The green leaf volatiles (Z)-3-hexenal and (E)-2-hexenal are key components of the characteristic strawberry aroma, an important determinant of consumer preferences. Green leaf volatiles (GLVs) are C6 compounds that impart fresh, green notes and are involved in plant wounding responses. GLV biosynthesis requires several enzymatic steps to convert polyunsaturated fatty acids to C6-aldehydes, alcohols, and esters, respectively. However, the biosynthesis of GLVs in strawberries, such as the isomerization of (Z)-3-hexenal to (E)-2-hexenal, remains poorly understood. In this study, we identified a (Z)-3:(E)-2-hexenal isomerase gene (FvHI) using phylogenetic analysis, characterized its expression in different tissues, and characterized its function using stable transformation. Volatile analysis by gas chromatography–mass spectrometry (GC–MS) of fruits from a Fragaria vesca near-isogenic line (NIL) collection revealed a distinct ratio of (Z)-3-hexenal and (E)-2-hexenal in lines containing Fragaria bucharica introgressions in the distal end of linkage group 5. Consequently, FvHI was located within this genomic region. The coding sequence of FvHI was nearly identical between the recurrent parent and a selected NIL individual containing an introgression in the distal end linkage group 5, indicating that the contrasting ratio of (Z)-3 and (E)-2 GLV isomers may be attributed to transcriptional differences. Accordingly, FvHI expression in ripe fruits was lower in the selected NIL individual than in the recurrent parent. Lastly, FvHI overexpression decreased (Z)-3-hexenal accumulation and increased (E)-2-hexenal accumulation in the recurrent parent and the selected NIL individual. These results suggest that FvHI plays a role in producing the characteristic strawberry aroma by converting (Z)-3-hexenal to (E)-2-hexenal.

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