R2R3-MYB repressor, BrMYB32, regulates anthocyanin biosynthesis in Chinese cabbage

Anthocyanin-enriched Chinese cabbage has health-enhancing antioxidant properties. Although various regulators of anthocyanin biosynthesis have been identified, the role of individual repressors in this process remains underexplored. This study identifies and characterizes the R2R3-MYB BrMYB32 in Chinese cabbage (Brassica rapa), which acts as a repressor in anthocyanin biosynthesis. BrMYB32 expression is significantly upregulated under anthocyanin inductive conditions, such as sucrose and high light treatment. Transgenic tobacco plants overexpressing BrMYB32 show decreased anthocyanin levels and downregulation of anthocyanin biosynthesis genes in flowers, highlighting BrMYB32's repressive role. Located in the nucleus, BrMYB32 interacts with the TRANSPARENT TESTA 8 (BrTT8), a basic helix-loop-helix protein, but no interaction was detected with the R2R3-MYB protein PRODUCTION OF ANTHOCYANIN PIGMENT 1 (BrPAP1). Functional assays in Chinese cabbage cotyledons and tobacco leaves demonstrate that BrMYB32 represses the transcript level of anthocyanin biosynthesis genes, thereby inhibiting pigment accumulation. Promoter activation assays further reveal that BrMYB32 inhibits the transactivation of CHALCONE SYNTHASE and DIHYDROFLAVONOL REDUCTASE through the C1 and C2 motifs. Notably, BrMYB32 expression is induced by BrPAP1, either alone or in co-expression with BrTT8, and subsequently regulates the expression of these activators. It verifies that BrMYB32 not only interferes with the formation of an active MYB-bHLH-WD40 complex but also downregulates the transcript levels of anthocyanin biosynthesis genes, thereby fine-tuning anthocyanin biosynthesis. Our findings suggest a model in which anthocyanin biosynthesis in Chinese cabbage is precisely regulated by the interplay between activators and repressors.