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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.