The synthetic and/or catabolic pathways of the amino acids valine, leucine, isoleucine, methionine, phenylalanine, and alanine contribute to the formation of odor-active alcohols, aldehydes, carbonyls, and esters in edible plant parts (Azevedo et al., 1997; Baldwin et al., 2002: Gonda et al., 2010; Pérez et al., 1992; Reineccius, 2006; Salunkhe and Do, 1976; Sanz et al., 1997). Further, other amino acids act as precursors to these odor-contributing amino acids. Aspartate, for instance, is consumed in the synthesis of several amino acids including lysine, methionine, and threonine, the latter of which is a precursor to isoleucine (Azevedo et al., 1997).
Branched-chain (BC) esters are important contributors to the fruity aroma of several important horticultural crops. The pathway for their formation has been previously described (Fig. 1), but new aspects continue to emerge. In apple (Malus ×domestica) fruit, for instance, 2-methylbutyl ester accumulation during ripening is associated with the increased synthesis of citramalic acid, which may contribute to a new pathway for isoleucine and BC ester synthesis in apple (Sugimoto et al., 2011, 2015). In banana fruit, the amino acids valine and leucine have been proposed to be used, respectively, in the formation of 2-methylpropyl and 3-methylbutyl esters (Tressl and Jennings, 1972), which account for about 70% of the volatile compounds produced by banana (Seymour, 1993).
Fruit esters are formed by the reaction between alcohols and acyl CoA derivatives of carboxylic acids. Labeling studies for BC esters have shown that leucine feeding yields 3-methylbutanol and 3-methylbutanoate, which can then be used to form 3-methylbutyl esters (e.g., 3-methylbutyl acetate, 3-methylbutyl butanoate, ethyl 3-methylbutanoate, butyl 3-methylbutanoate, and 3-methylbutyl 3-methylbutanoate); valine feeding produces 2-methylpropanol and 2-methylproponoate and their ester derivatives; isoleucine feeding yields 2-methylbutanol and 2-methylbutanoate and their ester derivatives (Pérez et al., 2002; Rowan et al., 1996, 1998; Tressl and Drawert, 1973; Wyllie and Fellman, 2000; Wyllie et al., 1996).
The banana ester profile has several 3-methylbutyl esters and a smaller number of 2-methylpropyl esters arising, respectively, from the pathways leading to the synthesis of leucine and valine (Tressl and Jennings, 1972; Wyllie and Fellman, 2000). Of these esters, 3-methylbutyl butanoate is the predominant ester of ‘Cavendish’ banana (Nogueira et al., 2003) and it acts with 3-methybutyl acetate and 3-methylbutyl 3-methylbutanoate to constitute the core components of the characteristic odor of ripened banana fruit (Jordan et al., 2001; Quast, 1976; Schiota, 1993).
Pools of free BC amino acids accumulate during ripening of apple, banana, melon (Cucumis melo), and strawberry (Fragaria ×ananassa) fruit (Pérez et al., 2002; Schieberle et al., 1990; Sugimoto et al., 2011; Tressl and Drawert, 1973; Yoshioka et al., 1982) and largely reflect the different BC aroma profile patterns found in those fruit. However, it is relevant to note that these BC amino acids are not directly converted to the alcohol and acid precursors of BC esters. Rather, the BC α-keto precursors to isoleucine, valine, and leucine (i.e., α-keto-β-methylvalerate, α-ketoisovalerate, and α-ketoisocaproate, respectively) are positioned more directly in the pathway of ester formation (Sugimoto et al., 2011) (Fig. 1). Tewari et al. (2000) noted that the BC α-keto acids are in approximate equilibrium with their respective BC amino acids, suggesting that the pools of BC amino acids mirror the pools of their respective α-keto acid precursors. In fact, BC esters can be produced directly from exogenously supplied BC α-keto acids (Gonda et al., 2010). Notably, in apple, there was no up-regulation in 10 branched-chain aminotransferase (BCAT) genes during ripening (Sugimoto et al., 2008, 2011), suggesting a lack of regulation at the point of interconversion of BC amino acids and their respective BC α-keto acid precursor. Additionally, BC α-keto decarboxylase and BC α-keto dehydrogenase activities may also be important limiting steps in the interconversion of the BC α-keto acid pools to ester precursors (Gonda et al., 2010; Sugimoto et al., 2008, 2011).
In apple, the greater portion of aroma biosynthesis occurs in the peel (Guadagni et al., 1971). However, the relationship between banana peel and aroma production is less clear. Ji and Srzednicki (2015) found that the peel of ripe banana emitted many of the same volatiles produced by the pulp, with the most abundant esters being 3-methylbutyl acetate and 3-methylbutyl butanoate. However, multiple stages of ripening were not evaluated and the production of aroma compounds by the pulp was not determined in this study. Emaga et al. (2007) documented total amino acid concentrations of the banana peel, but did not evaluate free amino acid pools. They found little change in the content of total amino acids during ripening. Thus, the relationship between the free amino acid pools and aroma formation in the peel is not known. If the relationship between the BC esters and free BC amino acid pools mirrors that of the pulp, the finding will help bolster the linkage between BC amino acid metabolism and BC ester formation. Even more to the point, while we believe that amino acid metabolism is related to the synthesis of the BC esters responsible for “banana” aroma, this is not known conclusively. Further, we do not know the details of the regulation of the pathways for their synthesis.
To our knowledge, detailed information about the concentration of free amino acids in banana fruit peel and pulp during ripening is not available. Most studies on fruit pulp aroma and free amino acid content report only one or two stages of development. This is, in part, due to challenges previously associated with analysis. Current high-throughput methods combining high-performance liquid chromatography (HPLC) with multiple reaction monitoring of selected mass spectra ions facilitate additional scrutiny of metabolites like amino acids. The aim of this work was to more precisely describe the relationship between the physiological measures of ripeness (e.g., internal ethylene, O2 and CO2 concentrations, aroma volatile formation, and peel color) and free amino acid content of the peel and pulp than has been previously accomplished for ripening fruit. This work expands on previous work detailing the ontogeny of aroma formation by whole fruit relative to the onset of autocatalytic ethylene formation (Jayanty et al., 2002).
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