Involvement of reactive oxygen species in the UV-B damage to the cyanobacterium Anabaena sp.

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Abstract

Reactive oxygen species (ROS) are involved the damage of living organisms under environmental stress including UV radiation. Cyanobacteria, photoautotrophic prokaryotic organisms, also suffer from increasing UV-B due to the depletion of the stratospheric ozone layer. The increased UV-B induces the production of ROS in vivo detected by using the ROS-sensitive probe 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA). Ascorbic acid and N-acetyl-l-cysteine (NAC) scavenged ROS effectively, while α-tocopherol acetate or pyrrolidine dithiocarbamate (PDTC) did not. The presence of rose bengal and hypocrellin A increased the ROS level by photodynamic action in the visible light. The presence of the herbicide, 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU), increased ROS production slightly, and ROS formation was greatly enhanced by the addition of methyl viologen due to the fact that this redox system diverts electrons from PSI to oxygen and thus forms ROS. UV-B induces ROS generation by photodynamic action and inhibition of the electron transport by damaging the electron receptors or enzymes associated with the electron transport chain during photosynthesis.

Introduction

The UV-B wavelength range (280–315 nm) of solar radiation has received much attention in recent years due to the fact that this spectral region increases as the stratospheric ozone concentration decreases [1]. The enhanced exposure to UV-B is potentially detrimental to all living organisms, including photoautotrophs due to their requirements for light [2], [3], [4]. Preliminary effects are DNA damage by dimerization of pyrimidine bases [5], direct photosynthetic damage by destruction of the D1 reaction center protein of photosystem II (PSII) [6], membrane damage, protein destruction and hormone inactivation [5].

Reactive oxygen species (ROS), including superoxide radical (O2⋅−), hydroxyl radical (OH), hydrogen peroxide (H2O2) and singlet oxygen (1O2) are thought to be induced in plants under environmental stress, including UV-B radiation [7], [8], [9], [10]. First of all, highly energetic photons in the UV-B range are absorbed by the chromophoric groups of many biologically important molecules such as chlorophylls, phycobiliproteins and quinones. These molecules can act as photosensitizers for the production of ROS [11], [12], [13], [14]. In addition, the intracellular accumulation of these potentially harmful ROS can be generated either by donation of energy or electrons directly to oxygen during photosynthetic energy transfer and electron transport as well as photorespiration and respiration [15], [16]. It is likely that oxidative damage is an early consequence of UV-B damage [7]. Furthermore, ROS are known to activate genes, the product of which in turn can affect the expression of other genes [8], [9], [10], [17], [18].

Cyanobacteria are oxygenic photosynthetic prokaryotes dominating the microbial communities of the most extreme environments on earth and important contributors to global photosynthetic biomass production and biofertilizers. Like plants, they cannot avoid the damage from the enhanced UV-B irradiation and the resultant oxidative damage [19], [20]. Due to the inherent instability and reactivity of most ROS and their low steady state levels, determination of the ROS concentration is more difficult than that of antioxidants and of activities of antioxidant enzymes. Among the ROS determination approaches, the fluorogenic method has been very successful and used to monitor the in vivo generation of ROS and the resulting oxidative stress [17], [18], [21], [22]. It was assumed previously that UV-A is more prone to induce ROS generation and to result in oxidative damage than UV-B; therefore more work on oxidative stress and oxidative damage has focused on UV-A, and more attention had been directed to the UV-B induced dimerization of pyrimidines in DNA molecules [7].

In this paper we report on UV-B-induced ROS production in the cyanobacterium Anabaena sp., isolated from rice fields in India [23], using 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) as the fluorometric probe and the effect of exogenous antioxidants as well as photosensitizers and herbicides to determine the involvement of ROS in the UV-B damage in this cyanobacterium. To our knowledge, this is the first report demonstrating directly the UV-B induced ROS production in cyanobacteria.

Section snippets

Chemicals

DCFH-DA, N-acetyl-l-cysteine (NAC), pyrrolidine dithiocarbamate (PDTC), ascorbic acid and α-tocopherol acetate were purchased from Sigma (St. Louis, MO, USA). Rose bengal (RB) was obtained from Merck. Hypocrellin A (HA) was a kind gift from Professor Li-Jin Jiang (Institute of Photographic Chemistry, Academia Sinica, Beijing, China). The DCFH-DA stock solution (2 mM) in ethanol was kept under nitrogen at −74 °C in the dark to avoid the autooxidation of DCFH-DA.

Organism and growth conditions

Anabaena sp., a filamentous and

ROS generation under artificial solar irradiation

To test whether UV-B induces the generation of ROS, 295-, 320- and 395-nm cut-off filters were used to obtain irradiation with or without UV-B or only PAR. The exponentially growing organisms were exposed to these three radiation regimes for different times (Fig. 1). After PAR radiation for up to 2 h, a strong DCF fluorescence can be detected (relative to the control baseline fluorescence). When UV-A was present in the radiation, ROS production increased approximately twofold. When UV-B was

Discussion

Reactive oxygen species (ROS) are inevitable byproducts of biological redox reactions; they can inactivate enzymes and damage important cellular components. Furthermore, singlet oxygen and hydroxyl radical are highly destructive. They initiate lipid peroxidation and the produced lipid peroxyl radicals and lipid hydroperoxides are also very reactive [25]. The increased production of ROS is considered to be a universal feature of stress conditions [7], [15], [35]. The formation of ROS is

Abbreviations

    DCF

    2′,7′-dichlorofluorescein

    DCFH

    2′,7′-dichlorodihydrofluorescein

    DCFH-DA

    2′,7′-dichlorodihydrofluorescein diacetate

    ROS

    reactive oxygen species

    PSII

    photosystem II

    UV-B

    ultraviolet B (280–315 nm)

    UV-A

    ultraviolet A (320–400 nm)

    PAR

    photosynthetically active radiation (400–700 nm)

    NAC

    N-acetyl-l-cysteine

    PDTC

    pyrrolidine dithiocarbamate

    RB

    rose bengal

    HA

    hypocrellin A

    DCMU

    3-(3,4-dichlorophenyl)-1,1-dimethyl urea

    MV

    methyl viologen

Acknowledgements

This work was financially supported by the Alexander von Humboldt Research Fellowship to Y.-Y. He and by the European Union (DG XII, Environment programme, ENV4-CT97-0580) to D.-P. Häder. We would like to thank M. Schuster and S. Seeler for their technical assistance.

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