The safflower MBW complex regulates HYSA accumulation through degradation by the E3 ligase CtBB1

Abstract The regulatory mechanism of the MBW (MYB‐bHLH‐WD40) complex in safflower ( Carthamus tinctorius ) remains unclear. In the present study, we show that the separate overexpression of the genes CtbHLH41 , CtMYB63 , and CtWD40‐6 in Arabidopsis thaliana increased anthocyanin and procyanidin contents in the transgenic plants and partially rescued the trichome reduction phenotype of the corresponding bhlh41 , myb63 , and wd40‐6 single mutants. Overexpression of CtbHLH41 , CtMYB63 , or CtWD40‐6 in safflower significantly increased the content of the natural pigment hydroxysafflor yellow A (HYSA) and negatively regulated safflower petal size. Yeast‐two‐hybrid, functional, and genetic assays demonstrated that the safflower E3 ligase CtBB1 (BIG BROTHER 1) can ubiquitinate CtbHLH41, marking it for degradation through the 26S proteasome and negatively regulating flavonoid accumulation. CtMYB63/CtWD40‐6 enhanced the transcriptional activity of CtbHLH41 on the CtDFR ( dihydroflavonol 4‐reductase ) promoter. We propose that the MBW‐ CtBB1 regulatory module may play an important role in coordinating HYSA accumulation with other response mechanisms.