Identification and functional characterisation of MYB and bHLH transcription factors which activate anthocyanin biosynthesis in banana ( Musa spp.)

Anthocyanins are flavonoid pigments that contribute to fruit quality, plant stress tolerance, and potential health benefits for consumers, yet they are absent from the fruit of Cavendish banana (Musa spp., AAA subgroup), the world’s most widely cultivated banana. Here, we identified and functionally characterised endogenous transcription factors that activate anthocyanin biosynthesis in banana. Comparative transcriptomic analysis of anthocyanin-rich purple and adjacent green sectors of juvenile Cavendish leaf tissue revealed coordinated upregulation of flavonoid biosynthetic genes and several transcription factors. Among these, two previously uncharacterised candidates, an R2R3-MYB and a basic helix–loop–helix (bHLH) protein, were prioritised and designated MaMYBA3 and MabHLHIIIf1, respectively. Stable co-expression of MaMYBA3 and MabHLHIIIf1 in Cavendish bananas resulted in strong anthocyanin accumulation across multiple vegetative tissues, with total concentrations reaching over 1300 mg per 100 g dry weight and profiles dominated by cyanidin glycosides. Anthocyanin accumulation correlated with transcriptional activation of key structural genes in the biosynthetic pathway. In contrast, fruit tissues of transgenic lines accumulated substantially lower anthocyanin levels and displayed a shift toward delphinidin-type anthocyanins despite strong transgene expression and pathway induction. These differences were associated with differential expression of transcriptional repressors and biosynthetic branch-point genes, suggesting tissue-specific regulatory influence. Collectively, our results demonstrate that Cavendish banana possesses an intact but tightly regulated anthocyanin biosynthetic pathway that can be activated through endogenous MYB-bHLH transcription factors. These findings provide evidence for tissue-dependent constraints on flavonoid pathway activation in Musa and establishes a molecular framework for developing visually distinctive and nutritionally enhanced banana cultivars.