Introduction

BRs are the most extensively dispensed plant growth-regulating steroidal phytohormones and play a critical role in developmental activities apart from other regulator hormones like cytokinin and gibberellin1,2,3. Brassinosteroid functioning is specific toward plant germination, growth, development, and reproductivity4. Various studies indicated the fundamental role of BRs in response to sudden temperature fluctuations and other abiotic stresses like frost and salinity5,6. Brassinosteroids (BRs) engage with kinase receptors on the membrane, giving rise to form tierce complexes involving BRI1 (brassinosteroid insensitive)- RK1 (receptor kinase)-and LR (leucine-rich) repeat receptors, serve as the starting point for the initiation of BR signaling within the plant cellular environment7,8. Subsequent pathways of kinases are progressively activated to carry out their function, which leads to the dephosphorylation and expression of BZR1 transcription factors that ultimately modulate the expression of various genes via binding to brassinosteroid responsive elements (BRRE)9,10,11.

BZR1 protein and BES1 protein, both belonging to the same family, are integral constituents of the brassinosteroid signaling in various pathways in plants. These proteins display a notable similarity in their sequences, sharing an overall 88% homology. Notably, the domains of both BZR1 and BES1 share 97% sequence consistency. This close sequence resemblance underscores their functional relatedness in mediating the plant’s response to brassinosteroid signaling10. BZR1 is involved in the modulation of gene expression through BR signaling pathways12. The BZR1 protein contains a greatly conserved DNA-binding domain, the N-terminal contains that nuclear localization sequence11. The BZR1 proteins exhibit 22–24 amino acids sequence spanning glycosylation sites targeted by Glycogen Synthase Kinase 3. Additionally, certain members of the BZR family possess a PEST (Proline-Glutamic acid-serine-threonine) motif, serving as a regulatory element for modulating protein stability11. The control of downstream BR response genes is maintained via BR through the specific binding to BRRE13,14. The interaction between BZR1/BES1 and PIF4 involves their ability to associate with the auxin response element ARF6. This interaction facilitates the promotion of ARF6 binding to SAUR15 and SAUR18, regulating cell elongation15. Ultimately, BZR1 is significantly involved in adapting plants to abiotic stresses16,17.

A highly susceptible to fungal infection mutant bes1-D provides compelling evidence of BES1’s direct involvement in the defense against pathogens18. BES1 cooperated with RD26, leading toward the inhibition of RD26 expression, subsequently suppressing drought responses19. Alternatively, PIF4 activates BR synthesis, leading to elevated BES1 levels and promoting thermosmorphogenesis20. Moreover, BZR1 contributes to enhanced freezing tolerance in Arabidopsis through both CBF-dependent & independent mechanisms21. This intricate network of interactions highlights the regulatory mechanisms orchestrated by BZR1 in regulating plant responses to drought, freezing, and temperature fluctuations.

B. oleracea holds significant importance in the realm of nutrition and agricultural practices22,23. Rich in essential components, white cabbage contributes to attaining a harmonious vegetable crop and is recognized for its potential health benefits, including antioxidant properties24,25,26. To date, BZR1 gene analysis has been reported in B. rapa27, Pyrus bretschneideri28, Nicotiana benthamiana29, potato30, maize31 and Arabidopsis32. Yet, our knowledge regarding the evolutionary history and expression profiles of Brassinazole-resistant proteins (BZR1) in B. oleracea is currently constrained. Exploration through genome-wide analysis is indispensable for understanding the growth and development of B. oleracea. Hence, this investigation undertook to identify and characterize the BZR1 family in B. oleracea. The findings of this research establish a foundation for subsequent functional investigations of BZR1 genes in B. oleracea.

Materials and methods

BZR1 genes identification in B. oleracea

The amino acid sequence corresponding to the BZR1 was identified from the tomato peptide genome using the identifier PF05687, obtained from the Pfam database (http://pfam.xfam.org/)33. The Phytozome platform (https://phytozome-next.jgi.doe.gov) was utilized to locate BZR1 genes within the genomes of B. oleracea, employing this peptide sequence. Redundant genes were then filtered out to ensure non-redundancy. Subsequently, the non-redundant protein sequence was validated using the Motif-Finder online database and NCBI-CDD (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi)34. To eliminate redundancy, we removed duplicate genes. Subsequently, the selected genes underwent additional validation to ensure the presence of the BES_N domain utilizing default parameters on both the Motif-Finder online database and NCBI-CDD (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi)35.

Determining physio-chemical properties and sub-cellular localization

Data regarding transcript IDs, gene localization, and gene and protein sequences were sourced from Phytozome. The Prot-Param online tool (http://web.expasy.org/protparam/) is employed to retrieve physio-chemical characters of BoBZR1 family genes, encompassing molecular weight, protein length, isoelectric point36, and instability index37. The forecasted subcellular localizations of BoBZR1 genes were determined utilizing WoLF PSORT (https://wolfpsort.hgc.jp/). The heat map, created through TBTools, allows for visual inspection of localization features across different cell organelles, using output data from cello-life (https://cello.life.nctu.edu.tw/) for enhanced representation38.

Evolutionary analysis and classification of BZR1

To observe the evolutionary dynamics of BZR1 proteins in Arabidopsis, tomato, cucumber, potato and cabbage, the full amino acid sequences of these genes were aligned using MUSCLE, employing default settings39. A neighbor-joining tree was constructed using MEGA11, incorporating statistically reliable bootstrap replication no. of 100040. The resulting phylogenetic tree facilitated the classification of BZR1 transcription factors into various groups based on the tree’s topology. For an enhanced visual representation, the aligned dataset was taken to iTol (https://itol.embl.de/personal_page.cgi) for the creation of a visually enriched and informative display41.

Exon–intron distribution of BoBZR1 and chromosomal positioning

To delineate the intron/exon architecture of the BoBZR1 gene, genomic and CDS files were utilized that were sourced through Phytozome. Utilizing these sequences, an online server (http://gsds.cbi.pku.edu.cn/) was employed to visually represent and analyze the gene structure of the BoBZR1 family genes42.

Chromosomal localization of all genes denoted by their chromosome number and precise start & end position was obtained using Phytozome and ProtParam. All chromosome length was evaluated from the genome file using TBtools. Additionally, a comprehensive chromosomal map illustrating the positioning of the BoBZR1 genes was generated using an online website (http://mg2c.iask.in/mg2c_v2.0/), following default settings for optimal visualization and analysis.

Comparative domain and conserved motif analysis

Comparative domain within the BoBZR1 family was analyzed using the NCBI (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi), employing default settings. Motif analysis of proteins within the BoBZR1 was carried out utilizing the MEME suite (https://meme-suite.org/meme/)43, and all other settings were kept at their default encompassing a lowest motif width of six and a highest width of fifty44. The TBTool software was utilized to visualize conserved domains and motifs, incorporating the hit-data, phylogenic data, and MEME-suite data45.

Assessment of Cis-regulatory elements (CREs) in BoBZR1 family gene promoters

Promoter regions were retrieved from phytozome to extract cis-regulatory elements. These elements have regulatory control over the functionality of target genes46. PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) was utilized to take CREs, spanning 5–20 base pairs within the promoter region47. Subsequently, the obtained data of measured specific regulatory elements were mapped in TBtools and Venn diagram was created using an online website Venny2.0.2 (https://bioinfogp.cnb.csic.es/tools/venny/index2.0.2.html).

Gene-duplication study and synteny analysis

BoBZR1 genes divergence time was assessed through Ka/Ks values. The software TBtools was utilized to ascertain the quantity of Ka/Ks substitution ratios. Paralogous gene pairs were subjected to Ka/Ks ratio calculations, providing insights into each gene pair’s pace of evolutionary progression. To estimate the time of divergence (T), the Ks value was input into the equation T = Ks / 2λ, where λ equaled 9 × 10− 948. For the synteny analysis, TBtools was utilized with default parameters, showing a comprehensive exploration of various gene duplication events.

Enrichment analysis and gene ontology

Gene Ontology analysis was conducted to validate the functions of BoBZR1 genes for additional confirmation of their functional roles49. Arabidopsis online database (https://www.arabidopsis.org/) was employed to elucidate the molecular functioning and their participation in diverse biological processes of genes. Additionally, a web tool (http://bioinformatics.sdstate.edu/go/) was employed to comprehend the roles played by BZR1 genes in B. oleracea. A p-value of 0.01 was employed to determine the significance of gene enrichment in the analysis50.

BoBZR1 genes expression analysis

Transcriptome analysis of BoBZR1 genes was undertaken to assess their involvement in cuticular wax development and expression patterns across various tissues examined to verify their functional relevance in response to both stress and growth stimuli.

Expression of BoBZR1 genes in glossy mutant Nwgl and wild-type

A cabbage (Brassica oleracea L. var. capitata) plant with a non-wax glossy (nwgl) phenotype was obtained from the inbred line G287. To produce F₂ mapping populations, this plant was crossed with the inbred lines G306 and G274. The cultivation occurred under natural field circumstances at the Shanghai Academy of Agricultural Sciences in Shanghai, China, throughout the regular growing season. High-throughput RNA sequencing data was utilized to assess the expression profiles in leaf samples obtained from a glossy mutant and its corresponding wild-type in cabbage. The experiment employed three replications for data collection. Subsequent analysis facilitated the identification of differentially expressed genes (DEGs) in nwgl leaves in comparison to the WT51.

Tissue-specific expression of BoBZR1 genes

Genes that were differently expressed that could be related to the developmental processes of plants were found using RNA-Seq expression data from seven different B. oleracea tissues52. To examine variations in BoBZR1s, expression was measured in numbers of fragments per kilobase of transcript per million mapped reads (FPKM) across seven distinct tissues.

Protein–protein (PPI) interaction

Utilizing STRING (https://string-db.org/), protein interaction (PPI) was analyzed through setting a 0.7 stringent confidence score53. The PPI network was established by integrating active interactions with a score of 0.4 from various sources. This interactome map facilitated the identification of key candidate genes participating in both physical and functional interactions54.

Results

Identification of BoBZR1 family gene members

Through this analysis, twelve BoBZR1 genes in B. oleracrea were successfully identified. Subsequent to the exclusion of redundant sequences, the dataset was refined by eliminating repetitive elements. The 12 BZR1 genes in B. oleracea were denoted as BoBZR1-1 to BoBZR1-12, with each designation corresponding to its specific chromosomal location. The comprehensive analysis of their physiochemical characteristics encompassed an in-depth analysis. Parameters scrutinized included transcript ID, isoelectric point, molecular weight, polypeptide length (Table 1), and the forecasted sub-cellular location of these B. oleracea proteins (Fig. 1). The B. oleracrea protein length exhibited variation, spanning from 94 amino-acid (AA) in BoBZR1-4 to 700 amino-acid (AA) in BoBZR1-3. Correspondingly, the molecular weights (MW) associated with these proteins ranged from 10.880 kDa to 79.09 kDa. The isoelectric point (pI) values showed diversity, ranging from 5.88 in BoBZR1-1 to 11.11 in BoBZR1-10.

Table 1 A comprehensive examination of various attributes of BZR1 genes discovered in B.oleracea.
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Fig. 1
figure 1

Heat Map showing all 12 BoBZR1 genes Sub-cellular localization. White-red represents the lowest to highest value of the gene in that specific organelle.

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Phylogenetic relationship, structure and motif analysis of BoBZR1 genes

A phylogenetic tree was constructed to clarify the phylogenetic relationships among BZR1 proteins and explore the evolutionary dynamics of genes (Fig. 2a). This tree encompasses BZR1 proteins from B. oleracea, as well as from Arabidopsis (16 proteins), tomato (9 proteins), cucumber (6 proteins), and potato (11 proteins). Utilizing the Arabidopsis BZR1 families as a reference, we classified BoBZR1 into 3 distinct classes: A, B, and C. Class-B turned out to be the biggest, having a total of 40 members. Proteins within the same clade generally share structural and functional similarities. Accordingly, the identified BZR1 proteins are expected to exhibit comparable structures and functions, indicating functional coherence among clade members.

The structural organization of BoBZR1 genes was examined by analyzing the intron–exon structures of 12 BoBZR1 gene members (Fig. 2b). Exons and introns function as pivotal structural components that influence the intricate tapestry of evolutionary interrelations among genes55. The BoBZR1 genes exhibited inconsistency in the number of introns and exons, ranging from one to ten. BoBZR1-1 contained 10 exons (52.63%), BoBZR1-3 had 9 exons (52.94%), while BoBZR1-4 was entirely composed of exons (100%). Additionally, nine genes contained 2 exons (66.66%).

Domain structure was scrutinized inherent in their protein sequences (Fig. 2c). BoBZR1 proteins analysis involved a detailed analysis of 15 identified motifs through the MEME program. Analyzing the BZR1 protein family showed that the same group of proteins share the same motifs. Some genes contained fewer motifs, i.e. BoBZR1-10, BoBZR1-4, BoBZR1-1 and BoBZR1-3 contained 2, 2, 3 and 3 motifs, respectively. All the genes contained Motif 1 and Motif 3. The existence of similar motifs among various members of the BZR1 proteins points towards the impact of gene expansion as a driving force in the evolutionary dynamics of these genes56.

Fig. 2
figure 2

(A) Evolutionary relationship in BZR1 genes of Solenum tuberosum, (A) thaliana, S. lycopersicum, C. sativus, and (B) oleracea. (B) Intron-Exon structures of the BoBZR1 genes. Red boxes show exons, while black lines show introns. (C) Motif distribution on BZR1 proteins in B. oleracea with a phylogenetic tree. Each bar in the graph is assigned a distinct color code to differentiate between the various types of motifs.

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Analysis of promoter Cis-element of BoBZR1

Cis-acting elements are non-coding DNA sequences in gene promoters that regulate gene transcription57. Twelve specific CREs within this region were identified that are highly involved in plant growth and developmental activities, stress and hormone responsiveness (Fig. 3). These include core elements, MBS (linked to drought inducibility), TC-rich repeats (involved in defense and stress responses), and ARE (responsible for anaerobic induction). Regarding hormonal regulation, there are elements such as TGACG-motif and CGTCA-motif (associated with methyl-jasmonate), ABRE (related to abscisic acid) and TGA-element (responsive to auxin). Additionally, gibberellin-related motifs (P-box) are identified, along with regulatory motifs for tissue-specific expression (e.g., GCN4_motif and CAT-box) and those associated with promoter and enhancer regions (e.g., CAAT-box). Notably, the element in promoter and enhancer regions emerges as the most abundant cis-acting element in the BoBZR1 promoter region.

Fig. 3
figure 3

(a) 12 Cis-regulatory elements in putative BoBZR1 promoters which are linked to different plant developmental processes (b) Venn diagram depicting the ratio of growth and development, stress-responsive and hormone-responsive Cis-regulatory elements. Plant development responses involve elements associated with light responsiveness, elements in promoter and enhancer regions, endosperm expression and meristem expression. Hormone response elements exhibit reactions to abscisic acid-responsive elements (ABA), gibberellin-responsive element, auxin-responsive elements and MeJA-responsiveness elements, while stress responses include elements related to anaerobic induction, defense and stress-responsive elements, wound responsive elements (MYB) and drought-inducibility. (c) Heatmap of 12 specific cis-regulatory elements related to growth and development, harmones and stress.

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Chromosomal distribution and duplication analysis

The genomic positioning analysis of BoBZR1 genes in B. oleracea revealed a markedly uneven distribution across eight chromosomes (Fig. 4). Chromosome 07 showed a notable concentration with three BoBZR1 genes, while chromosomes 1, 6, and 8 each contained two genes. The remaining chromosomes predominantly harbored a single gene.

Fig. 4
figure 4

Distribution of BoBZR1 genes on B.oleracea chromosomes. The scale on left side in Mb indicates various chromosome lengths.

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Synteny analysis of BoBZR1 genes revealed a non-uniform distribution of most paralogous gene pairs across the B. oleracea genome, suggesting their emergence from whole-genome duplication or segmental duplication (Fig. 5a). However, three paralogous gene pairs situated closely on the same chromosome likely resulted from tandem duplication. Specifically, BoBZR1-5, BoBZR1-6, BoBZR1-11, and BoBZR1-12 genes were identified as products of tandem duplication, while others were inferred to have arisen from whole-genome duplication or segmental duplication events. Multiple synteny analyses were conducted comparing the whole genomes of B. oleracea, Solanum tuberosum, and Arabidopsis (Fig. 5b). The duplicated genes between the chromosomes of these crops were highlighted by connecting threads. Seven BoBZR1 genes were duplicated between B. oleracea and Arabidopsis, while six were duplicated between B. oleracea and S. tuberosum.

We utilized Ks (synonymous substitution rate) as a metric to track the dates of duplication blocks, employing it as a means to estimate the timing of duplication events. This approach enabled us to trace the origin of segmental duplications in BoBZR1 genes (Fig. 5c), spanning from BoBZR1-2_BoBZR1-12 around 18.47 million years ago (Ks = 0.332) to BoBZR1-6_BoBZR1-12 approximately 259.61 million years ago (Ks = 4.673). For all four of the paralogous pairings in B. oleracea, the Ka/Ks ratio was found to be less than 1.

Fig. 5
figure 5

(A) Genome-wide synteny analysis of BoBZR1 genes showing the dominance of segmental duplication and rare occurrence of tandem duplication. (B) Multiple synteny analysis of B.oleracea, Arabidopsis and Potato. Blue lines showing the duplicated genes in the respective genomes. (C) The time of gene duplication for different paralogous pairs of BoBZR1 genes was estimated using TBtool software based on Ka (nonsynonymous substitutions) and Ks (synonymous substitutions) values.

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Orthologue identification and enrichment analysis

The functions and subcellular localization of BoBZR1 genes orthologous with Arabidopsis were determined at the molecular level. Simultaneously, broader biological processes involving these genes were identified through available datasets (Table 2). A fold enrichment graph was generated using ShinyGO v0.741 to visualize the overlapping effects of these genes on various biological processes (Fig. 6). The chart was employed to acknowledge genes’ involvement in different biological functions in the B. oleracea. In the biological process category, most of the BZR1 proteins were found to be involved in regulating DNA-templated transcription. In terms of molecular functions, these genes predominantly participate in sequence-specific DNA binding, protein binding, and transcription regulator activity.

Table 2 Arabidopsis orthologue of B.oleracea and their expression pattern analysis in BoBZR1 gene family.
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Fig. 6
figure 6

The Fold Enrichment chart illustrates the shared functions of BoBZR1 genes, with red-colored dots indicating a higher number of genes involved in a specific process, while smaller blue dots represent fewer genes associated with that process. The chart provides a visual representation of the enrichment of functions among the overlapping BoBZR1 genes.

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Transcriptomic analysis

BoBZR1 genes expression towards cuticular wax biosynthesis

In this experiment, two varieties of B. oleracea, namely the wild-type and glossy mutant (nwgl), were cultivated. Comparative analysis was done between both varieties, involving the calculation of p-values and log2fold change. BoBZR1-7 depicted significant differential expression. Genes like BoBZR1-1, BoBZR1-6, BoBZR1-7 and BoBZR1-10 showed considerable upregulation while the other genes were downregulated from wild-type and glossy mutant (Fig. 7a).

Tissue-specific BoBZR1 genes expression and correlation analysis

A heatmap of RNA-Seq gene expression profile in different tissues obtained for all 12 B. oleracea BZR1 genes was constructed (Fig. 7b). The tissues included in the analysis are callus, root, stem, leaf, flower, and silique. In all seven tested tissues, the BoBZR1 member’s expression was consistently observed. All the genes depicted significant expression across various tissues. The results depicted that the genes BoBZR1-1, BoBZR1-3, BoBZR1-4, BoBZR1-7, BoBZR1-8, BoBZR1-10 and BoBZR1-11 were highly expressed in stem while lowest expression in flowers. Moreover, BoBZR1-2 exhibited maximum expression levels in the leaf compared to other tissues. Conversely, BoBZR1-6 demonstrated lower expression in the root.

Fig. 7
figure 7

(A) Relative expression profile of BoBZR1 genes from wild type to mutant type B.oleracea varieties. (B) Relative expression profile in FPKM of the BoBZR1 genes in seven different tissues. (C) The correlation of expression data for different genes. Red hues represent positive correlations, while blue hues represent negative correlations.

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Protein–protein interaction

The interaction among proteins plays a crucial role in facilitating various essential plant functions and processes, including signal transduction pathways, plant development, and physiological and pathological processes58. In the case of BoBZR1 proteins, their protein-protein interactions were investigated using the STRING database. The analysis revealed interactions among BZR1 proteins themselves as well as with various other proteins in B. oleracea, indicating their involvement in diverse cellular processes and pathways within the plant.

PPI network exhibits an average local clustering coefficient of 0.269 (Fig. 8). This value depicts a medium clustering level, indicating that proteins in the network tend to interact with other proteins that are also interconnected. Seven genes were interrelated forming one cluster while BoBZR1-1 and BoBZR1-3 were interrelated to each other forming a separate cluster. The genes like BoBZR1-4 and BoBZR1-10 show no relation with other genes.

Fig. 8
figure 8

Protein–protein interactions within the BZR1 genes of Brassica oleracea are represented by colored lines, indicating different types of interactions among the proteins. The identification of biosynthesis proteins is based on predictions from the STRING database.

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Discussion

BR, a plant-specific steroidal hormone, exhibits a vast range of stress-resistance functioning that influences different growth processes59. Additionally, the BR signal transduction system is essential for plant development. BZR1 proteins maintain interlinks in BR and other different pathways of signaling60. PhBEH2 is suggested to serve as a key component facilitating crosstalk between signaling pathways in the Petunia hybrid61. The BZR1 gene family has been documented in several horticultural crops such as Chinese cabbage, tomato, potato, and peas. Although the regulatory mechanisms of BZR1 transcription factors have been well studied in (A) thaliana and potato, their characterization in (B) oleracea remains unexamined. Investigating these genes in B. oleracea is essential for understanding their evolutionary patterns and functional roles for deeper insights into their regulatory functions. A comprehensive genome-wide analysis of BoBZR1 genes has been conducted, encompassing their identification, chromosomal localization, evolutionary relationships, structural organization, and gene duplication or loss events. Additionally, this study explored their functional aspects and expression patterns across specific tissues.

In the entire genome of B. oleracea, twelve BZR1 family genes have been identified. Notably, Arabidopsis contains six BZR members62. 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 brassinosteroids63. The physicochemical elements of the BoBZR1 proteins were carefully investigated. 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. These proteins had a lower molecular weight than BZR gene families from other crops, such as cucumber. All identified BZR1 proteins exhibited a conserved hydrophilic nature, indicating their potential role in water-associated cellular processes64. The placement of BoBZR1 genes across the genome was discovered to be uneven, similar to that seen in Chinese cabbage. Furthermore, the subcellular localization of these genes revealed information about their prospective cellular activities, as proteins frequently execute jobs unique to their organelle of localization. Notably, 83% of BoBZR1 genes were found in the nucleus, implying a primary involvement in transcriptional control, while the remaining 17% were found in the cytoplasm, indicating potential supplementary functional contributions outside the nucleus.

Comparative analysis of BZR1 genes across different crops provides valuable insights into the evolutionary relationships within the BoBZR1 gene family. Genes clustering within the same phylogenetic clade are likely to share similar functional roles, allowing predictions about the function of less-characterized genes based on their well-studied counterparts. To explore these evolutionary connections, a phylogenetic tree was constructed, revealing that BoBZR1 genes were grouped into three distinct clades. Notably, one clade included AtBZR1 (Arabidopsis), BoBZR1 (B. oleracea), and SlBZR1 (Tomato), suggesting that BoBZR1 genes may exhibit functional similarities with their Arabidopsis and tomato homologs, given their close evolutionary association. Earlier studies have emphasized the significance of the exons-introns arrangement of a gene in the evolutionary process65. 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-introns66. 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 features67. Furthermore, motif analysis revealed the presence of Motifs 1 and 3 in all BoBZR1 genes, which was similarly detected in CsBZR and AtBZR genes. The conservation of these motifs implies a functional relationship between these genes, as such motifs frequently play an important role in gene regulation and activity. Their continuous existence across species demonstrates their importance in regulating gene function, probably contributes to the systemic regulation of BoBZR1 genes.

The Brassica lineage is believed to have diverged from a common ancestor with Arabidopsis around 20 million years ago (Mya), followed by a Whole Genome Triplication event approximately 15.9 Mya, which led to extensive gene loss and rearrangement68. To explore the evolutionary history of BoBZR1 genes, a comparative syntenic analysis was performed among B. oleracea, Arabidopsis, and potato, identifying conserved collinear regions. A notably higher number of BoBZR1 gene copies were detected in Arabidopsis, suggesting a closer evolutionary relationship among these species. This indicates that these plants may have descended from a shared ancestor, with subsequent gene expansion and variation influenced by mutations and environmental adaptations, leading to species-specific differences in BoBZR1 gene distribution. 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. The Ka/Ks ratio helps in understanding the evolutionary pressures acting on genes. A Ka/Ks value below 1 indicates purifying selection, which removes harmful mutations to preserve gene function. In B. oleracea, the analysis of duplicated BoBZR1 genes showed Ka/Ks values below 1, suggesting they have mostly undergone purifying selection over time. Further examination of gene divergence and duplication events revealed variations in Ka/Ks ratios among different BoBZR1 genes, especially those that resulted from WGT. These differences indicate that BoBZR1 genes have evolved under varying selection pressures, leading to different rates of evolution within B. oleracea48.

Gene expression is crucial in the manifestation of functioning, and promoters play a direct role in influencing gene expression69. The promoter region of a gene includes specific elements that help regulate its activity in response to different factors. 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 mechanisms21,70, hormone signaling71, and the regulation of plant growth72. The analysis of BoBZR1 genes in relation to cuticular wax development revealed that BoBZR1-6, BoBZR1-7, and BoBZR1-10 showed over 1.2-fold higher expression compared to other genes. This increased expression may be associated with the presence of ABA-responsive cis-regulatory elements (CREs) in their promoter regions, suggesting their significant role in regulating growth and drought defense mechanisms. Similarly, in Arabidopsis, BZR1 supports growth maintenance by binding to the BRRE box, highlighting its role in improving growth adaptation73 and BES1 plays a crucial regulatory role in cellulose biosynthesis by binding to the promoter regions of cellulose synthase genes, suggesting its involvement in stress adaptation and cell wall remodeling under environmental challenges74. In wheat, the transcription factor TaBZR2, belonging to the BES/BZR family, plays a key role in drought response by reducing the accumulation of reactive oxygen species through the activation of TaGST, a gene involved in detoxification and stress tolerance75. RNA-Seq analysis demonstrated that BoBZR1 genes were consistently expressed across all examined tissues, indicating their fundamental role in cellular processes. This widespread expression suggests that BoBZR1 genes are key regulators in plant growth, development, and defense mechanisms, contributing to essential physiological functions throughout different stages of plant life76. The BoBZR1 genes displayed widespread expression across all plant organs and were significantly regulated by developmental signals. Notably, BoBZR1-6, 5, 9, 2, and 12 showed higher expression levels in leaves, while BoBZR1-11, 4, 8, 10, 1, and 3 were more predominantly expressed in stems. This organ-specific expression pattern suggests that BoBZR1 proteins play essential roles in plant growth and development. Similarly, in maize, the expression of ZmBES1/BZR1 genes varied under ABA, far-red, red, and dark light treatments, highlighting their involvement in adaptive responses to environmental stimuli31. Higher expression levels of BoBZR1-5, BoBZR1-9, BoBZR1-11, and BoBZR1-12 were observed during both vegetative and reproductive growth stages, suggesting their possible involvement in later developmental processes Additionally, the observed expression correlations among these genes (Fig. 7c) suggest potential interactions of BoBZR1 genes. These findings provide insights into the potential roles of BoBZR1 genes, laying the groundwork for future studies on gene regulatory networks and their roles in stress responses.

Conclusion

This study identified twelve BoBZR1 genes in B. oleracea through in-silico analysis and classified them into three subgroups based on their evolution. These genes were unevenly distributed across chromosomes, and their promoter regions contained regulatory elements associated with growth, stress responses, light signaling, and hormone regulation. Expression analysis showed that BoBZR1-2 had consistently high expression across multiple tissues, indicating its potential role in plant growth and development. Additionally, the involvement of BoBZR1 genes in cuticular wax biosynthesis suggests their contribution to plant defense against environmental stresses. These findings offer a foundation for future research to further explore the functional significance of BoBZR1 genes in stress adaptation and development.