Molecular Breeding

, Volume 19, Issue 3, pp 227–239 | Cite as

Enhanced tolerance of transgenic sweetpotato plants that express both CuZnSOD and APX in chloroplasts to methyl viologen-mediated oxidative stress and chilling

  • Soon Lim
  • Yun-Hee Kim
  • Sun-Hyung Kim
  • Suk-Yoon Kwon
  • Haeng-Soon Lee
  • Jin-Seog Kim
  • Kwang-Yun Cho
  • Kee-Yoeup Paek
  • Sang-Soo Kwak


Oxidative stress is one of the major factors causing injury to plants exposed to environmental stress. Transgenic sweetpotato [Ipomoea batatas (L.) Lam. cv. Yulmi] plants with an enhanced tolerance to multiple environmental stresses were developed by expressing the genes of both CuZn superoxide dismutase (CuZnSOD) and ascorbate peroxidase (APX) under the control of an oxidative stress-inducible SWPA2 promoter in the chloroplasts of sweetpotato plants (referred to as SSA plants). SSA plants were successfully generated by the particle bombardment method and confirmed by PCR analysis. When leaf discs of SSA plants were subjected to 5 μM methyl viologen (MV), they showed approximately 45% less damage than non-transformed (NT) plants. When 200 μM MV was sprayed onto the whole plants, SSA plants showed a significant reduction in visible damage compared to leaves of NT plants, which were almost destroyed. The expression of the introduced CuZnSOD and APX genes in leaves of SSA plants following MV treatment was significantly induced, thereby reflecting increased levels of SOD and APX in the chloroplasts. APX activity in chloroplast fractions isolated from SSA plants was approximately 15-fold higher than that in their counterparts from NT plants. SSA plants treated with a chilling stress consisting of 4°C for 24 h exhibited an attenuated decrease in photosynthetic activity (Fv/Fm) relative to NT plants; furthermore, after 12 h of recovery following chilling, the Fv/Fm of SSA plants almost fully recovered to the initial levels, whereas NT plants remained at a lower level of Fv/Fm activity. These results suggest that SSA plants would be a useful plant crop for commercial cultivation under unfavorable growth conditions. In addition, the manipulation of the antioxidative mechanism in chloroplasts can be applied to the development of various other transgenic crops with an increased tolerance to multiple environmental stresses.


Ascorbate peroxidase Chilling stress Chloroplast Oxidative stress Superoxide dismutase Sweetpotato 



Ascorbate peroxidase


CuZn superoxide dismutase


Methyl viologen






Sweetpotato peroxidase anionic 2



This research was supported by grants from the BioGreen21 Program, Rural Development Administration, Korea, from the Environmental Biotechnology National Core Research Center, KOSEF/MOST, Korea, and from the International Collaboration Project, Ministry of Science and Technology (MOST), Korea. We are grateful to Prof. Ray A. Bressan, Purdue University for his valuable comments to the manuscript.


  1. Allen RD, Webb RP, Schake SA (1997) Use of transgenic plants to study antioxidant defenses. Free Radical Biol Med 23:473–479CrossRefGoogle Scholar
  2. Aoyama T, Chua NH (1997) A glucocortcoid-mediated transcriptional induction system in transgenic plants. Plant J 11:605–612PubMedCrossRefGoogle Scholar
  3. Aono M, Saji H, Sakamoto A, Tanaka K, Kondo N (1995) Paraquat tolerance of transgenic Nicotiana tabacum with enhanced activities of glutathione reductase and superoxide dismutase. Plant Cell Physiol 36:1687–1691PubMedGoogle Scholar
  4. 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 50:601–639PubMedCrossRefGoogle Scholar
  5. Badawi GH, Kawano N, Yamauchi Y, Shimada E, Sasaki R, Kubo A, Tanaka K (2004) Over-expression of ascorbate peroxidase in tobacco chloroplasts enhances the tolerance to salt stress and water deficit. Physiol Plant 121:231–238PubMedCrossRefGoogle Scholar
  6. Bowler C, Slooten L, Vandenbranden S, De Rycke R, Botterman J, Sybesma C, Van Montagu M, Inzé D (1991) Manganese superoxide dismutase can reduce cellular damage mediated by oxygen radicals in transgenic plants. EMBO J 10:1723–1732PubMedGoogle Scholar
  7. 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 72:248–254PubMedCrossRefGoogle Scholar
  8. Chen THH, Murata N (2002) Enhancement of tolerance of abiotic stress by metabolic engineering of betaines and other compatible solutes. Curr Opin Plant Biol 5:250–257PubMedCrossRefGoogle Scholar
  9. Dat J, Vandenabeele S, Vranova E, Van Montagu M, Inze D, Van Breusegem F (2000) Dual action of the active oxygen species during plant stress responses. Cell Mol Life Sci 57:779–795PubMedCrossRefGoogle Scholar
  10. Foyer CH, Descourvieres P, Kunert KJ (1994) Protection against oxygen radicals: an important defence mechanism studied in transgenic plants. Plant Cell Environ 17:507–523CrossRefGoogle Scholar
  11. Gama MICS, Leite RP, Cordeiro AR, Cantliffe DJ (1996) Transgenic sweetpotato plants obtained by Agrobacterium tumefaciens-mediated transformation. Plant Cell Tissue Organ Cult 46:237–244CrossRefGoogle Scholar
  12. Kasuga M, Liu Q, Miura S, Yamaguchi-Shinozaki K, Shinozaki K (1999) Improving plant drought, salt, and freezing tolerance by gene transfer of a single stress-inducible transcription factor. Nat Biotechnol 17: 287–291PubMedCrossRefGoogle Scholar
  13. Kim KY, Hur KH, 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 261:941–947PubMedCrossRefGoogle Scholar
  14. 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 51:831–838PubMedCrossRefGoogle Scholar
  15. Kimura T, Otani M, Noda T, Ideta O, Shimada T, Saito A (1999) Decrease of amylase content in transgenic sweetpotato. Breed Res 1:142Google Scholar
  16. Kwak SS, Kim SK, Lee MS, Jung KH, Park IH, Liu JR (1995) Three acidic peroxidases from suspension-cultures of sweetpotato. Phytochemistry 39:981–984CrossRefGoogle Scholar
  17. Kwon EJ, Kwon SY, Kim MZ, Lee JS, Ahn YS, Jeong BC, Kwak SS, Lee HS (2002a) Plant regeneration of major cultivars of sweetpotato (Ipomoea batatas) in Korea via somatic embryogenesis. Korean J Plant Biotechnol 29:189–192Google Scholar
  18. Kwon SY, Jeong YZ, Lee HS, Kim JS, Cho KY, Allen RD, Kwak SS, (2002b) Enhanced tolerance of transgenic tobacco plants expressing both superoxide dismutase and ascorbate peroxidase in chloroplasts against methyl viologen-mediated oxidative stress. Plant Cell Environ 25:873–882CrossRefGoogle Scholar
  19. Lee HS, Kim KY, You SH, Kwon SY, Kwak SS (1999) Molecular characterization and expression of a cDNA encoding copper/zinc superoxide dismutase from cultured cells of cassava (Manihot esculenta Crantz). Mol Gen Genet 262:807–814PubMedGoogle Scholar
  20. Li Y, Deng XP, Kwak SS, Tanaka K (2006) Drought tolerance of transgenic sweetpotato expressing both Cu/Zn superoxide dismutase and ascorbate peroxidase. J Plant Physiol Mol Biol 32:451–457Google Scholar
  21. McCord JM, Fridovich I (1969) Superoxide dismutase: an enzymatic function for erythrocuprein (hemocuprein). J Biol Chem 244: 6049–6055PubMedGoogle Scholar
  22. McKersie BD, Murnaghan J, Jones KS, Bowley SR (2000) Iron-superoxide dismutase expression in transgenic alfalfa increases winter survival without a detectable increase in photosynthetic oxidative stress tolerance. Plant Physiol 122:1427–1437PubMedCrossRefGoogle Scholar
  23. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiol Plant 15:473–497CrossRefGoogle Scholar
  24. Nakano Y, Asada K (1981) Hydrogen peroxidase is scavenged by ascorbate-specific peroxidase in spinach chloroplasts. Plant Cell Physiol 22:867–880Google Scholar
  25. Noctor G, Foyer CH (1998) Ascorbate and glutathine: keeping active oxygen under control. Annu Rev Plant Physiol Plant Mol Biol 49:249–279PubMedCrossRefGoogle Scholar
  26. O’Hara-Mays EP, Capwell JC (1992) Miniprep for chloroplast DNA isolation. Microchem J 47:245–250CrossRefGoogle Scholar
  27. Otani M, Shimada T, Kimura T, Saito A (1998) Transgenic plants production from embryogenic callus of sweetpotato (Ipomoea batatas (L.) Lam.) using Agrobacterium tumefaciens. Plant Biotechnol 15:11–16Google Scholar
  28. Payton P, Allen R, Trolinder N, Holaday A (1997) Over-expression of chloroplast-targeted Mn superoxide dismutase in cotton does not alter the reduction of photosynthesis after short exposures to low temperature and high light intensity. Phytosynth Res 52:233–244CrossRefGoogle Scholar
  29. Perl A, Perl-Treves R, Dalili S (1993) Enhanced oxidative stress defence in transgenic potato expressing Cu, Zn superoxide dismutase. Theor Appl Genet 85:568–576CrossRefGoogle Scholar
  30. Pitcher LH, Brennan E, Hurley A, Dunsmuir P, Tepperman JM, Zilinskas BA (1991) Overproduction of petunia copper/ zinc superoxide dismutase does not confer ozone tolerance in transgenic tobacco. Plant Physiol 97:452–455PubMedCrossRefGoogle Scholar
  31. Prakash CS (1994) Sweet potato biotechnology: progress and potential. Biotechnol Dev Mon 18:19–22Google Scholar
  32. Sanford JC, Smith FD, Russell JA (1992) Optimizing the biolistic process for different biological applications. Methods Enzymol 217:485–509Google Scholar
  33. Sen Gupta A, Heinon J, Holaday A, Burke J, Allen R (1993) Increased resistance to oxidative stress in transgenic plants that overexpress chloroplastic Cu/Zn superoxide dismutase. Proc Natl Acad Sci USA 90:1629–1633CrossRefGoogle Scholar
  34. Torsethaugen G, Lynne HP, Zilinskas A, Eva JP (1997) Overproduction of ascorbate peroxidase in the tobacco chloroplast does not provide protection against ozone. Plant Physiol 114:529–537PubMedGoogle Scholar
  35. Van Camp W, Capiau K, Van Montagu M, Inze D, Slooten L (1996) Enhancement of oxidative stress tolerance in transgenic tobacco plants overproducing Fe-superoxide dismutase in chloroplasts. Plant Physiol 112: 1703–1714PubMedCrossRefGoogle Scholar
  36. Wang FZ, Wang QB, Kwon SY, Kwak SS, Su WA (2005) Enhanced drought tolerance of transgenic rice plants expressing a pea manganese superoxide dismutase. J Plant Physiol 162:465–472PubMedCrossRefGoogle Scholar
  37. Yoshida K, Shinmyo A (2000) Transgene expression systems in plant, a natural bioreactor. J Biosci Bioeng 90:353–362PubMedGoogle Scholar
  38. 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 peroxidase. J Plant Physiol 156:504–509Google Scholar

Copyright information

© Springer Science+Business Media B.V. 2007

Authors and Affiliations

  • Soon Lim
    • 1
    • 3
  • Yun-Hee Kim
    • 1
  • Sun-Hyung Kim
    • 1
  • Suk-Yoon Kwon
    • 1
  • Haeng-Soon Lee
    • 1
  • Jin-Seog Kim
    • 2
  • Kwang-Yun Cho
    • 2
  • Kee-Yoeup Paek
    • 3
  • Sang-Soo Kwak
    • 1
  1. 1.Environmental Biotechnology Research CenterKorea Research Institute of Bioscience and Biotechnology (KRIBB)Yuseong-gu, DaejeonKorea
  2. 2.Biofunction Research Team, Bioorganic Science DivisionKorea Research Institute of Chemical Technology (KRICT)Yuseong-gu, DaejeonKorea
  3. 3.Department of HorticultureChungbuk National UniversityCheongjuKorea

Personalised recommendations