The balance between antioxidants, such as ascorbate (ASC) and glutathione, and oxidative reactive oxygen species (ROS) is known to play a pivotal role in the response of plant cells to abiotic stress. Here cell cultures of Arabidopsis thaliana were investigated with regard to their response to elevated levels of cadmium. At concentrations <100 microM, Cd induces a rapid and concentration-dependent H(2)O(2) accumulation. This response could be inhibited by diphenylene iodonium (DPI, 20 microM). Reverse transcription-PCR analysis of three RBOH (respiratory burst oxidase homologues) genes showed an increased transcription of RBOHF after 15 min. No change in ASC concentration was observed during the first 3 h after Cd addition. In contrast, glutathione levels completely diminished within 1 h. This drop could be attributed to an increase in phytochelatin 4. At the plasma membrane, Cd further induced a significant decrease in dehydroascorbate (DHA) uptake activity (up to 90% inhibition after 4 h). This decrease is not present when cells are treated with LaCl(3) before exposure to CdCl(2). LaCl(3) is a typical inhibitor of Ca channels and prevents Cd uptake in these cells as well as the Cd-induced ROS production. Therefore, these results appear to indicate that Cd uptake is a prerequisite for the change in DHA transport activity. However, DPI did not prevent the drop in DHA uptake activity present in Cd-treated Arabidopsis cells, indicating that this response seems to be independent of the Cd-induced H(2)O(2) production.

In cell suspension cultures of Nicotiana tabacum L. cv. Bright Yellow 2 (BY-2) a rapid and concentration-dependent accumulation of H(2)O(2) is induced by excess concentrations of copper (up to 100 microM). This specific and early response towards copper stress was shown to be extracellular. Addition of 300 U of catalase per ml decreased the level of H(2)O(2). Superoxide dismutase (5 U/ml) induced an increase in H(2)O(2) production by 22.2%. This indicates that at least part of the H(2)O(2) is produced by dismutation of superoxide. Pretreatment of the cell cultures with the NAD(P)H oxidase inhibitors diphenylene iodonium (2 and 10 microM) and quinacrine (1 and 5 mM) prevented the generation of H(2)O(2) under copper stress for 90%. The influence of the pH on the H(2)O(2) production revealed the possible involvement of cell-wall-dependent peroxidases in the generation of reactive oxygen species after copper stress.

The possible involvement of glucose (Glc) carriers in the uptake of vitamin C in plant cells is still a matter of debate. For the first time, it was shown here that plant cells exclusively take up the oxidised dehydroascorbate (DHA) form. DHA uptake is not affected by 6-bromo-6-deoxy-ascorbate, an ascorbate (ASC) analogue, specifically demonstrating ASC uptake in animal cells. There is no competition between Glc and DHA uptake. Moreover, DHA and Glc carriers respond in the opposite manner to different inhibitors (cytochalasin B, phloretin and genistein). In conclusion, the plant plasma membrane DHA carrier is distinct from the plant Glc transporters.