Reactive Oxygen Species Formation and Cell Death in Catalase-Deficient Tobacco Leaf Discs Exposed to Paraquat


In the present work, the response of tobacco (Nicotiana tabaccum L.) wild-type SR1 and transgenic CAT1AS plants (with a basal reduced CAT activity) was evaluated after exposure to the herbicide paraquat (PQ). Superoxide anion (O .−2 ) formation was inhibited at 3 or 21 h of exposure, but H2O2 production and ion leakage increased significantly, both in SR1 or CAT1AS leaf discs. NADPH oxidase activity was constitutively 57% lower in non-treated transgenic leaves than in SR1 leaves and was greatly reduced both at 3 or 21 h of PQ treatment. Superoxide dismutase (SOD) activity was significantly reduced by PQ after 21 h, showing a decrease from 70% to 55%, whereas catalase (CAT) activity decreased an average of 50% after 3 h of treatment, and of 90% after 21 h, in SR1 and CAT1AS, respectively. Concomitantly, total CAT protein content was shown to be reduced in non-treated CAT1AS plants compared to control SR1 leaf discs at both exposure times. PQ decreased CAT expression in SR1 or CAT1AS plants at 3 and 21 h of treatment. The mechanisms underlying PQ-induced cell death were possibly not related exclusively to ROS formation and oxidative stress in tobacco wild-type or transgenic plants.

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  1. 1.

    Mittler R, Vanderauwera S, Gollery M, Van Breusegem F (2004) Reactive oxygen gene network of plants. Trends Plant Sci 9:490–498

    PubMed  Article  CAS  Google Scholar 

  2. 2.

    Sagi M, Fluhr R (2006) Production of reactive oxygen species by plant NADPH oxidases. Plant Physiol 141:336–340

    PubMed  Article  CAS  Google Scholar 

  3. 3.

    Van Breusegem F, Bailey-Serres J, Mittler R (2008) Unraveling the tapestry of networks involving reactive oxygen species in plants. Plant Physiol 47:978–984

    Article  Google Scholar 

  4. 4.

    Bechtold U, Karpinski S, Mullineaux PM (2005) The influence of the light environment and photosynthesis on oxidative signalling responses in plant–biotrophic pathogen interactions. Plant Cell Environ 28:1037–1045

    Article  Google Scholar 

  5. 5.

    Dat JF, Pellinen R, Beeckman T, Van De Cotte B, Langebartels C, Kangasjärvi J, Inzé D, Van Breusegem F (2003) Changes in hydrogen peroxide homeostasis trigger an active cell death process in tobacco. Plant J 33:621–632

    PubMed  Article  CAS  Google Scholar 

  6. 6.

    Scandalios JG, Acevedo A, Ruzsa S (2000) Catalase gene expression in response to chronic high temperature stress in maize. Plant Sci 156:103–110

    PubMed  Article  CAS  Google Scholar 

  7. 7.

    Groppa MD, Tomaro ML, Benavides MP (2007) Polyamines and heavy metal stress: the antioxidant behavior of spermine in cadmium- and copper-treated wheat leaves. Biometals 20:185–195

    PubMed  Article  CAS  Google Scholar 

  8. 8.

    Azpilicueta CE, Benavides MP, Tomaro ML, Gallego SM (2007) Mechanism of CATA3 induction by cadmium in sunflower leaves. Plant Physiol Biochem 45:589–595

    PubMed  Article  CAS  Google Scholar 

  9. 9.

    Willekens H, Chamnongpol S, Davey M, Schraudner M, Langebartels C, Van Montagu C, Inzé D, Van Camp W (1997) Catalase is a sink for H2O2 and is indispensable for stress defence in C3 plants. EMBO J 16:4806–4816

    PubMed  Article  CAS  Google Scholar 

  10. 10.

    Willekens H, Inzé D, Van Montagu M, Van Camp W (1995) Catalases in plants. Mol Breed 1:207–228

    Article  CAS  Google Scholar 

  11. 11.

    Chamnongpol S, Willekens H, Langebartels C, Van Montagu M, Inzé D, Van Camp W (1996) Transgenic tobacco with a reduced catalase activity develops necrotic lesions and induces pathogenesis related expression under high light. Plant J 10:491–503

    Article  CAS  Google Scholar 

  12. 12.

    Chamnongpol S, Willekens H, Moeder W, Langebartels C, Sandermann H, Van Montagu M, Inzé D, Van Camp W (1998) Defense activation and enhanced pathogen tolerance induced by H2O2 in transgenic plants. Proc Natl Acad Sci USA 95:5818–5823

    PubMed  Article  CAS  Google Scholar 

  13. 13.

    Timbrell JA (1996) Introduction to toxicology. Taylor and Francis, London

    Google Scholar 

  14. 14.

    Suntres ZE (2002) Role of antioxidants in paraquat toxicity. Toxicology 180:65–77

    PubMed  Article  CAS  Google Scholar 

  15. 15.

    Benavides MP, Gallego SM, Comba ME, Tomaro ML (2000) Relationship between polyamines and paraquat toxicity in sunflower leaf discs. Plant Growth Regul 31:215–224

    Article  CAS  Google Scholar 

  16. 16.

    Qian H, Chen W, Sun L, Jin Y, Liu W, Fu Z (2009) Inhibitory effects of paraquat on photosynthesis and the response to oxidative stress in Chlorella vulgaris. Ecotoxicology 18:537–543

    PubMed  Article  CAS  Google Scholar 

  17. 17.

    Willats WG, Orfila C, Limberg G, Buchholt HC, van Alebeek GJ, Voragen AG, Marcus SE, Christensen TM, Mikkelsen JD, Murray BS, Knox JP (2001) Modulation of the degree and pattern of methyl-esterification of pectic homogalacturonan in plant cell walls. Implications for pectin methyl esterase action, matrix properties, and cell adhesion. J Biol Chem 276:19404–19413

    PubMed  Article  CAS  Google Scholar 

  18. 18.

    Cummins I, Burnet M, Edwards R (2001) Biochemical characterization of esterases active in hydrolyzing xenobiotics in weath and competing weeds. Physiol Plant 113:477–485

    Article  CAS  Google Scholar 

  19. 19.

    Stuhlfelder C, Lottspeich F, Mueller MJ (2002) Purification and partial amino acid sequences of an esterase from tomato. Phytochemistry 60:233–240

    PubMed  Article  CAS  Google Scholar 

  20. 20.

    Steward N, Martin R, Engasser JM, Goergen JL (1999) A new methodology for plant cell viability assessment using intracellular esterase activity. Plant Cell Rep 19:171–176

    Article  CAS  Google Scholar 

  21. 21.

    Frahry G, Schopfer P (1998) Inhibition of O2-reducing activity of horseradish peroxidase by diphenyleneiodonium. Phytochemistry 48:223–227

    PubMed  Article  CAS  Google Scholar 

  22. 22.

    Thordal-Christensen H, Zhang Z, Wei Y, Collinge DB (1997) Subcellular localization of H2O2 in plants. H2O2 accumulation in papillae and hypersensitive response during the barley–powdery mildew interaction. Plant J 11:1187–1194

    Article  CAS  Google Scholar 

  23. 23.

    Baker CJ, Mock NM (1994) An improved method for monitoring cell death in cell suspension and leaf disc assays using Evans blue. Plant Cell Tiss Org Cult 39:7–12

    Article  Google Scholar 

  24. 24.

    Shou H, Bordallo P, Fan JB, Yeakley JM, Bibikova M, Sheen J, Wang K (2004) Expression of an active tobacco mitogen-activated protein kinase enhances freezing tolerance in transgenic maize. Proc Natl Acad Sci USA 101:3298–3303

    PubMed  Article  CAS  Google Scholar 

  25. 25.

    Steward N, Martin R, Engasser JM, Goergen JL (1999) Determination of growth and lysis kinetics in plant cell suspension cultures from the measurement of esterase release. Biotech Bioeng 66:114–121

    Article  CAS  Google Scholar 

  26. 26.

    Heath RL, Packer L (1968) Photoperoxidation in isolated chloroplasts. I. Kinetics and stoichiometry of fatty acid peroxidation. Arch Biochem Biophys 125:189–198

    PubMed  Article  CAS  Google Scholar 

  27. 27.

    Van Gestelen P, Asard H, Caubergs RJ (1997) Solubilization and separation of a plant plasma membrane NADPH-O2-synthase from other NAD(P)H oxidoreductases. Plant Physiol 115:543–550

    PubMed  Google Scholar 

  28. 28.

    Nakano Y, Asada K (1981) Hydrogen peroxide is scavenged by ascorbate-specific peroxidase in spinach choroplast. Plant Cell Physiol 22:867–880

    CAS  Google Scholar 

  29. 29.

    Maehly AC, Chance B (1954) The assay of catalase and peroxidase. Meth Biochem Anal 1:357–424

    Article  CAS  Google Scholar 

  30. 30.

    Becana M, Aparicio-Tejo P, Irigoyen JJ, Sánchez-Díaz M (1986) Some enzymes of hydrogen peroxide metabolism in leaves and root nodules of Medicago sativa. Plant Physiol 82:1169–1171

    PubMed  Article  CAS  Google Scholar 

  31. 31.

    Chance B, Sies H, Boveris A (1979) Hydroperoxide metabolism in mammalian organs. Physiol Rev 59:527–605

    PubMed  CAS  Google Scholar 

  32. 32.

    Laemmli UK (1970) Clivage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    PubMed  Article  CAS  Google Scholar 

  33. 33.

    Bradford MM (1956) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  Google Scholar 

  34. 34.

    Vranová E, Atichartpongkul S, Villarroel R, Van Montagu M, Inzé D, Van Camp W (2002) Comprehensive analysis of gene expression in Nicotiana tabacum leaves acclimated to oxidative stress. Proc Natl Acad Sci USA 99:10870–10875

    PubMed  Article  Google Scholar 

  35. 35.

    Gechev TS, Gadjev I, Van Breusegem F, Inzé D et al (2002) Hydrogen peroxide protects tobacco from oxidative stress by inducing a set of antioxidant enzymes. Cell Mol Life Sci 59:708–714

    PubMed  Article  CAS  Google Scholar 

  36. 36.

    Gechev TS, Ferwerda MA, Mehterov N et al (2008) Arabidopsis AAL-toxin-resistant mutant atr1 shows enhanced tolerance to programmed cell death induced by reactive oxygen species. Biochem Biophys Res Commun 375:539–544

    Article  Google Scholar 

  37. 37.

    Kornbrust DJ, Mavis RD (1980) The effect of paraquat on microsomal lipid peroxidation in vitro and in vivo. Toxicol Appl Pharmacol 53:323–332

    PubMed  Article  CAS  Google Scholar 

  38. 38.

    Peixoto F, Vicente J, Madeira VMC (2004) A comparative study of plant and animal mitochondria exposed to paraquat reveals that hydrogen peroxide is not related to the observed toxicity. Toxicol In Vitro 18:733–739

    PubMed  Article  CAS  Google Scholar 

  39. 39.

    Bindschedler LV, Dewdney J, Blee KA, Stone JM, Asai T, Plotnikov J, Denoux C, Hayes T, Gerrish C, Davies DR (2006) Peroxidase dependent apoplastic oxidative burst in Arabidopsis required for pathogen resistance. Plant J 47:851–863

    PubMed  Article  CAS  Google Scholar 

  40. 40.

    Allan AC, Fluhr R (1997) Two distinct sources of elicited reactive oxygen species in tobacco epidermal cells. Plant Cell 9:1559–1572

    PubMed  Article  CAS  Google Scholar 

  41. 41.

    De Biasi MG, Astolfi S, Acampora A, Zuchi S, Fonzo V, Santangelo E, Caccia R, Badiani M, Soressi JP (2003) A H2O2-forming peroxidase rather than a NAD(P)H-dependent O 2 synthase may be the major player in cell death responses controlled by the PtoFen complex following fenthion treatment. Func Plant Biol 30:409–417

    Article  Google Scholar 

  42. 42.

    Iturbe-Ormaetxe I, Escuredo PR, Arrese-Igor C, Becana M (1998) Oxidative damage in pea plants exposed to water deficit or paraquat. Plant Physiol 116:173–181

    Article  CAS  Google Scholar 

  43. 43.

    Babbs CF, Pham JA, Coolbaugh RC (1989) Lethal hydroxyl radical production in paraquat-treated plants. Plant Physiol 90:1267–1270

    PubMed  Article  CAS  Google Scholar 

  44. 44.

    Montillet JL, Chamnongpol S, Rustéucci C, Dat J, van de Cotte B, Agnel JP, Battesti C, Inzé D, Van Breusegem F, Triantaphylidés C (2005) Fatty acid hydroperoxides and H2O2 in the execution of hypersensitive cell death in tobacco leaves. Plant Physiol 138:1516–1526

    PubMed  Article  CAS  Google Scholar 

  45. 45.

    Vicente JA, Peixoto F, Lopes ML, Madeira VM (2001) Differential sensitivities of plant and animal mitochondria to the herbicide paraquat. J Biochem Mol Toxicol 15:322–330

    PubMed  Article  CAS  Google Scholar 

  46. 46.

    Murgia I, Tarantino D, Vannini C, Bracale M, Carravieri S, Soave C (2004) Arabidopsis thaliana plants overexpressing thylakoidal ascorbate peroxidase show increased resistance to paraquat-induced photooxidative stress and to nitric oxide-induced cell death. Plant J 38:940–953

    PubMed  Article  CAS  Google Scholar 

  47. 47.

    Mano J, Ohno C, Domae Y, Asada K (2001) Chloroplastic ascorbate peroxidase is the primary target of methylviologen-induced photooxidative stress in spinach leaves: its relative relevance to monodehydroascorbate radical detected with in vivo ESR. BBA Bioenerg 1504:275–287

    Article  CAS  Google Scholar 

  48. 48.

    Miyake C, Shinzaki Y, Nishioka M, Horiguchi S, Tomizawa KI (2006) Photoinactivation of ascorbate peroxidase in isolated tobacco chloroplasts: Galdieria partita apx maintains the electron flux through the water–water cycle in transplastomic tobacco plants. Plant Cell Physiol 47:200–210

    PubMed  Article  CAS  Google Scholar 

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This work was supported by the University of Buenos Aires (Grant B044). Benavides MP and Groppa MD are researchers of the Consejo Nacional de Investigaciones Científicas y Técnicas (IQUIFIB-CONICET) and MF Iannone and EP Rosales have fellowships from CONICET. We thank Dr. Frank van Breusegem, Ghent University, Belgium, for providing SR1 and CAT1AS tobacco seeds.

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Correspondence to María Patricia Benavides.

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Iannone, M.F., Rosales, E.P., Groppa, M.D. et al. Reactive Oxygen Species Formation and Cell Death in Catalase-Deficient Tobacco Leaf Discs Exposed to Paraquat. Biol Trace Elem Res 146, 246–255 (2012).

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  • Cell death
  • Heavy metals
  • Hydrogen peroxide
  • Nicotiana tabacum
  • Reactive oxygen species