MaMYB12 and MaMYB308 antagonistically regulate flavonoid biosynthesis in mulberry (Morus alba): Implications for...

Mulberry (Morus alba), a tree with multipurpose applications, is a promising source of bioactive flavonoids for functional foods. Mulberry-derived flavonoids possess prominent antioxidant, anti-inflammatory, and cardioprotective activities, making them valuable functional ingredients for nutraceutical products. Elucidating the transcriptional regulation of flavonoid biosynthesis is essential for targeted molecular breeding to enhance flavonoid accumulation in mulberry, thereby supporting sustainable production of high-value food ingredients. In this study, five R2R3-MYB transcription factors whose expression correlates with flavonoid accumulation during mulberry fruit development were identified. Among these, MaMYB12 (subgroup 7, SG7) functions as a transcriptional activator. It transient overexpression in mulberry leaves significantly elevated the content of 30 flavonoid metabolites by directly activating the promoters of key flavonoid biosynthetic genes. In contrast, MaMYB308 (SG4) functions as a strong repressor, reducing the accumulation of 106 flavonoid metabolites through directly suppressing of key flavonoid biosynthetic genes. The direct binding of both MaMYB12 and MaMYB308 to target gene promoters was verified by yeast one-hybrid assays and electrophoretic mobility shift assays. Their antagonistic regulatory roles were further confirmed in stably transformed mulberry hairy roots. Additionally, extracts from MaMYB12-overexpressing hairy roots exhibited significantly enhanced antioxidant activity, directly linking this genetic manipulation to improved functional food properties. Our findings uncover a key transcriptional module governing flavonoid metabolism in mulberry and present practical genetic tools for the molecular breeding of mulberry varieties with optimized flavonoid profiles, advancing their utility as functional food ingredients.