Apricot (Prunus armeniaca L.) is a widely cultivated temperate fruit tree species with fruit containing many phytochemicals that are nutritionally valuable components of the human diet (Aubert and Chanforan, 2007). The fruits are appreciated by consumers for their unique flavor, to which aroma volatiles make an important contribution, together with sugars and organic acids (Balbontín et al., 2010; Chen J. et al., 2020). More than 300 volatiles have been identified from apricot fruit, including aldehydes, alcohols, ketones, lactones, terpenes, and esters (Cumplido-Laso et al., 2012). Among them, C6 aldehydes and alcohols offer a green-note aroma, while esters and lactones are responsible for fruity aromas. Esters such as (E)-2-hexenyl acetate and (Z)-3-hexenyl acetate are considered to be key odorants influencing the flavor quality of apricot fruit (D’Auria, 2006; Fu et al., 2017). Changes in aroma-related volatiles were previously reported during apricot fruit development and postharvest ripening (Gonzalez et al., 2009; Xi et al., 2016). Aldehydes tend to decline in the fruit while esters increase under post-harvest treatments (Cumplido-Laso et al., 2012; Galaz et al., 2013).

Volatile esters can be divided into two groups: straight-chain esters and branched-chain esters. In general, straight-chain esters are mainly synthesized via the fatty acid lipoxygenase (LOX), α-oxidation, and β-oxidation pathways (Schwab et al., 2008), while branched-chain esters are derived from the amino acid pathway. In the LOX pathway, unsaturated fatty acids, such as linoleic (18:2) and linolenic acid (18:3), are regio- and enantio-selectively deoxygenated by LOX and produce hydroperoxide isomers, which are subsequently cleaved by hydroperoxide lyase (HPL) to form hexanal and hexenal, respectively. The aldehydes can then be reduced to the corresponding C6 alcohols by alcohol dehydrogenase (ADH). Alcohol acyltransferase (AAT) catalyzes the final linkage of an acyl moiety and an alcohol to form esters and, thus, is directly responsible for the production of esters (Goff and Klee, 2006). The β-oxidation of saturated fatty acids is the primary biosynthetic process providing short-chain alcohols and acyl coenzyme A (CoA) for ester formation (Schwab et al., 2008). During the process, acyl CoAs can be reduced by acyl CoA reductase to aldehyde, which is continually reduced by ADH to alcohol. Finally, the alcohols can be used to produce esters by alcohol acyltransferase (AAT) (Bartley et al., 1985; Kruse et al., 2020; Liu et al., 2020). As for the amino acid pathway, amino acids such as L-isoleucine, L-leucine, L-valine, and L-methionine are the precursors of acyl-CoAs, involved in alcohol esterification reactions catalyzed by AATs to produce branched-chain esters (D’auria et al., 2007; Gonda et al., 2010; El Hadi et al., 2013). Therefore, alcohol acyltransferase enzymes catalyze the last decisive step in volatile ester formation in fruit (Schwab et al., 2008).

AATs from plant species belong to the BAHD superfamily of acyltransferases (ATs), which are important tailoring enzymes that contribute to the biosynthesis of several terpenoids, esters, alkaloids, and flavonoids (Kruse et al., 2020). BAHD-AT was named after the first four biochemically characterized ATs, namely benzylalcohol O-acetyltransferase (BEAT), anthocyanin O-hydroxycinnamoyltransferase (AHCTs), anthranilate N-hydroxycinnamoyl/benzoyltransferase (HCBT), and deacetylvindoline 4-O-acetyltransferase (DAT) (Bontpart et al., 2015). The proteins contain one 14–17 β-strand and one 13–17 α-helix domain connected to a crossover loop, and a solvent channel runs through the protein molecule. During the reaction, the donor binds to the front side, while the acceptor binds to the back side (Wang et al., 2021). Both of the most conserved motifs HXXXD and DFGWG are necessary for the catalytic function. HXXXD is exposed to the solvent channel and directly participates in the reaction. The catalytic His residue is accessible from both sides of the channel, while the Asp residue points away from the active site (Ma et al., 2005; Molina and Kosma, 2015). During the catalytic reaction process, the acyl acceptor and donor firstly approach the protein through the solvent channel and bind initially to the active site. Then, the basic His residue (H162) deprotonates the hydroxyl or amino group of the acceptor to form an oxyanion. Next, the oxyanion conducts a nucleophilic attack on the carbonyl carbon of the acyl donor to form a tetrahedral intermediate. Finally, the intermediate deprotonates to release a CoA-SH molecule and to form a new ester or amide (Walker et al., 2013). As for the functional protein, His-162 plays a key role in triggering the reaction, while Thr-384 and Trp-386 are vital for stabilizing the tetrahedral transition state through hydrogen bonds.

The products of AATs are mostly volatile esters, which are the main source of fruit flavors (Wang et al., 2021). During ripening of Korla fragrant pears, the production of hexyl acetate was induced and positively corelated with AAT enzyme activity and expression level of the AAT gene (Chen J. et al., 2020). A relatively high level of enzyme activity in apple fruit at harvest was observed to be accompanied by high levels of ester accumulation (Defilippi et al., 2005a). In peach, positive correlations between ester contents and AAT activity were also observed during low temperature storage plus shelf-life at 20°C (Xi et al., 2012). With increasing genome data of fruit trees, several AATs have been identified, and many AATs related to ester metabolism have been functionally characterized, such as in apple (Gonzalez et al., 2009), strawberry (Gonzalez-Agueero et al., 2009), melon (Guenther et al., 2011), papaya (Kader, 2008; Balbontín et al., 2010), and kiwifruit (Li et al., 2006). Although the role of the AAT protein in regulation of volatile ester content has been most extensively investigated in these fruits, no information is available on the function and regulation of enzymes in apricot. In our previous study, we observed that the AAT enzyme activities of apricot fruit are related to ester formation (Xi et al., 2016). However, the specific gene family member responsible for ester formation is still not identified in apricot, and the specific mechanism is poorly understood.

In the present study, we performed a genome-wide analysis of AAT family genes to identify to the specific gene impacting volatile esters in apricot fruit. PaAAT1 was ascertained as a new candidate gene through bioinformation and expression-pattern analysis. The function of cloning full-length cDNAs was verified by transient overexpression, and recombinant expression in Escherichia coli was used to assay the abilities of these proteins for different alcohol substrates. Ultimately, we demonstrated that PaAAT1 can function in the biosynthesis of C6 volatile esters and contribute to the aroma of apricot.

Volatile esters play an important role in determining for flavor of a number of fruits, and are positive with consumer preference (Cao et al., 2019; Qian et al., 2019). Among them, short-chain esters that are derived fatty acids are the major class of volatile organic compounds (VOCs) contributing to the overall characteristic of ripe apricots (Livak and Schmittgen, 2001; Liu et al., 2019). However, considering the range of esters found in various fruit species, a wide variety of alcohol acyltransferases with various specificities are yet to be isolated, and little is known about the genes controlling ester biosynthesis in apricot fruit. The identification of an alcohol acyltransferase responsible for ester synthesis in apricot is a key step in understanding metabolic basis for regulatory aroma quality. Recently, the release of the genome facilitated the discovery of the key genes involved in the biosynthesis of these compounds in apricot. The present study was focused on identifying the AAT genes on a genome-wide scale, as well as characterizing their functions for volatile ester formation.

Plant BAHD-ATs constitute a large family of acyl CoA-utilizing enzymes, which contribute to the diversity of natural products, including small volatile esters and modified anthocyanins, as well as constitutive defense compounds and phytoalexins (Schwab et al., 2008; Peng et al., 2020). Most of these enzymes are O-acetyltransferases (O-ATs), and a few are N-acetyltransferases. O-ATs are further classified according to their acceptors: alcohols, flavonoids, quinic/shikimic acids, terpenoids, alkaloids, lipids, and sucrose (Wang et al., 2021). Many plant species genomes were published, and a number of BAHD-ATs have been identified. There are estimated to be about 88 genes encoding members of the BAHD family in Arabidopsis (Luo et al., 2007), and Oryza sativa contains more than 120 BAHD-ATs (Kumar et al., 2021). Xu et al. (2021) identified and characterized the expression of the 46 BAHD family genes during development, ripening, and stress response in banana (Musa acuminata Colla). A total of 717 BAHD-AT genes were identified from seven Rosaceae species, including the genomes of Pyrus bretschneideri, Fragaria vesca, Malus domestica, Prunus avium, Pyrus communis, Prunus persica, and Rubus occidentalis, producing final lists of 114, 89, 140, 124, 86, 81, and 68 putative BAHD genes (Liu et al., 2020). In the present study, based on conserved motif analysis, a total of 153 BAHD-AT genes were identified from the apricot genome (Supplementary Table 2). Recently, Wang et al. (2021) summarized biochemically characterized catalytic function and mechanisms of 141 ATs in plants. Among them, only 11 ATs can utilize benzoyl-containing alcohols or short/medium-chain alcohols as acyl acceptors. These results suggested that even the BAHD-AT family contains a large number of gene members in plants, and the members of this family have some unique roles. Only several AAT paralogs exist in many plant genomes, producing diverse ester volatiles associated with fruit flavor and flower aroma.

So far, many AAT genes expressed in fruit from this family have been earlier implicated in the biosynthesis of volatile esters (Goulet et al., 2015). The expression of SlAAT1 substantially increases during ripening of the tomato fruit, in parallel with genes encoding other volatile-related enzymes, including SlCXE1 and lipoxygenase C (Goulet et al., 2012). Correlations between expression of flavor-related AAT genes and fruit ripening have also been established in other species including melon (El-Sharkawy et al., 2005), grape (Kalua and Boss, 2009), papaya (Balbontín et al., 2010), apple (Li et al., 2006), peach (Xi et al., 2012), Fragaria ananassa, and Fragaria chiloensis (Cumplido-Laso et al., 2012). However, of the five SlAAT genes, only SlAAT1 is highly expressed in the ripe fruit. In the present study, three PaAATs were identified from the apricot genome. To demonstrate the contribution of three PaAATs to ester production in apricot fruit, their expression levels were detected by qPCR. The results revealed that only PaAAT1 was highly expressed in fruit. Simultaneously, the expression level of PaAAT1 increased rapidly during the fruit ripening in parallel with volatile ester production. The expression pattern of PaAAT1 was strongly linked with the accumulation of six esters, especially with the accumulation of hexyl acetate, (Z)-3-hexenyl acetate, and (E)-2-hexenyl acetate, which suggested that PaAAT1 might play a vital role in the synthesis of esters in apricots.

Despite the low sequence homology found among the AAT genes identified to date, AAT proteins exhibited some common characteristics. All the fruit AAT genes identified to date encode proteins ranging from 419 to 479 amino acid residues, which corresponds to an average molecular weight of 48–55 kDa (Wang et al., 2021). The present study found that PaAAT1s are globular proteins with a size of 49.905 kDa, which is in good agreement with the molecular weight of the native AAT proteins purified in banana (40 kDa), strawberry (48 kDa), grape (50 kDa), and melon (50 kDa) (Hui et al., 2010). As for the complete protein, conserved motifs are generally the basis for recognition of homologous proteins across species boundaries. Most functionally characterized BAHD acyltransferases share two conserved motifs: a conserved HXXXD(G) motif and a less conserved DFGWG motif (D’Auria, 2006; Unno et al., 2007). For VlAMAT of the Concord grape cultivar, however, besides the basic motifs, another conserved LXXYYPXAGR motif existed near the N terminus of the sequence (75–84 residues). In the present study, PaAAT1 not only contained two common HTMCD (165–169 residues) in motif 1 and DVGWG (386–340 residues) conserved motifs in motif 7 but also contained the AAT-specific conserved motif LVYYYPLAGR (74–83 residues) in motif 8 (Figures 1, 2). Even so, residue differences might significantly alter the catalytic activity of the AAT protein and dramatically affected the esterification reactions as reported for SAAT, VAAT, CmAAT1-4, and peach (D’Auria, 2006; Song et al., 2021). Based on this, we inferred that the difference of two conserved motifs HXXXD and LXXYYPXAGR for position X might be a main reason for the difference in esterification ability of PaAAT1-3.

Plants are diverse in volatile esters, and the diversity mainly depends on substrate specificities of their corresponding proteins. Similar to BAHD enzymes, most of the native and recombinant AAT proteins exhibit broad specificities of many substrates. In many fruits, different specificities of the multiple AAT proteins codified by the genes have been identified. In strawberry, FaAAT2 and FvAAT1 both influence the ester content of the fruit. FaAAT2 has been identified to be responsible for ester biosynthesis in cultivated strawberry (Fragaria × ananassa); the enzyme had activity with C1–C8 straight-chain alcohols and aromatic alcohols in the presence of acetyl-CoA (Cumplido-Laso et al., 2012), whilst FvAAT function in wild strawberry (Fragaria vesca) and the FaAAT2 enzyme have a preference for C6–C10 aliphatic alcohols (Beekwilder et al., 2004). In apple (Malus pumila), at least two AAT enzymes are thought to produce ester volatiles (Souleyre et al., 2005; Li et al., 2006), and silencing AAT1 results in a strong decrease in some individual compound levels of propyl and butyl 2-methylbutanoate as well as propyl propanoate in ripe fruit (Souleyre et al., 2014; Yauk et al., 2017). Overexpressing MdAAT2 in transgenic tobacco leaves significantly increased the concentrations of methyl benzoate and methyl tetradecanoate, suggesting that MdAAT2 may use medium-chain fatty acyl CoA and benzoyl-CoA as acyl donors together with methanol acceptors as substrates (Li et al., 2008). Melon CmAAT1, CmAAT3, and CmAAT4 effectively account for the great diversity of esters formed in the fruit (El-Sharkawy et al., 2005; Galaz et al., 2013). As an aromatic alcohol AT, PhBPBT in Petunia hybrida displayed a broad spectrum for 13 acceptors, including: benzyl alcohol, 3-hydroxybenzyl alcohol, and 2-phenylethanol (Wang et al., 2021). PtSABT and PtBEBT from Populus trichocarpa catalyzed the formation of salicyl benzoate and benzyl benzoate with 75 and 70% conversion rates, respectively (Wang et al., 2021). All these examples illustrate the specificity in aroma production that each species can acquire while maintaining an overall ability to produce an array of esters. Peach PpAAT1 can produce esters and lactones by different acetyl (Song et al., 2021). In the present study, phylogenetic analysis revealed that PpAAT1 was close to PpAAT1, and overexpression PaAAT1 led to the remarkable content increase in hexyl acetate, (Z)-3-hexenyl acetate, and (E)-2-hexenyl acetate. Therefore, PpAAT1 might be the most important enzyme for volatile C6 ester biosynthesis in apricot fruit.

Previous work suggested that volatile esters or the expression of AAT genes is influenced by plant hormone treatment and specific transcription factors. The expression of genes from wild and cultivated strawberries (Aharoni et al., 2004), banana (Beekwilder et al., 2004), melon (El-Sharkawy et al., 2005), and grape (Wang and De Luca, 2005) is strongly induced during fruit ripening. Ethylene has proved to be a major regulator of the AAT activity in apple (Defilippi et al., 2005b; Schaffer et al., 2007), melon (Flores et al., 2002), kiwifruit (Zhang et al., 2009; Günther et al., 2015), pear (Li G. et al., 2014) and peach (Ortiz et al., 2010). Abscisic acid is also reported to be involved in aromatic ester biosynthesis related to ethylene in green apples (Wang et al., 2018). Salicylic acid and methyl jasmonate enhance ester regeneration in 1-MCP-treated apple fruit after long-term cold storage (Li et al., 2006). The expression of apple AAT (MdAAT2) is induced by salicylic acid (SA) in apple fruits and the MdAAT2 promoter’s response to SA and ethylene in transgenic tobacco (Li P.-C. et al., 2014). Influence of methyl jasmonate (MeJA) on production of aroma-related esters in “Nanguo” pears was recently closely connected to ethylene biosynthesis and signal transduction (Yin et al., 2021). In the present study, an ABA, MeJA, and salicylic acid-response element (TCA) was found in the promoter of PaAATs (Figure 1B), suggesting that the three plant hormones might play a potential regulatory role in PaAAT expression and the ester biosynthesis pathway. At present, only few transcription factors have been found to regulate ester formation and AAT expression. In strawberry, overexpression of FveERF could activate the expression of the AAT gene and ester accumulation (Li et al., 2020), and volatile esters in postharvest strawberry were affected by the interaction of light and temperature (Fu et al., 2017). In the study, cis-elements of light response and temperature were found in the promoters of three PaAAT1s, suggesting that PaAAT1s are also involved in light and temperature regulation. These studies suggested that the ester metabolism was finely controlled by the complex network of environment factors, transcription factors, and AAT expression.

In the study, three AAT genes were identified in the apricot genome. The gene structure and gene expression analysis indicated that PpAAT1 is putatively associated with ester formation in apricots. The transient overexpression of PaAAT1 in apricot fruit resulted in a remarkable decrease in hexenol, (E)-2-hexenol, and (Z)-3-hexenol levels while significantly increasing the corresponding acetate production (p < 0.01). The substrate assay revealed that the PaAAT1 enzyme can produce hexenyl acetate, (E)-2-hexenyl acetate, and (Z)-3-hexenyl when C6 alcohols are used as substrates for the reaction. These results suggest that PpAAT1 is responsible for the biosynthesis of C6 esters in apricot during fruit ripening. However, the regulation mechanism of esters needs to be further explored in the future work.

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s.

WX conceptualized the experiments, reviewed, and edited the manuscript. WZ and WK performed the experiments. CY analyzed the data. WZ and RF prepared the manuscript. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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