The HSF-DREB-MYB transcriptional regulatory module regulates flavonol biosynthesis and flavonoid B-ring hydroxylation in banana ( Musa acuminata )

Abstract Plant flavonols act primarily as ultraviolet radiation absorbers, reactive oxygen species scavengers, and phytoalexins, and they contribute to biotic and abiotic stress tolerance in plants. Banana ( Musa acuminata ), an herbaceous monocot and important fruit crop, accumulates flavonol derivatives in different organs, including the edible fruit pulp. Although flavonol content varies greatly in different organs, the molecular mechanisms involving transcriptional regulation of flavonol synthesis in banana are not known. Here, we characterized three SG7-R2R3 MYB transcription factors (MaMYBFA1, MaMYBFA2, and MaMYBFA3) and their upstream regulators, heat shock transcription factor (MaHSF11) and dehydration responsive element binding factor (MaDREB1), to elucidate the molecular mechanism involved in transcriptional regulation of flavonol biosynthesis in banana. MaMYBFA positively regulate flavonol synthase 2 ( MaFLS2) and downregulates MaFLS1 . We show these transcription factors to be weak regulators of flavonol synthesis. Overexpression of MaHSF11 enhances flavonol contents, particularly that of myricetin, and promotes flavonol B-ring hydroxylation, which contributes to the diversity of flavonol derivatives. MaHSF11 directly interacts with the MaFLS1 and flavonoid 3 ′ , 5 ′ -hydroxylase 1 ( MaF3 ′ 5 ′ H1) promoters, both in vitro and in vivo . MaHSF11 activates the expression of MaDREB1 directly, which in turn regulates the expression of MaMYBFA3 . Overall, our study elucidates a novel regulatory mechanism for flavonol synthesis in banana and suggests possible targets for genetic optimization to enhance nutritional value and stress responses in this globally important fruit crop. Significance statement Our study reveals that the three stress-responsive SG7 MYB factors, MaMYBFA1-3, are modest regulators of flavonol biosynthesis in banana. In contrast, the drought– and heat-responsive MaHSF11 plays a strong, positive role in this pathway, enhancing flavonol biosynthesis and flavonoid B-ring hydroxylation by upregulating key biosynthetic genes, such as MaFLS1 and MaF3’5’H1 , as well as MaDREB1 , another regulator of flavonol biosynthesis in banana.