FhMYB4 : a dual‐pathway repressor coordinating floral color and scent in Freesia hybrida

Floral color and scent often covary during development (e.g. peaking at anthesis to attract pollinators), suggesting shared regulatory mechanisms. However, transcription factors simultaneously repressing anthocyanin and volatile terpene biosynthesis are rarely reported. This study explores the versatile roles of FhMYB4, a subgroup 4 R2R3-MYB repressor in Freesia hybrida. The study employed integrated approaches including gene expression analysis, protein-protein/promoter interaction assays, and transient petal/leaf/protoplast experiments to investigate the functional mechanisms of FhMYB4 and the potential applications of its engineered variants underlying anthocyanin and linalool biosynthesis. FhMYB4 simultaneously inhibits anthocyanin and linalool biosynthesis via dual mechanisms: direct binding to MYB cis-elements in the promoters of FhDFR3 (anthocyanin) and FhTPS1 (linalool), and interaction with the bHLH factor FhTT8L to disrupt the anthocyanin-related MYB-bHLH-WD40 (MBW) activation complex. Intriguingly, FhMYB4 also forms a negative feedback loop by being transcriptionally activated by upstream MYB activators (FhPAP1 and FhMYB21L2) while autorepressing its own promoter. Engineering FhMYB4 variants (e.g. ethylene-responsive element binding factor-associated amphiphilic repression motif mutation fused to VP16) convert them into transcriptional activators, enhancing both anthocyanin and linalool pathways. This study reveals FhMYB4 as a novel integrator of floral secondary metabolism, providing tools for biotechnological improvement of ornamental traits in flower crops.