Skip to main content
SpringerLink
Log in

Photooxidative reactions in chloroplast thylakoids. Evidence for a Fenton-type reaction promoted by superoxide or ascorbate

  • Regular Paper
  • Published:
Photosynthesis Research Aims and scope Submit manuscript

Abstract

A methyl viologen (MV)* mediated Mehler reaction was studied using Type C and D chloroplasts (thylakoids) from spinach. The extent of photooxidative reactions were measured as (a) rate of ethylene formation from methional oxidation indicating the production of oxygen radicals, and (b) rate of malondialdehyde (MDA) formation as a measure of lipid peroxidation. Without added ascorbate, 1 μM FerricEDTA increased ethylene formation by greater than 4-fold, but had no effect on MDA production. Ascorbate (1 mM) produced a tripling of ethylene while it reduced MDA formation in the presence of iron. Radical scavengers diethyldithiocarbamate (DDTC), formate, 1,4-diazabicyclo (2.2.2octane) (DABCO), inhibited ethylene formation. Using 0,4 M mannitol to scavenge hydroxyl radicals, the rates of ethylene formation were reduced 40 to 60% with or without 1 μM Fe(III) EDTA. The strong oxidant(s) not scavenged by mannitol are hypothesized to be either alkoxyl radicals from lipid peroxidation, or ‘site specific’ formation of hydroxyl radicals in a lipophillic environment not exposed to mannitol. Singlet oxygen does not appear to be a significant factor in this system. Catalase strongly inhibited both ethylene and MDA synthesis under all conditions; 1 mM ascorbate did not reverse this inhibition. However, the strong superoxide dismutase (SOD) inhibition of ethylene and MDA formation was completely reversed by 1 mM ascorbate. This suggests that superoxide was functioning as an iron reducing agent and that in its absence, ascorbate was similarly promoting oxidations. Therefore, these oxidative processes were dependent on the presence of H2O2 and a reducing agent, suggesting the involvement of a Fenton-type reaction.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

Abbreviations

DABCO:

1,4-diazabicyclo(2.2.2.octane)

DCMU:

3-(3′,4 Dichlorophenyl). 1,1′-dimethyl urea

DDTC:

diethyldithiocarbamate

EDTA:

ethylenediamine-tetraacetic acid

MDA:

malondialdehyde

MV:

methyl viologen

SOD:

superoxide dismutase

TBA:

thiobarbituric acid

TCA:

trichloroacetic acid

References

  1. Anderson RF and Patel KB (1978) The effect of 1,4-diazabicyclo [2.2.2]-octane on the radiosensitivity of bacteria. Photochem and Photobiol 28:881–885

    Google Scholar 

  2. Arnon DI (1949) Copper enzymes in isolated chloroplasts. Polyphenol oxidase in Beta vulgaris. Plant Physiol 20:1–15

    Google Scholar 

  3. Aust SD and Svingen BA (1982) The role of iron in enzymatic lipid peroxidation. In Pryor W (ed) Free radicals in biology, vol. V, pp 1–28 New York: Academic Press, Inc

    Google Scholar 

  4. Sadger MR (1985) Photosynthetic oxygen exchange, Ann Rev Plant Physiol 36:27–53

    Google Scholar 

  5. Beauchamp C and Fridovich I (1970) Mechanism for the production of ethylene from methional. The generation of the hydroxyl radical by xanthine oxidase. J Biol Chem 245:4641–4646

    Google Scholar 

  6. Borg DC et al. (1984) Autooxidative cytotoxicity: is there metal-independent formation of hydroxyl radicals? Are there ‘crypto-hydroxyl radicals? In Bors W, Saran M, and Tait D (eds) Oxygen radicals in chemistry and biology. pp 123–129. New York: Walter de Gruyter

    Google Scholar 

  7. Bors W et al. (1979) On the nature of biochemically generated hydroxyl radicals. Studies using the bleaching of p-nitrosodimethylaniline as a direct assay method. Eur J Biochem 95:621–627

    Google Scholar 

  8. Bors W et al. (1979) Superoxide anions do not react with hydroperoxides. FEBS Letters 107:403–406

    Google Scholar 

  9. Bors W et al. (1980) The nature of intermediates during biological oxygen activation. In Bannister WH and Bannister JV (eds) Biological and clinical aspects of superoxide and superoxide dismutase, pp 1–31. New York: Elsevier/North-Holland

    Google Scholar 

  10. Bors W et al. (1981) Organic oxygen radicals in biology generation and reactions. In: Rodgers M and Powers E (eds) Oxygen and oxy-radicals in chemistry and biology. pp 75–81. New York: Academic Press

    Google Scholar 

  11. Buege JA and Aust SD (1978) Microsomal lipid peroxidation. In Colowick SP and Kaplan NO (eds) Methods in enzymology Vol 52c:302–310. New York: Academic Press

    Google Scholar 

  12. Cohen G and Sinet PM (1980) Fenton's reagent-once more revisisted. In Bannister JV and Hall HAO (eds) Chemical and biochemical aspects of superoxide and superoxide dismutase, Vol. IIA pp. 27–37. New York: Elsevier/North Holland

    Google Scholar 

  13. Dorfman LM and Adams GE (1973) Reactivity of the hydroxyl radical in aqueous solutions. In NSRDS-NBS 46, pp 1–59. Washington: National Bureau of Standards, US Department of Commerce

    Google Scholar 

  14. Elstner EF and Heupel A (1973) On the decarboxylation of α-keto acids by isolated chloroplasts. Biochim Biophys Acta 325:182–188

    Google Scholar 

  15. Elstner EF and Konze JR (1974) Light-dependent ethylene production by isolated chloroplasts. FEBS Lett 45:18–21

    Google Scholar 

  16. Elstner EF Saran M Bors W and Lengfelder E (1978) Oxygen activation in isolated chloroplasts. Mechanism of ferredoxin-dependent ethylene evolution from methionine. Eur J Biochem 89:61–66

    Google Scholar 

  17. Fee J (1980) Is superoxide toxic? In Bannister JV and Hall HAO (eds) Chemical and biochemical aspects of superoxide and superoxidedismutase, Vol IIA, pp 41–48. New York: Elsevier/North Holland

    Google Scholar 

  18. Foote CS (1976) Photosensitized oxidation and singlet oxygen: consequences in biological systems. In Pryor W (ed) Free radicals in biology vol. II, pp 85–133. New York: Academic Press

    Google Scholar 

  19. Foyer C Rowell J and Walker D (1983) Measurement of the ascorbate content of spinach leaf protoplasts and chloroplasts during illumination. Planta 157:239–244

    Google Scholar 

  20. Furbank RT Badger MR Osmond CB (1982) Photosynthetic oxygen exchange in isolated cells and chloroplasts of C3 plants. Plant Physiol 70:927–931

    Google Scholar 

  21. Girotti AW and Thomas JP (1984) Superoxide and hydrogen peroxide-dependent lipid peroxidation in intact and triton-dispersed erythrocyte membranes. Biochem Biophys Res Comm 18:474–480

    Google Scholar 

  22. Gutteridge JMC and Wilkins S (1982) Copper-dependent hydroxyl radical damage to ascorbic acid. Formation of thiobarbituric acid-reactive product. FEBS Lett 132:327–330

    Google Scholar 

  23. Halliwell B (1975) Hydroxylation of p-coumaric acid by illuminated chloroplasts. Eur J Biochem 55:355–360

    Google Scholar 

  24. Halliwell B (1978a) Superoxide-dependent formation of hydroxyl radicals in the presence of iron chelates. FEBS Lett 92:321–326

    Google Scholar 

  25. Halliwell B (1982) Ascorbic acid and the illuminated chloroplast. In Seib PA ed. Ascorbic acid:chemistry, metabolism and uses, pp 263–274. New York: Am Chem Soc.

    Google Scholar 

  26. Hosein B and Palmer G (1983) The kinetics and mechanisms of oxidation of reduced spinach ferredexin by molecular oxygen and its reduced products. Biochim Biophys Acta 723:383–390

    Google Scholar 

  27. Hossain et al. (1984) Monodehydroascorbate reductase in spinach chloroplasts and its participation in regeneration of ascorbate for scavenging hydrogen peroxide. Plant and Cell Physiol 25:385–395

    Google Scholar 

  28. Klebanoff SJ and Rosen H (1978) Ethylene formation by polymorphonuclear leukocytes; role of myeloperoxidase. J Exp Med 148:490–506

    Google Scholar 

  29. Kutsuki H and Gold MH (1982) Generation of hydroxyl radical and its involvement in lignin degradation by Phanerochaete chrysosporium. Biochem Biophys Res Comm 109:320–327

    Google Scholar 

  30. Lutz et al. (1973) Protection by diethyldithiocarbamate against carbon tetrachloride lethality in rats and against carbon tetrachloride-induced lipid peroxidation in vitro. Biochem Pharmacol 22:1729–1734

    Google Scholar 

  31. Marsho TV Behrens PW Radmer RJ (1979) Photosynthetic oxygen reduction in isolated intact chloroplasts and cells from spinach. Plant Physiol 64:656–659

    Google Scholar 

  32. McCord JM Day DD (1978) Superoxide-dependent production of hydroxyl radical catalyzed by iron-EDTA complex. FEBS Lett 86:139–142

    Google Scholar 

  33. McVetty PBE and Canvin DT (1982) Inhibition of photosynthesis by low oxygen concentrations. Can J Bot 59:721–725

    Google Scholar 

  34. Nakatani HY and Barber J (1977) An improved method for isolating chloroplasts retaining their outer membranes. Biochim Biophys Acta 461:510–512

    Google Scholar 

  35. Peiser GD and Yang SF (1978) Chlorophyll destruction in the presence of bisulfite and linoleic acid hydroperoxide. Phytochem 17:79–84

    Google Scholar 

  36. Powles SB Osmond CB and Thorne SW (1979) Photoinhibition of intact attached leaves of C3 plants illuminated in the absence of both carbon dioxide and of photorespiration. Plant Physiol 64:982–988

    Google Scholar 

  37. Pryor WA and Tang RH (1978) Ethylene formation from methional. Biochem Biophys Res Commun 81:498–503

    Google Scholar 

  38. Reeves SG and Hall DO (1980) Higher plant chloroplasts and grana. General preparative procedures (excluding high carbon dioxide fixation ability chloroplasts). In Colowick SP and Kaplan NO (eds) Methods in enzymology Vol 69, pp 85–94. New York: Academic Press

    Google Scholar 

  39. Rigo A Stevanato R Finazzi-Argo A and Rotilio G (1977) An attempt to evaluate the rate of the Haber-Weiss reaction by using OH radical scavengers. FEBS Lett 80:130–132

    Google Scholar 

  40. Saran M et al. (1983) Formation and reactivities of alkoxy radicals. In Cohen G and Greenwald R (eds) Oxy radicals and their scavenger systems. Vol I: Molecular Aspects, pp 20–25

  41. Shinar E et al. (1983) The analogous mechanisms of enzymatic inactivation induced by ascorbate and superoxide in the presence of copper. J Biol Chem 258:14778–14783

    Google Scholar 

  42. Sawyer DT Gibian MJ (1979) The chemistry of the superoxide ion. Tetrahedron 35:1471–1481

    Google Scholar 

  43. Takahama U and Nishimura M (1976) Effects of electron donor and acceptors, electron transfer mediators, and superoxide dismutase on lipid peroxidation in illuminated chloroplast fragments. Plant & Cell Physiol. 17:111–118

    Google Scholar 

  44. Thomas MJ et al. (1982) The role of superoxide in xanthine oxidase-induced autooxidation of linoleic acid. J Biol Chem 257:8343–8347

    Google Scholar 

  45. Walling C (1975) Fenton's reagent revisited. Acc Chem Res 8:125–131

    Google Scholar 

  46. Weinstein J and Bielski BHJ (1979) Kinetics of the interaction of HO2 and O2 radicals with hydrogen peroxide. The Haber-Weiss reaction. J Amer Chem Soc 101:58–62

    Google Scholar 

  47. Winterbourn CC (1979) Comparison of superoxide with other reducing agents in the biological production of hydroxyl radicals. Biochem J 182:625–628

    Google Scholar 

  48. Winterbourn CC (1981b) Hydroxyl radical production in body fluids. Role of metal ions, ascorbate and superoxide. Biochem J 198:125–131

    Google Scholar 

  49. Winterbourn CC (1983) Lactoferrin-catalyzed hydroxyl radical production. Additional requirements for a chelating agent. Biochem J 210:15–19

    Google Scholar 

Download references

Author information

Author notes

  1. Brad L. Upham

    Present address: Department of Plant Pathology, Physiology and Weed Science, Virginia Polytechnic Institute and State University, 24061, Blacksburg, VA, USA

Authors and Affiliations

  1. Department of Botany and Plant Pathology, University of New Hampshire, 03824, Durham, N.H., USA

    Brad L. Upham & Leland S. Jahnke

Authors
  1. Brad L. Upham
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Leland S. Jahnke
    View author publications

    You can also search for this author in PubMed Google Scholar

Additional information

Scientific contribution number 1315 from the New Hampshire Agriculture Experiment Station.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Upham, B.L., Jahnke, L.S. Photooxidative reactions in chloroplast thylakoids. Evidence for a Fenton-type reaction promoted by superoxide or ascorbate. Photosynth Res 8, 235–247 (1986). https://doi.org/10.1007/BF00037131

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00037131

Key words

Search

Navigation