Reprint of: 1-Aminocyclopropanecarboxylate Synthase, a Key Enzyme in Ethylene Biosynthesis.

1-Aminocyclopropanecarboxylate (ACC) synthase, which catalyzes the conversion of S-adenosylmethionine (SAM) to ACC and methylthioadenosine, was demonstrated in tomato extract. Methylthioadenosine was then rapidly hydrolyzed to methylthioribose by a nucleosidase present in the extract. ACC synthase had an optimum pH of 8.5, and a Km of 20 µM with respect to SAM. S-Adenosylethionine also served as a substrate for ACC synthase, but at a lower efficiency than that of SAM. Since S-adenosylethionine had a higher affinity for the enzyme than SAM, it inhibited the reaction of SAM when both were present. S-Adenosylhomocysteine was, however, an inactive substrate. The enzyme was activated by pyridoxal phosphate at a concentration of 0.1 µM or higher, and competitively inhibited by aminoethoxyvinylglycine and aminooxyacetic acid, which are known to inhibit pyridoxal phosphate-mediated enzymic reactions. These results support the view that ACC synthase is a pyridoxal enzyme. The biochemical role of pyridoxal phosphate is catalyzing the formation of ACC by α,γ-elimination of SAM is discussed.

[Promoter specificity of LE-ACS6 gene in LE-ACS6::GUS transgenic arabidopsis plants].

The LE-ACS6 gene encodes ACC synthase, the key enzyme of ethylene biosynthesis pathway. Accumulation of LE-ACS6 transcripts is concomitant with the system 1 ethylene production in the pre-climacteric tomato fruit, and both are down regulated by exogenous ethylene treatment. To elucidate the possible role of system 1 ethylene in plant development and investigate the promoter tissue specificity of LE-ACS6 gene, stable transformation of Arabidopsis with a LE-ACS6 promoter-GUS fusion construct by Agrobacterium method has been done. Histochemical localization of GUS activity and beta-glucuronidase enzyme assay in transgenic LE-ACS6::GUS plants showed strong expression of GUS in cotyledons and hypocotyls of 6 d seedlings, but no GUS activity was detected in roots. The GUS activity of 6 d seedling was increased significantly when treated with NAA 10(-4)mol/L. In 40 d rosette leaf LE-ACS6 promoter driven GUS gene was predominantly expressed in mature leaves. Lower level of GUS expression was detected in younger and older leaves. Wounding was found to increase GUS gene expression in transgenic leaves. Again, exogenous NAA was found to increase the GUS activity in wounded leaf tissue. Strong staining reaction was observed in the top part of rapidly growing stems. In different developmental stages of Arabidopsis seeds, "mature green" pods were strongly stained, but "ripening fruits" were not colored. These observations are concomitant with the system 1 ethylene production, suggesting a popular mechanism in regulating ethylene biosynthesis in different plants, at least in tomato and Arabidopsis.

Novel broccoli 1-aminocyclopropane-1-carboxylate oxidase gene (Bo-ACO3) associated with the late stage of postharvest floret senescence.

A novel 1-aminocyclopropane-1-carboxylate (ACC) oxidase gene (Bo-ACO3) was first isolated from senescing broccoli florets by subtractive hybridization. The cDNA clone comprised a 963 bp open reading frame encoding a protein of 321 amino acids. The predicted molecular mass and pI were 36 kDa and 5.42, respectively. Bo-ACO3 shares 68% identity in the coding region with Bo-ACO1 (ACC Ox1) and Bo-ACO2 (ACC Ox2) and is quite divergent from the 3' untranslated regions. Bo-ACO3 transcript was accumulated to high levels only at the late stage of senescence after harvest. Southern blot hybridization using full-length cDNA as a probe suggested that the Bo-ACO3 gene is a single-copy gene in the broccoli genome. The deduced 321 amino acid sequence of Bo-ACO3 shares 70% identity with either Bo-ACO1 or Bo-ACO2. The BO-ACO3 gene was expressed in Escherichia coli as a 38 kDa active ACO enzyme. It was concluded that Bo-ACO3 is a senescence-associated gene involved in the late-phase senescence of postharvest broccoli.

Subcellular localization of 1-aminocyclopropane-1-carboxylic acid oxidase in apple fruit.

1-Aminocyclopropane-1-carboxylic acid (ACC) oxidase catalyzes the oxidation of ACC to the gaseous plant hormone, ethylene. Although the enzyme does not contain a typical N-terminal consensus sequence for the transportation across the endoplasmic reticulum (ER), it has recently been shown to locate extracellularly by immunolocalization study. It was of interest to examine whether the enzyme contains a signal peptide that is overlooked by structure prediction. We observed that the in vitro translated apple ACC oxidase was not co-processed or imported by the canine pancreatic rough microsomes, a system widely used to identify signal peptide for protein translocation across ER, suggesting that apple ACC oxidase does not contain a signal peptide for ER transport. A highly specific polyclonal antibody raised against the recombinant apple ACC oxidase was used to examine the subcellular localization of the enzyme in apple fruit (Malus domestica, var. Golden Delicious). The location of ACC oxidase appeared to be mainly in the cytosol of the apple fruit pericarp tissue as was demonstrated by electron microscopy using immunogold-labeled antibodies. The pre-immune serum or pre-climacteric fruit control gave essentially no positive signal. Based on these observations, we conclude that ACC oxidase is a cytosolic protein.