Emitted and endogenous volatiles in ‘Tsugaru’ apple: The mechanism of ester and (E,E)-α-farnesene accumulation

Abstract

Volatile compound production was studied in terms of biosynthetic gene expression in apple (‘Tsugaru’). To this end, we first analysed the endogenous and emitted volatiles in the skin of ripened apple fruit. GC–MS and GC analyses suggested that the boiling point of the endogenous compounds in apple skin is an important determinant of the composition and amount of emitted volatiles. Since esters and (E,E)-α-farnesene are the major endogenous volatiles in apple, key biosynthetic genes were isolated from apple skin and their expression patterns were analysed with ethylene biosynthetic genes. During fruit development, the onset of alcohol acyltransferase (pMdAAT) expression in the skin, which is responsible for ester biosynthesis, coincided with the accumulation of 1-aminocyclopropane-1-carboxylate synthase (MdACSs, MdACS5B) mRNA. Thereafter, pMdAAT expression remained at a high level, even when no MdACS transcripts were observed. On the other hand, the accumulation of (E,E)-α-farnesene synthase (pMdAFS1) transcripts in the skin was associated with the expression of MdACSs and 1-aminocyclopropane-1-carboxylate oxidase (MdACO1). After harvest, the inhibition of pMdAAT expression in 1-methylcyclopropene (1-MCP)-treated apple skin was incomplete. In contrast, the expression of pMdAFS1 was repressed by 1-MCP treatment concomitant with considerable inhibition of ethylene production. These results suggest that pMdAFS1 expression is controlled by ethylene, whereas pMdAAT expression is developmentally regulated in the skin of ‘Tsugaru’.

Introduction

Volatile compounds are one of the factors that determine apple (Malus × domestica) fruit quality (Kondo, Setha, Rudell, Buchanan, & Mattheis, 2005). Apples emit a complex mixture of volatile compounds including alcohols, esters, aldehydes, ketones, and sesquiterpenes (Dimick & Hoskin, 1983). The major aromatic compounds in apples are volatile esters (Brackmann et al., 1993, Olías et al., 1992). Esters are, associated with the ‘fruity’ attributes of a fruit’s flavour and, are important aromatic components in many ripened fruits (Aharoni et al., 2000). Esters are derived from amino acids and fatty acids (Dixon & Hewett, 2000). Alcohol acyltransferase (AAT), a BAHD enzyme superfamily, is a key enzyme in ester biosynthesis, catalysing the final step in ester formation by linkage of an acyl moiety from acyl-CoA to the appropriate alcohol (Fellman, Miller, Mattison, & Mattheis, 2000). Studies have reported that apple AAT (MdAAT) is expressed at a later stage during fruit ripening (Defilippi et al., 2005, Souleyre et al., 2005), and studies based on in situ hybridization suggested that MdAAT2 is primarily located in the fruit skin (Li et al., 2006). MdAAT is able to use a range of alcohols and CoA acids to produce the range of esters found in apple fruit (Souleyre et al., 2005). Apart from esters, apples also produce a large amount of (E,E)-α-farnesene (Girard & Lau, 1995), a sesquiterpene that is biosynthesized via the mevalonate pathway (Pechous & Whitaker, 2004). The products of (E,E)-α-farnesene oxidation can induce superficial scald disorder, an important disease in the apple industry; several studies have conducted comparative analyses of (E,E)-α-farnesene biosynthesis in resistant and susceptible cultivars (Lurie et al., 2005, Pechous et al., 2005, Tsantili et al., 2007). (E,E)-α-farnesene synthase (AFS), encoded by MdAFS1 in apple, is a key enzyme in (E,E)-α-farnesene biosynthesis, catalysing the conversion of farnesyl diphosphate to (E,E)-α-farnesene in the final steps of sesquiterpene biosynthesis (Pechous & Whitaker, 2004).

Ethylene is a key regulatory molecule for ripening and senescence and it is thought to regulate fruit ripening by coordinating the expression of many genes, which are responsible for chlorophyll degradation, carotenoid synthesis, conversion of starch to sugars, cell-wall modulation, and so on (Theologis, 1992). It is well-known that volatile biosynthesis is also regulated by ethylene. Kondo et al. (2005) showed that chemical inhibition of ethylene action suppresses volatile production. Moreover, several studies show that the genes responsible for volatile biosynthesis are regulated by ethylene. For example, an antisense experiment with MdACO1 shows that MpAAT1 and MdAFS1 were expressed only after exogenous ethylene application in MdACO1-antisense apple transformants (Schaffer et al., 2007).

To date, research aimed at identifying the volatiles and their biosynthetic regulation in apple (e.g. Young, Chu, Lu, & Zhu, 2004) has focused on the relationship between the emitted volatile compounds and their related gene expression, despite the fact that the quality and quantity of endogenous volatiles may influence the emitted volatiles such as in petunia (Oyama-Okubo et al., 2005) and that gene expression should be directly related to endogenous, rather than emitted, volatiles in apple, because the genes function in the tissues in which endogenous volatiles biosynthesis took place. In addition, information regarding the volatile biosynthesis in ‘Tsugaru’ apple is not available, despite being a major cultivar in Japan. Therefore, in this study, we investigated the emitted and endogenous volatiles and the expression of pMdAAT, pMdAFS1 and ethylene biosynthetic genes in ‘Tsugaru’ apple skin to gain insight into the regulatory mechanism underlying volatile compound production via ethylene action in this cultivar. To this end, we analysed the emitted and endogenous volatiles using capillary gas chromatography–mass spectrometry (GC–MS) and GC. Furthermore, we analysed the expressions of pMdAAT, pMdAFS1, apple 1-aminocyclopropane-1-carboxylate synthase (MdACSs), and apple 1-aminocyclopropane-1-carboxylate oxidase (MdACO1) in 1-methylcyclopropene (1-MCP)-treated skins as well as during fruit development. On the basis of the results, we discuss the possible regulation of volatile compound production via ethylene action in ‘Tsugaru’ apple skin.