BR, a plant-specific steroidal hormone, exhibits a vast range of stress-resistance functioning that influences different growth processes [58]. Additionally, the BR signal transduction system is essential for plant development. BZR1 proteins maintain interlinks in BR and other different pathways of signaling [59]. PhBEH2 is suggested to serve as a key component facilitating crosstalk between signaling pathways in the Petunia hybrid [60]. The characterization of BR signaling in B. oleracea is currently lacking. It seems to be essential to identify the integral component of BR-signaling, reveal evolutionary aspects, and explore its functioning. These steps are important in unraveling the BR signaling networks. They represent the starting necessary for future genetics work efforts related to BR in a broader range of vegetables [61]. As of now, BoBZR1 gene genome-wide analysis has been conducted. The whole analysis included identification, localization, evolutionary relationships, structural analysis and assessments of gene duplication and loss. Additionally, our study delved into the functional exploration, incorporating transcriptional activation of genes, and their expression profiling specific tissue.
In the entire genome of B. oleracea, twelve BZR1 family genes have been identified. Notably, Arabidopsis contains six BZR members [62]. Additionally, beyond the initially recognized BZR genes in Arabidopsis, research has revealed that BAM7 & BAM8 of the BAM family have DNA binding domain similar to that of BRASSINAZOLE RESISTANT1. This specific domain is a characteristic feature of transcription factors to maintain response with brassinosteroids [63]. The molecular weights (Mw) of all BZR1 proteins in B. oleracea were observed to fall within two distinct ranges: from 10.88 to 36.18 kDa and from 75.87 to 79.09 kDa. Importantly, it is noteworthy that all the identified BZR1 proteins seem to share a conserved role as hydrophilic proteins [64].
To elucidate the evolutionary relationships in BZR1 across various species, a phylogenetic tree was constructed. The analysis revealed that BoBZR1 genes were categorized into three distinct groups. The first group contains AtBZR1 and BoBZR1, with SlBZR1 also belonging to the same group. Tandem repeat and fragment replication are essential mechanisms for gene family duplication, contributing to the generation and functional diversification of novel genes [65]. Earlier studies have emphasized the significance of the exons-introns arrangement of a gene in the evolutionary process [66]. Through the structural and motifs analysis, it became clear that genes belonging to a similar clade and population exhibited similarities in terms of the numbers and positions of motifs and exons-introns [67]. Structural analysis of BZR1 genes revealed that except of BoBZR1-1, BoBZR1-3, and BoBZR1-4, all the other genes share the same number of exons and introns. This observation suggests a potential conserved structural pattern among the majority of BoBZR1 genes, highlighting the importance of structural features [68]. Furthermore, motif analysis revealed that BoBZR1-2 and BoBZR1-1 share the same number of motifs with their closest homologs BoBZR1-11, BoBZR1-12, and BoBZR1-3, a potential functional similarity or conservation of motifs among these specific BoBZR1 genes [55].
The Brassica ancestor diverged from a shared ancestor with Arabidopsis around 20 Mya (million years ago) and experienced a Whole Genome Triplication roughly 15.9 million years ago, subsequently undergoing significant gene loss [69]. The identification of duplicated genes on the same chromosome implies a potential tandem duplication event, while the presence of duplicated genes on various chromosomes suggests a segmental duplication. Focusing on B. oleracea, we conducted an analysis of the Ka/Ks ratio for expected duplicated genes, revealing values below 1. These findings suggest that over their evolutionary history, selection for purification has mostly affected the BoBZR1 proteins. Our examination of the anticipated time of divergence or duplication in the homologous genes uncovered notable disparities in Ka/Ks ratios among various BoBZR1 genes, particularly those triplicated as a result of Whole Genome Triplication, suggests diverse evolutionary pressures. These distinct selection pressures have led to varying rates of evolution among the BZR1 genes in B. oleracea [47].
Gene expression is crucial in the manifestation of functioning, and promoters play a direct role in influencing gene expression [70]. Among 12 BoBZR1, each member is associated with stress responsiveness, hormone responsiveness, and plant development responsive CREs. BZR1 gene is likely to contain a significant role in governing growth and response to different kinds of stress, indicates the BZR1 gene family serves as an essential element in the interacting BR signaling and other hormonal signals. Various reports confirmed the active participation of the BZR family in a diverse range of stress response mechanisms [21], hormone signaling [71], and the regulation of plant growth [72]. The BoBZR1 genes analysis towards cuticular wax development depicted that expression in BoBZR1-6, 7 and 10 were more than1.2 folds as compared to other genes which could be linked to the existence of ABA-responsive CREs in their promoters, indicating the considerably positive contribution of these genes to the coordinated regulation of growth and defense mechanisms. In Arabidopsis, BZR1 facilitates growth maintenance by binding to the BRRE box [73]. BES1 plays a regulatory role in cellulose biosynthesis by binding to cellulose synthase genes promoter region [74]. In wheat, a transcription factor TaBZR2 from the BES/BZR family plays an essential role in responding to drought through decreasing the buildup of reactive oxygen species with the activation of TaGST [75]. RNA-Seq revealed that the BoBZR1 genes exhibited constitutive expression across all tested tissues. This suggests that these BZR1 genes likely serve essential roles in fundamental cellular processes, acting as integral components of major defense machinery throughout various stages of plant growth and development [76]. The BoBZR1 genes were expressed across all organs and significantly induced by developmental signals. Generally, BoBZR1-6, 5, 9, 2 and 12 genes exhibited higher expression levels in leaves, while others BoBZR1-11, 4, 8, 10, 1 and 3 were more prominent in stems compared to other organs. This observation implies that BoBZR1 proteins have a crucial role in the growth and development of various organs. In maize, ABA, far-red, red, and dark light treatments showed variations in gene expression in both the root and shoot, revealing that all ZmBES1/BZR1 genes were influenced by these treatments [31]. Increased expression levels of BoBZR1-5, BoBZR1-9, BoBZR1-11, and BoBZR1-12 were observed in vegetative and reproductive growth stages, suggesting their potential contribution to subsequent developmental stages. Based on these findings, it is conceivable to anticipate the potential functions of the BoBZR1 genes. Furthermore, the expression correlation among genes indicates potential interactions between different genes (Fig. 12), and provides a foundation for research into the results of gene interactions in response to various stresses.