BnaPAP2.C2 plays a novel inhibitory role in anthocyanin accumulation compared to its paralogs in rapeseed ( Brassica napus L.)

Tissue-specific anthocyanin pigmentation is observed in rapeseed (Brassica napus L. AACC, 2n = 38) as well as in its ancestral diploids Brassica rapa (AA, 2n = 20) and Brassica oleracea (CC, 2n = 18). We previously identified the MYB genes BnaPAP2.A7b and BnaPAP2.C6a as key regulators of anthocyanin biosynthesis. Here we uncover an antagonistic regulatory mechanism in leaves involving their paralog BnaPAP2.C2. Unlike the pigmentation-associated genes, BnaPAP2.C2 is constitutively expressed in both green and purple leaves, regardless of anthocyanin levels. Its promoter contains two enhancers (463 and 486 bp) that synergistically regulate transcription. Competitive binding studies reveal that BnaPAP2.C2, although lacking activation capacity, sequesters BnaTT8 and outcompetes BnaPAP2.A7b, thereby suppressing anthocyanin biosynthesis. Under environmental stress, elevated expression of BnaPAP2.A7b promotes anthocyanin biosynthesis, whereas BnaPAP2.C2 is downregulated. This paralog-specific molecular antagonism provides new insight into the evolution of MYB-bHLH interaction specificity. Together, these findings uncover a novel inhibitory mechanism within the anthocyanin regulatory hierarchy of polyploid rapeseed, highlighting competitive binding as an evolutionary innovation driving functional diversification of duplicated MYB regulators.