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The biological function of ascorbate oxidase (AAO) was not yet clarified, although it was suggested that AAO may be involved in cell growth. We investigated AAO expression and ascorbate metabolism during non‐synchronous, synchronous, and elongation cultures of tobacco BY‐2 cells. In non‐synchronous culture, AAO mRNA was abundant in logarithmic growth phase. Ascorbate content greatly increased during the growth, whereas dehydroascorbate content was slightly increased. In synchronous division culture, AAO mRNA was detected in all phases, but the levels were quite low in G1 phase. Ascorbate content was high in all phases, whereas dehydroascorbate content was low, especially in G1 phase. In elongation culture, the levels of AAO mRNA increased during elongation of the cells. AAO activity in the culture medium, as well as ascorbate and dehydroascorbate contents in the cells, also increased during the elongation. We propose that AAO expression and production of dehydroascorbate are under the control of the cell cycle and that AAO may function apoplastically as an ascorbate oxidizer in the process of cell elongation.

Transgenic tobacco plants expressing the ascorbate oxidase (AAO) gene in sense and antisense orientations, and an Arabidopsis mutant in which the T-DNA was inserted into a putative AAO gene, were used to examine the potential roles of AAO for salt-stress tolerance in plants. AAO activities in the transgenic tobacco plants expressing the gene in sense and antisense orientations were, respectively, about 16-fold and 0.2-fold of those in the wild type. Under normal growth conditions, no significant differences in phenotypes were observed, except for a delay in flowering time in the antisense plants. However, at high salinity, the percentage germination, photosynthetic activity, and seed yields were higher in antisense plants, with progressively lower levels in the wild type and the sense plants. The redox state of apoplastic ascorbate in sense plants was very low even under normal growth conditions. Upon salt stress, the redox state of symplastic and apoplastic ascorbate decreased among the three types of plants, but was lowest in the sense plants. The hydrogen peroxide contents in the symplastic and apoplastic spaces were higher in sense plants, progressively lower in the wild type, followed by the antisense plants. The Arabidopsis T-DNA inserted mutant exhibited very low ascorbate oxidase activity, and its phenotype was similar to that of antisense tobacco plants. These results suggest that the suppressed expression of apoplastic AAO under salt-stress conditions leads to a relatively low level of hydrogen peroxide accumulation and a high redox state of symplastic and apoplastic ascorbate which, in turn, permits a higher seed yield.

In higher plants, the terminal step of L-ascorbic acid (AsA) biosynthesis is catalyzed by the enzyme L-galactono-1,4-lactone dehydrogenase (EC 1.3.2.3, GalLDH). We generated AsA-de®cient transgenic tobacco BY-2 cell lines by antisense expression of the GalLDH cDNA that was ampli®ed from BY-2 cells using PCR. Two transgenic cell-lines, AS1±1 and AS2±2, having a marked expression of antisense RNA were analyzed. Antisense suppression of GalLDH mRNA led to a signi®cant decline in the GalLDH activity. The AsA levels in the transgenic cell lines were found to be 30% lower than the wild-type BY-2 cells. In synchronous cultures, division of AS1±1 and AS2±2 cells was restrained with a concomitant decrease in mitotic index that was probably due to a decline in AsA levels. The rate of cell growth was also found to be less than that of the wild-type cells. Interestingly, there was a signi®cant phenotypic difference between the transgenic and wild-type cells. The calli of AS1±1 and AS2±2 appeared to be sticky and soft. Back extrusion method also showed that AsA-de®cient BY-2 callus was rheologically soft. Furthermore, microscopic analysis revealed that AS1±1 and AS2±2 cells were abnormally slender, suggesting a potential for a signi®cant and a uni-axial elongation. Thus, we observed that decline in the AsA levels has an adverse effect on the division, growth and structure of a plant cell.

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...

L-Galactono-1,4-lactone dehydrogenase (GalLDH; EC 1.3.2.3) is the last enzyme in the putative L-ascorbic acid (AsA) biosynthetic pathway of plants. Here, we show for the first time that the overexpression of GalLDH can increase AsA content in tobacco (Nicotiana tabacum L.) BY-2 cells. To see the effect, we analyzed the properties of these AsA-overproducing transgenic cell lines, especially in relation to AsA content of cells, cell division, senescence and resistance to oxidative stress. The mitotic index in AsA-overproducing cells was higher than in wild-type cells. Moreover, the browning of these cells was markedly restrained, and the proportion of dead cells was reduced, especially in the later period of culture. These AsA-overproducing cells also acquired resistance to paraquat (methyl viologen), which produces active oxygen species. These results contribute to the previous insights about AsA and raise the possibility of the generation of plants that have resistance to environmental stresses by increasing their AsA content.

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