Ascorbate oxidase expression in pumpkin (Cucurbita spp.) tissues was studied. Specific ascorbate oxidase activities in pumpkin leaf and stem tissues were about 2 and 1.5 times that in the fruit tissues, respectively. In seeds, little ascorbate oxidase activity was detected. Northern blot analyses showed an abundant ascorbate oxidase mRNA in leaf and stem tissues. Fruit tissues had lower levels of ascorbate oxidase mRNA than leaf and stem tissues. Ascorbate oxidase mRNA was not detected in seeds. Specific ascorbate oxidase activity gradually increased during early seedling growth of pumpkin seeds. The increase was accompanied by an increase in ascorbate oxidase mRNA. When ascorbate oxidase activity in developing pumpkin fruits was investigated, the activities in immature fruits that are rapidly growing at 0, 2, 4, and 7 d after anthesis were much higher than those in mature fruits at 14 and 30 d after anthesis. The specific activity and mRNA of ascorbate oxidase markedly increased after inoculation of pumpkin fruit tissues into Murashige and Skoog's culture medium in the presence of an auxin such as 2,4-dichlorophenoxyacetic acid (2,4-D) but not in the absence of 2,4-D. In the presence of 10 mg/L of 2,4-D, ascorbate oxidase mRNA was the most abundant. Thus, ascorbate oxidase is induced by 2,4-D. These results indicate that ascorbate oxidase is involved in cell growth. In pumpkin callus, ascorbate oxidase activity could be markedly increased by adding copper. Furthermore, immunological blotting showed that the amount of ascorbate oxidase protein was also increased by adding copper. However, northern blot analyses showed that ascorbate oxidase mRNA was not increased by adding copper. We suggest that copper may control ascorbate oxidase expression at translation or at a site after translation.Ascorbate oxidase (EC 1.10.3.3) is a copper-containing blue enzyme that has been studied in plant tissues such as pumpkin (17, 29), cucumber (24), and orange (31). The enzyme catalizes the oxidation of ascorbic acid to dehydroascorbic acid. Recently, the enzyme has been used for clinical and food analyses of L-ascorbic acid (8,20).The definitive biological function of ascorbate oxidase is not clear, although it has been reported that the enzyme may participate in a redox system involving ascorbic acids (32). Furthermore, the subcellular localization of ascorbate oxidase is uncertain. The enzyme may be localized in the cell wall 1 This work was supported in part by Grants-in-Aid for Scientific Research (No. 02261214) from the Ministry of Education, Science, and Culture of Japan. (13,22,25), although there have been some reports that the enzyme is localized in the cytoplasm (33), microsomes (33), or vacuole (31). Recently, Lin and Varner (18) studied the expression of ascorbate oxidase in zucchini and reported that the enzyme may be involved in reorganization of the cell wall.We have studied the ascorbate oxidase in cultured pumpkin (Cucurbita spp.) cells and in a previous paper reported that ascorbate oxidase activity ra...

A Agtl 1 cDNA expression library was constructed from size-fractionated poly(A)-rich RNA of cultured pumpkin cells. A full-length cDNA clone for ascorbate oxidase mRNA was selected from the library by screening with synthetic oligonucleotides designed from the amino-terminal sequence of ascorbate oxidase protein. The identity of the clone was confirmed by comparing the amino acid sequence deduced by nucleotide sequence analysis with that determined for the amino-terminal sequence of pumpkin ascorbate oxidase. The nucleotide sequence of the cDNA insert was found to contain an open reading frame of 579 codons corresponding to a signal peptide of 30 amino acids and the mature 549-residue ascorbate oxidase. Furthermore, it was found that the amino acid sequence deduced from the nucleotide sequence of the cDNA insert contained four potential Nglycosylation sites and copper-binding amino acid residues located in four regions where the sequence was identical or nearly identical to those of the other known blue multicopper oxidases Neurospora crassa laccase and human ceruloplasmin.Ascorbate oxidase is a copper-containing enzyme that occurs in many plant tissues such as pumpkin [l, 21, cucumber [3] and orange [4], and plays a role in secondary metabolism. The precise biological function of ascorbate oxidase has not been clarified, although it has been reported that the enzyme may participate in a redox system involving ascorbic acid IS]. However, the enzyme has recently been used as a valuable reagent for clinical and food analyses of L-ascorbic acid [6, 71.We have studied the ascorbate oxidase in cultured pumpkin cells and in a previous paper [8] reported that ascorbate oxidase activity rapidly increased during growth of pumpkin callus. We have found that ascorbate oxidase activity in cultured pumpkin cells can be markedly increased by adding copper, a prosthetic metal of the enzyme, and that this increase is accompanied by an increase in the enzyme protein, suggesting that copper controls the amount of ascorbate oxidase protein [9]. Furthermore, we have shown that ascorbate oxidase is secreted into the culture medium in the cell suspension cultures [lo] and that calcium ions markedly stimulate the secretion of ascorbate oxidase [ll]. Further studies are required to elucidate the molecular mechanisms responsible for regulating ascorbate oxidase gene expression.Recently, Ohkawa et al. [12] have isolated the cDNA for ascorbate oxidase from cucumber fruit tissues and determined the nucleotide sequence of the cDNA insert. So far, however, the cDNA for pumpkin ascorbate oxidase has not been cloned. In the present study, we constructed a cDNA expression library from poly(A)-rich RNA of cultured pumpkin cells and selected a full-length cDNA clone for ascorbateCorrespondence to

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