Apoplastic ascorbate does not prevent the oxidation of fluorescent amphiphilic dyes by ambient and elevated concentrations of ozone in leaves

Cut leaves of spinach were infiltrated with solutions containing oxidation-sensitive fluorescent dyes. Excess solution was removed from the intercellular space by centrifugation. Fluorescence of the dyes D494 and D283 started to decrease immediately after the onset of fumigation with ozone at concentrations similar to or not much higher than ambient concentrations in air on sunny days. Only part of ozone entering the leaves was intercepted by the dye. The major part was degraded by unspecified reactions. Photosynthesis was not inhibited while the introduced dye was oxidized by ozone, showing that open stomata facilitated gas exchange after the introduction of dye into the leaf interior. Feeding of ascorbate to the leaves via the petiole failed to affect the ozone-dependent decrease in fluorescence emission from the dye. Likewise, infiltration of leaves by solutions containing dye and 10 mM ascorbate did not produce significant protection of the dye against oxidation by ozone. However, such protection was observed in vitro, when solutions of dye and ascorbate were bubbled with air containing ozone. Although there is little doubt that apoplastic ascorbate occupies a central position in the antioxidative defense of leaf tissue, we are surprised to find that it is much less effective than expected to decrease the oxidation of fluorescent lipophilic probes which had been introduced into the leaf interior. The data suggest that ascorbate is not a primary reductant of ozone in the apoplast. With a microscope-mounted CCD-camera connected to the gas exchange equipment we obtained spatial information on the fluorescence signal and present first results on an heterogeneous distribution of ozone action.