Abstract
Volatile C6-aldehydes contribute green/grassy notes to the aroma of many unripe fruits. C6-aldehydes are also likely important contributors to flavor intensity in fruits that remain green when ripe, including green-fleshed kiwifruit (Actinidia spp.). Here, we investigated the genetic basis for aldehyde production in kiwifruit in an A. chinensis mapping population. A major quantitative trait locus for producing multiple aldehydes was identified on chromosome 28 and named the aldehyde (ALD) locus. This locus co-located with 3 tandemly arrayed A. chinensis LIPOXYGENASE (AcLOX4a-c) genes in the Red5 genome. Expression of the ALD 13-LOX genes and aldehyde production decreased as the fruit developed and ripened in multiple Actinidia spp. In planta transient overexpression and biochemical analysis indicated that AcLOX4a and AcLOX4c produce hexanal and hexenal isomers. The third gene, AcLOX4b, was inactive and likely a pseudogene. The ALD LOX genes were targeted for CRISPR-Cas9 knockout, generating A. chinensis kiwifruit lines that contained insertions/deletions in all 3 target genes. Gas chromatography-mass spectrometry analysis showed that C6-aldehyde levels were reduced in leaves and fruit of the CRISPR-Cas9 lines, with a >90% reduction in line CAL5-2 compared with the control. Sensory aroma analysis showed that consumers could readily discriminate unripe CAL5-2 fruit from controls, describing the fruit as less grassy. Consumer discrimination was weaker in ethylene-ripened CAL5-2 fruit, likely due to high levels of fruity esters. Our results validate the importance of C6-aldehydes in kiwifruit flavor, and our characterization of the ALD locus is a critical step toward maintaining and improving flavor intensity in kiwifruit.