Hydroxyl radicals are not the protagonists of UV-B-induced damage in isolated thylakoid membranes
Iva Šnyrychová A B C , Péter B. Kós A and Éva Hideg A C DA Institute of Plant Biology, Biological Research Centre, Szeged, Hungary.
B Laboratory of Biophysics, Department of Experimental Physics, Faculty of Science, Palacký University, Olomouc, Czech Republic.
C These authors contributed equally.
D Corresponding author. Email: ehideg@brc.hu
Functional Plant Biology 34(12) 1112-1121 https://doi.org/10.1071/FP07151
Submitted: 18 June 2007 Accepted: 17 October 2007 Published: 27 November 2007
Abstract
The production of reactive oxygen species (ROS) was studied in isolated thylakoid membranes exposed to 312 nm UV-B irradiation. Hydroxyl radicals (•OH) and hydrogen peroxide were measured directly, using a newly developed method based on hydroxylation of terephthalic acid and the homovanillic acid/peroxidase assay, respectively. At the early stage of UV-B stress (doses lower than 2.0 J cm–2), •OH were derived from superoxide radicals via hydrogen peroxide. Production of these ROS was dependent on photosynthetic electron transport and was not exclusive to UV-B. Both ROS were found in samples exposed to the same doses of PAR, suggesting that the observed ROS are by-products of the UV-B-driven electron transport rather than specific initiators of the UV-B-induced damage. After longer exposure of thylakoids to UV-B, leading to the inactivation of PSII centres, a small amount of •OH was still observed in thylakoids, even though no free hydrogen peroxide was detected. At this late stage of UV-B stress, •OH may also be formed by the direct cleavage of organic peroxides by UV-B. Immunodetection showed that the presence of the observed ROS alone was not sufficient to achieve the degradation of the D1 protein of PSII centres.
Additional keywords: D1 protein, hydrogen peroxide, oxidative stress, PSII, terephthalate.
Acknowledgements
Iva Šnyrychová was supported by a Visegrád Post-Graduate Scholarship (VPSP, S-023–2006) from the International Visegrád Fund and by the grant of The Ministry of Education, Youth and Sports of the Czech Republic (MSM 6198959215). Terephthalic acid was a kind gift from Dr Tamás Kálai (Department of Medicinal and Organic Chemistry, University of Pécs, Hungary). We thank Dr Virpi Paakkarinen and Prof. Eva-Mari Aro (Department of Biology, University of Turku, Finland) for the D1-protein-specific polyclonal antibody.
A-H-Mackerness S,
Surplus SL,
Jordan BR, Thomas B
(1998) Effects of supplementary ultraviolet-B radiation on photosynthetic transcripts at different stages of leaf development and light levels in pea (Pisum sativum L.): role of active oxygen species and antioxidant enzymes. Photochemistry and Photobiology 68, 88–96.
| Crossref | GoogleScholarGoogle Scholar |
A-H-Mackerness S,
John CF,
Jordan B, Thomas B
(2001) Early signalling components in ultraviolet-B responses: distinct roles for different reactive oxygen species and nitric oxide. FEBS Letters 489, 237–242.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Ananyev GM,
Renger G,
Wacker U, Klimov VV
(1994) Photoreduction of superoxide radicals and the superoxide dismutase activity of Photosystem II. The possible involvement of cytochrome b559. Photosynthesis Research 41, 327–338.
| Crossref | GoogleScholarGoogle Scholar |
Aro EM,
Virgin I, Andersson B
(1993) Photoinhibition of Photosystem II. Inactivation, protein damage and turnover. Biochimica et Biophysica Acta 1143, 113–134.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Aro EM,
Hundal T,
Carlberg I, Andersson B
(1990) In vitro studies on light-induced inhibition of Photosystem-II and D1-protein degradation at low temperatures. Biochimica et Biophysica Acta 1019, 269–275.
| Crossref | GoogleScholarGoogle Scholar |
Barbato R,
Friso G,
Giardi MT,
Rigoni F, Giacometti GM
(1991) Breakdown of the photosystem II reaction centre D1 protein under photoinhibitory conditions: identification and localization of the C-terminal degradation products. Biochemistry 30, 10220–10226.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Barbato R,
Frizzo A,
Friso G,
Rigoni F, Giacometti GM
(1995) Degradation of the D1 protein of photosystem II reaction centre by ultraviolet-B radiation requires the presence of functional manganese on the donor side. European Journal of Biochemistry 227, 723–729.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Barényi B, Krause GH
(1985) Inhibition of photosynthetic reactions by light. Planta 163, 218–226.
| Crossref | GoogleScholarGoogle Scholar |
Barta C,
Kálai T,
Hideg K,
Vass I, Hideg É
(2004) Differences in the ROS-generating efficacy of various ultraviolet wavelengths in detached spinach leaves. Functional Plant Biology 31, 23–28.
| Crossref | GoogleScholarGoogle Scholar |
Bauer G
(2000) Reactive oxygen and nitrogen species: efficient, selective, and interactive signals during intercellular induction of apoptosis. Anticancer Research 20(6B), 4115–4139.
| PubMed |
Beers RF, Sizer IW
(1952) A spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase. The Journal of Biological Chemistry 195, 133–140.
| PubMed |
Bilger W, Björkman O
(1990) Role of the xanthophyll cycle in photoprotection elucidated by measurements of light-induced absorbency changes, fluorescence and photosynthesis in leaves of Hedera canariensis. Photosynthesis Research 25, 173–185.
| Crossref | GoogleScholarGoogle Scholar |
Brosché M, Strid Å
(2003) Molecular events following perception of ultraviolet-B radiation by plants. Physiologia Plantarum 117, 1–10.
| Crossref | GoogleScholarGoogle Scholar |
Burke JJ, Oliver MJ
(1992) Differential temperature sensitivity of pea superoxide dismutases. Plant Physiology 100, 1595–1598.
| PubMed |
Candeias LP,
Patel KB,
Stratford MRL, Wardman P
(1993) Free hydroxyl radicals are formed on reaction between the neutrophil-derived species superoxide anion and hypochlorous acid. FEBS Letters 333, 151–153.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Cheregi O,
Sicora C,
Kós PB,
Barker M,
Nixon PJ, Vass I
(2007) The role of the FtsH and Deg proteases in the repair of UV-B radiation-damaged Photosystem II in the cyanobacterium Synechocystis PCC 6803. Biochimica et Biophysica Acta – Bioenergetics 1767, 820–828.
| Crossref |
Fee JA, Bull C
(1986) Steady-state kinetic studies of superoxide dismutases. Saturative behavior of the copper- and zinc-containing protein. The Journal of Biological Chemistry 261, 13000–13005.
| PubMed |
Gómez JM,
Jiménez A,
Olmos E, Sevilla F
(2004) Location and effects of long-term NaCl stress on superoxide dismutase and ascorbate peroxidase isoenzymes of pea (Pisum sativum cv. Puget) chloroplasts. Journal of Experimental Botany 55, 119–130.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Green R, Fluhr R
(1995) UV-B-induced PR-1 accumulation is mediated by active oxygen species. The Plant Cell 7, 203–212.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Greenberg BM,
Gaba V,
Canaani O,
Malkin S,
Mattoo AK, Edelman M
(1989) Separate photosensitizers mediate degradation of the 32-kDa Photosystem-II reaction center protein in the visible and UV spectral regions. Proceedings of the National Academy of Sciences of the United States of America 86, 6617–6620.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Guilbault GG,
Brignac PJ, Zimmer M
(1966) Homovanillic acid as a fluorometric substrate for oxidative enzyme. Analytical Chemistry 38, 190–196.
Hakala M,
Rantamäki S,
Puputti EM,
Tyystjärvi T, Tyystjärvi E
(2006) Photoinhibition of manganese enzymes: insights into the mechanism of photosystem II photoinhibition. Journal of Experimental Botany 57, 1809–1816.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Haußühl K,
Andersson B, Adamska I
(2001) A chloroplast DegP2 protease performs the primary cleavage of the photodamaged D1 protein in plant photosystem II. The EMBO Journal 20, 713–722.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
He J-M,
Xu H,
She X-P,
Song X-G, Zhao W-M
(2005) The role and interrelationship of hydrogen peroxide and nitric oxide in the UV-B-induced stomatal closure in broad bean. Functional Plant Biology 32, 237–247.
| Crossref | GoogleScholarGoogle Scholar |
He Y-Y, Häder D-P
(2002) UV-B-induced formation of reactive oxygen species and oxidative damage of the cyanobacterium Anabaena sp.: protective effects of ascorbic acid and N-acetyl-L-cysteine. Journal of Photochemistry and Photobiology. B, Biology 66, 115–124.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Henzler T,
Ye Q, Steudle E
(2004) Oxidative gating of water channels (aquaporins) in Chara by hydroxyl radicals. Plant, Cell & Environment 27, 1184–1195.
| Crossref | GoogleScholarGoogle Scholar |
Hideg É, Vass I
(1996) UV-B induced free radical production in plant leaves and isolated thylakoid membranes. Plant Science 115, 251–260.
| Crossref | GoogleScholarGoogle Scholar |
Hideg É,
Mano J,
Ohno C, Asada K
(1997) Increased levels of monodehydroascorbate radical in UV-B irradiated broad bean leaves. Plant & Cell Physiology 38, 684–690.
Jakob B, Heber U
(1996) Photoproduction and detoxification of hydroxyl radicals in chloroplasts and leaves and relation to photoinactivation of photosystems I and II. Plant & Cell Physiology 37, 629–635.
Klimov V,
Ananyev G,
Zastryzhnaya O,
Wydrzynski T, Renger G
(1993) Photoproduction of hydrogen-peroxide in photosystem-II membrane-fragments – a comparison of 4 signals. Photosynthesis Research 38, 409–416.
| Crossref | GoogleScholarGoogle Scholar |
Kobiler O,
Koby S,
Teff D,
Court D, Oppenheim AB
(2002) The phage lambda CII transcriptional activator carries a C-terminal domain signalling for rapid proteolysis. Proceedings of the National Academy of Sciences of the United States of America 99, 14964–14969.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Kramer GF,
Norman HA,
Krizek DT, Mirecki RM
(1991) Influence of UV-B radiation on polyamines, lipid peroxidation and membrane lipids in cucumber. Phytochemistry 30, 2101–2108.
| Crossref | GoogleScholarGoogle Scholar |
Kubo A,
Aono M,
Nakajima N,
Saji H,
Tanaka K, Kondo N
(1999) Differential responses in activity of antioxidant enzymes to different environmental stresses in Arabidopsis thaliana. Journal of Plant Research 112, 279–290.
| Crossref | GoogleScholarGoogle Scholar |
Kumagai J,
Katoh H,
Miyazaki T,
Hidema J, Kumagai T
(1999) Differences in the sensitivity to UVB radiation of two cultivars of rice (Oryza sativa L.) based on observation of long-lived radicals. Journal of Radiation Research 40, 303–310.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Larkum AWD,
Karge M,
Reifarth F,
Eckert HJ,
Post A, Renger G
(2001) Effect of monochromatic UV-B radiation on electron transfer reactions of Photosystem II. Photosynthesis Research 68, 49–60.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Leffers GG, Gottesman S
(1998) Lambda Xis degradation in vivo by Lon and FtsH. Journal of Bacteriology 180, 1573–1577.
| PubMed |
Malanga G, Puntarulo S
(1995) Oxidative stress and antioxidant content in Chlorella vulgaris after exposure to ultraviolet-B radiation. Physiologia Plantarum 94, 672–679.
| Crossref | GoogleScholarGoogle Scholar |
Malanga G,
Calmanovici G, Puntarulo S
(1997) Oxidative damage to chloroplasts from Chlorella vulgaris exposed to ultraviolet-B radiation. Physiologia Plantarum 101, 455–462.
| Crossref | GoogleScholarGoogle Scholar |
Mishra NP,
Mishra RK, Singhal GS
(1993) Involvement of active oxygen species in photoinhibition of photosystem II: protection of photosynthetic efficiency and inhibition of lipid peroxidation by superoxide dismutase and catalase. Journal of Photochemistry and Photobiology. B, Biology 19, 19–24.
| Crossref | GoogleScholarGoogle Scholar |
Miyao M
(1994) Involvement of active oxygen species in degradation of the D1 protein under strong illumination in isolated subcomplexes of photosystem-II. Biochemistry 33, 9722–9730.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Miyao M,
Ikeuchi M,
Yamamoto N, Ono T
(1995) Specific degradation of the D1 protein of photosystem-II by treatment with hydrogen-peroxide in darkness – implications for the mechanism of degradation of the D1 protein under illumination. Biochemistry 34, 10019–10026.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Panagopoulos I,
Bornman JF, Björn LO
(1990) Effects of ultraviolet-radiation and visible-light on growth, fluorescence induction, ultraweak luminescence and peroxidase-activity in sugar-beet plants. Journal of Photochemistry and Photobiology. B, Biology 8, 73–87.
| Crossref | GoogleScholarGoogle Scholar |
Paździoch-Czochra M, Wideńska A
(2002) Spectrofluorometric determination of hydrogen peroxide scavenging activity. Analytica Chimica Acta 452, 177–184.
| Crossref | GoogleScholarGoogle Scholar |
Pospíšil P,
Arató A,
Krieger-Liszkay A, Rutherford AW
(2004) Hydroxyl radical generation by photosystem II. Biochemistry 43, 6783–6792.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Rao MV,
Paliyath G, Ormrod DP
(1996) Ultraviolet-B and ozone-induced biochemical changes in antioxidant enzymes of Arabidopsis thaliana. Plant Physiology 110, 125–136.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Renger G,
Völker M,
Eckert HJ,
Fromme R,
Hohm-Veit S, Graber P
(1989) On the mechanism of photosystem II deterioration by UV-B irradiation. Photochemistry and Photobiology 49, 97–105.
| Crossref | GoogleScholarGoogle Scholar |
Renger G
(2001) Photosynthetic water oxidation to molecular oxygen: apparatus and mechanism. Biochimica et Biophysica Acta – Bioenergetics 1503, 210–228.
| Crossref | GoogleScholarGoogle Scholar |
Richter M,
Rühle W, Wild A
(1990) Studies on the mechanism of photosystem II photoinhibition. II. The involvement of toxic oxygen species. Photosynthesis Research 24, 237–243.
| Crossref | GoogleScholarGoogle Scholar |
Saran M,
Beck-Speier I,
Fellerhoff B, Bauer G
(1999) Phagocytic killing of microorganisms by radical processes: consequences of the reaction of hydroxyl radicals with chloride yielding chlorine atoms. Free Radical Biology & Medicine 26, 482–490.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Schopfer P,
Liszkay A,
Bechtold M,
Frahry G, Wagner A
(2002) Evidence that hydroxyl radicals mediate auxin-induced extension growth. Planta 214, 821–828.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Shibata H,
Baba K, Ochiai H
(1991) Near-UV irradiation induces shock proteins in Anacystis nidulans R-2: possible role of active oxygen. Plant & Cell Physiology 32, 771–776.
Shirasaka N,
Ohnishi H,
Sato K,
Miyamoto R,
Terashita T, Yoshizumi H
(2005) Horseradish peroxidase degrades lipid hydroperoxides and suppresses lipid peroxidation of polyunsaturated fatty acids in the presence of phenolic antioxidants. Journal of Bioscience and Bioengineering 100, 653–656.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Šnyrychová I, Hideg É
(2007) The first application of terephthalate fluorescence for highly selective detection of hydroxyl radicals in thylakoid membranes. Functional Plant Biology 34, 1105–1111.
Šnyrychová I,
Pospíšil P, Nauš J
(2006) Reaction pathways involved in the production of hydroxyl radicals in thylakoid membrane: EPR spin-trapping study. Photochemical & Photobiological Sciences 5, 472–476.
| Crossref | GoogleScholarGoogle Scholar |
Sopory SK,
Greenberg BM,
Mehta RA,
Edelman M, Mattoo AK
(1990) Free radical scavengers inhibit light-dependent degradation of the 32 kDa Photosystem II reaction center protein. Zeitschrift für Naturforschung 45c, 412–417.
Surplus SL,
Jordan BR,
Murphy AM,
Carr JP,
Thomas B, A-H-Mackerness S
(1998) Ultraviolet-B-induced responses in Arabidopsis thaliana: role of salicylic acid and reactive oxygen species in the regulation of transcripts encoding photosynthetic and acidic pathogenesis-related proteins. Plant, Cell & Environment 21, 685–694.
| Crossref | GoogleScholarGoogle Scholar |
Takahashi M, Asada K
(1982) Dependence of oxygen affinity for Mehler reaction on photochemical activity of chloroplast thylakoids. Plant & Cell Physiology 23, 1457–1461.
Takeuchi I,
Fukumoto R,
Kasahara H,
Sakaki T, Kitao M
(1995) Peroxidation of lipids and growth inhibition induced by UV-B radiation. Plant Cell Reports 14, 566–570.
| Crossref | GoogleScholarGoogle Scholar |
Terashima I,
Noguchi K,
Itoh-Nemoto T,
Park YM,
Kubo A, Tanaka K
(1998) The cause of PSI photoinhibition at low temperatures in leaves of Cucumis sativus, a chilling-sensitive plant. Physiologia Plantarum 103, 295–303.
| Crossref | GoogleScholarGoogle Scholar |
Vass I,
Sass L,
Spetea C,
Bakou A,
Ghanotakis D, Petrouleas V
(1996) UV-B induced inhibition of photosystem II electron transport studied by EPR and chlorophyll fluorescence. Impairment of donor and acceptor side components. Biochemistry 35, 8964–8973.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Vass I,
Kirilovski D, Etienne AL
(1999) UV-B radiation-induced donor- and acceptor-side modifications of photosystem II in the cyanobacterium Synechocystis sp PCC 6803. Biochemistry 38, 12786–12794.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Vass I,
Turcsányi E,
Touloupakis E,
Ghanotakis D, Petrouleas V
(2002) The mechanism of UV-A radiation-induced inhibition of photosystem II electron transport studied by EPR and chlorophyll fluorescence. Biochemistry 41, 10200–10208.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Wang Y,
Feng H,
Qu Y,
Cheng J,
Zhao Z,
Zhang M,
Wang X, An L
(2006) The relationship between reactive oxygen species and nitric oxide in ultraviolet-B-induced ethylene production in leaves of maize seedlings. Environmental and Experimental Botany 57, 51–61.
| Crossref | GoogleScholarGoogle Scholar |
Wilson KE,
Thompson JE,
Huner NPA, Greenberg BM
(2001) Effects of ultraviolet-A exposure on ultraviolet-B-induced accumulation of specific flavonoids in Brassica napus. Photochemistry and Photobiology 73, 678–684.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Yan EB,
Unthank JK,
Castillo-Melendez M,
Miller SL,
Langford SJ, Walker DW
(2005) Novel method for in vivo hydroxyl radical measurement by microdialysis in fetal sheep brain in utero. Journal of Applied Physiology 98, 2304–2310.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Yumoto I,
Iwata H,
Sawabe T,
Ueno K,
Ichise N,
Matsuyama H,
Okuyama H, Kawasaki K
(1999) Characterization of a facultatively psychrophilic bacterium, Vibrio rumoiensis sp. nov., that exhibits high catalase activity. Applied and Environmental Microbiology 65, 67–72.
| PubMed |
