Brassinolide inhibits flavonoid biosynthesis and red-flesh coloration via the MdBEH2.2-MdMYB60 complex in apple

Flavonoid content, which is an important indicator of the nutritional value of fruits and vegetables, directly determines the marketability of many fruit crops, including apple (Malus domestica). Brassinosteroids (BRs) are steroid hormones that affect flavonoid biosynthesis in plants, but the underlying regulatory mechanism remains unclear. In this study, treatments with brassinolide (the most active BR) and brassinazole (a BR biosynthesis inhibitor) decreased and increased, respectively, the flavonoid, anthocyanin, and proanthocyanidin (PA) content in red-fleshed apple seedlings and calli. We subsequently demonstrated that a BZR (BRI1-EMS-suppressor (BES)/brassinazole-resistant) family transcription factor, MdBEH2.2, participates in BR-regulated flavonoid biosynthesis. Specifically, MdBEH2.2 inhibits the accumulation of flavonoids, anthocyanins, and PAs in apple seedlings; however, brassinazole treatment weakens the inhibitory effect. Additionally, we confirmed that a BR-induced MYB TF, MdMYB60, interacts with MdBEH2.2. The resulting MdBEH2.2-MdMYB60 complex further enhances the inhibitory effect of MdBEH2.2 or MdMYB60 on the transcription of flavonoid biosynthesis-related genes. These results indicate that brassinolide decreases flavonoid content through the MdBEH2.2-MdMYB60 regulatory module. Our findings further clarify the molecular mechanism mediating the regulation of flavonoid biosynthesis by BR signals in horticultural crops.