Volatile esters are important to wine

Volatile esters are important to wine quality and to fresh berry grape flavor, especially in fox grapes (V. labrusca) [16]. Volatile esters such as Z-3-hexyl acetate can be found in V. vinifera ‘Cabernet Sauvignon’ berries albeit at low levels and their accumulation diminishes to below detection levels during ripening [17]. Conversely, in ‘Muscat Hamburg’ berries the concentration of benzyl acetate and benzyl benzoate remained low during ripening [7]. Although plants produce thousands of different volatile compounds, ample accumulating evidence indicates that tissues possess a hidden potential capacity to produce even more volatiles [13]. Biosynthetic barriers preventing the accumulation of additional volatiles include differential spatial or temporal gene expression patterns, substrate availability, as well as enzyme or substrate compartmentalization [18]. Moreover, tissues that normally do not produce a given metabolite, often express genes and display enzyme activities that could catalyze the synthesis of given metabolites if the proper substrates would be available. These potential and concealed pathways have collectively been termed "silent" or "concealed" metabolism [18]. For example, although tomato fruit normally lack volatile esters, the enzymes potentially producing these esters are active in tomato fruit [6,19]. Furthermore, transgenic tomato fruits overexpressing the Ocimum basilicum geraniol synthase gene (GES) accumulated more than 10 novel geraniol-derived products, including geranyl, neryl and citronellyl acetate produced apparently by endogenous previously concealed enzymes [20]. The last biosynthetic step in the formation of esters in the plant kingdom is largely catalyzed by a group of enzyme members of the BADH gene family termed alcohol acyl transferases (AAT). These enzymes transfer an acyl group from acyl-CoA derivatives to an alcohol acceptor generating an ester [21]. The origin of the alcohol precursors leading to the production of the acetyl ester include amino Pioglitazone derivatives, such as 2-phenylethanol and 3-methylbutanol [22], fatty acid derived C6 aliphatic alcohols, such as hexanol [23] or oxygenated monoterpenes, such as geraniol [24]. Esters are important in processed grape berry products such as wines but mainly originate from yeast metabolism during fermentation [25]. The potential involvement of AAT enzyme activities in acetyl ester production in V. vinifera fresh berries is not fully understood. Therefore, our hypothesis is that grape berries possess a concealed potential to produce acetate esters. In the present study, we characterized the metabolism of exogenous L-Leu, L-Phe and L-Met in mature grape berry tissues to evaluate the possible concealed ability of the fruit to accumulate volatile esters. Berries supplemented with amino acids accumulated volatile compounds including the acetyl esters 3-methylbutyl acetate and 2-phenylethyl acetate, absent in intact berries. We isolated a new member of the BAHD gene family (VvAAT2) encoding for a protein possessing AAT activity in vitro. VvAAT2 catalyzes the esterification of either amino- or fatty acid- derived volatile alcohols in the presence of acetyl-CoA. Our study is an attempt to evaluate biological barriers limiting the accumulation of acetyl esters in grape berries.
Material and methods
Results
Discussion The initial step in amino acid metabolism into acetate esters is their conversion to the corresponding volatile aldehydes [39]. Our results demonstrate that grape berry sections readily support the conversion of L-Leu, L-Phe and L-Met into their corresponding aldehydes and the aldehydes are often further metabolized into the corresponding alcohols and acetate esters (Fig. 1). Interestingly, the levels of 2-phenylethanal were generally more than ten-fold higher than the levels of the benzaldehyde accumulated in L-Phe treated berries. Benzaldehyde is a common volatile present in many fruits and flowers and contributes to a typical almond and cherry notes to fruit aromas. 2-Phenylethanal is a common constituent of passion fruit [40] and in excess it causes detrimental flavors in tomatoes [41]. Although benzaldehyde has been reported to occur in ‘Muscat Hamburg’ grape berries [7], to the best of our knowledge the occurrence of 2-phenylethanal has not been reported before in V. vinifera grape berries.