- Ethylene biosynthesis and action in tomato: a model for climacteric fruit ripening
The concentration of organic acids and sugars has an important influence on the taste of ripening fruits. The characteristic flavour of edible fruits results from the aroma volatiles produced within the fruit during ripening and on maceration. The volatile profile of fruits determined by gas chromatography and mass spectroscopy is complex, including many alcohols, aldehydes and esters. Previous studies indicate that the differences in flavour between tomato varieties is due, at least in part, to variation in aroma volatile production (Brauss et al., 1998). Over 400 volatile compounds are detected in tomato fruit (Hobson and Grierson, 1993), although a group of seven including hexanal, hexenal, hexenol, 3-methylbutanal, 3-methylbutanol, methylnitrobutane, and isobutylthiazole are amongst the most important contributors to fruit aroma. These flavour volatiles are formed by several different pathways: 3-methylbutanal and 3-methylbutanol are formed by the deamination and decarboxylation of amino acids whereas hexanal, hexenal and hexenol are formed by lipid oxidation of unsaturated fatty acids on the maceration of fruit. Hexanal and hexenal arise through the activity of lipoxygenases (LOX), which catalyse the hydroperoxidation of polyunsaturated fatty acids containing a cis,cis-pentadiene structure. In plants, reaction intermediates are unsaturated fatty acid hydroperoxides (HPOs), which give rise to the C6 aldehydes through the action of hydroperoxide lyases (HPO-lyase). Two groups of HPO-lyases cleave either 9-(S)-HPOs or 13-(S)-HPOs, generating two C9 fragments, or a C6 and a C12 fragment, respectively (Hatanaka, 1993).
In tomato fruit, linoleic and linolenic acid are the main LOX substrates, and the majority of HPOs found in tomato fruit, however, are 9-isomers. It appears that the 13-isomers, which are produced in a much smaller proportion, are metabolized further to produce flavour volatiles and compounds involved in defence, such as jasmonic acid (JA) (Galliard and Matthew, 1977; Regdel et al., 1994; Smith et al., 1997). The main aldehydes produced are hexanal and hexenal (Galliard and Matthew, 1977), and these aldehydes can then be further transformed into hexenol and hexanol by the action of alcohol dehydrogenase (ADH).
Tomato LOX consists of a family of at least five genes, TomloxA and TomloxB (Ferrie et al., 1994), TomloxC and Tomlox D (Heitz et al., 1997), and TomloxE (NCBI Accession AY008278). Analysis of the role that ethylene plays in the regulation of TomloxA, TomloxB and TomloxC during tomato ripening has shown that the individual isoforms are differentially regulated and may have different functions (Griffiths et al., 1999a). Levels of TomloxA mRNA decrease as ripening progresses and this is delayed in the mutants, Nr and rin as well as sense suppressed ACO1 (low ethylene) fruit, indicating that this gene is regulated by both ethylene and developmental factors (Griffiths et al., 1999a). TomloxB expression increases during ripening and is regulated by ethylene, since the mutant and low ethylene transgenic fruit show reduced expression. TomloxC transcripts increase in response to ethylene, however, ethylene treatment of mature green fruit does not induce expression. This would indicate the presence of a developmental pathway that initiates expression and an ethylene component that enhances mRNA levels once expression has been initiated by the developmental pathway (Griffiths et al., 1999a).
Silencing of TomloxA and TomloxB, by antisense gene knockout, failed to reduce flavour volatiles in ripening fruit and did not alter the levels of TomloxC mRNA (Griffiths et al., 1999b), suggesting that TomloxC may encode the major fruit lipoxygenase involved in flavour volatile production. TomloxC and the mainly leaf expressed TomloxD differ from the other LOX enzymes in that they are chloroplast targeted (Heitz et al., 1997). It therefore seems likely that during ripening TomloxC utilizes the polyunsaturated fatty acids from the redundant thylakoid structures as a substrate to produce the aroma volatiles hexanal and hexanol. TomloxD is thought to function in the octadecanoid defence signalling pathway which is activated in response to herbivore and pathogen attack (Heitz et al., 1997). Suppression of the Arabidopsis chloroplast atLOX2 gene, which is most similar to TomloxD, resulted in the absence of wound-inducible jasmonic acid (JA) accumulation and reduced expression of the wound- and JA-inducible vsp gene (Bell et al., 1995). Therefore, it appears that LOX has a dual role in both volatile production and defence signalling. Recent work with Arabidopsis has provided evidence that atLOX2 is a translation initiation factor-4e-binding protein and that this interaction may play a regulatory role given the numerous examples of products of LOX activity, such as JA, found to be involved in translational activation (Freire et al., 2000). Therefore the ethylene-dependent and independent regulation of LOX genes may orchestrate many aspects of fruit ripening and defence against pathogens.
Alcohol dehydrogenase (ADH) has also been shown to play a role in hexanol and hexenol accumulation in ripening tomato fruit (Speirs et al., 1998). Two ADHs have been indentified in tomato, ADH1 which is found only in pollen, seeds and young seedlings (Tanksley, 1979) and ADH2 which accumulates during the later stages in ripening commitant with the accumulation of flavour volatiles (Chen and Chase, 1993; Longhurst et al., 1994). Genetic manipulation of ADH2 levels in ripening tomato fruit has been shown to affect the balance of some flavour aldehydes and alcohols and fruits with increased ADH2 levels had a more intense ‘ripe fruit’ flavour (Speirs et al., 1998). Tomato ADH2 has not been identified as ethylene inducible. However, it is induced by low oxygen stress and it is likely that increasing ADH2 activity during ripening is a function of decreasing oxygen concentration within ripening fruit (Speirs et al., 2002).
Ethylene has also been shown to be important in the production of aroma volatiles in Charentais melon fruit, as antisense suppression of ethylene production results in strong inhibition of aroma (Ayub et al., 1996; Bauchot et al., 1998). The melon aroma volatile profile mainly consists of volatile esters and, although little information exists regarding the biosynthetic pathways involved, the last step in their formation is catalysed by acyl-transferases (AAT) (Fellman et al., 2000). AATs are a super family of multifunctional AATs and are implicated in diverse biochemical pathways such as fruit ripening, the production of epicuticular waxes and benzoyltransfer reactions (St-Pierre et al., 1998). A ripening-related gene, MEL2, isolated from melon fruit (Aggelis et al., 1997), has been identified as an ATT by expression in yeast (Yahyaoui et al., 2002). Recent analysis of antisense ACO melon has shown that fruit treated with the ethylene antagonist 1-methylcyclopropane have a 50% reduction in AAT activity. This indicates that the last step of alcohol acetylation comprises ethylene-independent and ethylene-dependent AATs (Flores et al., 2002). MEL2 also shows similarity to pTOM36 isolated from a tomato fruit-ripening library (Davies and Grierson, 1989). Although the function of this gene has not been fully investigated, ripening tomato does produces aromatic esters. An AAT gene that plays a crucial role in flavour biogenesis has also been recently cloned from strawberry, a non-climacteric fruit, indicating that AATs are not exclusively regulated by ethylene (Aharoni et al., 2000).
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