Browsing by Author "Bisht, Naveen C."

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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, Meetu
Arsenic (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.
Biofortification of oilseed Brassica juncea with the anti-cancer compound glucoraphanin by suppressing GSL-ALK gene family
(Nature Publishing Group, 2015) Augustine, Rehna; Bisht, Naveen C.
Glucosinolates are amino acids derived secondary metabolites, invariably present in Brassicales, which have huge health and agricultural benefits. Sulphoraphane, the breakdown product of glucosinolate glucoraphanin is known to posses anti-cancer properties. AOP (2-oxoglutarate-dependent dioxygenases) or GSL-ALK enzyme catalyzes the conversion of desirable glucoraphanin to deleterious gluconapin and progoitrin, which are present in very high amounts in most of the cultivable Brassica species including Brassica juncea. In this study we showed that B. juncea encodes four functional homologs of GSL-ALK gene and constitutive silencing of GSL-ALK homologs resulted in accumulation of glucoraphanin up to 43.11 μmoles g(-1) DW in the seeds with a concomitant reduction in the anti-nutritional glucosinolates. Glucoraphanin content was found remarkably high in leaves as well as sprouts of the transgenic lines. Transcript quantification of high glucoraphanin lines confirmed significant down-regulation of GSL-ALK homologs. Growth and other seed quality parameters of the transgenic lines did not show drastic difference, compared to the untransformed control. High glucoraphanin lines also showed higher resistance towards stem rot pathogen Sclerotinia sclerotiorum. Our results suggest that metabolic engineering of GSL-ALK has huge potential for enriching glucoraphanin content, and improve the oil quality and vegetable value of Brassica crops.
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.
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.
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, Deepak
Brassica 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.
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.
Cloning, in silico characterization and prediction of three dimensional structure of SbDof1, SbDof19, SbDof23 and SbDof24 proteins from sorghum [Sorghum bicolor (L.) Moench]
(Springer, 2013) Kushwaha, Hariom; Gupta, Shubhra; Singh, Vinay Kumar; Bisht, Naveen C.; Sarangi, Bijaya K.; Yadav, Dinesh
In the present study, four full-length Dof (DNA-binding with one finger) genes from Sorghum bicolor namely SbDof1, SbDof19, SbDof23, and SbDof24 were PCR amplified, gel eluted, cloned, and sequenced (accession number HQ540084, HQ540085, HQ540086, and HQ540087, respectively). These sequences were further characterized in silico by subjecting them to homology search, multiple sequence alignment, phylogenetic tree construction, and protein functional analysis, revealing their identity to Dof like proteins. Phylogenetic analysis of cloned SbDof genes along with other reported Dof proteins revealed existence of two major groups A and B, while group A was further bifurcated into two sub-groups (viz., I and II). Motif scan analysis of SbDof proteins revealed the presence of glycine- and alanine-rich profiles in SbDof1, while proline-rich profile was observed in SbDof23. Asparagines, methionine, and serine-rich profiles were common in case of both SbDof19 and SbDof24 proteins. The three dimensional structures of SbDof proteins were predicted by I-TASSER server based on multiple threading method. The modeled structures were refined by energy minimization and their stereo chemical qualities were validated by PROCHECK and QMEAN server indicating the acceptability of the predicted models. The final models were submitted to PMDB database with assigned PMDB IDs, i.e., PM0077395, PM0077396, PM0077397, PM0077398, and PM0076448 for SbDof1, SbDof19, SbDof23, SbDof24, and Dof domain, respectively. Based on gene ontology (GO) terms in I-TASSER server putative functions of modeled SbDof proteins were also predicted.
Comparative mapping of Brassica juncea and Arabidopsis thaliana using intron polymorphism (IP) markers: homeologous relationships, diversification and evolution of the A, B and C Brassica genomes
(BioMed Central, 2008) Panjabi, Priya; Jagannath, Arun; Bisht, Naveen C.; Padmaja, K Lakshmi; Sharma, Sarita; Gupta, Vibha; Pradhan, Akshay K; Pental, Deepak
Background: Extensive mapping efforts are currently underway for the establishment of comparative genomics between the model plant, Arabidopsis thaliana and various Brassica species. Most of these studies have deployed RFLP markers, the use of which is a laborious and time-consuming process. We therefore tested the efficacy of PCR-based Intron Polymorphism (IP) markers to analyze genome-wide synteny between the oilseed crop, Brassica juncea (AABB genome) and A. thaliana and analyzed the arrangement of 24 (previously described) genomic block segments in the A, B and C Brassica genomes to study the evolutionary events contributing to karyotype variations in the three diploid Brassica genomes. Results: IP markers were highly efficient and generated easily discernable polymorphisms on agarose gels. Comparative analysis of the segmental organization of the A and B genomes of B. juncea (present study) with the A and B genomes of B. napus and B. nigra respectively (described earlier), revealed a high degree of colinearity suggesting minimal macro-level changes after polyploidization. The ancestral block arrangements that remained unaltered during evolution and the karyotype rearrangements that originated in the Oleracea lineage after its divergence from Rapa lineage were identified. Genomic rearrangements leading to the gain or loss of one chromosome each between the A-B and A-C lineages were deciphered. Complete homoeology in terms of block organization was found between three linkage groups (LG) each for the A-B and A-C genomes. Based on the homoeology shared between the A, B and C genomes, a new nomenclature for the B genome LGs was assigned to establish uniformity in the international Brassica LG nomenclature code. Conclusion: IP markers were highly effective in generating comparative relationships between Arabidopsis and various Brassica species. Comparative genomics between the three Brassica lineages established the major rearrangements, translocations and fusions pivotal to karyotype diversification between the A, B and C genomes of Brassica species. The inter-relationships established between the Brassica lineages vis-à-vis Arabidopsis would facilitate the identification and isolation of candidate genes contributing to traits of agronomic value in crop Brassicas and the development of unified tools for Brassica genomics.
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.
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.
Conventional and novel Gγ protein families constitute the heterotrimeric G-protein signaling network in soybean
(Public Library of Science, 2011) Choudhury, Swarup Roy; Bisht, Naveen C.; Thompson, Rheannon; Todorov, Oleg; Pandey, Sona
Heterotrimeric G-proteins comprised of Gα, Gβ and Gγ proteins are important signal transducers in all eukaryotes. The Gγ protein of the G-protein heterotrimer is crucial for its proper targeting at the plasma membrane and correct functioning. Gγ proteins are significantly smaller and more diverse than the Gα and Gβ proteins. In model plants Arabidopsis and rice that have a single Gα and Gβ protein, the presence of two canonical Gγ proteins provide some diversity to the possible heterotrimeric combinations. Our recent analysis of the latest version of the soybean genome has identified ten Gγ proteins which belong to three distinct families based on their C-termini. We amplified the full length cDNAs, analyzed their detailed expression profile by quantitative PCR, assessed their localization and performed yeast-based interaction analysis to evaluate interaction specificity with different Gβ proteins. Our results show that ten Gγ genes are retained in the soybean genome and have interesting expression profiles across different developmental stages. Six of the newly identified proteins belong to two plant-specific Gγ protein families. Yeast-based interaction analyses predict some degree of interaction specificity between different Gβ and Gγ proteins. This research thus identifies a highly diverse G-protein network from a plant species. Homologs of these novel proteins have been previously identified as QTLs for grain size and yield in rice.
Defense versus growth trade-offs: insights from glucosinolates and their catabolites
(John Wiley & Sons, 2023) Malhotra, Bhanu; Kumar, Pawan; Bisht, Naveen C.
Specialized metabolites are a structurally diverse group of naturally occurring compounds that facilitate plant-environment interactions. Their synthesis and maintenance in plants is overall a resource-demanding process that occurs at the expense of growth and reproduction and typically incurs several costs. Evidence emerging on different specialized compounds suggests that they serve multiple auxiliary functions to influence and moderate primary metabolism in plants. These new functionalities enable them to mediate trade-offs from defenses to growth and also to offset their production and maintenance costs in plants. Recent research on glucosinolates (GSLs), which are specialized metabolites of Brassicales, demonstrates their emerging multifunctionalities to fine-tune plant growth and development under variable environments. Herein, we present findings from the septennium on individual GSLs and their catabolites (GHPs) per se, that work as mobile signals within plants to mediate precise regulations of their primary physiological functions. Both GSLs and GHPs calibrate growth-defense trade-off interactions either synergistically or directly when they function as storage compounds, abiotic stress alleviators, and one-to-one regulators of growth pathways in plants. We finally summarize the overall lessons learned from GSLs and GHPs as a model and raise the most pressing questions to address the molecular-genetic intricacies of specialized metabolite-based trade-offs in plants.
Differential expression and interaction specificity of heterotrimeric G-protein family in Brassica nigra reveal their developmental-and condition-specific roles
(Oxford University Press, 2014) Kumar, Roshan; Arya, Gulab C.; Bisht, Naveen C.
Heterotrimeric G-proteins, comprised of α, β and γ subunits, are important signal transducers across phyla. The G-proteins are well characterized in the model plants Arabidopsis and rice, and their inventories are possible from a few other plant species; however, information about the roles played by G-proteins in regulating various growth and developmental traits particularly from polyploid crops is still awaited. In this study, we have isolated one Gα (BniB.Gα1), three Gβ (BniB.Gβ1-BniB.Gβ3) and four Gγ (BniB.Gγ1-BniB.Gγ4) coding sequences from the paleopolyploid Brassica nigra, a major condiment crop of the Brassicaceae family. Sequence and phylogenetic analysis revealed that whole-genome triplication events in the Brassica lineage had proportionally increased the inventory of the Gβ subunit, but not of the Gα and Gγ subunits in B. nigra. Real-time quantitative reverse transcription-PCR (qRT-PCR) analysis showed that members of the G-protein subunit genes have distinct temporal and spatial expression patterns and were differentially altered in response to various stress and phytohormone treatments, thereby suggesting differential transcriptional regulation of G-protein genes in B. nigra. Interestingly, specific members of G-protein subunits were co-expressed across plant developmental stages, and in response to different elicitor treatments. Yeast-based interaction screens further predicted that the B. nigra G-protein subunits interacted in most of the possible combinations, although showing a high degree of interaction specificity between different G-protein subunits. Our data on physical interactions coupled with the co-expression pattern of the multiple G-protein subunit genes suggested that tissue- and condition-specific functional combinations of Gαβγ heterotrimers may exist in paleopolyploid B. nigra, to control diverse growth and development processes.
Duplicated RGS (Regulator of G-protein signaling) proteins exhibit conserved biochemical but differential transcriptional regulation of heterotrimeric G-protein signaling in Brassica species
(Nature Publishing Group, 2018) Kumar, Roshan; Bisht, Naveen C.
G-alpha (Gα) and 'Regulator of G-protein Signaling (RGS)' proteins are the two key components primarily involved in regulation of heterotrimeric G-proteins signaling across phyla. Unlike Arabidopsis thaliana, our knowledge about G-protein regulation in polyploid Brassica species is sparse. In this study, we identified one Gα and two RGS genes each from three species of Brassica 'U' triangle and assessed the effects of whole genome triplication on the divergence of gene sequence and structure, protein-protein interaction, biochemical activities, and gene expression. Sequence and phylogenetic analysis revealed that the deduced Gα and RGS proteins are evolutionarily conserved across Brassica species. The duplicated RGS proteins of each Brassica species interacted with their cognate Gα but displayed varying levels of interaction strength. The Gα and the duplicated RGS proteins of Brassica species exhibited highly conserved G-protein activities when tested under in-vitro conditions. Expression analysis of the B. rapa RGS genes revealed a high degree of transcriptional differentiation across the tested tissue types and in response to various elicitors, particularly under D-glucose, salt and phytohormone treatments. Taken together, our results suggest that the RGS-mediated regulation of G-protein signaling in Brassica species is predominantly governed by stage and condition-specific expression differentiation of the duplicated RGS genes.
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.
Effective restoration of male-sterile (barnase) lines requires overlapping and higher levels of barstar expression: A multi-generation field analysis in Brassica juncea
(Springer, 2015) Bisht, Naveen C.; Jagannath, Arun; Augustine, Rehna; Burma, Pradeep K.; Gupta, Vibha; Pradhan, Akshay K.; Pental, Deepak
We have earlier reported the deployment of an effective pollination control system for hybrid seed production in Brassica juncea based on use of the barnase-barstar system. The current study analyses the stability and performance of four male-sterile (barnase) and 34 fertility restorer (barstar) lines under containment field conditions. Line (barnase) x tester (barstar) crosses were studied for effective fertility restoration over three successive growing seasons by screening the F1 and representative F2 progeny for three major parameters, namely segregation of Basta resistance and sensitive plants, distribution of male-sterile and fertile plants (vis-à-vis segregation of the barnase and the barstar genes) and pollen viability of the restored events. A total of 29 male sterile/restorer combinations out of 53 obtained in the first growing season were stable for their restoration ability when tested for two subsequent growing seasons. Stable restorers with high pollen viability were obtained at a significantly higher frequency for one of the barnase lines, bn 3.6, out of the four lines tested. Among the barstar lines, constructs carrying two transcription units of the barstar gene provided more effective fertility restoration. Quantitative real-time PCR analysis of restored progeny obtained for all the four barnase lines showed that three barnase lines (bn 3.23, bn 3.4 and bn 3.48) had an early and much higher level of barnase expression than the line bn 3.6 indicating that the level of barnase expression in the line bn 3.6 is optimal for better and stable restoration. It was observed that a high barstar : barnase transcript ratio and an extended window of barstar gene expression are critical parameters for the development of stable male-sterile (barnase) and fertility-restorer (barstar) combinations.
Evaluation of candidate reference genes for gene expression normalization in Brassica juncea using real time quantitative RT-PCR
(Public Library of Science, 2012) Chandna, Ruby; Augustine, Rehna; Bisht, Naveen C.
The real time quantitative reverse transcription PCR (qRT-PCR) is becoming increasingly important to gain insight into function of genes. Given the increased sensitivity, ease and reproducibility of qRT-PCR, the requirement of suitable reference genes for normalization has become important and stringent. It is now known that the expression of internal control genes in living organism vary considerably during developmental stages and under different experimental conditions. For economically important Brassica crops, only a couple of reference genes are reported till date. In this study, expression stability of 12 candidate reference genes including ACT2, ELFA, GAPDH, TUA, UBQ9 (traditional housekeeping genes), ACP, CAC, SNF, TIPS-41, TMD, TSB and ZNF (new candidate reference genes), in a diverse set of 49 tissue samples representing different developmental stages, stress and hormone treated conditions and cultivars of Brassica juncea has been validated. For the normalization of vegetative stages the ELFA, ACT2, CAC and TIPS-41 combination would be appropriate whereas TIPS-41 along with CAC would be suitable for normalization of reproductive stages. A combination of GAPDH, TUA, TIPS-41 and CAC were identified as the most suitable reference genes for total developmental stages. In various stress and hormone treated samples, UBQ9 and TIPS-41 had the most stable expression. Across five cultivars of B. juncea, the expression of CAC and TIPS-41 did not vary significantly and were identified as the most stably expressed reference genes. This study provides comprehensive information that the new reference genes selected herein performed better than the traditional housekeeping genes. The selection of most suitable reference genes depends on the experimental conditions, and is tissue and cultivar-specific. Further, to attain accuracy in the results more than one reference genes are necessary for normalization.
Evolution, expression differentiation and interaction specificity of heterotrimeric G-protein subunit gene family in the mesohexaploid Brassica rapa
(PLOS, 2014) Arya, Gulab C.; Kumar, Roshan; Bisht, Naveen C.
Heterotrimeric G-proteins, comprising of Gα, Gβ, and Gγ subunits, are important signal transducers which regulate many aspects of fundamental growth and developmental processes in all eukaryotes. Initial studies in model plants Arabidopsis and rice suggest that the repertoire of plant G-protein is much simpler than that observed in metazoans. In order to assess the consequence of whole genome triplication events within Brassicaceae family, we investigated the multiplicity of G-protein subunit genes in mesohexaploid Brassica rapa, a globally important vegetable and oilseed crop. We identified one Gα (BraA.Gα1), three Gβ (BraA.Gβ1, BraA.Gβ2, and BraA.Gβ3), and five Gγ (BraA.Gγ1, BraA.Gγ2, BraA.Gγ3, BraA.Gγ4, and BraA.Gγ5) genes from B. rapa, with a possibility of 15 Gαβγ heterotrimer combinations. Our analysis suggested that the process of genome triplication coupled with gene-loss (gene-fractionation) phenomenon have shaped the quantitative and sequence diversity of G-protein subunit genes in the extant B. rapa genome. Detailed expression analysis using qRT-PCR assays revealed that the G-protein genes have retained ubiquitous but distinct expression profiles across plant development. The expression of multiple G-protein genes was differentially regulated during seed-maturation and germination stages, and in response to various phytohormone treatments and stress conditions. Yeast-based interaction analysis showed that G-protein subunits interacted in most of the possible combinations, with some degree of subunit-specific interaction specificity, to control the functional selectivity of G-protein heterotrimer in different cell and tissue-types or in response to different environmental conditions. Taken together, this research identifies a highly diverse G-protein signaling network known to date from B. rapa, and provides a clue about the possible complexity of G-protein signaling networks present across globally important Brassica species.
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.
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.