Apple aroma: alcohol acyltransferase, a rate limiting step for ester biosynthesis, is regulated by ethylene
Introduction
Key components of the fruit flavor complex are the volatile compounds that constitute aroma. These include a broad group of metabolites that are important components of flavor in fruit and vegetables and in addition regulate the interactions of plants with other organisms [1]. In addition to the four basic tastes that the human palate can recognize, aroma exerts an important influence on the final consumer acceptance of a fruit/vegetable commodity [2]. The aroma properties of fruits depend upon the combination of volatiles and the concentration, and threshold of individual volatile compounds. In apple, the typical aroma compounds are the fruity esters that develop during ripening with a maximum endogenous ester concentration occurring at the climacteric peak [3], [4]. The gaseous plant hormone ethylene is associated with many physiological processes in plants, and plays an especially important role in the ripening process of climacteric fruit, initiating and enhancing ripening-related changes including decreased firmness, increased soluble solids content and enhanced flavor [5], [6], [7]. The association between ethylene and aroma production has been shown through the use of both ethylene action and ethylene biosynthesis inhibitors that result in a reduction in levels of ester volatiles in apple fruit [8], [9]. Similarly, in climacteric ACC-oxidase antisense transgenic melons, ripening parameters including color of the rind and aroma (especially esters) production were strongly reduced at low levels of endogenous ethylene [10], [11], suggesting that these parameters are physiologically regulated by ethylene during fruit development. However, little is known of the underlying mechanisms that regulate this relationship between ethylene biosynthesis and ester biosynthesis. It is also not clear if the enzymes responsible for aroma components are constitutive or induced during the climacteric response [4].
Earlier studies have established that the beta-oxidation of fatty acids is the primary biosynthetic process that provides alcohols and acyl co-enzyme A (CoA) for ester formation [12]. Acyl CoAs are reduced by acyl CoA reductase to aldehydes, which are in turn reduced by the alcohol dehydrogenase (ADH) enzyme to form alcohols that are converted to esters via the action of alcohol acyltransferase (AAT) enzyme [13]. The AAT enzyme catalyzes the linkage of an acetyl moiety from acetyl CoA to the appropriate alcohol. This enzyme has been studied in some detail in ripe fruit, including apple [3], [14], banana [15], melon [16], [17], and especially strawberry where the enzyme has been purified and characterized, and the gene cloned [18], [19]. Experiments performed with banana and strawberry fruit indicate a correlation between substrate specificity and volatile esters present in each fruit's aroma, suggesting a significant role of AAT enzyme in flavor biogenesis in these species [4], [20]. In preliminary experiments performed in apples, the activity of AAT appears to increase with the onset of ripening followed by a decrease in extractable activity [3]. However, despite the importance of AAT as a key enzyme in aroma synthesis in fruits, many aspects, such as the mechanism of action, substrate specificity, and physiological relevance, remain unclear [16], [20]. Ester biosynthesis can also be limited by the concentration of precursor alcohols [21], which suggests that critical steps for ester formation may be located upstream in the pathway. The enzyme ADH has been associated with fruit ripening and has been shown to be responsible for the interconversion of aldehyde and alcohol forms of flavor volatiles [22], [23]. Mature green tomato contained lower levels of ADH2 transcripts as compared to ripened fruits, which was correlated with lower levels of alcohols and higher levels of aldehydes [24]. Similarly, differences in the accumulation of lipoxygenase-derived volatiles were observed in an Arabidopsis ADH mutant that lacked ADH activity, which resulted in the accumulation of C6 aldehydes and a reduction of alcohols [25]. This evidence suggests that ethylene plays an important role in apple as a regulator of ester biosynthesis with AAT and/or ADH enzymes as key enzymes that modulate the biochemical steps in flavor biogenesis. The main objective of this research was to outline the underlying mechanism of ester biosynthesis and its regulation in apple fruit.
Section snippets
Plant material and treatments
Transgenic Greensleeves (GS) apple fruits suppressed for ethylene biosynthesis were obtained from different lines grown in an experimental orchard in Northern CA [26]. Fruits of selected GS apple lines (GS, 67G, 68G, 103Y, and 130Y) were evaluated in 2001 and 2002, and the ones with the highest level of ethylene suppression (67G and 68G) were selected for further studies in 2003. Fruits were harvested at a preclimacteric stage relative to the non-transformed line, prior to initiation of
Ethylene biosynthesis
Fruit obtained from the selected transgenic lines showed a 95% reduction in ethylene production relative to the non-transformed line, with an absence or delay of the climacteric peak (Fig. 1).
This decrease was related to a reduction in either ACS enzyme activity in the ACS-silenced line (103Y) or ACO enzyme activity in the ACO-silenced lines (67G and 68G) (Fig. 2). In the ACS-silenced line there was a 90% reduction in ACS enzyme activity relative to that of the control line. This resulted in a
Conclusions
The availability of the transgenic Greensleeves apples suppressed in ethylene biosynthesis and the ethylene inhibitor 1-MCP allowed the identification and characterization of an important biochemical step modulating aroma via ester production during fruit ripening. Under these conditions, the last step of ester biosynthesis seems to be an important control point, in which AAT enzyme activity and transcripts were highly modulated by ethylene. However, the molecular and functional
Acknowledgements
We acknowledge the Washington Fruit Tree Research Commission for their support of this research. We also thank Christian M. Leutenegger, DVM, Ph.D., FVH, Lucy Whittier Molecular and Diagnostic Core Facility, UC Davis, for expert real-time TaqMan PCR analysis.
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