The regulation of light-dependent anthocyanin biosynthesis in Brassica rapa subsp. rapa cv. Tsuda turnip was investigated using an ethyl methanesulfonate (EMS)-induced mutant R30 with light-independent pigmentation. TILLING (targeting induced local lesions in genomes) and subsequent analysis showed that a stop codon was inserted in the R2R3-MYB transcription factor gene BrMYB4 and that the encoded protein (BrMYB4mu) had lost its C-terminal region. In R30, anthocyanin accumulated in the below-ground portion of the storage root of 2-month-old plants. In 4-day-old seedlings and 2-month-old plants, expression of BrMYB4 was similar between R30 and the wild type (WT), but the expression of the cinnamate 4-hydroxylase gene (BrC4H) was markedly enhanced in R30 in the dark. In turnip seedlings, BrMYB4 expression was suppressed by UV-B irradiation in the WT, but this negative regulation was absent in R30. Concomitantly, BrC4H was repressed by UV-B irradiation in the WT, but stayed at high levels in R30. A gel-shift assay revealed that BrMYB4 could directly bind to the promoter region of BrC4H, but BrMYB4mu could not. The BrMYB4-enhanced green fluorescent protein (eGFP) protein could enter the nucleus in the presence of BrSAD2 (an importin b-like protein) nuclear transporter, but BrMYB4mu-eGFP could not. These results showed that BrMYB4 functions as a negative transcriptional regulator of BrC4H and mediates UV-B-dependent phenylpropanoid biosynthesis, while BrMYB4mu has lost this function. In the storage roots, the expression of anthocyanin biosynthesis genes was enhanced in R30 in the dark and in sunlight in both the WT and R30. However, in the WT, anthocyanin-inducing sunlight did not suppress BrMYB4 expression. Therefore, sunlight-induced anthocyanin biosynthesis does not seem to be regulated by BrMYB4.
