Browsing by Author "Kumar, Roshan"

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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.
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.
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.
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.
Interacting partners of Brassica juncea regulator of G-protein signaling protein suggest its role in cell wall metabolism and cellular signaling
(Portland Press, 2022) Kumar, Roshan; Bisht, Naveen C.
Heterotrimeric G-proteins interact with various upstream and downstream effectors to regulate various aspects of plant growth and development. G-protein effectors have been recently reported in Arabidopsisthaliana, however, less information is available from polyploid crop species having complex networks of G-protein components. Regulator of G-protein signaling (RGS) is a well-characterized GTPase accelerating protein, which plays an important role in the regulation of the G-protein cycle in plants. In this study, four homologs encoding RGS proteins were isolated from the allotetraploid Brassica juncea, a globally important oilseed, vegetable, and condiment crop. The B. juncea RGS proteins were grouped into distinct BjuRGS1 and BjuRGS2 orthologous clades, and the expression of BjuRGS1 homologs was predominantly higher than BjuRGS2 homologs across the tested tissue types of B. juncea. Utilizing B. juncea Y2H library screening, a total of 30 non-redundant interacting proteins with the RGS-domain of the highly expressed BjuA.RGS1 was identified. Gene ontology analysis indicated that these effectors exerted various molecular, cellular, and physiological functions. Many of them were known to regulate cell wall metabolism (BjuEXP6, Bju-α-MAN, BjuPGU4, BjuRMS3) and phosphorylation-mediated cell signaling (BjuMEK4, BjuDGK3, and BjuKinase). Further, transcript analysis indicated that the identified interacting proteins have a co-expression pattern with the BjuRGS homologs. These findings increase our knowledge about the novel targets of G-protein components from a globally cultivated Brassica crop and provide an important resource for developing a plant G-protein interactome network.
Isopropylmalate synthase regulatory domain removal abolishes feedback regulation at the expense of leucine homeostasis in plants
(Oxford University Press, 2025) Varghese, Mohan; Kumar, Roshan; Sharma, Aprajita; Lone, Asif; Gershenzon, Jonathan; Bisht, Naveen C.
In the leucine (Leu) biosynthesis pathway, homeostasis is achieved through a feedback regulatory mechanism facilitated by the binding of the end-product Leu at the C-terminal regulatory domain of the first committed enzyme, isopropylmalate synthase (IPMS). In vitro studies have shown that removing the regulatory domain abolishes the feedback regulation on plant IPMS while retaining its catalytic activity. However, the physiological consequences and underlying molecular regulation on Leu flux upon removing the IPMS C-terminal domain remain to be explored in plants. Here, we removed the IPMS C-terminal regulatory domain using a CRISPR/Cas9-based gene editing system and studied the resulting impact on the Leu biosynthesis pathway under in planta conditions. Absence of the IPMS regulatory domain unexpectedly reduced the formation of the end product Leu but increased the levels of Leu pathway intermediates in mustard (Brassica juncea). Additionally, delayed growth was observed when IPMS devoid of the regulatory domain was introduced into IPMS-null mutants of Escherichia coli and Arabidopsis thaliana. Further, a detailed biochemical analysis showed that in the absence of the C-terminal regulatory domain, a Leu pathway intermediate (α-ketoisocaproate) could compete with the native IPMS substrate (2-oxoisovalerate) for the active site. Combining these metabolomic, biochemical, and in planta analyses, we demonstrate that the C-terminal regulatory domain of IPMS is critical for maintaining Leu-Val homeostasis in plants.
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.
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.
Pattern of expression and interaction specificity of multiple G-protein beta (Gβ) subunit isoforms with their potential target proteins reveal functional dominance of BjuGβ1 in the allotetraploid Brassica juncea
(Elsevier B.V., 2017) Kumar, Roshan; Sharma, Aprajita; Chandel, Ishita; Bisht, Naveen C.
Heterotrimeric G-protein, consisting Gα, Gβ and Gγ subunits, interacts with various upstream and downstream effector (target) proteins to regulate a large array of conserved and species-specific biological functions. The targets of G-protein components are recently reported in model plant Arabidopsis thaliana; however limited information is available from crop species. In this study, we utilized yeast two-hybrid (Y2H) assay to screen the diversity of interacting partners of multiple Gβ subunit isoforms from allotetraploid Brassica juncea, a globally important oilseed and vegetable crop. The three BjuGβ genes (BjuGβ1-3), resulted from whole genome triplication event in Brassica lineage, showed distinct expression profile during plant developmental stages with maximal transcript abundance during reproductive stages. Protein-protein interaction of three BjuGβ proteins (bait) against the Y2H cDNA library (prey) identified a total of 14 and 1 non-redundant targets for BjuGβ1 and BjuGβ2, whereas BjuGβ3 screening surprisingly did not yield any genuine target, thereby suggesting functional dominance of BjuGβ1. The triplicated BjuGβ isoforms showed a high degree of interaction strength and specificity with the identified target proteins, which are known to be involved in diverse biological functions in plants. qRT-PCR analysis further indicated that the expression of BjuGβ-target genes was developmentally regulated under various tissue types studied and showed a high degree of co-expression pattern with the BjuGβ genes, particularly during flower and silique development in B. juncea. Taken together, our data provides novel insights on pattern of expression and interaction specificity governing functional divergence of multiple Gβ subunit proteins in polyploid B. juncea.
Targeted editing of multiple homologues of GTR1 and GTR2 genes provides the ideal low-seed, high-leaf glucosinolate oilseed mustard with uncompromised defence and yield
(John Wiley & Sons, 2023) Mann, Avni; Kumari, Juhi; Kumar, Roshan; Kumar, Pawan; Pradhan, Akshay K.; Pental, Deepak; Bisht, Naveen C.
Glucosinolate content in the two major oilseed Brassica crops-rapeseed and mustard has been reduced to the globally accepted Canola quality level (<30 μmoles/g of seed dry weight, DW), making the protein-rich seed meal useful as animal feed. However, the overall lower glucosinolate content in seeds as well as in the other parts of such plants renders them vulnerable to biotic challenges. We report CRISPR/Cas9-based editing of glucosinolate transporter (GTR) family genes in mustard (Brassica juncea) to develop ideal lines with the desired low seed glucosinolate content (SGC) while maintaining high glucosinolate levels in the other plant parts for uncompromised plant defence. Use of three gRNAs provided highly efficient and precise editing of four BjuGTR1 and six BjuGTR2 homologues leading to a reduction of SGC from 146.09 μmoles/g DW to as low as 6.21 μmoles/g DW. Detailed analysis of the GTR-edited lines showed higher accumulation and distributional changes of glucosinolates in the foliar parts. However, the changes did not affect the plant defence and yield parameters. When tested against the pathogen Sclerotinia sclerotiorum and generalist pest Spodoptera litura, the GTR-edited lines displayed a defence response at par or better than that of the wild-type line. The GTR-edited lines were equivalent to the wild-type line for various seed yield and seed quality traits. Our results demonstrate that simultaneous editing of multiple GTR1 and GTR2 homologues in mustard can provide the desired low-seed, high-leaf glucosinolate lines with an uncompromised defence and yield.
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.