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Item High-level production of health-beneficial glucoraphanin by multiplex editing of AOP2 gene family in mustard(John Wiley & Sons, 2025) Kumar, Pravin; Bisht, Naveen C.Intake of glucosinolates through the consumption of cruciferous vegetables has been associated with numerous health benefits. In recent decades, glucosinolate glucoraphanin has gained a lot of attention, as its hydrolysis product (sulforaphane) is known to possess numerous health-promoting benefits, including anti-cancer and chemopreventive activities. However, due to the low availability of glucoraphanin in most of the cultivated Brassica crops (except broccoli), there is an increasing interest in many laboratories around the world to manipulate the glucosinolate profile for human benefit. Here, we report the high-level production of health-beneficial glucoraphanin by CRISPR/Cas9 editing of the ALKENYL HYDROXALKYL PRODUCING 2 (BjuAOP2) gene family, displaying distinct expression profiles in the allotetraploid mustard, Brassica juncea. Multiplex editing of five BjuAOP2 homologues, using four gRNAs, provided glucoraphanin accumulation up to 41.60, 75.10, 59.21 and 27.64 μmoles/g dry weight in sprouts, microgreens, seeds and leaves, respectively, of the transgene-free BjuAOP2-edited lines, while providing a significant reduction of the anti-nutritional and goitrogenic alkenyl glucosinolates including progoitrin. The glucoraphanin enhancement in BjuAOP2-edited lines was found to be dose-dependent, wherein loss-of-function mutations in BjuAOP2.A09 and BjuAOP2.B01 homologues had a more prominent effect. The transgene-free BjuAOP2-edited lines were stable for high glucoraphanin and performed at par with the wild-type plants for various seed quality and yield parameters when tested under containment conditions in the field. The development of high-glucoraphanin mustard will help its adoption as a global superfood with health-promoting benefits and as a bioactive source of high-value sulforaphane for industrial production.Item Extra-large G-proteins influence plant response to Sclerotinia sclerotiorum by regulating glucosinolate metabolism in Brassica juncea(John Wiley & Sons, 2021) Tiwari, Ruchi; Kaur, Jagreet; Bisht, Naveen C.Heterotrimeric G-proteins are one of the highly conserved signal transducers across phyla. Despite the obvious importance of G-proteins in controlling various plant growth and environmental responses, there is no information describing the regulatory complexity of G-protein networks during pathogen response in a polyploid crop. Here, we investigated the role of extra-large G-proteins (XLGs) in the oilseed crop Brassica juncea, which has inherent susceptibility to the necrotrophic fungal pathogen Sclerotinia sclerotiorum. The allotetraploid B. juncea genome contains multiple homologs of three XLG genes (two BjuXLG1, five BjuXLG2, and three BjuXLG3), sharing a high level of sequence identity, gene structure organization, and phylogenetic relationship with the progenitors’ orthologs. Quantitative reverse transcription PCR analysis revealed that BjuXLGs have retained distinct expression patterns across plant developmental stages and on S. sclerotiorum infection. To determine the role of BjuXLG genes in the B. juncea defence response against S. sclerotiorum, RNAi-based suppression was performed. Disease progression analysis showed more rapid lesion expansion and fungal accumulation in BjuXLG-RNAi lines compared to the vector control plants, wherein suppression of BjuXLG3 homologs displayed more compromised defence response at the later time point. Knocking down BjuXLGs caused impairment of the host resistance mechanism to S. sclerotiorum, as indicated by reduced expression of defence marker genes PDF1.2 and WRKY33 on pathogen infection. Furthermore, BjuXLG-RNAi lines showed reduced accumulation of leaf glucosinolates on S. sclerotiorum infection, wherein aliphatic glucosinolates were significantly compromised. Overall, our data suggest that B. juncea XLG genes are important signalling nodes modulating the host defence pathways in response to this necrotrophic pathogen.Item A complex interplay of Gβ and Gγ proteins regulates plant growth and defence traits in the allotetraploid Brassica juncea(Springer Nature Publishing AG, 2021) Arya, Gulab Chand; Tiwari, Ruchi; Bisht, Naveen C.Plant heterotrimeric G-proteins regulate a wide range of responses despite their limited repertoire of core components. The roles and functional interactions between different G-protein subunits are quite perplexing, which get further complicated with polyploidy. Here, we show that the allotetraploid Brassica juncea comprises multiple homologs of G-protein genes, encoding six BjuGβ and ten highly divergent BjuGγ subunit proteins, later being classified into type-A1, type-A2 and type-C Gγ proteins. The encoded BjuGβ and BjuGγ proteins shared close evolutionary relationship and have retained distinct spatio-temporal expression patterns during plant developmental stages and in response to the necrotrophic pathogen, Sclerotinia sclerotiorum. RNAi based suppression of BjuGβ and BjuGγ genes suggested functional overlap and selectivity of BjuGβs with three distinct BjuGγ type subunits, to regulate plant height (BjuGβγA2 and BjuGβγC), seed weight (BjuGβGγA1 and BjuGβGγC), silique size (BjuGβGγC) and pathogen response (BjuGβGγA1 and BjuGβGγC). Further, the triplicated BjuGβ genes, formed due to Brassica specific whole-genome-triplication event, showed differential involvement during pathogen response, wherein overexpression of BjuGβ2 displayed higher resistance to Sclerotinia infection. Taken together, our study demonstrates that multiple BjuGβ and BjuGγ proteins have retained distinct spatio-temporal expression and functional selectivity to regulate specific plant growth and defence traits in the oilseed B. juncea.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 A chromosome-scale assembly of allotetraploid Brassica juncea (AABB) elucidates comparative architecture of the A and B genomes(John Wiley & Sons, 2021) Paritosh, Kumar; Yadava, Satish Kumar; Singh, Priyansha; Bhayana, Latika; Mukhopadhyay, Arundhati; Gupta, Vibha; Bisht, Naveen C.; Zhang, Jianwei; Kudrna, David A; Copetti, Dario; Wing, Rod A; Reddy, Vijay Bhaskar; Pradhan, Akshay Kumar; Pental, DeepakBrassica juncea (AABB), commonly referred to as mustard, is a natural allopolyploid of two diploid species – B. rapa (AA) and B. nigra (BB). We report a highly contiguous genome assembly of an oleiferous type of B. juncea variety Varuna, an archetypical Indian gene pool line of mustard, with ~100x PacBio single‐molecule real‐time (SMRT) long‐reads providing contigs with an N50 value of >5Mb. Contigs were corrected for the misassemblies and scaffolded with BioNano optical mapping. We also assembled a draft genome of B. nigra (BB) variety Sangam using Illumina short‐read sequencing and Oxford Nanopore long‐reads and used it to validate the assembly of the B genome of B. juncea. Two different linkage maps of B. juncea, containing a large number of genotyping‐by‐sequencing markers were developed and used to anchor scaffolds/contigs to the 18 linkage groups of the species. The resulting chromosome‐scale assembly of B. juncea Varuna is a significant improvement over the previous draft assembly of B. juncea Tumida, a vegetable type of mustard. The assembled genome was characterized for transposons, centromeric repeats, gene content, and gene block associations. In comparison to the A genome, the B genome contains a significantly higher content of LTR/Gypsy retrotransposons, distinct centromeric repeats, and a large number of B. nigra specific gene clusters that break the gene collinearity between the A and the B genomes. The B. juncea Varuna assembly will be of major value to the breeding work on oleiferous types of mustard that are grown extensively in south Asia and elsewhere.Item Heterotrimeric Gα subunit regulates plant architecture, organ size and seed weight in the oilseed Brassica juncea(Springer Nature Publishing AG, 2020) Kumar, Roshan; Bisht, Naveen C.Key message Two BjuGα proteins exhibit conserved GTP-binding and GTP-hydrolysis activities, and function in maintaining overall plant architecture and controlling multiple yield-related traits in the oilseed Brassica juncea. Abstract Heterotrimeric G-protein (Gα, Gβ and Gγ) are key signal transducers, well characterized in model plants Arabidopsis and rice. However, our knowledge about the roles played by G-proteins in regulating various growth and developmental traits in polyploid crops, having a complex G-protein signalling network, is quite sparse. In the present study, two Gα encoding genes (BjuA.Gα1 and BjuB.Gα1) were isolated from the allotetraploid Brassica juncea, a globally cultivated oilseed crop of the Brassicaceae family. BjuGα1 genes share a close evolutionary relationship, and the encoded proteins exhibit highly conserved G-protein activities while showing expression diferentiation, wherein BjuA.Gα1 was the highly abundant transcript during plant growth and developmental stages. RNAi based suppression of BjuGα1 displayed compromised efects on most of the tested vegetative and reproductive parameters, particularly plant height (32–58%), fower and siliques dimensions, and seed weight (11–13%). Further, over-expression of a constitutively active Gα, lacking the GTPase activity, produced plants with increased height, organ size and seed weight (7–25%), without altering seed quality traits like fatty acid composition, glucosinolates, oil and protein contents. Our study demonstrates that BjuGα1 proteins control overall plant architecture and multiple yield-related traits in the oilseed B. juncea, suggesting that BjuGα1 could be a promising target for crop improvement.Item Ectopic overexpression of cytosolic ascorbate peroxidase gene (Apx1) improves salinity stress tolerance in Brassica juncea by strengthening antioxidative defense mechanism(Springer Nature, 2020) Saxena, Saurabh C.; Salvi, Prafull; Kamble, Nitin Uttam; Joshi, Pankaj K.; Majee, Manoj; Arora, SandeepSalinity stress is considered to be a key constrain that reduces the crop productivity by impairing plant growth and development. During salt stress condition, an underlying mechanism for reduction in crop yield is increase in ROS level that can potentially harm cellular macromolecules, leading to disruption of essential physiological and biochemical processes. Plants possess a complex antioxidative defense machinery for scavenging these ROS. Ascorbate peroxidase (APX, E.C. 1.11.1.11), is a crucial antioxidant enzyme involved in Ascorbate–Glutathione pathway that primarily detoxifies the negative impact of H2O2 in cell. The efficient scavenging of H2O2 is a prerequisite for enhanced tolerance to salinity stress. Here, we have inspected whether over-expression of APX could provide protection against salinity stress. Cytosolic ascorbate peroxidase (Apx1) gene, isolated from Arabidopsis thaliana, was chosen as the candidate gene for strengthening the antioxidative defense system of Brassica juncea. Physiological parameters were employed to analyze the growth status of transgenic plants. Leaf disc assay was done to evaluate the salinity stress tolerance potential of transgenic plants, using several physiological and biochemical parameters. Under salinity stress, the transgenic plants performed well as compared to their non-transgenic counterparts; as revealed through greater proline accumulation, increased chlorophyll stability index, lower chlorophyll a/b ratio, and higher antioxidative enzyme activities. Further, the lower H2O2 levels were well correlated with lesser membrane damage as measured through MDA content. Collectively, our results clearly depicted that ectopic overexpression of AtApx1 gene was able to confer salinity stress tolerance by strengthening the antioxidative defense system in B. juncea.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 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.