Biochemistry of xanthophyll-dependent non-photochemical fluorescence quenching in isolated chloroplasts

Abstract

Higher plants possess several mechanisms that protect the photosynthetic apparatus against damage from excess-light. One mechanism dissipates supersaturating light levels thermally or nonradiatively. Although studies with intact leaves and isolated chloroplasts have shown that the nonradiative dissipation or NRD phenomenon is related to the light-induced transthylakoid pH-gradient (ΔpH) and zeaxanthin formed by violaxanthin de-epoxidation in the xanthophyll-cycle, the biochemical relationship remains unclear. The research plan of this dissertation was based on the premises that the biochemical relationship can be characterized in isolated chloroplasts and that the results of in vitro studies may explain the NRD mechanism in intact leaves. The experimental approach was to probe the NRD mechanism using various mediators, inhibitors, and uncouplers of light-driven electron-transport, the xanthophyll-cycle, and NRD, as well as dark ATP-induced proton pumping. Chloroplasts were isolated from Pisum sativum L. cv. Oregon and Lactuca sativa L. cv. Romaine. The results suggest that all NRD relates to a common mechanism, occurs in the photosystem II pigment bed, and depends on lumen proton concentration. The data further suggest that the violaxanthin de-epoxidation products, antheraxanthin and zeaxanthin, contribute equally to NRD and that no NRD occurs in their absence. These results contrast earlier studies, which suggest that NRD actually comprises of two mechanisms, one zeaxanthin-dependent in the light-harvesting pigment bed and one constitutive in the photosystem II reaction center. The work investigating ATP-induced L\pH showed that NRD is actually a 'dark-reaction', only indirectly related to actinic light. These results appear to exclude the previous suggestions that NRD involves light-dependent changes in the redox-state of electron-transport components. The ATP-induced NRD may also explain the mechanism behind dark-sustained NRD observed in leaves under photoinhibitory conditions where CO2-fixing capacity was limited and ATPase activation and ATP accumulation were possible.