C. Neubauer et Hy. Yamamoto, MEMBRANE BARRIERS AND MEHLER-PEROXIDASE REACTION LIMIT THE ASCORBATE AVAILABLE FOR VIOLAXANTHIN DE-EPOXIDASE ACTIVITY IN INTACT CHLOROPLASTS, Photosynthesis research, 39(2), 1994, pp. 137-147
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.