Regulation of Ascorbate Oxidase Expression in Pumpkin by Auxin and Copper

Abstract

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...

Auxin Metabolism in the Root Apical Meristem

“…Whether AAO could regulate endogenous auxin levels within the QC has not yet been proven. However, its in vitro ability to oxidize auxin, its high concentration within the QC, and the property of auxin to regulate AAO mRNA and protein levels and activity (Esaka et al, 1992;Kerk and Feldman, 1995) argue in favor of in vivo catabolic interactions between AAO and auxin.…”

“…1B). Esaka et al (1992) have demonstrated a marked increase in ascorbate oxidase protein in pumpkin cells by adding copper to the culture medium. We used this method to cause a 1.7-fold increase in AAO in seedling radish roots grown in culture supplemented with 10 m CuSO 4 .…”

Ascorbate oxidase is the potential conductor of a symphony of signaling pathways

“…The "mysterious enzyme" ascorbate oxidase (AO; EC 1.10.3.3) is a blue copper oxidase displaying several interesting features: (1) it is only found in plants and fungi; 1 (2) it is associated to the cell wall; 2 (3) it catalyzes oxygen reduction to water preferentially using ascorbate (AsA) as the electron donor; 3 (4) its expression and activity are induced by auxin, 4 and light. 5 All this information, suggesting in part a possible role of AO in signal perception/transduction, has been known for decades, but the function of this remarkable enzyme remained controversial.…”

Constitutive overexpression of GmDof17-1, a putative DOF transcription factor from soybean causing growth inhibition in tobacco

“…The expression of the pumpkin ascorbate oxidase gene (AOBP), which binds to the AGTA repeat through the Dof domain, is regulated by auxin (Kisu et al, 1998;Esaka et al, 1992). PtDof1 was isolated from Populus tomentosa, which exhibits tissue-specifi c expression (Xin et al, 2009).…”