Brassica species include important oil crops and vegetables in the world. The R2R3-MYB gene participates in a variety of plant functions, including the activation or inhibition of anthocyanin biosynthesis. Although previous studies have reported its phylogenetic relationships, gene structures, and expression patterns in Arabidopsis, the number and sequence variation of this gene family in Brassica crops and its involvement in the natural quantitative variation in anthocyanin biosynthesis regulation are still largely unknown. In this study, by using whole genome sequences and comprehensive genome-wide comparative analysis among the six cultivated Brassica species, 2120 R2R3-MYB genes were identified in six Brassica species, in total These R2R3-MYB genes were phylogenetically clustered into 12 groups. The R2R3-MYB family between A and C subgenomes showed better collinearity than between B and C and between A and B. From comparing transcriptional changes of five Brassica species with the purple and green leaves for the detection of the R2R3-MYB genes associated with anthocyanin biosynthesis, 7 R2R3-MYB genes were co-differentially expressed. The promoter and structure analysis of these genes showed that some variations between non-coding region, but they were highly conserved at the protein level and spatial structure. Co-expression analysis of anthocyanin-related genes and R2R3-MYBs indicated that MYB90 was strongly co-expressed with TT8, and they were co-expressed with structural genes F3H, LDOX, ANS and UF3GT at the same time. These results further clarified the roles of the R2R3-MYBs for leaf coloration in Brasica species, which provided new insights into the functions of the R2R3-MYB gene family in Brasica species.

Brassica napus is one of the most important oil crops in the world. Breeding oilseed rape with colorful flowers can greatly enhance the ornamental value of B. napus and thus improve the economic benefits of planting. As water-soluble flavonoid secondary metabolites, anthocyanins are very important for the synthesis and accumulation of pigments in the petals of plants, giving them a wide range of bright colors. Despite the documentation of over 60 distinct flower shades in B. napus, the intricacies underlying flower color variation remain elusive. Particularly, the mechanisms driving color development across varying flower color backgrounds necessitate further comprehensive investigation. This research undertook a comprehensive exploration through the integration of transcriptome and metabolome analyses to pinpoint pivotal genes and metabolites underpinning an array of flower colors, including beige, beige-red, yellow, orange-red, deep orange-red, white, light-purple, and purple. First, we used a two-way BLAST search to find 275 genes in the reference genome of B. napus Darmor v10 that were involved in making anthocyanins. The subsequent scrutiny of RNA-seq outcomes underscored notable upregulation in the structural genes F3H and UGT, alongside the MYB75, GL3, and TTG1 transcriptional regulators within petals, showing anthocyanin accumulation. By synergizing this data with a weighted gene co-expression network analysis, we identified CHS, F3H, MYB75, MYB12, and MYB111 as the key players driving anthocyanin synthesis in beige-red, orange-red, deep orange-red, light-purple, and purple petals. By integrating transcriptome and weighted gene co-expression network analysis findings with anthocyanin metabolism data, it is hypothesized that the upregulation of MYB75, which, in turn, enhances F3H expression, plays a pivotal role in the development of pigmented oilseed rape flowers. These findings help to understand the transcriptional regulation of anthocyanin biosynthesis in B. napus and provide valuable genetic resources for breeding B. napus varieties with novel flower colors.

Introduction: Cauliflower is widely cultivated all over the world is attributed to its palatable flavor, high levels of anti-cancer compounds, and diverse array of nutrients. Exposure to extremely cold stress during production can result in a more frequent occurrence of purple discoloration in cauliflower curds. In response to cold stress, plants naturally produce anthocyanins to eliminate reactive oxygen species (ROS) generated as a defense mechanism.Methods: This research involved conducting mRNA sequencing analysis on cauliflower curds both before and after exposure to cold stress treatment.Results: It was determined that the up-regulation of anthocyanin biosynthesis-related genes CHS, CHI, DFR, ANS, UGFT, PAP1/2, and MYBL2 occurred significantly in response to cold stress, resulting in a significant increase in total anthocyanin content. Subsequently, miRNA sequencing was employed to identify miRNAs in cauliflower curds, followed by differential expression analysis. The results showed that Bna-miR289 and Ath-miR157a may play a key role in regulating the accumulation of anthocyanin in cauliflower curds. Furthermore, we utilized degradome sequencing data to predict the target genes of the identified miRNAs, resulting in the identification of BolK_3g48940.1, BolK_9g11680.1, BolK_7g41780.1, BolK_3g68050.1, and BolK_3g729700.1 as targets. Subsequently, the expression patterns of the miRNAs and their target genes were validated using qRT-PCR, the results showed that Ath-miR157a and its target genes BolK_3g68050.1 and BolK_3g72970.1 may be the key to the purple of cauliflower curds under cold stress. Discussion: Our preliminary findings identified key miRNAs and their target genes that may be involved in regulating anthocyanin synthesis, thereby enhancing the cold tolerance of cauliflower through mRNA, miRNA, and degradome sequencing. Overall, our study sheds light on the activation of anthocyanin synthesis in flower curds under cold stress conditions as a mechanism to enhance resilience to adverse environmental conditions.

The Brassicaceae family includes the model plant Arabidopsis thaliana, along with various vegetables and oil crops, which possess significant economic and scientific value. Notably, three diploid species within the U’s Triangle of Brassica have undergone natural hybridization, resulting in the formation of three allotetraploid species, which provides an excellent model for investigating the phylogenetic, evolutionary, and functional differentiation of plant homologous genes. In this study, we systematically identified MYBL2 homologous genes within the 31 Brassicaceae species, with a total of 48 homologous genes identified from 30 species, and phylogenetic analysis revealed the presence of six subfamilies, Ka/Ks analysis showed that only 10 MYBL2 homologous gene were positively selected during evolution. We subsequently concentrated on the evolution, gene structure, and conserved domain analysis of 16 MYBL2 homologous genes across six Brassica crops found in U’s Triangle. Our findings indicated that these 16 MYBL2 homologous genes predominantly clustered into two branches and exhibited a high degree of evolutionary conservation. Further RNA-seq analysis of various tissues and organs from Brassica crops demonstrated that MYBL2 homologous genes were significantly up-regulated in tissues with anthocyanin accumulation. Concurrently, we employed Weighted Gene Co-expression Network Analysis (WGCNA) to identify hub genes regulated by anthocyanin in different tissues of B. napus, revealing that BnaMYBL2.C06 exhibited a strong repressor with anthocyanin biosynthetic genes (ABGs) in petals. Finally, quantitative reverse transcription PCR (qRT-PCR) analysis of B. napus leaves, stems, and petals indicated that four MYBL2 homologous genes were significantly up-regulated in leaves and petals, with expression patterns consistent with those of ABGs. Our results contribute new insights into the transcriptional regulatory mechanisms of anthocyanin in Brassica crops.

Brassica species include important oil crops and vegetables in the world. The R2R3-MYB gene participates in a variety of plant functions, including the activation or inhibition of anthocyanin biosynthesis. Although previous studies have reported its phylogenetic relationships, gene structures, and expression patterns in Arabidopsis, the number and sequence variation of this gene family in Brassica crops and its involvement in the natural quantitative variation in anthocyanin biosynthesis regulation are still largely unknown. In this study, by using whole genome sequences and comprehensive genome-wide comparative analysis among the six cultivated Brassica species, 1990 R2R3-MYB genes were identified in six Brassica species, respectively. These R2R3-MYB genes were phylogenetically clustered into 12 groups. The R2R3-MYB family between A and C subgenomes showed better collinearity than between B and C and between A and B. From comparing transcriptional changes of five Brassica species with the purple and green leaves for the detection of the R2R3-MYB genes associated with anthocyanin biosynthesis, 7 R2R3-MYB genes were co-differentially expressed. The promoter and structure analysis of these genes showed that some variations between non-coding region, but they were highly conserved at the protein level and spatial structure. Co-expression analysis of anthocyanin-related genes and R2R3-MYBs indicated that MYB90 was strongly co-expressed with TT8, and they were co-expressed with structural genes F3H, LDOX, ANS and UF3GT at the same time. These results further clarified the roles of the R2R3-MYBs for leaf coloration in Brasica species, which provided new insights into the functions of the R2R3-MYB gene family in Brasica species.