Formation kinetics and H2O2 distribution in chloroplasts and protoplasts of photosynthetic leaf cells of higher plants under illumination.
ABSTRACT The dye H(2)DCF-DA, which forms the fluorescent molecule DCF in the reaction with hydrogen peroxide, H(2)O(2), was used to study light-induced H(2)O(2) 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 H(2)O(2) 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 H(2)O(2) 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.
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ABSTRACT: Cytoplasmic streaming occurs in most plant cells and is vitally important for large cells as a means of long-distance intracellular transport of metabolites and messengers. In internodal cells of characean algae, cyclosis participates in formation of light-dependent patterns of surface pH and photosynthetic activity, but lateral transport of regulatory metabolites has not been visualized yet. Hydrogen peroxide, being a signaling molecule and a stress factor, is known to accumulate under excessive irradiance. This study was aimed to examine whether H2O2 produced in chloroplasts under high light conditions is released into streaming fluid and transported downstream by cytoplasmic flow. To this end, internodes of Chara corallina were loaded with the fluorogenic probe dihydrodichlorofluorescein diacetate and illuminated locally by a narrow light beam through a thin optic fiber. Fluorescence of dihydrodichlorofluorescein (DCF), produced upon oxidation of the probe by H2O2, was measured within and around the illuminated cell region. In cells exhibiting active streaming, H2O2 first accumulated in the illuminated region and then entered into the streaming cytoplasm, giving rise to the expansion of DCF fluorescence downstream of the illuminated area. Inhibition of cyclosis by cytochalasin B prevented the spreading of DCF fluorescence along the internode. The results suggest that H2O2 released from chloroplasts under high light is transported along the cell with the cytoplasmic flow. It is proposed that the shift of cytoplasmic redox poise and light-induced elevation of cytoplasmic pH facilitate the opening of H(+)/OH(-)-permeable channels in the plasma membrane.Protoplasma 06/2013; 250:1339. DOI:10.1007/s00709-013-0517-8 · 3.17 Impact Factor
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ABSTRACT: Emerging evidence suggests that cytoplasmic streaming can regulate the plasma-membrane H+ transport and photosynthetic electron flow. Microfluorometric and surface pH measurements on Chara corallina internodes revealed the transmission of photoinduced signals by the cytoplasmic flow for a distance of few millimeters from the site of stimulus application. When a 30-s pulse of bright light was locally applied, the downstream cell regions responded with either release or enhancement of non-photochemical quenching of chlorophyll fluorescence, depending on the background irradiance of the analyzed cell area. Under dim background irradiance (<20 μmol m-2 s-1), the arrival of the distant signal from the brightly illuminated 400-μm-wide zone elevated the maximal fluorescence F m ' in the analyzed downstream area, whereas at higher background irradiances it induced strong quenching of F m ' . At intermediate irradiances the increase and decrease in F m ' appeared as two successive waves. The transition between the F m ' responses of opposite polarities occurred at a narrow threshold range of irradiances. This indicates that inevitable slight variations in irradiance at the bottom chloroplast layer combined with the cyclosis-transmitted signals may contribute to the formation of a photosynthetic activity pattern. The rapid cyclosis-mediated release of non-photochemical quenching, unlike the delayed response of opposite polarity, was associated with opening of H+ (OH-)-conducting plasma membrane channels, as evidenced by the concurrent alkaline pH shift on the cell surface. It is proposed that the initial increase in F m ' after application of a distant photostimulus is determined, among other factors, by the wave of alkaline cytoplasmic pH.Biophysics of Structure and Mechanism 03/2013; 42(6). DOI:10.1007/s00249-013-0895-z · 2.47 Impact Factor