense response in B. juncea seedlings. In contrast, the higher amounts of aliphatic glucosinolates were less regulated by the tested biotic elicitors in B. juncea. Expression profiling of multiple homologs of key transcriptional regulators of glucosinolate biosynthesis further showed that a complex interplay of these regulators exists in polyploid B. juncea where they exert co-ordinated and overlapping effects toward altering glucosinolate accumulation. This study has a significant role toward understanding and augmenting plant defense mechanisms in B. juncea, a globally important oilseed crop of genus Brassica.Item 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.