Expression and of Putative Lipoxygenase (LOX) Genes Relative to LOX-dependent Aroma Volatile Emissions in Apple Fruit

The involvement of LOX pathway activity in the formation of aroma compounds in apple has been suggested in numerous studies, but the nature of that involvement is incompletely understood.  While there is little disagreement in the contribution of LOXs to the formation of C-6 aldehydes induced by the disruption of apple tissue, it is not clear whether LOXs contribute significantly to autonomously-produced volatiles from intact fruit.  We explored the expression of all 22 LOX genes in apple for 8 time points throughout ripening (immature to senescent stage).  Only 16 LOX genes could be amplified in the fruit. Phylogenetic analysis of the 22 LOX gene sequences, classified 13 of them with a 13-LOX predicted function, 8 with a 9-LOX predicted function and 1 with unknown function. The expression of most of genes exhibited no discernable pattern during ripening; only 6 LOXs were expressed in a ripening-dependent manner. Quantitative RT-PCR was performed on these 6 LOX candidates. Of these, 2 13-LOX genes were down-regulated during ripening, one 13-LOX gene underwent a brief transient rise at the onset of ripening, one 9-LOX gene underwent a decline throughout ripening, and two 9-LOX genes were sharply up-regulated as ripening progressed. Changes in the cis-3-hexenal production correlated with the expression of the 13-LOX genes undergoing a decline.  A rise in the production of hexanal, hexanol and hexyl esters paradoxically correlated with the putative 9-LOX genes.  Confocal microscopy analysis of transiently expressed 13- and 9-LOXs suggests that the 13-LOXs are localized in the chloroplasts or cell wall/plasmalemma while the 9-LOXs are localized in the cell wall/plasmalemma and the nucleus.