Abstract
Formed during the reduction of molecular oxygen or water oxidation, reactive oxygen species (ROS) are produced by a variety of enzymes and redox reactions in almost every compartment of the plant cell. In addition to causing cellular damage, these ROS play a role in signaling networks. Many factors contribute to and, simultaneously, control their metabolism, and it is difficult to detect individual ROS accurately. This is due to several challenges inherent to ROS—their relatively short half-lives, low intracellular concentrations, enzymatic and non-enzymatic scavenging capacity of the cells, and the absence of absolutely selective probes for ROS. Here, we describe the common approaches taken for detecting primary ROS, singlet oxygen, superoxide, and hydrogen peroxide as we discuss their advantages and limitations. We can conclude that using two or more independent methods that yield similar results for detection is a reliable means for studying ROS in intact plant tissues.
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References
Afanas’ev IB (2001) Lucigenin chemiluminescence assay for superoxide detection. Circ Res 89:E46
Asada K (1999) The water-water cycle in chloroplasts: scavenging of active oxygens and dissipation of excess photons. Annu Rev Plant Physiol Plant Mol Biol 50:601–639
Asada K (2006) Production and scavenging of reactive oxygen species in chloroplasts and their functions. Plant Physiol 141:391–396
Asada K, Takahashi M (1987) Production and scavenging of active oxygen in photosynthesis. In: Kyle DJ, Osmond CB, Arntzen CJ (eds) Topics in photosynthesis, photoinhibition, vol 9. Elsevier, Amsterdam, pp 227–287
Auclair C, Voisin E (1985) Nitro blue tetrazolium reduction. In: Greenwald RA (ed) CRC handbook of methods for oxygen radical research. CRC, Boca Raton, pp 123–132
Azzi A, Montecucco C, Richter C (1975) The use of acetylated ferricytochrome c for the detection of superoxide radicals produced in biological membranes. Biochem Biophys Res Commun 65:597–603
Benov L, Sztejnberg L, Fridovich I (1998) Critical evaluation of the use of hydroethidine as a measure of superoxide anion radical. Free Radic Biol Med 25:826–831
Blokhina O, Virolainen E, Fagerstedt KV (2003) Antioxidants, oxidative damage and oxygen deprivation stress: a review. Ann Bot 91:179–194
Boveris A (1984) Determination of the production of superoxide radicals and hydrogen peroxide in mitochondria. Meth Enzymol 105:429–435
Boveris A, Martino E, Stoppani AO (1977) Evaluation of the horseradish peroxidase–scopoletin method for the measurement of hydrogen peroxide formation in biological systems. Anal Biochem 80:145–158
Cleland RE, Grace SC (1999) Voltammetric detection of superoxide production by photosystem II. FEBS Lett 457:348–352
Dambrova M, Baumane L, Kalvinsh I, Wikberg JE (2000) Improved method for EPR detection of DEPMPO-superoxide radicals by liquid nitrogen freezing. Biochem Biophys Res Commun 275:895–898
Dikalov S, Skatchkov M, Bassenge E (1997) Spin trapping of superoxide radicals and peroxynitrite by 1-hydroxy-3-carboxy-pyrrolidine and 1-hydroxy-2,2,6,6-tetramethyl-4-oxo-piperidine and the stability of corresponding nitroxyl radicals towards biological reductants. Biochem Biophys Res Commun 231:701–704
Flors C, Fryer MJ, Waring J, Reeder B, Bechtold U, Mullineaux PM, Nonell S, Wilson MT, Baker NR (2006) Imaging the production of singlet oxygen in vivo using a new fluorescent sensor, Singlet Oxygen Sensor Green. J Exp Bot 57:1725–1734
Földes T, Čermák P, Macko M, Veis P, Macko P (2009) Cavity ring-down spectroscopy of singlet oxygen generated in microwave plasma. Chem Phys Lett 467:233–236
Foyer CH, Noctor G (1999) Leaves in the dark see the light. Science 284:599–601
Frejaville C, Karoui H, Tuccio B, le Moigne F, Culcasi M, Pietri S, Lauricella R, Tordo P (1995) 5-(Diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide: a new efficient phosphorylated nitrone for the in vitro and in vivo spin trapping of oxygen-centered radicals. J Med Chem 38:258–265
Fridovich I (1997) Superoxide anion radical (O2), superoxide dismutases, and related matters. J Biol Chem 272:18515–18517
Fryer MJ, Oxborough K, Mullineaux PM, Baker NR (2002) Imaging of photo-oxidative stress responses in leaves. J Exp Bot 53:1249–1254
Geerts A, Roels F (1981) Quantitation of catalase activity by microspectrophotometry after diaminobenzidine staining. Histochem Cell Biol 72:357–367
Georgiou AD, Papapostolou I, Patsoukis N, Tsegenidis T, Sideris T (2005) An ultrasensitive fluorescent assay for the in vivo quantification of superoxide radical in organisms. Anal Biochem 347:144–151
Halliwell B, Gutterdge JMC (1989) Free radicals in biology and medicine. Clarendon, Oxford
Hempel SL, Buettner GR, O’Malley YQ, Wessels DA, Flaherty DM (1999) Dihydrofluorescein diacetate is superior for detecting intracellular oxidants: comparison with 2′7′-dichlorodihydrofluorescein diacetate, 5 (and 6)-carboxy-2′7′-dichlorodihydrofluorescein diacetate, and dihydrorhodamine 123. Free Radical Bio Med 270:146–159
Hideg É, Kálai T, Hideg K, Vass I (1998) Photoinhibition of photosynthesis in vivo results in singlet oxygen production: detection via nitroxide-induced fluorescence quenching in broad bean leaves. Biochemistry 37:11405–11411
Hideg É, Barta C, Kálai T, Vass I, Hideg K, Asada K (2002) Detection of singlet oxygen and superoxide with fluorescent sensors in leaves under stress by photoinhibition or UV radiation. Plant Cell Physiol 43:1154–1164
Hideg E, Kálai T, Kós PB, Asada K, Hideg K (2006) Singlet oxygen in plants—its significance and possible detection with double (fluorescent and spin) indicator reagents. Photochem Photobiol 82:1211–1218
Hinkle PC, Butow RA, Racker E, Chance B (1967) Partial resolution of the enzymes catalyzing oxidative phosphorylation. XV Reverse electron transfer in the flavin-cytochrome beta region of the respiratory chain of beef heart submitochondrial particles. J Biol Chem 242:5169–5173
Jabs T (1999) Reactive oxygen intermediates as mediators of programmed cell death in plants and animals. Biochem Pharmacol 57:231–245
Janiszewski M, Souza HP, Liu X, Pedro MA, Zweier JL, Laurindo FR (2002) Overestimation of NADH-driven vascular oxidase activity due to lucigenin artifacts. Free Radic Biol Med 32:446–453
Kariola T, Brader G, Li J, Palva ET (2005) Chlorophyllase 1, a damage control enzyme, affects the balance between defense pathways in plants. Plant Cell 17:282–294
Kearns DR (1971) Physical and chemical properties of singlet molecular oxygen. Chem Rev 71:395–427
Keren N, Berg A, van Kann PJM, Levanon H, Ohad I (1997) Mechanism of photosystem II inactivation and D1 protein degradation at low light: the role of back electron flow. Proc Natl Acad Sci USA 94:1579–1584
Keston AS, Brandt R (1965) The fluorometric analysis of ultramicro quantities of hydrogen peroxide. Anal Biochem 11:1–5
Laloi C, Apel K, Danon A (2004) Reactive oxygen signaling: the latest news. Curr Opin Plant Biol 7:323–328
Landmesser U, Dikalov S, Price SR, McCann L, Fukai T, Holland SM, Mitch WE, Harrison DG (2003) Oxidation of tetrahydrobiopterin leads to uncoupling of endothelial cell nitric oxide synthase in hypertension. J Clin Invest 111:1201–1209
Lion Y, Delmelle M, van de Vorst A (1976) New method of detecting singlet oxygen production. Nature 263:442–443
Lvovich V, Scheeline A (1997) Amperometric sensors for simultaneous superoxide and hydrogen peroxide detection. Anal Chem 69:454–462
Mahalingam R, Jambunathan N, Gunjan SK, Faustin E, Weng H, Ayoubi P (2006) Analysis of oxidative signaling induced by ozone in Arabidopsis thaliana. Plant Cell Environ 29:1357–1371
Mahler H, Wuennenberg P, Linder M, Przybyla D, Zoerb C, Langraf F, Forreiter C (2007) Singlet oxygen affects the activity of the thylakoid ATP synthase and has a strong impact on its γ subunit. Planta 225:1073–1083
Makino K, Hagiwara T, Hagi A, Nishi M, Murakami A (1990) Cautionary note for DMPO spin trapping in the presence of iron ion. Biochem Biophys Res Commun 172:1073–1080
McCord JM, Fridovich I (1968) The reduction of cytochrome c by milk xanthine oxidase. J Biol Chem 243:5753–5760
Mittler R (2002) Oxidative stress, antioxidants and stress tolerance. Trends Plant Sci 7:405–410
Mittler R, Vanderauwera S, Gollery M, Breusegem V (2004) Reactive oxygen gene network of plants. Trends Plant Sci 9:490–498
Nakano M (1990) Assay for superoxide dismutase based on chemiluminescence of luciferin analog. Meth Enzymol 186:227–232
Nappy AJ, Vass E (2000) Hydroxyl radical production by ascorbate and hydrogen peroxide. Neurotoxicity Res 2:343–355
Niyogi KK (1999) Photoprotection revisited: genetic and molecular approaches. Annu Rev Plant Physiol Plant Mol Biol 50:333–359
Peshavariya HM, Dusting GG, Selemidis S (2007) Analysis of dihydroethidium fluorescence for the detection of intracellular and extracellular superoxide produced by NADPH oxidase. Free Radical Res 41:699–712
Pospíšil P, Šnyrychova I, Kruk J, Strzałka K, Nauš J (2006) Evidence that cytochrome b559 is involved in superoxide production in photosystem II: effect of synthetic short-chain plastoquinone in a cytochrome b559 tobacco mutant. Biochem J 397:321–327
Ragàs X, Jiménez-Banzo A, Sánchez-Garcıá D, Batllori X, Nonell S (2009) Singlet oxygen photosensitisation by the fluorescent probe Singlet Oxygen Sensor Green. Chem Commun 20:2920–2922
Robinson KM, Janes MS, Pehaf M, Monette JS, Ross MF, Hagen TM, Murphy MP, Beckman JS (2006) Selective fluorescent imaging of superoxide in vivo using ethidium-based probes. Proc Natl Acad Sci USA 103:15038–15043
Schweitzer C, Schmidt R (2003) Physical mechanisms of generation and deactivation of singlet oxygen. Chem Rev 103:1685–1757
Sies H (1981) Measurements of hydrogen peroxide formation in situ. Meth Enzymol 77:15–20
Sonoike K (2006) Photoinhibition and protection of photosystem I. In: Golbeck GH (ed) Photosystem I: the plastocyanin: ferredoxin oxidoreductase in photosynthesis. Springer, Dordrecht, pp 657–668
Staniek K, Nohl H (1999) H2O2 detection from intact mitochondria as a measure for one-electron reduction of dioxygen requires a non-invasive assay system. Biochim Biophys Acta 1413:70–80
Tanaka K, Kobayashi F, Isogai Y, Iizuka T (1991) Electrochemical determination of superoxide anions generated from a single neutrophil. Bioelectrochem Bioenerg 26:413–421
Tarpey MM, Fridovich I (2001) Methods of detection of vascular reactive species: nitric oxide, superoxide, hydrogen peroxide, and peroxynitrite. Circ Res 89:224–236
Telfer A, Bishop SM, Phillips D, Barber J (1994) Isolated photosynthetic reaction-center of photosystem-II as a sensitizer for the formation of singlet oxygen—detection and quantum yield determination using a chemical trapping technique. J Biol Chem 269:13244–13253
Thomson L, Trujillo M, Telleri R, Radi R (1995) Kinetics of cytochrome c2+ oxidation by peroxynitrite: implications for superoxide measurements in nitric oxide-producing biological systems. Arch Biochem Biophys 319:491–497
Trebst A (2003) Function of β-carotene and tocopherol in photosystem II. Z Naturforsch 58c:609–620
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This work was supported for 2 years by a Pusan National University Research Grant.
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Zulfugarov, I.S., Tovuu, A., Kim, JH. et al. Detection of Reactive Oxygen Species in Higher Plants. J. Plant Biol. 54, 351–357 (2011). https://doi.org/10.1007/s12374-011-9177-4
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DOI: https://doi.org/10.1007/s12374-011-9177-4