Molecular mechanism controlling anthocyanin composition and content in radish plants with different root colors

Radish (Raphanus sativus) roots exhibit various colors that reflect their anthocyanin compositions and contents. However, the details of the mechanism linking the expression of anthocyanin biosynthesis and their transcriptional regulators to anthocyanin composition in radish roots remained unknown. Here, we characterized the role of the anthocyanin biosynthetic enzyme flavonoid 3'-hydroxylase (RsF3'H), together with the R2R3 MYB transcription factor (TF) RsMYB1 and the basic helix-loop-helix (bHLH) TF TRANSPARENT TESTA 8 (RsTT8), in four radish plants with different root colors: white (W), deep red (DR), dark purple (DP), and dark greyish purple (DGP). The DR plant contained heterozygous for RsF3'H with low expression level and accumulated a large amount of pelargonidin, resulting in deep red color. While, the DP and DGP plants accumulated the cyanidin due to the higher expression level of functional RsF3'H. Notably, RsMYB1 and RsTT8 transcripts were abundant in all pigmented roots, but not in white roots. To investigate the differential expression of RsMYB1 and RsTT8, we compared the sequences of their promoter regions among the four radish plants, revealing variations in the numbers of cis-elements and in promoter architecture. Promoter activation assays demonstrated that variation in the RsMYB1 and RsTT8 promoters may contribute to the expression level of these genes, and RsMYB1 can activate its own expression as well as promote the RsTT8 expression. These results suggested that RsF3'H plays a vital role in anthocyanin composition and the expression level of both RsMYB1 and RsTT8 are crucial determinants for anthocyanin content in radish roots. Overall, these findings provide insight into the molecular basis of anthocyanin composition and level in radish roots.