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Proanthocyanidins are oligomeric flavonoids that promote plant disease resistance and benefit human health. Banana is one of the world's most extensively farmed crops and its fruit pulp contain proanthocyanidins. However, the transcriptional regulatory network that fine tunes proanthocyanidin biosynthesis in banana remains poorly understood.We characterised two proanthocyanidin-specific R2R3 MYB activators (MaMYBPA1-MaMYBPA2) and four repressors (MaMYBPR1-MaMYBPR4) to elucidate the mechanisms underlying the transcriptional regulation of proanthocyanidin biosynthesis in banana.Heterologous expression of MaMYBPA1 and MaMYBPA2 partially complemented the Arabidopsis thaliana proanthocyanidin-deficient transparent testa2 mutant. MaMYBPA1 and MaMYBPA2 interacted physically with MaMYCs to transactivate anthocyanin synthase, leucoanthocyanidin reductase, and anthocyanidin reductase genes in vitro and form functional MYB-bHLH-WD Repeat (MBW) complexes with MaTTG1 to transactivate these promoters in vivo.Overexpression of MaMYBPAs alone or with MaMYC in banana fruits induced proanthocyanidin accumulation and transcription of proanthocyanidin biosynthesis-related genes. MaMYBPR repressors are also shown to interact with MaMYCs forming repressing MBW complexes, and diminished proanthocyanidin accumulation. Interestingly overexpression of MaMYBPA induces the expression of MaMYBPR, indicating an agile regulation of proanthocyanidin biosynthesis through the formation of competitive MBW complexes. Our results reveal regulatory modules of R2R3 MYB-that fine tune proanthocyanidin biosynthesis and offer possible targets for genetic manipulation for nutritional improvement of banana.

SummaryProanthocyanidins are oligomeric flavonoid pigments that promote plant disease resistance and benefit human health. However, the transcriptional regulatory network that fine-tunes proanthocyanidin biosynthesis in banana (Musa acuminata) fruit remains poorly understood.We characterized two proanthocyanidin-specific R2R3 MYB activators (MaMYBPA1-MaMYBPA2) and four repressors (MaMYBPR1–MaMYBPR4) to elucidate the mechanisms underlying the transcriptional regulation of proanthocyanidin biosynthesis in banana.Heterologous expression of MaMYBPA1 and MaMYBPA2 partially complemented the Arabidopsis thaliana proanthocyanidin-deficient transparent testa2 mutant. MaMYBPA1 and MaMYBPA2 interacted physically with MaMYCs and bound the promoters of the genes encoding anthocyanin synthase, leucoanthocyanidin reductase, and anthocyanidin reductase in vitro and form functional MBW complexes with MaTTG1 in A. thaliana protoplasts to transactivated these promoters in vivo.Overexpression of MaMYBPAs alone or with MaMYC in banana fruits induced proanthocyanidin accumulation and the transcription of proanthocyanidin biosynthesis– related genes. MaMYBPR repressors are also shown to interact with MaMYCs, forming repressing MBW complexes, and diminished proanthocyanidin accumulation. Interestingly the overexpression of MaMYBPA induces the expression of MaMYBPR, indicating an agile regulation of proanthocyanidin biosynthesis via the formation of competitive MBW complex. Taken together, our results reveal regulatory modules of R2R3 MYB- that fine-tune proanthocyanidin biosynthesis and offer possible targets for genetic manipulation in banana.

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