Structural, Evolutionary, and Regulatory Divergence of FAH12 from FAD2 Reveals Recurrent Independent Neofunctionalization Underlying Ricinoleic Acid Biosynthesis in Ricinus communis

Ricinus communis is the primary commercial source of ricinoleic acid (RA), a hydroxy fatty acid (HFA) synthesized by the fatty acid hydroxylase FAH12, which evolved from the Δ12-oleate desaturase FAD2. However, the evolutionary origins and diversification mechanisms of FAH12 across HFA-producing plants remain poorly understood. Here, we performed a comprehensive cross-species analysis of FAH12 and FAD2 homologs by integrating sequence analysis, structural modeling, phylogenetic reconstruction, and transcriptomic profiling. Across all currently available HFA-producing plant lineages, we found that amino acid substitutions associated with hydroxylase activity exhibit strong lineage-specific patterns rather than universal conservation, indicating multiple evolutionary solutions to catalytic divergence. Phylogenetic and synteny analyses further suggest that FAH12 arose independently from ancestral FAD2 duplications in distinct plant lineages, supporting a model of recurrent independent neofunctionalization. Transcriptomic and qRT-PCR analyses reveal that FAH12 exhibits a highly specialized endosperm-preferential expression pattern and is embedded within a regulatory network that is partially decoupled from that of FAD2. Together, these findings demonstrate that FAH12 evolution is driven by recurrent independent origins coupled with transcriptional specialization, providing a framework linking structural variation, evolutionary history, and regulatory divergence for understanding and engineering hydroxy fatty acid biosynthesis in plants.