FORMATION AND DECAY OF MONODEHYDROASCORBATE RADICALS IN ILLUMINATED THYLAKOIDS AS DETERMINED BY EPR SPECTROSCOPY

H2O2 is reduced by an ascorbate-specific peroxidase (APX) in chloroplasts, generating the monodehydroascorbate (MDA) radical as the primary oxidation product. Using EPR spectroscopy we have measured the light-driven formation and decay of this species in thylakoids containing active APX. Illumination caused a rapid exponential rise in the steady-state MDA radical concentration in the absence of added electron accepters other than O-2. This increase was sensitive to KCN and catalase and was prevented by anaerobic conditions, demonstrating the requirement for APX activity and endogenously generated H2O2, i.e., the Mehler reaction. When the illumination was removed, a second, transient increase in the radical signal was observed, indicating that photoreduction of the MDA radical and O-2 were occurring simultaneously in the light. This interpretation is also supported by the sigmoidal behavior of the chlorophyll dependence of MDA radical formation in illuminated thylakoids. Ferredoxin lowered the light-induced, steady-state MDA radical concentration, and is thus implicated as the physiological photoreductant for this Hill acceptor. In the absence of uncoupler, NADP(+) prevented formation of the MDA radical by lowering the flux to molecular O-2. However, in the presence of uncoupler (5 mM NH4Cl) this constraint was apparently overcome, i.e., net formation of the radical occurred. The EPR method represents a novel approach to investigating the interaction of O-2 and ascorbate metabolism in chloroplasts under a variety of physiologically relevant conditions, to be applied in future studies of plant response to environmental stress.