Monodehydroascorbate radical detected by electron paramagnetic resonance spectrometry is a sensitive probe of oxidative stress in intact leaves

Monodehydroascorbate radical (MDA) was determined in leaf segments of several plant species using electron paramagnetic resonance spectrometry. When the leaves were young and healthy, MDA was often below detection level in both the light and dark. However, in senescent leaves, higher levels of MDA were observed in the light than in the dark. After removal of ascorbate by infiltration of such leaves with ascorbate oxidase, dark-signals of MDA did not disappear; thus the apoplast was not a major source of MDA in the dark. Methylviologen induced high levels of light-dependent MDA, indicating the participation of the photoproduced superoxide and the hydrogen peroxide derived from the superoxide in the production of MDA in chloroplasts. The photoproduction of MDA was greatly enhanced by intense light, water stress, and suppression of the photosynthetic reactions due to either infiltration or cyanide. Thus, MDA is a sensitive endogeneous probe of oxidative stress in leaf tissues in the sense that increased MDA levels indicate either increased oxidation of ascorbate or decreased efficiency of ascorbate regeneration, or a combination of both.