Integrated expression and structural analysis of FaHPL genes reveals developmental regulation of green volatile biosynthesis in strawberry

Commercial strawberries (Fragaria x ananassa) are recognized worldwide for their exceptional fruit quality, which includes outstanding aroma, flavor, color, and nutritional value. Hydroperoxide lyase (HPL) enzymes are central to the biosynthesis of green leaf volatiles (GLVs), which contribute to fruit aroma and stress responses. Here, we identify and characterize four FaHPL genes in commercial strawberry using integrative genomic, structural, and expression approaches. Phylogenetic and motif analyses reveal evolutionary conservation of catalytic domains across isoforms, while promoter analysis suggests differential transcriptional regulation in response to hormonal and environmental cues. All four FaHPL genes are predominantly expressed during early fruit development, with FaHPL4 showing the highest expression during small green stage and with FaHPL3 displayed the strongest predicted substrate interaction based on molecular docking and dynamics simulations. This isoform exhibits enhanced stability in complex with HPOD and HPOT, consistent with an active enzymatic role. These data suggest a model in which FaHPL3 and FaHPL4 mediates GLV production in green-stage fruits, shaping the aroma profile and potentially contributing to defense. Finally, we propose a conceptual model in which GLVs predominate during early fruit development, mediated by high FaHPL expression, while volatile esters become dominant in ripe stages, associated with increased expression of alcohol acyltransferases (AATs) genes. Additionally, we propose that FaHPL3 and FaHPL4 are a promising target for functional studies aimed at modulating volatile composition in strawberry. This work contributes to our understanding of aroma biosynthesis dynamics and provides a foundation for future targeted breeding strategies in strawberry.