El teorema de incompletez de Gödel y la diagonalización

ACC oxidase (ACO; EC 1.14.17.4) is the enzyme that catalyses the final step of ethylene biosynthesis, which is the conversion of ACC into ethylene, and was initially called the ethylene forming enzyme (EFE). Identification and characterisation of ACO has lagged behind ACS, since it was initially proposed that the enzyme was unstable and difficult to purify in vitro (Cameron et al., 1979).

Introduction

12 EFE was first identified from cDNA clones of ripening-related tissues in tomato, where one clone was homologous to mRNA which accumulated prior to a wounding- induced ethylene peak in the unripe fruit and leaf tissue of the tomato (Slater et al.,

1985). This clone was designated pTOM13 (now called LE-ACO1) and it was shown that it was involved in ethylene production (Slater et al., 1985) and was induced by ethylene in mature green tomato fruit (Maunders et al., 1987). To show this, Hamilton et al. (1990) transformed tomato plants with an antisense construct of

pTOM13 and they observed a great reduction in ethylene production in these plants. They suggested that the pTOM13 gene encoded an enzyme involved in ethylene biosynthesis, but the coded protein is too small to be an ACC synthase (Hamilton et al., 1990). The translated product of pTOM13 had a molecular mass of ca. 35 kDa (Smith et al., 1986), whilst partially purified ACC synthase from zucchini fruit had a molecular mass of ca. 53 kDa (Sato et al., 1991). Further confirmation that the

pTOM13 transcript encoded EFE was achieved by comparing the in vivo activity of the enzyme obtained from leaf disks of wild type and pTOM13 transformed plants in the antisense orientation and it was shown that the ethylene-forming activity in the antisense plants was reduced in a dosage-dependent manner (Hamilton et al., 1990). Finally, Hamilton et al. (1991) showed that a full length clone of pTOM13 (pRC13)

cDNA expressed heterologously in yeast had an EFE activity.

The deduced amino acid sequence of pTOM13 was shown to have high homology to flavonone-3-hydroxylase (Hamilton et al., 1990), a member of the 2-oxoglutarate- dependant dioxygenases (2-ODDs) (Prescott and John, 1996; Iturriagagoitia-Bueno

et al., 1996), which is a non-heme iron enzyme family requiring aerobic conditions during extraction. The majority of its members require Fe2+ _{and 2-oxoglutarate, as}

co-substrates for activity in vitro (Britsch and Griserbach, 1986), but ACO seems to be unique in this family, since it requires ascorbate but not 2-oxoglutarate as co- substrate (Escribano et al., 1996; Schofield and Zhang, 1999). However, when Fe2+

and ascorbate were added into a soluble fraction of melon fruit protein, EFE activity could be detected and measured (Ververidis and John, 1991). Subsequently, confirmation that the EFE activity measured in vitro was representative of the in vivo

Introduction

13 (Fernandez-Maculet and Yang, 1992). Following stereospecificity analysis of the reaction, the stoichiometry of the EFE catalysed reaction was determined (Figure 1.3.) (Dong et al., 1992; Fernandez-Macule and Yang, 1992; Kuai and Dilley, 1992; Schofield and Zhang, 1999) and the enzyme was renamed ACC oxidase.

Figure 1.3 Stoichiometry of the reaction catalysed by ACC oxidase.

Reaction catalysed by ACC Oxidase (here named as ACCO) (A), and proposed metal coordination chemistry of ACO based on spectroscopic studies, showing the Fe, dioxide binding sites and the amino acid residues, in the active site of the enzyme (B) (Modified from Schofield and Zhang, 1999).

Following the discovery that ACC oxidase activity in vitro, with the addition of appropriate cofactors, resembled the in vivo activity (Fernandez-Maculet and Yang, 1992), purification and characterisation of the enzyme from many plant species progressed quickly, including that in avocado (McGarvey and Christoferson, 1992), tomato (Zhang et al., 1995), pear (Fonseca et al., 2004) and white clover (Gong and McManus, 2000). Kinetic analysis of ACO activity demonstrated that the ACO enzyme exists as more than one isoform, for example, in apple (Binnie and McManus, 2009).

In comparison to ACS, ACO is encoded by a smaller multi-gene family in many plant species. Four members of ACO have been identified and isolated from tomato,

LE-ACO1, LE-ACO2, LE-ACO3 and LE-ACO4 (Bouzayen et al., 1993; Barry et al.,

1996; Nakatsuka et al., 1998), whilst three members were isolated and identified from melon, CM-ACO1, CM-ACO2 and CM-ACO3 (Tang et al., 1993). Similarly, two members of ACO have been identified in peach, PP-ACO1 and PP-ACO2

Introduction

14 (Binnie and McManus, 2009). Members of the ACO gene family share a high nucleotide sequence identity (more than 70%) in their coding regions, both within the same family and between families from different plant species (Lasserre et al., 1996), but these genes have more distinct sequences in the 5´ and 3´UTRs.

Initially ACS was thought to have been the key step which limited ethylene biosynthesis, and ACO was considered to be expressed constitutively during plant development which led to the suggestion that ACO did not play a part in the crucial regulation of ethylene biosynthesis (Yang and Hoffmann, 1984; Theologies et al.,

1993). However, evidence is now accumulating to show that ACO also contributes towards the fine tuning of ethylene production in higher plants. As with ACS, members of ACO gene families have been reported to be regulated at both the transcriptional and post-transcriptional levels.

Studies of ACO gene expression and ACO protein accumulation in many plant species have shown these to be developmentally regulated (Barry et al., 1996; Alonso et al., 2003; Shan and Goodwin, 2006; Higgins et al., 2006, Binnie and McManus, 2009). In relation to the four members in the tomato gene family,

LEACO1 and LEACO3 are expressed in the floral and senescent leaf tissues,

LEACO1 and LEACO4 are found in the fruit and their expression increases during fruit ripening, while LEACO2 is expressed in the anther (Barry et al., 1996; Nakatsuka et al., 1998; Anjanasree et al., 2005). Similarly, the lowest level of the

Ca-ACO1 transcript has been found during the early ripening stages of coffee fruit development (Pereira et al., 2005). In apple, three MD-ACO genes are also differentially expressed where MD-ACO1 is only expressed in mature apple fruit, whilst MD-ACO2 is expressed predominantly in younger fruit tissues, but it is also expressed in young leaf tissues, while MD-ACO3 is predominantly expressed in young and mature leaf tissues, with less expression in young (pre-ripening) fruit tissues (Binnie and McManus, 2009).

In addition to the developmental regulation of the ACO multi-gene family, evidence is now emerging to suggest that ACO gene expression is responsive to abiotic or environmental cues, in addition to exogenously applied hormones. For example,

Introduction

15 following wounding (at post-harvest), elevated ethylene production in broccoli has shown to be associated with expression of BO-ACO1 and BO-ACO2 (Higgins et al.,

2006). In melon, only CM-ACO1 was responsive to wounding and ethylene treatment, whilst both CM-ACO2 and CM-ACO3 were not (Laserre et al., 1996). Similarly, there are two members of PP-ACO in peach where expression of PP- ACO1 is induced by wounding and propylene (ethylene) treatment in young fully- expanded leaves, but expression of PP-ACO2 transcript is absent in leaves and is not induced by wounding and ethylene treatment (Ruperti et al., 2001). In addition, Shan and Goodwin (2006) reported that NbACO1 expression is induced by fungal infection in Nicotiana banthamiana, whilst expression of RP-ACO1 in rumex (Rumex palustris) is induced by submergence (Rieu et al., 2005).

This evidence supports the view that ACO may also regulate ethylene biosynthesis in plants, both during plant development and in response to environmental cues. However, involvement of ACO has not yet been studied as extensively as ACS, as so less evidence is currently available to support the idea that only a specific ACO isoform is regulated by certain environmental cues and at which levels (transcriptional, post-transcriptional and/or post-translational) this regulation occurs .