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Overexpression of sweetpotato swpa4 peroxidase results in increased hydrogen peroxide production and enhances stress tolerance in tobacco

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Plant peroxidases (POD) reduce hydrogen peroxide (H2O2) in the presence of an electron donor. Extracellular POD can also induce H2O2 production and may perform a significant function in responses to environmental stresses via the regulation of H2O2 in plants. We previously described the isolation of 10 POD cDNA clones from cell cultures of sweetpotato (Ipomoea batatas). Among them, the expression of the swpa4 gene was profoundly induced by a variety of abiotic stresses and pathogenic infections (Park et al. in Mol Gen Genome 269:542–552 2003; Jang et al. in Plant Physiol Biochem 42:451–455 2004). In the present study, transgenic tobacco (Nicotiana tabacum) plants overexpressing the swpa4 gene under the control of the CaMV 35S promoter were generated in order to assess the function of swpa4 in planta. The transgenic plants exhibited an approximately 50-fold higher POD specific activity than was observed in control plants. Both transient expression analysis with the swpa4-GFP fusion protein and POD activity assays in the apoplastic washing fluid revealed that the swpa4 protein is secreted into the apoplastic space. In addition, a significantly enhanced tolerance to a variety of abiotic and biotic stresses occurred in the transgenic plants. These plants harbored increased lignin and phenolic content, and H2O2 was also generated under normal conditions. Furthermore, they showed an increased expression level of a variety of apoplastic acidic pathogenesis-related (PR) genes following enhanced H2O2 production. These results suggest that the expression of swpa4 in the apoplastic space may function as a positive defense signal in the H2O2-regulated stress response signaling pathway.

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Green fluorescent protein


Methyl viologen


Pathogenesis related




  1. Amaya I, Botella MA, Calle MD, Medina MI, Heredia A, Bressan RA, Hasegawa PM, Quesada MA, Valpuesta V (1999) Improved germination under osmotic stress of tobacco plants overexpressing a cell wall peroxidase. FEBS Lett 457:80–84

  2. Beauchamp C, Fridovich I (1971) Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Anal Biochem 44:276–287

  3. Bindschedler LV, Minibayeva F, Gardner SL, Gerrish C, Davies DR, Bolwell GP (2001) Early signaling events in the apoplastic oxidative burst in suspension cultured French bean cells involved cAMP and Ca2+. New Phytol 151:185–194

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

  5. Bolwell GP (1995) The origin of the oxidative burst in plants. Free Rad Res 23:517–532

  6. Bolwell GP (1999) Role of active oxygen species and NO in plant defense responses. Curr Opin Plant Biol 2:287–294

  7. Bolwell GP, Bindschedler LV, Blee KA, Butt VS, Davies DR, Gardner SL, Gerrish C, Minibayeva F (2002) The apoplastic oxidative burst in response to biotic stress in plants: a three-component system. J Exp Bot 53:1367–1376

  8. Bradford MM (1976) 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

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

  10. Dani V, Simon WJ, Duranti M, Croy RRD (2005) Changes on the tobacco leaf apoplast proteome in response to salt stress. Proteomics 5:737–745

  11. Desikan R, Mackerness SAH, Hancock JT, Neill SJ (2001) Regulation of the Arabidopsis transcriptome by oxidative stress. Plant Physiol 127:159–172

  12. Dowd PF, Lagrimini LM (1997) Examination of different tobacco (Nicotiana spp.) types under- and overproducing tobacco anionic peroxidase for their leaf resistance to Helicoverpa zea. J Chem Ecol 23:2357–2370

  13. Dowd PF, Lagrimini LM, Nelsen TC (1998) Relative resistance of transgenic tomato tissues expressing high levels of tobacco anionic peroxidase to different insect species. Nat Toxins 6:241–249

  14. Dowd PF, Lagrimini LM (2006) Examination of the biological effects of high anionic peroxidase production in tobacco plants grown under field conditions. I. Insect pest damage. Trans Res 15:197–204

  15. Duffey SS, Stout MJ (1996) Anti-nutritive and toxic components of plant defense against insects. Arch Insect Biochem Physiol 32:3–37

  16. Duroux L, Welinder KG (2003) The peroxidase gene family in plants: a phylogenetic overview. J Mol Evol 57:397–407

  17. Elfstrand M, Sitbon F, Lapierre C, Bottin A, Arnold SV (2002) Altered lignin structure and resistance to pathogens in spi 2-expressing tobacco plants. Planta 214:708–716

  18. Ellis WD, Duford HB (1968) The kinetics of cyanide and floride binding by ferric horseradish peroxidase. Biochemistry 7:205–2062

  19. Fecht-Christoffers MM, Braun HP, Lemaitre-Guillier C, VanDorsselaer A, Horst WJ (2003) Effect of manganese toxicity on the proteome of the leaf apoplast in cowpea. Plant Physiol 133:1935–1946

  20. Guo ZJ, Chen XJ, Wu XL, Ling JQ, Xu P (2004) Overexpression of the AP2/EREBP transcription factor OPBP1 enhances disease resistance and salt tolerance in tobacco. Plant Mol Biol 55:607–618

  21. Hatfield R, Fukushima RS (2005) Can lignin be accurately measured? Crop Sci 45:832–839

  22. Hiraga S, Sasaki K, Ito H, Ohashi Y, Matsui H (2001) A large family of class III plant peroxidases. Plant Cell Physiol 42:462–468

  23. Hu JP, Kulkarni A (2000) Metabolic fate of chemical mixtures. I. “Shuttle oxidant” effect of lipoxygenase-generated radical of chlorpromazine and related phenothiazines on the oxidation of benzidine and other xenobiotics. Teratog Carcinog Mutagen 20:195–208

  24. Huh GH, Lee SJ, Bae YS, Liu JR, Kwak SS (1997) Molecular cloning and characterization of cDNAs for anionic and neutral peroxidases from suspension-cultured cells of sweetpotato and their differential expression in response to stress. Mol Gen Genet 255:382–391

  25. Jang IC, Park SY, Kim KY, Kwon SY, Kim GK, Kwak SS (2004) Differential expression of 10 sweetpotato peroxidase genes in response to bacterial pathogen, Pectobacterium chrysanthemi. Plant Physiol Biochem 42:451–455

  26. Kawano (2003) Roles of the reactive oxygen species-generating peroxidase reactions in plant defense and growth induction. Plant Cell Rep 21:829–837

  27. Kim SK, Kwak SS, Jung KH, Min SR, Park IH, Liu JR (1994) Selection of plant cell lines for high yields of peroxidase. J Biochem Mol Biol 27:132–137

  28. Kim KY, Huh GH, Lee HS, Kwon SY, Hur Y, Kwak SS (1999) Molecular characterization of two anionic peroxidase cDNAs isolated from suspension cultures of sweetpotato. Mol Gen Genet 261:941–947

  29. Kim KY, Huh GH, Kwon SY, Lee HS, Hur Y, Bang JW, Choi KS, Kwak SS (2000) Differential expression of four sweetpotato peroxidase genes in response to abscisic acid and ethephon. Phytochemistry 54:19–22

  30. Kim KY, Kwon SY, Lee HS, Hur Y, Bang JW, Kwak SS (2003) A novel oxidative stress inducible peroxidase promoter from sweetpotato: molecular cloning and characterization in transgenic tobacco plants and cultured cells. Plant Mol Biol 51:831–838

  31. Koch E, Slusarenko A (1990) Arabidopsis is susceptible to infection by a downy mildew fungus. Plant Cell 2:437–445

  32. Kwak SS, Kim SK, Lee MS, Jung KH, Park IH, Liu JR (1995) Acidic peroxidase from suspension cultures of sweetpotato. Phytochemistry 39:981–984

  33. Kwon SY, Choi SM, Ahn YO, Lee HS, Lee HB, Park YM, Kwak SS (2003) Enhanced strese tolerance of transgenic tobacco plants expressing a human dehydroascorbate reductase gene. J Plant Physiol 160:347–353

  34. Lagrimini LM (1991) Wound-induced deposition of polyphenols in transgenic plants overexpressing peroxidase. Plant Physiol 96:577–583

  35. Lavid N, Schwartz A, Yarden O, Tel-Or E (2001) The involvement of polyphenols and peroxidase activities in heavy metal accumulation by epidermal glands of the water lily (Nymphaeaceae). Planta 212:323–331

  36. Lee BR, Kim KY, Jung WT, Avice JC, Ourry A, Kim TH (2007) Peroxidases and ligninfication in relation to the intensity of water-deficit stress in white clover (Trifolium repens L.). J Exp Bot 13:1–9

  37. Lim S, Kim YH, Kim SH, Kwon SY, Lee HS, Kim JG, Cho CY, Paek KY, Kwak SS (2007) Enhanced tolerance of transgenic sweetpotato plants expressing both superoxide dismutase and ascorbate peroxidase in chloroplasts against methyl viologen-mediated oxidative stress. Mol Breed 19:227–239

  38. Marentes E, Griffith M, Mlynarz A, Brush RA (1993) Proteins accumulate in the apoplast of winter rye leaves during cold acclimation. Physiol Plant 87:499–507

  39. Mika A, Minibayeva F, Beckett R, Lüthje S (2004) Possible functions of extracellular peroxidases in stress-induced generation and detoxification of active oxygen species. Phytochem Rev 3:173–193

  40. Mitsuya S, Taniguchi M, Miyake H, Takabe T (2006) Overexpression of RCI2A decreases Na+ uptake and mitigates salinity-induced damages in Arabidopsis thaliana plants. Physiol Plant 128:95–102

  41. Mlickova K, Luhova L, Lebeda A, Mieslerova B, Pec P (2004) Reactive oxygen species generation and peroxidase activity during Oidium neolycopersici infection on Lycopersicon species. Plant Physiol Biochem 42:753–761

  42. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497

  43. Park SY, Ryu SH, Kwon SY, Lee HS, Kim GK, Kwak SS (2003) Differential expression of six novel peroxidase cDNAs from cell cultures of sweetpotato on response to stress. Mol Gen Genom 269:542–552

  44. Passardi F, Cosio C, Penel C, Dunand C (2005) Peroxidases have more functions than a Swiss army knife. Plant Cell Rep 24:255–265

  45. Passardi F, Penel C, Dunand C (2004) Performing the paradoxical: how plant peroxidases modify the cell wall. Trends Plant Sci 9:534–540

  46. Peever TL, Higgins VJ (1989) Electrolyte leakage, lipoxygenase, and lipid peroxidation induced in tomato leaf tissue by specific and nonspecific elicitors from Cladosporium fulvum. Plant Physiol 90:867–875

  47. Polle A, Otter T, Mehne-Jakobs B (1994) Effect of magnesium deficiency on antioxidative system in needles of Norway spruce [Picea abies (L.) Karst.] grown with different ratios of nitrate and ammonium as nitrogen sources. New Phytol 128:621–628

  48. Pomar F, Caballero N, Pedreno R, Barcelo A (2002) H2O2 generation during the auto-oxidation of coniferyl alcohol drives the oxidase activity of a highly conserved class III peroxidase involved in lignin biosynthesis. FEBS Lett 529:198–202

  49. Porra RJ, Thompson WA, Kriedemann PE (1989) Determination of accurate extinction coefficients and stimulaneouws equations for assaying chlorophyll a and b extracted with four different solvents: verification of the concentration of chlorophyll standards by atomic absorption spectroscopy. Biochim Biophys Acta 975:384–394

  50. Sgherri C, Stevanovic B, Navari-Izzo F (2004) Role of phenolics in the antioxidative status of the resurrection plant Ramonda serbica during dehydration and rehydration. Physiol Plant 122:478–485

  51. Shieh MW, Wessler SR, Raikhel NV (1993) Nuclear targeting of the maize R protein requires two nuclear localization sequences. Plant Physiol 101:353–361

  52. Snook ME, Mason PF, Sisson VA (1986) Polyphenols in the Nicotiana species. Tobacco Sci 30:43–49

  53. Stadnik MJ, Buchenauer H (2000) Inhibition of phenylalanine ammonia-lyase suppresses the resistance induced by benzothiadiazole in wheat to Blumeria graminis f. sp. tritici. Physiol Mol Plant Pathol 57:25–34

  54. Takahama U (2004) Oxidation of vacuolar and apoplastic phenolic substrates by peroxidase: physiological significance of the oxidation reactions. Phytochem Rev 3:207–219

  55. Talcott ST, Howard LR (1999) Chemical and sensory quality of processed carrot puree as influenced by stress-induced phenolic compounds. J Agric Food Chem 47:1362–1366

  56. 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–powder mildew interaction. Plant J 11:1187–1194

  57. Van Loon LC, Rep M, Pieterse CMJ (2006) Significance of inducible defense-related proteins in infected plants. Annu Rev Phytopathol 44:135–162

  58. Yun BW, Huh GH, Lee HS, Kwon SY, Jo JK, Kim JS, Cho KY, Kwak SS (2000) Differential resistance to methyl viologen in transgenic tobacco plants that express sweetpotato peroxidases. J Plant Physiol 156:504–509

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This work was supported by grants from the Environmental Biotechnology National Core Research Center, KOSEF/MOST, from the Korea Foundation for International Cooperation of Science and Technology (KICOS), MOST and from Biogreen21 Program (20070301034015), Rural Development Administration, Korea.

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Correspondence to Sang-Soo Kwak.

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Kim, Y., Kim, C.Y., Song, W. et al. Overexpression of sweetpotato swpa4 peroxidase results in increased hydrogen peroxide production and enhances stress tolerance in tobacco. Planta 227, 867–881 (2008). https://doi.org/10.1007/s00425-007-0663-3

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  • Acidic PR genes
  • Apoplastic space
  • Environmental stresses
  • Hydrogen peroxide
  • Ipomoea
  • Nicotiana
  • Peroxidase