Inactivation of Ascorbate Peroxidase in Spinach Chloroplasts on Dark Addition of Hydrogen Peroxide: Its Protection by Ascorbate

Abstract

Dark addition of hydrogen peroxide to intact spinach chloroplasts resulted in the inactivation of ascorbate peroxidase accompanied by a decrease in ascorbate contents. This was also the case in reconstituted chloroplasts containing ascorbate, NADP+, NAD+ and ferredoxin. The addition of hydrogen peroxide during light, however, showed little effect on ascorbate contents and ascorbate peroxidase activity in either the intact or reconstituted chloroplasts. In contrast to ascorbate peroxidase, the enzymes participating in the regeneration of ascorbate in chloroplasts (monodehydroascorbate reductase, dehydroascorbate reductase and glutathione reductase) were not affected by the dark addition of hydrogen peroxide. Ascorbate contents increased again by illumination of the chloroplasts after the dark addition of hydrogen peroxide. These results show that the inactivation of the hydrogen peroxide scavenging system on dark addition of hydrogen peroxide [Anderson et al. (1983) Biochim. Biophys. Acta 724: 69, Asada and Badger (1984) Plant & Cell Physiol. 25: 1169] is caused by the loss of ascorbate peroxidase activity. Ascorbate peroxidase activity was rapidly lost in ascorbate-depleted medium, and protected by its electron donors, ascorbate, isoascorbate, guaiacol and pyrogallol, but not by GSH, NAD(P)H and ferredoxin.