The pathway of ethylene synthesis is well established in higher plants (reviewed in Bleecker and Kende, 2000). Ethylene is formed from methionine via S-adenosyl-L-methionine (AdoMet) and the cyclic non-protein amino acid 1-aminocyclopropane-1-carboxylic acid (ACC). ACC is formed from AdoMet by the action of ACC synthase (ACS) and the conversion of ACC to ethylene is carried out by ACC oxidase (ACO) (Kende, 1993). In addition to ACC, ACS produces 5'-methylthioadenosine, which is utilized for the synthesis of new methionine via a modified methionine cycle. This salvage pathway preserves the methylthio group through every revolution of the cycle at the cost of one molecule of ATP. Thus high rates of ethylene biosynthesis can be maintained even when the pool of free methionine is small. Two other ethylene-regulated genes have been identified that may play a possible role in the methionine cycle, E4, a putative methionine sulphoxide reductase protein and ER69 a putative cobalamine-independent methionine synthase (Montgomery et al., 1993a; Zegzouti et al., 1999). In this pathway it is well known that biosynthesis is subject to both positive and negative feedback regulation (Kende, 1993). Positive feedback regulation of ethylene biosynthesis is a characteristic feature of ripening fruits and senescing flowers in which exposure to exogenous ethylene or propylene results in a large increase in ethylene production due to the induction of ACS and ACO. Both of these enzymes are encoded by small multigene families and their expression is differentially regulated by various developmental, environmental and hormonal signals (Kende, 1993; Zarembinski and Theologis 1994; Barry et al., 2000; Llop-Tous et al., 2000).
At least eight ACS genes have been identified in tomato (LEACS1A, LEACS1B and LEACS2–7), (Zarembinski and Theologis, 1994; Oetiker et al., 1997; Shiu et al., 1998) and many others have been identified in both climacteric and non-climacteric fruits such as melon, cucumber and citrus (Nakajima et al., 1990; Yamamoto et al., 1995; Wong et al., 1999). However, the role that ACS plays in ripening has been most widely studied in tomato. ACS shows homology to pyridoxal-5'-phosphate (PLP)-dependent aminotransferases and mutant complementation studies have shown that the enzyme can act as a dimer (Tarun and Theologis, 1998). Recent studies of the ACS crystal structure (Capitani et al., 1999) and PLP co-factor binding (Huai et al., 2001) have confirmed similarity between the ACS catalytic binding site and those of other PLP-dependent aminotransferases. The presence of LEACS2 and LEACS4 transcripts during ripening has been well documented (Rottmann et al., 1991; Olson et al., 1991; Yip et al., 1992; Lincoln et al., 1993; Barry et al., 2000). Recent work has also confirmed the presence of LEACS1A and LEACS6 in tomato fruit before the onset of ripening and shown that each ACS in fruit has a different expression pattern (Fig. 1B) (Barry et al., 2000).
Analysis of ACS gene induction in mutant fruit with disrupted ethylene signalling has been used to identify which ACS gene is ethylene-regulated. The Never ripe (Nr) mutant cannot perceive ethylene due to a mutation in the ethylene-binding domain of the NR ethylene receptor (Lanahan et al., 1994; Wilkinson et al., 1995). Fruit from the ripening inhibitor (rin) mutant do not show autocatalytic ethylene production (Herner and Sink, 1973) and cannot transmit the ethylene signal downstream to ripening genes due to a mutation in the RIN transcription factor (Vrebalov et al., 2002). Nr and rin mutant fruit have shown that LEACS2 expression requires ethylene whereas LEACS1A and LEACS4 exhibited only slightly delayed expression in Nr indicating that ethylene is not responsible for regulation of these genes (Barry et al., 2000). All four fruit ACS genes showed the same expression patterns in rin fruit as in mature green wild-type fruit, but did not show any ripening-related changes of expression (Barry et al., 2000). Therefore, it has been proposed that LEACS1A and LEACS6 are involved in the production of system 1 ethylene in green fruit (Barry et al., 2000). System 1 continues throughout fruit development until a competence to ripen is attained, whereupon a transition period is reached, during which LEACS1A expression increases and LEACS4 is induced. During this transition period, system 2 ethylene synthesis (autocatalysis) is initiated and maintained by ethylene-dependent induction of LEACS2 (Barry et al., 2000). Antisense inhibition of LEACS2, which also down-regulated LEACS4, reduced ripening-related synthesis of ethylene to 0.1% of control fruit. The antisense fruit displayed an abnormal pattern of ripening such as reduced lycopene accumulation, delayed softening and a much reduced climacteric peak (Oeller et al., 1991).
Some debate exists as to whether ACS enzymes are regulated transcriptionally, post-transcriptionally or post-translationally and whether ethylene plays a role in this regulation (Kende, 1993; Olson et al., 1995; Oetiker et al., 1997). In vitro analysis of LEACS2 enzyme activity has shown that the deletion of 52 amino acids from the C-terminus increases enzyme activity (Li and Mattoo, 1994; Li et al., 1996). However, it has recently been shown that LEACS2 is phosphorylated in wounded tomato fruit and is not truncated (Tatsuki and Mori, 2001). Sequence analyses have identified a conserved domain that is considered to be the phosphorylation site (F/L)RLS(F/L). Recombinant LEACS3 and LEACS2 containing this domain were phosphorylated in vitro whereas LEACS4 was not phosphorylated and does not contain this site (Tatsuki and Mori, 2001). It seems that the role of phosphorylation is not to regulate the specific activity of the enzyme but to control the rate of enzyme turnover (Spanu et al., 1994). The possibility that ACS phosphorylation regulates ethylene production is supported by the finding that mutation of the C-terminal domain of Arabidopsis ACS5 induces the eto2-1 mutant to overproduce ethylene (Vogel et al., 1998). Furthermore, observations by Ecker that the ETO1 protein bound to ACS5 in vitro and inhibited its activity has led to speculation that the ETO1 protein may be involved in a protein degradation pathway (Cosgrove et al., 2000; Tatsuki and Mori, 2001). Therefore; it is possible that phosphorylation of ACS protects the protein from degradation, which in turn could cause ACS to accumulate and ACS activity to increase, accounting for the burst of ethylene produced by ripening fruit (Tatsuki and Mori, 2001).
Initially it was thought that ACS activity was the key step in controlling the production of ethylene and that ACO activity was constitutive (Yang and Hoffman, 1984; Theologis et al., 1993). However, the role that ACO activity plays in the regulation of ethylene biosynthesis has become apparent in recent years. The rise in ACO activity precedes ACS activity in preclimacteric fruit in response to ethylene, indicating that ACO activity is important for controlling ethylene production (Lui et al., 1985). Examination of ACO mRNA expression patterns in various tissues and different developmental stages provided further evidence for the regulatory role that ACO plays in ethylene production during fruit ripening (Holdsworth et al., 1987; Hamilton et al., 1990; Balague et al., 1993; Barry et al., 1996). Historically, studying ACO has proved to be problematic due to the lack of an in vitro assay and difficulties encountered during purification (Kende, 1993). The first ACO gene was identified through antisense expression of a clone, pTOM13, then of unknown function (Holdsworth et al., 1987). mRNA expression patterns showed the pTOM13 gene was expressed in both ripening tomatoes and wounded leaves and down-regulation of this gene produced transgenic tomato plants with reduced levels of ethylene synthesis and ACO activity (Hamilton et al., 1990). The role of this enzyme in ethylene biosynthesis from ACC was confirmed by expression of pTOM13 in yeast and Xenopus oocytes, where the pTOM13-encoded protein was shown to convert ACC to ethylene, with the correct stereospecificity (Hamilton et al., 1991; Spanu et al., 1991). A further three ACO genes have been identified in tomato in response to wounding and during flower development, leaf and flower senescence and fruit ripening (Holdsworth et al., 1988; Barry et al., 1996; Blume and Grierson, 1997; Nakatsuka et al., 1998; Llop-Tous et al., 2000). ACO genes have also been identified in petunia, mung bean and other climacteric fruit such as melon, avocado, apples, and bananas (reviewed in Jiang and Fu, 2000).
ACO enzymes are members of the Fe(II-dependent family of oxidases/oxygenases (Hamilton et al., 1990; Prescott, 1993). In vitro ACO activity requires ascorbate as a substrate and the CO2 produced during the climacteric peak is thought to activate the enzyme in vivo (Dong et al., 1992; Smith and John, 1993). Two models have been proposed for the production of ethylene from ACC. In the first model the ascorbate association with the Fe(II) ion activates a bound O2 to yield high-valent iron-oxo species that oxidizes ACC to release ethylene (Zhang et al., 1997). More recently, it has been suggested that the role of the Fe(II) ion is to bind ACC and O2 simultaneously and promote electron transfer, which initiates catalysis of ACC to ethylene (Rocklin et al., 1999).
Analysis of ACO gene expression patterns in ripening fruit shows that each gene is highly regulated with transcripts of individual members accumulating to varying degrees at distinct developmental stages (Barry et al., 1996). LEACO1 and, at a lower level LEACO3, are expressed at the onset of fruit ripening. LEACO1 transcripts peak at breaker +3 and then fall back to levels observed at breaker, whereas LEACO3 transcripts are only transiently expressed at breaker before disappearing. Therefore it is likely that the first step in catalytic ethylene biosynthesis is the de novo synthesis of ACO1, the ethylene produced induces ACS gene expression, which in turn produces more ACC.