Comparative Genomics and Functional Characterization Reveal FaERF4 as a Key Regulator of Proanthocyanidin Biosynthesis in Strawberry.

Strawberry (Fragaria × ananassa) is an economically important berry rich in bioactive proanthocyanidins (PAs) with healthy benefits. The AP2/ERF gene family, one of the largest transcription factors (TFs) in plants, regulates diverse biological processes. However, its role in PA biosynthesis in strawberry remains unclear. In this study, comparative genomics of 39 species revealed lineage-specific expansion of AP2/ERF, primarily driven by whole-genome/segmental duplication. Integrated transcriptomic and metabolomic analyses of a high-PA natural mutant identified FaERF4 as a key positive regulator. Subcellular localization confirmed that FaERF4 localizes both the nucleus and cytoplasm. Transient/stable overexpression showed that FaERF4 significantly increased PA content and upregulated flavonoid genes without affecting anthocyanin accumulation. Mechanistically, FaERF4 specifically bound to and activated the promoters of FaLAR and FaANR, thereby promoting PA biosynthesis. This study reveals a novel mechanism by which FaERF4 specifically regulates PA biosynthesis and provides a theoretical basis for metabolic engineering of functional nutrients in strawberry.