Browsing by Author "Nambiar, Deepti M."

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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.
GTR1 and GTR2 transporters differentially regulate tissue-specific glucosinolate contents and defence responses in the oilseed crop Brassica juncea
(John Wiley & Sons, 2021) Nambiar, Deepti M.; Kumari, Juhi; Augustine, Rehna; Kumar, Pawan; Bajpai, Prabodh K.; Bisht, Naveen C.
GTR1 and GTR2 transporters are components of the source to sink translocation network of glucosinolates, which are major defence metabolites in the Brassicaceae. These transporters can be genetically manipulated for reduction of seed-glucosinolates without inhibiting glucosinolate biosynthesis, thereby maintaining the inherent defence potential of plants. However, the different roles of GTRs in influencing tissue-specific distribution of glucosinolates in agriculturally important Brassica crops are yet unknown. Here, we report functional characterization of two groups of glucosinolate transporters (GTR1 and GTR2) from Brassica juncea based on gene expression data, biochemical analysis, gene-complementation studies in GTR-deficient mutants and RNAi-based knockdown followed by insect feeding experiments. Although both GTRs showed ubiquitous expression patterns and broad substrate specificity, the single-gene knockdown lines displayed different phenotypes. The GTR2-knockdown plants showed a significant reduction of glucosinolates in seeds and a higher accumulation in leaves and pods, while the GTR1-knockdown plants displayed a smaller reduction of glucosinolates in seeds and significantly lower glucosinolate levels in leaves. Consequently, knockdown of GTR2 resulted in higher resistance towards the generalist pest, Spodoptera litura. Overall, our study highlights the distinctive roles of B. juncea GTRs in tissue-specific accumulation of glucosinolates and the potential for manipulating GTR2 for enhanced nutrition and plant defence.
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.