Banana, a globally important staple fruit, is naturally deficient in anthocyanins; however, successful engineering of anthocyanin-enriched banana has not been reported to date. Herein, a regulatory network of five R2R3-MYBs (MusaUP1, MusaUP2, MbaMA2, MusaMA4, and MusaMA8) differentially synchronizing anthocyanin biosynthesis in banana bract is reported. RNA-seq data of red bract revealed a web of regulatory and structural genes fine-tuning anthocyanin accumulation through amalgamation of MYBs and bHLHs activities. Yeast one-hybrid (Y1H) demonstrated differential affinities of these MusaMYBs to banana TT8, CHS, ANR, UFGT, FLS, ANS, and LAR, revealing an intricate pattern of layered regulation in bract pigmentation. Functional competence of this MYB network resulted in intense anthocyanin accumulation in whitish onion and restoration of pigmentation in myb90/tt8 Arabidopsis seedlings. Hierarchical regulation in this MYB network stemmed from contrasting control over early and late flavonoid structural genes as revealed by disparate orange fluorescence of myb90/tt8 Arabidopsis seedlings after DPBA staining. In banana, a distinctive requirement of TT8 for pigmentation was observed for MbaMA2 and MusaMA8, while MusaUP1, MusaUP2, and MusaMA4 were self-competent, although co-expression of MusaTT8 augmented the ectopic pigmentation effect. Transcript abundance of flavonoid structural genes in transgenic banana is in coherence with Y1H data, thus catalysing pigmentation up to 500-fold over control. This regulatory MYBs hierarchical framework manifested flux in a spectrum of distinct pigment metabolites, viz peonidin-3,5-diglucoside in MusaUP1 and MusaUP2, dalbergiodin in MbaMA2/TT8 lines (FLS-mediated pathway), leucodelphinidin and leucopelargonidin in MusaMA4 lines (DFR to ANS flux), and prodelphinidin B4 in MusaMA8 lines. This study will be a step forward towards metabolic engineering for bio-fortification of banana and development of functional foods, as evident by strong antioxidant activities of these MYB lines.

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