Age-dependent changes in levels of ascorbic acid and chlorogenic acid, and activities of peroxidase and superoxide dismutase in the apoplast of tobacco leaves: Mechanism of the oxidation of chlorogenic acid in the apoplast

In order to understand browning in tobacco plants during aging, age-dependent changes in the levels of ascorbic acid (AA) and chlorogenic acid (CGA) and its isomers were investigated in the apoplast and the symplast of the leaves, Also activities of peroxidase (POX) and superoxide dismutase (SOD) were determined. AA decreased during aging until it was no longer detectable in the apoplast, while symplastic AA remained although the level decreased on aging. In contrast, levels of CGA and its isomers and activity of POX in the apoplast increased on aging, while those in the symplast remained nearly constant in mature and old leaves. The activity of SOD in the apoplast increased during aging, while that in the symplast decreased. Oxidation of CGA by the apoplastic solution was observed in the absence of externally added H2O2, and the oxidation was inhibited by SOD and catalase. Brown components, which contained caffeic acid moieties, accumulated in the apoplast on aging and the components produced O-2(-) and H2O2 by autooxidation. From these results, we conclude (i) that brown components are formed in the apoplast by the CGA/POX system, (ii) that the H2O2 required for the reaction fan be provided by the CGA/POX system itself and by autooxidation of the brown components, and (iii) that apoplastic SOD functions to generate H2O2 from apoplastically formed O-2(-).