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  • AAT enzyme activity was readily detected

    2024-07-09

    AAT enzyme activity was readily detected in crude cell-free extracts obtained from berry tissues of all accessions but displayed different substrate selectivities (Fig. 2). Cell-free extracts derived from ‘Muscat Hamburg’ berries showed the highest AAT activity with all alcoholic substrates tested, displaying the highest AAT activity when benzyl alcohol was offered as a substrate together with acetyl-CoA (Fig. 2). These findings suggest that substrate specificity of AAT activity may play an important role in the concealed potential to accumulate acetate esters among the three accessions. The substrate specificity displayed suggests either the involvement of multiple AAT enzymes or alternatively the presence of a single AAT enzyme with different affinity to various alcohol substrates. A fifty-fold higher benzyl alcohol AAT activity as compared to 2-phenylethyl alcohol AAT activity was detected in ‘Muscat Hamburg’ berry extracts (Fig. 2). In view of this AAT substrate preference, and taking into account that benzyl alcohol accumulated in L-Phe-fed ‘Muscat Hamburg’ berries (Fig. 1B), benzyl acetate accumulation would be expected. Conversely, we did not detect benzyl acetate in L-Phe-fed ‘Muscat Hamburg’ berries (Fig. 1B). Although in many cases the alcohol substrate availability clearly limits ester formation, there must be other regulatory processes involved in acetate ester formation such as compartmentalization of the enzyme or the substrate. A search for AAT genes was performed to better understand the molecular regulation of the concealed acetate ester formation in grape berries. We identified seven AAT-like upregulated expressed sequences in ‘Muscat Hamburg’ berry transcriptome as compared to their expression in ‘3003’ and ‘Superior Seedless’ berries (Fig. 3). Interestingly, VvAAT2 expression was relatively high in early berry development stages and its levels dramatically diminished upon ripening (Fig. 4). Still, the low levels of expression are apparently sufficient to support acetylation activity in the ripe berries (Fig. 2) and accumulation of acetate derivatives upon exogenous amino sch772984 feeding (Fig. 1). Hence, the accumulation of volatile esters in intact berries is probably limited by substrate availability. In accordance to the AAT activity levels observed among the accessions and their annotations we isolated two transcripts termed VvAAT2 and VvAAT3. VvAAT2 is a V. vinifera gene previously annotated as a putative benzyl alcohol O-benzoyltransferase (accession XP_002264599) (https://blast.ncbi.nlm.nih.gov/). The ectopic expression of VvAAT2 suggests that the encoded protein supported the acetylation activity of branched chain, aliphatic and aromatic alcohol substrates originating either from amino or fatty acid metabolism (Fig. 5). VvAAT2 is the first BAHD gene family member characterized from V. vinifera displaying a broad substrate AAT activity. This is in contrast to other known Vitis AAT genes that display very narrow alcoholic substrate specificity [16,38]. VvAAT2 possesses a high AAT activity towards benzyl alcohol (Fig. 5), similarly to previously characterized Cucumis melo CmAAT3 gene [53] with 75% amino acid sequence similarly to VvAAT2. Interestingly, the Vitis VvAAT2 resembles both in substrate specificity and in its amino acids sequence to the melon CmAAT3 in comparison to other AAT genes in Vitis. Phylogenetic trees of AAT genes were shown to cluster by substrate affinity rather than genetic origin [21]. Studies performed in melon indicated that AAT gene expression was rate limiting for ester production, AAT gene expression was upregulated during fruit development and was modulated by ethylene [54]. The metabolism of exogenous L-Met followed a more complex pattern as compared to the relative direct metabolism of L-Leu and L-Phe. Although the accumulation of the aldehyde methional and its respective alcohol methionol was prominent, no methionyl acetate was detected (Fig. 1C). Nevertheless, incubation of grape berry sections with exogenous L-Met resulted in the accumulation of volatile thiol compounds previously unknown to occur in fresh grapes. A similar process was shown to occur in L-Met-fed melon fruit sections [14] and Arabidopsis tissues [55]. The enzymatic reactions were shown to be catalyzed by L-methionine-γ-lyase (MGL) enzyme activities [14,55]. MGL is a PLP dependent enzyme also prominent in microorganisms, generating methanethiol, ammonia and α–ketobutyrate. Although methanethiol was detected in all three accessions upon incubation with exogenous L-Met, its oxidation products DMDS and DMTS were only detected in ‘Muscat Hamburg’ (Fig. 1C). The above observations strengthen the notion that ‘Muscat Hamburg’ is not only more aromatic, but has a greater concealed potential to generate and accumulate volatile compounds than ‘3003’ or ‘Superior Seedless’. Methanethiol possess a strong cooked cabbage flavor and DMDS and DMTS are associated with sulfur and garlic notes that are probably detrimental to the acceptance of fresh grape berry aroma. Still, low levels of DMDS have a great impact on the typical flavor and aroma of Syrah and Grenache Noir wines, although its formation is probably related to yeast fermentation during wine manufacture [56].