Abstract
Proanthocyanidins (PAs), anthocyanins, and flavonols are key flavonoids that play diverse roles in plant physiology and human health. Despite originating from a shared biosynthetic pathway, the regulatory mechanisms of PA biosynthesis and the cooperative regulation of different kinds of flavonoids remain elusive, particularly in flower tissues or organs. Here, we elucidated the regulatory network governing PA biosynthesis in Freesia hybrida 'Red River®' by characterizing four TT2-type MYB transcription factors, designated FhMYBPAs. Phylogenetic analysis, subcellular localization, and transactivation assays predicted their roles as PA-related activators. Pearson correlation analysis revealed significant correlations between FhMYBPAs and PA accumulation in various floral tissues and development stages. Functional studies demonstrated that FhMYBPAs activated PA biosynthesis by directly binding to the promoters of target genes, which can be enhanced by FhTT8L. Additionally, a hierarchical and feedback regulatory model involving FhTTG1, FhMYB27, and FhMYBx was proposed for PA biosynthesis. Furthermore, comparative analysis of flavonoid-related MYB factors involving FhPAP1, FhMYB5, FhMYBF1, and FhMYB21L2 highlighted their roles in regulating PA, anthocyanin, and flavonol biosynthesis, with some exhibiting versatile regulations. Overall, our findings provide insights into the spatio-temporal regulation of flavonoids in flowers and expand our understanding of MYB-mediated transcriptional regulation of specialized metabolites in plants.