Characterization of ACC oxidase from the leaves of Malus domestica Borkh. (apple) : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Plant Biochemistry and Molecular Biology, Institute of Molecular BioSciences, Massey University, Palmerston North, New Zealand
The expression, accumulation and kinetic properties of 1 -aminocyclopropane- 1 -carboxylic acid (ACC) oxidase (ACO), the enzyme which catalyses the final step in the ACC-dependent pathway of ethylene biosynthesis in plants, is examined. The investigation is divided into three sections: (i) identification of two ACO genes in apple leaf tissue, designated MD-ACO2 and MD-ACO3, (ii) kinetic analyses of each of the three isoforms of ACO in apple (MD-ACO1, MD-ACO2 and MD-ACO3) expressed in E. coli, and (iii) temporal and developmental expression in vivo of each of the ACO genes and accumulation of the corresponding gene products in leaf and fruit tissue. The coding regions of putative ACO gene transcripts were generated from leaf tissue using RT-PCR. Sequence alignments and interrogation of the expressed sequence tags (ESTs) database indicated that the entire open reading frame (ORF) sequences were encoded by two distinct genes, and these are designated MD-ACO2 and MD-ACO3. A third ACO gene had been identified in apple by other research workers previously, and designated MD-ACO1. Differences are obvious in the number of base-pairs (bp) constituting the entire ORF of MD-ACO1 (942 bp), MD-ACO2 (990 bp) and MD-ACO3 (966 bp). MD-ACO1 and MD-ACO2 share a close nucleotide sequence identity of 93.9 % in the ORF but diverge in the 3' untranslated regions (3' -UTR) (69.5 %). In contrast, MD-ACO3 shares a lower sequence identity with both MD-ACO1 (78.5 %) and MD-ACO2 (77.8 %) in the ORF, and in the 3'-UTR (MD-ACO1, 68.4 %; MD-ACO2, 71 %). A comparison of the gene structures show that the endonuclease restriction sites are unique to each individual MD-ACO sequence. Genomic Southern analysis, using probes spanning the 3'-UTR and the 3'-end of the coding region confirmed that MD-ACO3 is encoded by a distinct gene. However, while the distinction between MD-ACO1 and MD-ACO2 is not as definitive, different gene expression patterns adds credence to their distinctiveness. Each of the three deduced amino acid sequences contain all of the residues hitherto reported to be necessary for maximal ACO activity. Expression of MD-ACO1, MD-ACO2 and MD-ACO3 as fusion proteins in E. coli was induced using isopropy1-β-D-thiogalactopyranoside (IPTG), the recombinant proteins purified by nickel-nitrilotriacetic acid (NiNTA) affinity chromatography and the products had predicted masses determined from the nucleotide sequences, including the His-tag of 38.53 kDa (MD-ACO1), 40.39 kDa (MD-ACO2) and 39.3 kDa (MD-ACO3). Polyclonal antibodies were raised against the MD-ACO3 fusion in rabbit for western blot analysis. Antibodies had been raised previously against recombinant MD-ACO1, and while it was considered likely the MD-ACO2 would be recognized by the MD-ACO1 antibodies, MD-ACO2 does not appear to be recognized in vivo by the antibody. Analyses of the kinetic properties of the three apple ACOs was undertaken. Apparent Michaelis constants (Km) of 89.39 μM (MD-ACO1), 401.03 μM (MD-ACO2) and 244.5 μM (MD-ACO3) have been determined which suggests differences in the affinity of each enzyme for the substrate ACC. Maximum velocity (Vmax) was determined for MD-ACO1 (15.15 nmol), MD-ACO2 (12.94 nmol) and MD-ACO3 (18.94 nmol). The catalytic constant (Kcat) was determined for MD-ACO1 (6.6 x 10-2), MD-ACO2 (3.44 x 10-2) and for MD-ACO3 (9.14 x 10-2), with kcat/Km(μM s-1) values of 7.38 x 10-4 μM s-1 (MD-ACO1), 0.86 x 10-4 M s-1 (MD-ACO2) and 3.8 x 10-4 μM s-1 (MD-ACO3). The optimal pH for MD-ACO1 was 7.0 - 7.5, MD-ACO2 7.5 - 8.0 and MD-ACO3 7.0 - 8.0. All three isoforms exhibited absolute requirements for the co-substrate ascorbate in vitro with optimal activity at 30 mM. Similarly, ferrous iron (FeSO4.7H20; of 15 - 25 μM) and sodium bicarbonate (NaHCO3; of 30 mM) were required for optimal activity, and were the same for all isoforms. No significant difference in thermostability was found in this study between the MD-ACO isoforms at the P = 0.05 level. However, the activities of the enzyme differed significantly between temperatures over time. In vivo expression of each of the ACO genes in leaf tissue determined using RT-PCR and cDNA Southern analysis reveal that both MD-ACO2 and MD-ACO3 are expressed in young leaf tissue and in mature leaf tissue. While MD-ACO3 is expressed predominantly in young leaf tissue, MD-ACO2 (in common with MD-ACO1) is expressed predominantly in mature fruit tissue. None of the MD-ACOs were observed to be senescence associated genes (SAG). MD-ACO3 protein accumulated predominantly in young leaf tissue and less intensely in both mature leaf tissue and young fruit tissue, while MD-ACO1 accumulated only in mature fruit tissue. For the developmental studies, samples were collected at approximately 11 am in this study. MD-ACO2 and MD-ACO3 were also expressed in leaf tissue collected over a 24 h period in the spring and also in the autumn. For both genes transcripts accumulated in the presence of fruit but tended to disappear in the absence of fruit. These results show that MD-ACO1, MD-ACO2 and MD-ACO3 are differentially expressed, and that MD-ACO3 is encoded by a gene distinct from MD-ACO1 and MD-ACO2. MD-ACO1 and MD-ACO2 are either allelic forms of the same gene or closely clustered. Although there is some variation in kinetic properties which may reflect different physiological environments, they do not vary greatly.