Glucosinolates (GSLs) are secondary plant metabolites that are enriched in rapeseed and related Brassica species, and they play important roles in defense due to their anti-nutritive and toxic properties. Here, we conducted a genome-wide association study of six glucosinolate metabolites (mGWAS) in rapeseed, including three aliphatic glucosinolates (m145 gluconapin, m150 glucobrassicanapin and m151 progoitrin), one aromatic glucosinolate (m157 gluconasturtiin) and two indole glucosinolates (m165 indolylmethyl glucosinolate and m172 4-hydroxyglucobrassicin), respectively. We identified 113 candidate intervals significantly associated with these six glucosinolate metabolites. In the genomic regions linked to the mGWAS peaks, 187 candidate genes involved in glucosinolate biosynthesis (e.g., BnaMAM1, BnaGGP1, BnaSUR1 and BnaMYB51) and novel genes (e.g., BnaMYB44, BnaERF025, BnaE2FC, BnaNAC102 and BnaDREB1D) were predicted based on the mGWAS, combined with analysis of differentially expressed genes. Our results provide insight into the genetic basis of glucosinolate biosynthesis in rapeseed and should facilitate marker-based breeding for improved seed quality in Brassica species.

The TIFY gene family plays important roles in various plant biological processes and responses to stress and hormones. The chromosome-level genome of the Brassiceae species has been released, but knowledge concerning the TIFY family is lacking in the Brassiceae species. The current study performed a bioinformatics analysis on the TIFY family comparing three diploid (B. rapa, B. nigra, and B. oleracea) and two derived allotetraploid species (B. juncea, and B. napus). A total of 237 putative TIFY proteins were identified from five Brassiceae species, and classified into ten subfamilies (six JAZ types, one PPD type, two TIFY types, and one ZML type) based on their phylogenetic relationships with TIFY proteins in A. thaliana and Brassiceae species. Duplication and synteny analysis revealed that segmental and tandem duplications led to the expansion of the TIFY family genes during the process of polyploidization, and most of these TIFY family genes (TIFYs) were subjected to purifying selection after duplication based on Ka/Ks values. The spatial and temporal expression patterns indicated that different groups of BnaTIFYs have distinct spatiotemporal expression patterns under normal conditions and heavy metal stresses. Most of the JAZIII subfamily members were highest in all tissues, but JAZ subfamily members were strongly induced by heavy metal stresses. BnaTIFY34, BnaTIFY59, BnaTIFY21 and BnaTIFY68 were significantly upregulated mostly under As³⁺ and Cd²⁺ treatment, indicating that they could be actively induced by heavy metal stress. Our results may contribute to further exploration of TIFYs, and provided valuable information for further studies of TIFYs in plant tolerance to heavy metal stress.

B-box (BBX) proteins, a subfamily of zinc-finger transcription factors, are involved in various environmental signaling pathways. In this study, we conducted a comprehensive analysis of BBX family members in Brassica crops. The 482 BBX proteins were divided into five groups based on gene structure, conserved domains, and phylogenetic analysis. An analysis of nonsynonymous substitutions and (Ka)/synonymous substitutions (Ks) revealed that most BBX genes have undergone purifying selection during evolution. An analysis of transcriptome data from rapeseed (Brassica napus) organs suggested that BnaBBX3d might be involved in the development of floral tissue-specific RNA-seq expression. We identified numerous light-responsive elements in the promoter regions of BnaBBX genes, which were suggestive of participation in light signaling pathways. Transcriptomic analysis under shade treatment revealed 77 BnaBBX genes with significant changes in expression before and after shading treatment. Of these, BnaBBX22e showed distinct expression patterns in yellow- vs. black-seeded materials in response to shading. UPLC-HESI-MS/MS analysis revealed that shading influences the accumulation of 54 metabolites, with light response BnaBBX22f expression correlating with the accumulation of the flavonoid metabolites M46 and M51. Additionally, BnaBBX22e and BnaBBX22f interact with BnaA10.HY5. These results suggest that BnaBBXs might function in light-induced pigment accumulation. Overall, our findings elucidate the characteristics of BBX proteins in six Brassica species and reveal a possible connection between light and seed coat color, laying the foundation for further exploring the roles of BnaBBX genes in seed development.

Calmodulin-binding transcription activators (CAMTAs) are pivotal regulators decoding calcium signals, with crucial roles in plant development, hormone responses, and adaptation to abiotic stresses. Although extensive research has been conducted on CAMTAs in model plants such as Arabidopsis thaliana, a comprehensive genome-wide analysis of the CAMTA gene family across the economically important Brassica U-triangle species has not been performed. In this study, we systematically identified and characterized 64 CAMTA genes from the genomes of Brassica U-triangle species. Phylogenetic analysis classified these genes into four conserved groups, a finding corroborated by analyses of gene structure and conserved motifs. These analyses revealed strong evolutionary preservation of functional domains, especially the calmodulin-binding domain (CaMBD). Chromosomal distribution and collinearity assessment highlighted the significant impact of polyploidization on the expansion of the CAMTA family, with most orthologous pairs being under purifying selection. Cis-element analysis in promoters uncovered an abundance of stress- and hormone-related elements, suggesting diverse regulatory roles for these genes. Furthermore, RNA-Seq and RT-qPCR expression profiling demonstrated that BnaCAMTA genes exhibit tissue-specific expression and are dynamically responsive to various phytohormones (ABA, JA, and GA) and abiotic stresses (salt and drought), particularly in the root. Notably, BnaCAMTA5.2, which was prioritized among several validated candidates, mediates the antagonistic regulation of hypocotyl and root growth under GA and salt stress, indicating its key role in balancing growth promotion and stress adaptation. Additionally, we identified a set of stress-related miRNAs that potentially target BnaCAMTAs, suggesting a potential layer of post-transcriptional regulation. Our results provide valuable insights into the evolutionary and functional diversity of CAMTA genes in Brassica U-triangle species and lay a foundation for further research into their roles in enhancing stress resistance in B. napus.