* To whom correspondence should be addressed.
Received August 16, 2011; Revision received September 6, 2011
The dye H2DCF-DA, which forms the fluorescent molecule DCF in the reaction with hydrogen peroxide, H2O2, was used to study light-induced H2O2 production in isolated intact chloroplasts and in protoplasts of mesophyll cells of Arabidopsis, pea, and maize. A technique to follow the kinetics of light-induced H2O2 production in the photosynthesizing cells using this dye has been developed. Distribution of DCF fluorescence in these cells in the light has been investigated. It was found that for the first minutes of illumination the intensity of DCF fluorescence increases linearly after a small lag both in isolated chloroplasts and in chloroplasts inside protoplast. In protoplasts of Arabidopsis mutant vtc2-2 with disturbed biosynthesis of ascorbate, the rate of increase in DCF fluorescence intensity in chloroplasts was considerably higher than in protoplasts of the wild type plant. Illumination of protoplasts also led to an increase in DCF fluorescence intensity in mitochondria. Intensity of DCF fluorescence in chloroplasts increased much more rapidly than in cytoplasm. The cessation of cytoplasmic movement under illumination lowered the rate of DCF fluorescence intensity increase in chloroplasts and sharply accelerated it in the cytoplasm. It was revealed that in response to switching off the light, the intensity of fluorescence of both DCF and fluorescent dye FDA increases in the cytoplasm in the vicinity of chloroplasts, while it decreases in the chloroplasts; the opposite changes occur in response to switching on the light again. It was established that these phenomena are connected with proton transport from chloroplasts in the light. In the presence of nigericin, which prevents the establishment of transmembrane proton gradients, the level of DCF fluorescence in cytoplasm was higher and increased more rapidly than in the chloroplasts from the very beginning of illumination. These results imply the presence of H2O2 export from chloroplasts to cytoplasm in photosynthesizing cells in the light; the increase in this export falls in the same time interval as does the cessation of cytoplasmic movement.
KEY WORDS: plants, protoplasts, chloroplasts, reactive oxygen species, hydrogen peroxide, confocal microscopy