Publications of NIPGR Scientists
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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 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.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 Heterotic patterns of primary and secondary metabolites in the oilseed crop Brassica juncea(Springer Nature, 2019) Bajpai, Prabodh K.; Reichelt, Michael; Augustine, Rehna; Gershenzon, Jonathan; Bisht, Naveen C.Heterosis refers to the superior performance of F1 hybrids over their respective parental inbred lines. Although the genetic and expression basis of heterosis have been previously investigated, the metabolic basis for this phenomenon is poorly understood. In a preliminary morphological study in Brassica juncea, we observed significant heterosis at the 50% flowering stage, wherein both the growth and reproduction of F1 reciprocal hybrids were greater than that of their parents. To identify the possible metabolic causes or consequences of this heterosis, we carried out targeted LC-MS analysis of 48 primary (amino acids and sugars) and secondary metabolites (phytohormones, glucosinolates, flavonoids, and phenolic esters) in five developmental tissues at 50% flowering in hybrids and inbred parents. Principal component analysis (PCA) of metabolites clearly separated inbred lines from their hybrids, particularly in the bud tissues. In general, secondary metabolites displayed more negative heterosis values in comparison to primary metabolites. The tested primary and secondary metabolites displayed both additive and non-additive modes of inheritance in F1 hybrids, wherein the number of metabolites showing an additive mode of inheritance were higher in buds and siliques (52.77–97.14%) compared to leaf tissues (47.37–80%). Partial least regression (PLS) analysis further showed that primary metabolites, in general, displayed higher association with morphological parameters in F1 hybrids. Overall, our results are consistent with a resource-cost model for heterosis in B. juncea, where metabolite allocation in hybrids appears to favor growth, at the expense of secondary metabolism.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 Feeding behaviour of generalist pests on Brassica juncea: implication for manipulation of glucosinolate biosynthesis pathway for enhanced resistance(John Wiley & Sons, 2017) Kumar, Pawan; Augustine, Rehna; Singh, Amarjeet Kumar; Bisht, Naveen C.Differential accumulation of plant defense metabolites has been suggested to have important ecological consequence in the context of plant-insect interactions. Feeding of generalist pests on Brassica juncea showed a distinct pattern with selective exclusion of leaf margins which are high in glucosinolates. Molecular basis of this differential accumulation of glucosinolates could be explained based on differential expression profile of BjuMYB28 homologs, the major biosynthetic regulators of aliphatic glucosinolates, as evident from quantitative real-time PCR and promoter:GUS fusion studies in allotetraploid B. juncea. Constitutive over-expression of selected BjuMYB28 homologs enhanced accumulation of aliphatic glucosinolates in B. juncea. Performance of two generalist pests, Helicoverpa armigera and Spodoptera litura larvae on transgenic B. juncea plants were poor compared to wild-type plants in a no-choice experiment. Correlation coefficient analysis suggested that weight gain of H. armigera larvae was negatively correlated with gluconapin (GNA) and glucobrassicanapin (GBN) whereas that of S. litura larvae was negatively correlated with GNA, GBN and sinigrin (SIN). Our study explains the significance and possible molecular basis of differential distribution of glucosinolates in B. juncea leaves and shows the potential of over-expressing BjuMYB28 for enhanced resistance of Brassica crops against the tested generalist pests.Item Arsenic affects the production of glucosinolate, thiol and phytochemical compounds: A comparison of two Brassica cultivars(Elsevier B.V., 2017) Pandey, Chandana; Augustine, Rehna; Panthri, Medha; Zia, Ismat; Bisht, Naveen C.; Gupta, MeetuArsenic (As), a non-essential metalloid, severely affects the normal functioning of plants, animals and humans. Plants play a crucial role in metabolic, physiological and numerous detoxification mechanisms to cope up with As induced stress. This study aimed to examine the differential response in two Brassica juncea cultivars, Varuna and Pusa Jagannath (PJn) exposed to different doses of As (50, 150, 300 μM) for 48 h duration. Change in morphological traits, concentration of individual as well as total GSL, sulfur related thiol proteins, sulfur content, and phytochemicals were analyzed in both cultivars. Accumulation pattern of As showed dose dependent accumulation in both the cultivars, being more in PJn. Our finding revealed that both cultivars were tolerant at low concentrations of As, while at higher concentration Varuna excelled over PJn. The increased tolerance of Varuna cultivar exposed to 150 and 300 μM concentration of As, correlated with its increased thiol related proteins, sulfur content and phytochemicals, which serves as defence strategy in the plant against oxidative stress. Differential pattern of total as well as individual GSLs content was observed in both Varuna and PJn cultivars. Varuna cultivar showed higher level of total and aliphatic GSLs, which serves as defence compound with other detoxification machineries to combat As stress. Our findings provide foundation for developing metalloid tolerant crops by analyzing the role of different genes involved in GSL mechanism and signaling pathways in different organs of plant.Item Class-specific evolution and transcriptional differentiation of 14-3-3 family members in mesohexaploid Brassica rapa(Frontiers Media S.A., 2016) Chandna, Ruby; Augustine, Rehna; Kanchupati, Praveena; Kumar, Roshan; Kumar, Pawan; Arya, Gulab C.; Bisht, Naveen C.14-3-3s are highly conserved, multigene family proteins that have been implicated in modulating various biological processes. The presence of inherent polyploidy and genome complexity has limited the identification and characterization of 14-3-3 proteins from globally important Brassica crops. Through data mining of Brassica rapa, the model Brassica genome, we identified 21 members encoding 14-3-3 proteins namely, BraA.GRF14.a to BraA.GRF14.u. Phylogenetic analysis indicated that B. rapa contains both ε (epsilon) and non-ε 14-3-3 isoforms, having distinct intron-exon structural organization patterns. The non-ε isoforms showed lower divergence rate (Ks < 0.45) compared to ε protein isoforms (Ks > 0.48), suggesting class-specific divergence pattern. Synteny analysis revealed that mesohexaploid B. rapa genome has retained 1–5 orthologs of each Arabidopsis 14-3-3 gene, interspersed across its three fragmented sub-genomes. qRT-PCR analysis showed that 14 of the 21 BraA.GRF14 were expressed, wherein a higher abundance of non-ε transcripts was observed compared to the ε genes, indicating class-specific transcriptional bias. The BraA.GRF14 genes showed distinct expression pattern during plant developmental stages and in response to abiotic stress, phytohormone treatments, and nutrient deprivation conditions. Together, the distinct expression pattern and differential regulation of BraA.GRF14 genes indicated the occurrence of functional divergence of B. rapa 14-3-3 proteins during plant development and stress responses.Item Genomic origin, expression differentiation and regulation of multiple genes encoding CYP83A1, a key enzyme for core glucosinolate biosynthesis, from the allotetraploid Brassica juncea(Springer, 2015) Meenu; Augustine, Rehna; Majee, Manoj; Pradhan, Akshay K.; Bisht, Naveen C.MAIN CONCLUSION: The multiple BjuCYP83A1 genes formed as a result of polyploidy have retained cell-, tissue-, and condition-specific transcriptional sub-functionalization to control the complex aliphatic glucosinolates biosynthesis in the allotetraploid Brassica juncea. Glucosinolates along with their breakdown products are associated with diverse roles in plant metabolism, plant defense and animal nutrition. CYP83A1 is a key enzyme that oxidizes aliphatic aldoximes to aci-nitro compounds in the complex aliphatic glucosinolate biosynthetic pathway. In this study, we reported the isolation of four CYP83A1 genes named BjuCYP83A1-1, -2, -3, and -4 from allotetraploid Brassica juncea (AABB genome), an economically important oilseed crop of Brassica genus. The deduced BjuCYP83A1 proteins shared 85.7-88.4 % of sequence identity with A. thaliana AtCYP83A1 and 84.2-95.8 % among themselves. Phylogenetic and divergence analysis revealed that the four BjuCYP83A1 proteins are evolutionary conserved and have evolved via duplication and hybridization of two relatively simpler diploid Brassica genomes namely B. rapa (AA genome) and B. nigra (BB genome), and have retained high level of sequence conservation following allopolyploidization. Ectopic over-expression of BjuCYP83A1-1 in A. thaliana showed that it is involved mainly in the synthesis of C4 aliphatic glucosinolates. Detailed expression analysis using real-time qRT-PCR in B. juncea and PromoterBjuCYP83A1-GUS lines in A. thaliana confirmed that the four BjuCYP83A1 genes have retained ubiquitous, overlapping but distinct expression profiles in different tissue and cell types of B. juncea, and in response to various elicitor treatments and environmental conditions. Taken together, this study demonstrated that transcriptional sub-functionalization and coordinated roles of multiple BjuCYP83A1 genes control the biosynthesis of aliphatic glucosinolates in the allotetraploid B. juncea, and provide a framework for metabolic engineering of aliphatic glucosinolates in economically important Brassica species.