Chloroplastic ascorbate peroxidase is the primary target of methylviologen-induced photooxidative stress in spinach leaves: its relevance to monodehydroascorbate radical detected with in vivo ESR

Methylviologen (MV) induces oxidative damages in leaves. In order to understand its mechanism we studied initial biochemical events under light in MV-fed spinach leaves. When isolated chloroplasts were illuminated in the presence of MV, both stromal and thylakoid-bound ascorbate peroxidases (APX) were inactivated rapidly at the same rates, and their inactivation was retarded by ascorbate (AsA) at higher concentrations. Since MV accelerates the photoproduction of O-2(-) in Photosystem (PS) I and simultaneously inhibits the photoreduction of monodehydroascorbate (MDA) to AsA, the inactivation of APX was attributed to the loss of AsA and accumulation of H2O2 in the stroma. Following APX, superoxide dismutase and NADP(+)-glyceraldehyde 3-phosphate dehydrogenase, both of which are vulnerable to H2O2, were inactivated by MV plus light. Dehydroascorbate reductase, monodehydroascorbate reductase, PS II, PS I and ferredoxin-NADP(+) reductase were far less sensitive to the treatment. In the treated leaves, cytosolic APX and guaiacol-specific peroxidase were also inactivated, but slower than chloroplastic APXs were. Catalase was not inactivated. Thus the MV-induced photooxidative damages of leaves are initiated with the inactivation of chloroplastic APXs and develop via the inactivation of other H2O2-sensitive targets. The decay half-life of the MDA signal after a short illumination in the leaves, as determined by in vivo electron spin resonance spectrometry (ESR), was prolonged when the H2O2-scavenging capacity of the leaf cells was abolished by the inactivation of chloroplastic and cytosolic APXs. The measurement of MDA in leaves by ESR, therefore, allows to estimate in vivo cellular capacity to scavenge the photoproduced H2O2 (C) 2001 Elsevier Science B.V. All rights reserved.