Systematic identification of R2R3-MYB S6 subfamily genes in Brassicaceae and its role in anthocyanin biosynthesis in Brassica crops

Abstract

Abstract The Brassicaceae family encompasses various plants, including the widely studied Arabidopsis thaliana and several vegetables and oil crops that hold significant economic importance as human food sources. The S6 subfamily's R2R3-MYB genes play a crucial role in regulating anthocyanin biosynthesis in plants, however, their systematic identification in Brassicaceae plants remains incomplete. Notably, Brassica crops have undergone significant genomic changes, including tripling and post-natural hybridization doubling events, during their long evolutionary journey after diverging from Arabidopsis. Consequently, the copy number of R2R3-MYB genes has experienced substantial alterations, and its functions may be significantly differentiated. Hence, Brassica crops present an optimal model for investigating copy number variation and functional divergence of S6 subfamily R2R3-MYB genes. In this investigation, we systematically identified 31 homologous genes of R2R3-MYB transcription factors belonging to the S6 subfamily in Brassicaceae. A total of 92 homologous genes were identified, with species representation ranging from 0 to 10. Phylogenetic analysis revealed the classification of these homologous genes into six distinct groups. Notably, approximately 70% of the homologous genes were found within the G6 group, suggesting a high degree of evolutionary conservation. Moreover, a phylogenetic analysis was performed on 35 homologous genes obtained from six species belonging to the Brassica U's triangle. The findings provided evidence of high conservation among orthologous genes across species and demonstrated strong collinearity on subgenomic chromosomes. However, notable tandem duplications were observed on chromosomes A7 and C6. Subsequently, the cis-acting elements of these 35 homologous genes were predicted, and their structures, conserved motifs, and characteristic conserved domains were analyzed. Once again, the results confirmed the significant similarities between orthologous genes. Simultaneously, we employed white and purple flower rapeseed specimens to perform qRT-PCR validation of the principal genes and transcriptional regulators associated with the anthocyanin synthesis pathway. The outcomes revealed a significant differential expression of BnaPAP2.A7.b in purple flowers, alongside the differential expression of BnaPAP2.C6.d. Ultimately, drawing upon prior research findings and the findings of this investigation, a transcriptional regulatory framework was proposed to govern anthocyanin accumulation in distinct tissues or organs of B. napus. The findings of our study offer novel perspectives on the functional diversification of R2R3-MYB transcription factors within the S6 subfamily homologous genes, while also shedding light on the regulatory network governing anthocyanin biosynthesis in species belonging to the Brassicaceae family.