Formation Kinetics and H2O2 Distribution in Chloroplasts and Protoplasts of Photosynthetic Leaf Cells of Higher Plants under Illumination

Institute of Basic Biological Problems, Russian Academy of Sciences, Pushchino, Moscow Region, Russia.
Biochemistry (Moscow) (Impact Factor: 1.3). 02/2012; 77(2):143-51. DOI: 10.1134/S0006297912020046
Source: PubMed


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.

  • Source
    • "To investigate the kinetics of H 2 O 2 production in chloroplasts, leaf protoplasts were exposed to 2′,7′-dichlorodihydrofluorescein diacetate (H 2 DCFDA). Enzymatic deacetylation and subsequent oxidation by H 2 O 2 generate 2′,7′-dichlorofluorescein (DCF), which was quantified by fluorescence microscopy (Naydov et al., 2012). Protoplasts were prepared from 30-day-old leaves (pre-senescence). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Senescence is a highly regulated process characterized by the active breakdown of cells, which ultimately leads to the death of plant organs or whole plants. In annual plants such as Arabidopsis thaliana senescence can be observed in each individual leaf. Whether deficiencies in photosynthesis promote the induction of senescence was investigated by monitoring chlorophyll degradation, photosynthetic parameters, and reactive oxygen species accumulation in photosynthetic mutants. Several mutations affecting components of the photosynthetic apparatus, including psal-2, psan-2, and psbs, were found to lead to premature or faster senescence, as did simultaneous inactivation of the STN7 and STN8 kinases. Premature senescence is apparently not directly linked to an overall reduction in photosynthesis but to perturbations in specific aspects of the process. Dark-induced senescence is accelerated in mutants affected in linear electron flow, especially psad2-1, psan-2, and pete2-1, as well as in stn7 and stn8 mutants and STN7 and STN8 overexpressor lines. Interestingly, no direct link with ROS production could be observed. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.
    Full-text · Article · Aug 2015 · Journal of Experimental Botany
  • Source
    • "Whilst acquisition of a photosynthetic endosymbiont may have been beneficial to the host cell in many ways, the plastid is also a major source of potentially damaging ROS (Dorrell and Howe, 2012). There is evidence for extensive leakage of H 2 O 2 out of plastids via aquaporins, particularly at high light intensities (Mubarakshina et al., 2010; Naydov et al., 2012). Plastid acquisition is therefore associated with a greatly increased requirement for cellular antioxidant systems to prevent photodamage. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Ascorbic acid (vitamin C) is an enzyme co-factor in eukaryotes that also plays a critical role in protecting photosynthetic eukaryotes against damaging reactive oxygen species derived from the chloroplast. Many animal lineages, including primates, have become ascorbate auxotrophs due to the loss of the terminal enzyme in their biosynthetic pathway, l-gulonolactone oxidase (GULO). The alternative pathways found in land plants and Euglena use a different terminal enzyme, l-galactonolactone dehydrogenase (GLDH). The evolutionary processes leading to these differing pathways and their contribution to the cellular roles of ascorbate remain unclear. Here we present molecular and biochemical evidence demonstrating that GULO was functionally replaced with GLDH in photosynthetic eukaryote lineages following plastid acquisition. GULO has therefore been lost repeatedly throughout eukaryote evolution. The formation of the alternative biosynthetic pathways in photosynthetic eukaryotes uncoupled ascorbate synthesis from hydrogen peroxide production and likely contributed to the rise of ascorbate as a major photoprotective antioxidant.
    Full-text · Article · Mar 2015 · eLife Sciences
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Chloroplasts have developed a light-dependent system for the control of the activities of key enzymes involved in assimilatory (photosynthetic) and dissimilatory pathways, which allows a switch between these opposing pathways to prevent futile cycling. This regulatory system, known as the ferredoxin/thioredoxin system, consists of several proteins constituting a redox cascade that transmits the light signal perceived by chlorophyll to selected target proteins, thereby influencing their activity. A central component of the redox cascade is a novel enzyme, the ferredoxin:thioredoxin reductase, which is capable of reducing a disulfide bridge with the help of an iron-sulfur cluster. Recent developments on the elucidation of the structures of several implicated proteins and on the mechanism of signal transfer have greatly improved our understanding of this regulatory mechanism. This review describes the components of the redox cascade, the principal target proteins, and the mechanism of action of the light-signal transfer.
    Preview · Article · Mar 2003 · Antioxidants and Redox Signaling
Show more