Genomic analyses of 238 seed plants reveal the evolutionary mechanisms driving specialization of F3H, ANS, and FLS in flavonoid biosynthesis

Introduction: Flavonoid metabolism innovation is critical for seed plants' terrestrial adaptation. Three key 2-oxoglutarate-dependent dioxygenase (2ODD) families-F3H, ANS, FLS-underpin flavonoid biosynthesis, but their evolutionary diversification mechanisms remain unclear. Methods: Comprehensive analyses were performed across 238 seed plant genomes, including phylogenetic/collinearity analyses, conserved motif detection, duplication-type classification, selection pressure assessment, and Brassica napus promoter/expression profiling. Results: These families originated from a common ancestral gene (ancestor X) and diversified via multiple duplications (TRD/WGD as primary drivers). Their evolution aligns with the EAC model, with subfunctionalization resolving ancestral multifunctionality. Structural conservation and lineage-specific motif variations support functional differentiation, and purifying selection predominates. Discussion: Our findings clarify the molecular evolutionary mechanisms of flavonoid pathway diversification, providing novel insights into the origin of flavonol and anthocyanin biosynthesis in seed plants.