Volatile esters, a major class of compounds contributing to the aroma of many fruit, are synthesized by alcohol acyl-transferases (AAT). We demonstrate here that, in Charentais melon (Cucumis melo var. cantalupensis), AAT are encoded by a gene family of at least four members with amino acid identity ranging from 84% (Cm-AAT1/Cm-AAT2) and 58% (Cm-AAT1/Cm-AAT3) to only 22% (Cm-AAT1/Cm-AAT4). All encoded proteins, except Cm-AAT2, were enzymatically active upon expression in yeast and show differential substrate preferences. Cm-AAT1 protein produces a wide range of short and long-chain acyl esters but has strong preference for the formation of E-2-hexenyl acetate and hexyl hexanoate. Cm-AAT3 also accepts a wide range of substrates but with very strong preference for producing benzyl acetate. Cm-AAT4 is almost exclusively devoted to the formation of acetates, with strong preference for cinnamoyl acetate. Site directed mutagenesis demonstrated that the failure of Cm-AAT2 to produce volatile esters is related to the presence of a 268-alanine residue instead of threonine as in all active AAT proteins. Mutating 268-A into 268-T of Cm-AAT2 restored enzyme activity, while mutating 268-T into 268-A abolished activity of Cm-AAT1. Activities of all three proteins measured with the prefered substrates sharply increase during fruit ripening. The expression of all Cm-AAT genes is up-regulated during ripening and inhibited in antisense ACC oxidase melons and in fruit treated with the ethylene antagonist 1-methylcyclopropene (1-MCP), indicating a positive regulation by ethylene. The data presented in this work suggest that the multiplicity of AAT genes accounts for the great diversity of esters formed in melon.

Alcohol dehydrogenases (ADH) participate in the biosynthetic pathway of aroma volatiles in fruit by interconverting aldehydes to alcohols and providing substrates for the formation of esters. Two highly divergent ADH genes (15% identity at the amino acid level) of Cantaloupe Charentais melon (Cucumis melo var. Cantalupensis) have been isolated. Cm-ADH1 belongs to the medium-chain zinc-binding type of ADHs and is highly similar to all ADH genes expressed in fruit isolated so far. Cm-ADH2 belongs to the short-chain type of ADHs. The two encoded proteins are enzymatically active upon expression in yeast. Cm-ADH1 has strong preference for NAPDH as a co-factor, whereas Cm-ADH2 preferentially uses NADH. Both Cm-ADH proteins are much more active as reductases with K (m)s 10-20 times lower for the conversion of aldehydes to alcohols than for the dehydrogenation of alcohols to aldehydes. They both show strong preference for aliphatic aldehydes but Cm-ADH1 is capable of reducing branched aldehydes such as 3-methylbutyraldehyde, whereas Cm-ADH2 cannot. Both Cm-ADH genes are expressed specifically in fruit and up-regulated during ripening. Gene expression as well as total ADH activity are strongly inhibited in antisense ACC oxidase melons and in melon fruit treated with the ethylene antagonist 1-methylcyclopropene (1-MCP), indicating a positive regulation by ethylene. These data suggest that each of the Cm-ADH protein plays a specific role in the regulation of aroma biosynthesis in melon fruit.

Mountain papaya ( Vasconcellea pubescens ) is a climacteric fruit that develops a strong and characteristic aroma during ripening. Esters are the main volatile compounds produced by the fruit, and most of them are dependent on ethylene. As esters are synthesized through alcohol acyltransferases (AAT), a full-length cDNA (VpAAT1) was isolated that displayed the characteristic motifs of most plant acyltransferases. The full-length cDNA sequence was cloned and expressed in yeasts, obtaining a functional enzyme with high AAT activity toward the formation of benzyl acetate. The transcript accumulation pattern provided by qPCR analysis showed that the VpAAT1 gene is expressed exclusively in fruit tissues and that a high level of transcripts is accumulated during ripening. The increase in VpAAT1 transcripts in fruit is coincident with the increase in AAT activity; transcript accumulation is induced by ethylene, and it is avoided by 1-methylcyclopropene (1-MCP) treatment. The data indicate that VpAAT1 is involved in aroma formation and that ethylene plays a major role in regulating its expression.

Flavor composition has been defined as a complex attribute of fruit quality, in which the mix of sugars, acids and volatiles play a primary role. In table grapes (Vitis vinifera L.), sweetness and sourness are the most important flavor attributes for fresh consumption. However, most of the studies available have been performed on wine grapes, which are grown, cultured and processed differently to table grapes. Therefore, the objective of this work was to characterize the changes in sugars and organic acids during the development of 'Thompson Seedless', 'Red Globe' and 'Crimson Seedless' grown under the same agroclimatic conditions. Each variety was sampled weekly from 2 wk before véraison until commercial harvest. Sugars and organic acids were quantified by high performance liquid chromatography (HPLC) equipped with an evaporative light scattering detector (ELSD) and ultra violet detector, respectively. The ranges of acid and sugars concentrations found in grapes were as follows: tartaric acid, 1. . Among sugars, glucose was the most abundant one in early stages and then it decreased until the harvest period, when the amount of fructose and glucose converged to an average of 47% for each sugar. Despite organic acids reaching steady levels 3-4 wk before commercial harvest, there were important differences in the organic acid profiles among varieties, with 'Thompson Seedless' showing the lowest tartaric/malic acid ratio of 1.19. These differences are an important aspect in terms of overall flavor.

Alcohol acyltransferases (AAT) play a key role in the biosynthesis of ester aroma volatiles in fruit. Three ripening-specific recombinant AATs of cantaloupe Charentais melon fruit (Cm-AAT1, Cm-AAT3, and Cm-AAT4) are capable of synthesizing thioether esters with Cm-AAT1 being by far the most active. All proteins, as well as AAT(s) extracted from melon fruit, are active as tetramers of around 200 kDa. Kinetic analysis demonstrated that CoA-SH, a product of the reaction, is an activator at low concentrations and an inhibitor at higher concentrations. This was confirmed by the addition of phosphotransacetylase at various concentrations, capable of modulating the level of CoA-SH in the reaction medium. Site-directed mutagenesis of some amino acids that were specific to the Cm-AAT sequences into amino acids that were consensus to other characterized AATs greatly affected the selectivity of the original protein and the number of esters produced.

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