A dual positional specific lipoxygenase functions in the generation of flavor compounds during climacteric ripening of apple

Lipoxygenase(LOX)is an important contributor to the formation of aroma-active C6 aldehydes in apple(Malus×domestica)fruit upon tissue disruption but little is known about its role in autonomously produced aroma volatiles from intact tissue.We explored the expression of 22 putative LOX genes in apple throughout ripening,but only six LOXs were expressed in a ripening-dependent manner.Recombinant LOX1:Md:1a,LOX1:Md:1c,LOX2:Md:2a and LOX2:Md:2b proteins showed 13/9-LOX,9-LOX,13/9-LOX and 13-LOX activity with linoleic acid,respectively.While products of LOX1:Md:1c and LOX2:Md:2b were S-configured,LOX1:Md:1a and LOX2:Md:2a formed 13(R)-hydroperoxides as major products.Site-directed mutagenesis of Gly567 to an alanine converted the dual positional specific LOX1:Md:1a to an enzyme with a high specificity for 9(S)-hydroperoxide formation.The high expression level of the corresponding MdLOX1a gene in stored apple fruit,the genetic association with a quantitative trait locus for fruit ester and the remarkable agreement in regio-and stereoselectivity of the LOX1:Md:1a reaction with the overall LOX activity found in mature apple fruits,suggest a major physiological function of LOX1:Md:1a during climacteric ripening of apples.While LOX1:Md:1c,LOX2:Md:2a and LOX2:Md:2b may contribute to aldehyde production in immature fruit upon cell disruption our results furnish additional evidence that LOX1:Md:1a probably regulates the availability of precursors for ester production in intact fruit tissue.

The carrot monoterpene synthase gene cluster on chromosome 4 harbours genes encoding flavour-associated sabinene synthases

In plants,low molecular weight terpenes produced by terpene synthases(TPS)contribute to multiple ecologically and economically important traits.The present study investigates a carrot terpene synthase gene cluster on chromosome 4 associated with volatile monoterpene production.Two carrot mutants,yellow and cola,which are contrasting in the content of low molecular weight terpenes,were crossed to develop an F2 mapping population.The mapping analysis revealed overlapping QTLs on chromosome 4 for sabinene,α-thujene,α-terpinene,γ-terpinene,terpinen-4-ol and 4-carene.The genomic region of this locus includes a cluster of five terpene synthase genes(DcTPS04,DcTPS26,DcTPS27,DcTPS54 and DcTPS55).DcTPS04 and DcTPS54 displayed genotype-and tissue-specific variation in gene expression.Based on the QTL mapping results and the gene expression patterns,DcTPS04 and DcTPS54 were selected for functional characterization.In vitro enzyme assays showed that DcTPS54 is a single-product enzyme catalysing the formation of sabinene,whereas DcTPS04 is a multiple-product terpene synthase producingα-terpineol as a major product and four additional products including sabinene,β-limonene,β-pinene and myrcene.Furthermore,we developed a functional molecular marker that could discriminate carrot genotypes with different sabinene content in a set of 85 accessions.