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Item Genome-wide identification, characterization, and expression analysis of Clavata3 Insensitive receptor Kinases (CIKs) on developmental tissues and under temperature stress conditions in Brassica oleracea(Springer Nature Publishing AG, 2025) Suresh, Gokul Babu; Baskar, Harssitha; Subramaniam, Geethanjali; Soundararajan, PrabhakaranUnprecedented climate changes are one of the major global threats to agriculture. Though cauliflower is grown worldwide in different conditions, it is sensitive to temperature fluctuation. Clavata3 Insensitive receptor Kinase (CIK) is one of the important co-receptors involved in WUS-CLV pathway for shoot apical meristem (SAM) maintenance, floral development and environment buffering. In this study we did genome-wide identification and characterization of CIKs in Brassica oleracea var. botrytis cv. Korso. Expression analysis was conducted on developmental tissues and short-term heat and cold stress conditions. Seven fulllength BolCIKs have been identified in the Korso genome through comparison against Arabidopsis thaliana CIKs. Gene structure and motifs are distinct between each sub-class. Ka/Ks ratios showed that all CIKs underwent purifying selection. Abundance of stress-responsive cis-elements, such as MYB and MYC recognition sites, showed the involvement of BolCIK in environmental stress regulation. Among the nine developmental tissues such as leaf, stem, root, curd, sepal, petal, stamen, pistil, and silique, except BolCIK4 and BolCIK5a, CIKs were merely detected in leaves. Contrastingly, expression of all BolCIKs was detected in stem, curd, and reproductive organs. Importantly, most of the BolCIKs were highly expressed in sepal, petal, pistil, and siliques. In SAM specific manner, BolCIK3 and BolCIK6 are upregulated in heat stress and cold stress, respectively. Other BolCIKs expressions are varying between tissues-types and stress conditions. Since CIKs can possibly be involved in thermomorphogenesis, results of this work may lead to further exploration of its function to improve climate resilience in cauliflower.Item Perspectives on the utilization of resistance mechanisms from host and nonhost plants for durable protection of Brassica crops against Alternaria blight(PeerJ, Inc., 2019) Fatima, Urooj; Bhorali, Priyadarshini; Borah, S.; Senthil-Kumar, MuthappaBackground: Alternaria brassicae, the causal organism of Alternaria blight, is a necrotroph infecting crops of the Brassicaceae family at all growth stages. To circumvent this problem, several disease management strategies are being used in the field, and disease-resistant varieties have also been developed. However, no strategy has proven completely successful, owing to the high variability in virulence among A. brassicae isolates, which causes a diverse spectrum of symptoms. Nonhost resistance (NHR) is a robust and broad-spectrum defense mechanism available in plants, and the exploitation of gene pools from plant species that are nonhost to A. brassicae could serve as novel sources of resistance. Methodology: We searched the literature using key words relevant to this study in various search engines, such as PubMed, Web of Science, and Google Scholar, as well as certain journal websites. The literature was retrieved, sorted, and mined to extract data pertinent to the present review. Results: In this review, we have comprehensively covered the recent progress made in developing Alternaria blight resistance in Brassica crops by exploiting host germplasm. We also enumerate the potential NHR sources available for A. brassicae and the NHR layers possibly operating against this pathogen. In addition, we propose different strategies for identifying NHR-related genes from nonhost plants and testing their relevance in imparting broad-spectrum resistance when transferred to host plants. Conclusion: This review will help broaden the current knowledge base pertaining to the resistance sources available in host germplasm, the exploitation of NHR mechanisms, and their applications in protecting Brassica crops from Alternaria blight. The insights might also be applicable to a wider repertoire of plant pathogens.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 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.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.Item 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.Item Analysis of a salinity induced BjSOS3 protein from Brassica indicate it to be structurally and functionally related to its ortholog from Arabidopsis(Elsevier, 2011) Kushwaha, Hemant R.; Kumar, Gautam; Verma, Praveen K.; Singla-Pareek, Sneh L.; Pareek, AshwaniArabidopsis has been a favorite model system for plant biologist. It is anticipated that comparative analysis of this plant with other members of Brassicaceae may aid in identification of orthologs playing role as key genetic determinants for salinity response. In this endeavor, we have recently identified SOS family members from Brassica juncea in our laboratory and reported their salinity responsive transcriptional induction in seedlings of various diploid and amphidiploids species. In the present study, we have carried out detailed time kinetics for BjSOS3 expression in a salinity tolerant B. juncea var. CS52. Transcript analysis at the sensitive growth stages of plants viz. seedling and reproductive stage indicated clear differential transcriptional regulation of BjSOS3 under non-induced as well as salinity induced conditions in a time and organ specific manner, mirroring their respective tolerance physiology. Similar to its ortholog from Arabidopsis thaliana, the modeled BjSOS3 protein show typical features of a Ca(2+) binding protein with four conserved EF-hands. We have also attempted to study the binding of SOS3 protein with the modeled SOS2 protein. It has been established that SOS3 protein senses Ca(2+) though the binding is very weak; we show the down regulation of BjSOS3 mRNA in presence of calcium chelator - EGTA under the various stress conditions including ABA. In situ localization of BjSOS3-GFP fusion protein in onion peel has shown its presence strongly in plasma membrane as well as cytosol. The leads presented in the paper will assist in understanding and establishing the SOS signaling machinery in B. juncea.