MEMBRANE BARRIERS AND MEHLER-PEROXIDASE REACTION LIMIT THE ASCORBATE AVAILABLE FOR VIOLAXANTHIN DE-EPOXIDASE ACTIVITY IN INTACT CHLOROPLASTS

The presence of an acidic lumen and the xanthophylls, zeaxanthin and a ntheraxanthin, are minimal requirements for induction of non-radiative dissipation of energy in the pigment bed of Photosystem II. We recent ly reported that ascorbate, which is required for formation for these xanthophylls, also can mediate the needed lumen acidity through the Me hler-peroxidase reaction [Neubauer and Yamamoto (1992) Plant Physiol 9 9: 1354-1361]. It is demonstrated that in non-CO2-fixing intact chloro plasts and thylakoids of Lactuca sativa, L. c.v. Romaine, the ascorbat e available to support de-epoxidase activity is influenced by membrane barriers and the ascorbate-consuming Mehler-peroxidase reaction. In i ntact chloroplasts, this results in biphasic kinetic behavior for ligh t-induced de-epoxidation. The initial relatively high activity is due to ascorbate preloaded into the thylakoid before light-induction and t he terminal low activity due to limiting ascorbate from the effects of chroroplast membranes barriers and a light-dependent process. A five- fold difference between the initial and final activities was observed for light-induced de-epoxidation in chloroplasts pre-incubated with 12 0 mM ascorbate for 40 min. The light-dependent activity is ascribed to the competitive use of ascorbic acid by ascorbate peroxidase in the M ehler-peroxidase reaction. Thus, stimulating ascorbic peroxidase with H2O2 transiently inhibited de-epoxidase activity and concomitantly inc reased photochemical quenching. Also, the effects inhibiting ascorbate peroxidase with KCN, and the K-M values for ascorbate peroxidase and violaxanthin de-epoxidase of 0.36 and 3.1 mM, respectively, support th is conclusion. These results indicate that regulation of xanthophyll-d ependent non-radiative energy dissipation in the pigment bed of Photos ystem II is modulated not only by lumen acidification but also by asco rbate availability.