EPR evidence for superoxide anion formation in leaves during exposure to low levels of ozone

Although the superoxide anion radical (O) has been implicated in the phytotoxicity of ozone, (O₃), its role has been inferred from indirect evidence based on the activity of oxyradical scavenging systems in the leaf, particularly superoxide dismutase (SOD). Direct observations of radical signals obtained by electron paramagnetic resonance spectrometry (EPR) of intact, attached leaves of bluegrass (Poa pratensis L.) and ryegrass (Lolium perenne L.) and leaf pieces of radish (Raphanus sativus L.) during exposure to 240 μg m^?3 O₃ in air flowing through the spectrometer cavity have revealed the appearance of a signal with the characteristics of O. The exposures used were insufficient to cause any necrotic injury to the leaves. The appearance of the signal is light-dependent, suggesting that it originates in the chloroplast, and its appearance is reduced in leaves in which the apoplastic pool of ascorbic acid has been enriched by prior vacuum infiltration. In each species, the signal only appeared after about 1 h of exposure to O₃, and then increased steadily over the next 4 h. The lability of the species responsible for the signal is such that it can no longer be reliably detected about 15 min after cessation of the exposure to O₃. These observations are interpreted as indicating that apoplastic ascorbate initially reduces the production of O, probably by reducing the penetration of O₃ into the cell, with any O produced being scavenged by the chloroplastic SOD-per-oxidase system, but its formation from O₃ then begins to exceed the capacity of the scavenging systems to remove it.