An alcohol acyl transferase from apple (cv. Royal Gala), MpAAT1, produces esters involved in apple fruit flavor

Abstract

Apples have long been cultivated by humans for their fruit. They produce a complex mixture of over 200 volatile compounds [1], including alcohols, aldehydes, ketones, sesquiterpenes and esters. Esters are associated with 'fruity' attributes of fruit flavor and typically increase to high levels late in the ripening process [2]. In the commercial apple cultivar, Malus pumila cv. Royal Gala, over 30 esters have been identified [3,4]. These can be broadly separated into straight chain esters and branched chain esters. In apples, straight chain esters are thought to be biosynthesized from fatty acids via the lipoxygenase pathway [5]. In contrast branched chain esters are thought to be produced from the metabolism of branched chain amino acids such as isoleucine [6]. Of the esters produced by Royal Gala, butyl acetate, hexyl acetate, and 2-methylbutyl acetate dominate the flavor of ripe fruit, with the latter two being identified by analytical sensory panels as having the greatest impact on the attractiveness of the fruit [4]. Apple flavor is characterized by combinations of ester compounds, which increase markedly during fruit ripening. The final step in ester biosynthesis is catalyzed by alcohol acyl transferases (AATs) that use coenzyme A (CoA) donors together with alcohol acceptors as substrates. The gene MpAAT1, which produces a predicted protein containing features of other plant acyl transferases, was isolated from Malus pumila (cv. Royal Gala). The MpAAT1 gene is expressed in leaves, flowers and fruit of apple. The recombinant enzyme can utilize a range of alcohol substrates from short to medium straight chain (C3-C10), branched chain, aromatic and terpene alcohols. The enzyme can also utilize a range of short to medium chain CoAs. The binding of the alcohol substrate is rate limiting compared with the binding of the CoA substrate. Among different alcohol substrates there is more variation in turnover compared with K m values. MpAAT1 is capable of producing many esters found in Royal Gala fruit, including hexyl esters, butyl acetate and 2-methylbutyl acetate. Of these, MpAAT1 prefers to produce the hexyl esters of C3, C6 and C8 CoAs. For the acetate esters, however, MpAAT1 preference depends upon substrate concentration. At low concentrations of alcohol substrate the enzyme prefers utilizing the 2-methylbutanol over hexanol and butanol, while at high concentrations of substrate hexanol can be used at a greater rate than 2-methylbutanol and butanol. Such kinetic characteristics of AATs may therefore be another important factor in understanding how the distinct flavor profiles of different fruit are produced during ripening.Abbreviations AAT, alcohol acyl transferase; coA, coenzyme A; IPTG, isopropyl thio-b-D-galactoside; MpAAT1, apple AAT1; SPME, solid phase microextraction.

Identification of Genes with Potential Roles in Apple Fruit Development and Biochemistry through Large-Scale Statistical Analysis of Expressed Sequence Tags

“…We did not observe significant ripening-related or skin-associated overrepresentation of ESTs representing the alcohol acyl transferase (AAT) clusters present in the libraries analyzed (data not shown). This observation is not inconsistent with expression data in studies by Defilippi et al (2005) and Souleyre et al (2005), demonstrating significant levels of AAT gene expression even in nonripe apple fruit tissues. We analyzed mRNA levels of genes corresponding to the LOX cluster MD187410 and acyl-CoA synthetase cluster MD042900 in relation to the ripening-related production of volatile esters in fruit from field-grown plants.…”

Analyses of Expressed Sequence Tags from Apple

“…MpAAT1 is up-regulated during ripening in response to ethylene (Defilippi et al, 2005b;Souleyre et al, 2005). The MpAAT1 enzyme is able to synthesize a wide range of esters, including many found in Royal Gala fruit (Souleyre et al, 2005). It is possible that the other alcohol acyl transferases found in the apple sequence dataset may also be contributing to the production of volatile esters.…”

“…An example of this type of analysis is shown for the acyl transferases (Fig. 8), a gene family that contains members that are involved in ester biosynthesis in apple (Souleyre et al, 2005). For this gene family, there is at least one clade with more representatives from apple than Arabidopsis.…”

“…One apple alcohol acyl transferase (MpAAT1) has been well characterized. MpAAT1 is up-regulated during ripening in response to ethylene (Defilippi et al, 2005b;Souleyre et al, 2005). The MpAAT1 enzyme is able to synthesize a wide range of esters, including many found in Royal Gala fruit (Souleyre et al, 2005).…”

Two QTL characterized for soft scald and soggy breakdown in apple (Malus × domestica) through pedigree-based analysis of a large population of interconnected families

“…However, markers for the identified founding sources of the HDI haplotype at LG2 are identical in state for a set of 21 SNP markers covering the first 2.03 cM of the linkage map. This area contains QTL identified for various alcohols and esters (Dunemann et al 2009;Souleyre et al 2014;Farneti et al 2017) and aromatic compounds (Souleyre et al 2014;Kumar et al 2015;Yauk et al 2015;Farneti et al 2017) found in fruit that were thought to be under regulation of MdAAT1 (MDP0000637737) (Souleyre et al 2014;Farneti et al 2017), which codes for an alcohol acyl-transferase that catalyzes the transacylation from acyl-CoA to alcohol (Souleyre et al 2005), the suppression of which led to increased levels of hexanol and other alcohols (Souleyre et al 2014). MdAAT1 is within the LG2 QTL identified in this study.…”