Lipoxygenase and Apple Aroma: Gene Expression of Lipoxygenase Family Members in Apple Peel

Many aroma volatiles in fresh apple are produced via cellular disruption due to cutting or mastication. Six-carbon (C₆) volatiles, including the aldehydes trans-2-hexenal, hexanal and cis-3-hexenal, as well as their corresponding alcohols, are produced from action of the lipoxygenase (LOX) pathway on substrates released by tissue disruption. LOX genes are sometimes classified based on function and are grouped into 13-LOX and the 9-LOX groups, which generate C₆ and C₉ aldehydes, respectively. Another classification system is based on structure with those having a putative chloroplast transit peptide called type-2 LOXs, and those that do not are called type-1 LOXs.  All 13 LOXs are thought to be type-2LOXs.  It has been proposed that a 13-LOX gene with a chloroplast transit peptide may be involved in apple aroma. In our work, twenty-two lipoxygenase gene sequences were retrieved from the apple genome to identify possible LOX gene candidates that might participate in the aroma production in apple. We isolated RNA from apple skin for 8 time points throughout ripening (immature to senescent stage), made cDNA and performed semi-quantitative RT-PCR for all 22 LOXs.  Amplified fragments from the coding sequence of each LOX were sequenced and compared to the published genomic sequence.  The expression of most of genes exhibited no discernable pattern during ripening; however, at least 6 LOXs were highly expressed and ripening-dependent. qRT-PCR was performed on these 6 LOX candidates. Of these, 4 LOX genes were down-regulated during ripening, and 2 LOX genes were up-regulated as ripening progressed. Changes in the lipid profile and C₆ aldehyde and alcohol production correlated (positively and negatively) with changes in gene expression data for the 6 LOX genes, suggesting they are good candidates for further investigation into their involvement in the biosynthesis of disruption-dependent aroma volatiles. Confocal microscopy analysis and biochemical characterization of apple LOX proteins is needed to interpret gene expression data and to more fully understand the role of LOX in aroma formation.