PLANT PHYSIOLOGY , Vol 103, Issue 2 575-583, Copyright © 1993 by American Society of Plant Biologists
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ENVIRONMENTAL AND STRESS PHYSIOLOGY |
Multiple Effects of Dithiothreitol on Nonphotochemical Fluorescence Quenching in Intact Chloroplasts (Influence on Violaxanthin De-epoxidase and Ascorbate Peroxidase Activity)
C. Neubauer
Julius von Sachs Institut fur Biowissenschaften mit Botanischem Garten, Lehrstuhl Botanik I, Universitat Wurzburg, Mittlerer Dallenbergweg 64, D-8700 Wurzburg, Germany
Reversible nonphotochemical fluorescence quenching depends on thylakoid
lumen acidification and violaxanthin de-epoxidation and is correlated with
photoprotection of photosynthesis. The O2-dependent electron flow in the
coupled Mehler-ascorbate peroxidase reaction (MP-reaction) mediates the
electron flow necessary for lumen acidification and violaxanthin
de-epoxidation in isolated, intact chloroplasts. Inhibition of violaxanthin
de-epoxidation by dithiothreitol (DTT) was correlated with suppression of
fluorescence quenching. In addition, DTT was also found to suppress
fluorescence quenching due to inhibition of ascorbate peroxidase activity,
a main enzyme of the MP-reaction, even in the presence of zeaxanthin. In
intact, non-CO2-fixing chloroplasts, violaxanthin and antheraxanthin
de-epoxidation and the ascorbate peroxidase activity show different
sensitivities to increasing DTT concentrations. Violaxanthin de-epoxidase
activity, measured as the sum of zeaxanthin and antheraxanthin formed, was
inhibited with an inhibitor concentration for 50% inhibition (I50) of 0.35
mM DTT. In contrast, inhibition of the O2-dependent electron flow and
corresponding lumen acidification occurred with higher I50 values of 2.5
and 3 mM DTT, respectively, and was attributed to inhibition of ascorbate
peroxidase activity (I50 = 2 mM DTT). Accordingly, the DTT-induced
inhibition of the nigericin-sensitive nonphotochemical fluorescence
quenching was correlated linearly with the decreasing concentrations of
zeaxanthin and antheraxanthin and was almost unaffected by DTT inhibition
of the MP-reaction and correlated [delta]pH. The nigericin-insensitive,
photoinhibitory kind of nonphotochemical fluorescence quenching up to 1 mM
was mainly correlated with inhibition of violaxanthin de-epoxidation. At
higher DTT concentrations, it was attributed to inhibition of both
violaxanthin de-epoxidation and MP-reaction. The results show that DTT has
multiple, but distinguishable, effects on nonphotochemical fluorescence
quenching in isolated chloroplasts, necessitating careful interpretation.
