Mechanism of Growth Inhibition in Herbicide-Resistant Transgenic Rice Overexpressing Protoporphyrinogen Oxidase (Protox) Gene

Protoporphyrinogen Oxidase (Protox) 유전자 과다발현 제초제 저항성 형질전환 벼의 생육저해 기작

  • Kuk, Yong-In (Dept. of Development in Resources, College of Life Science and Natural Resources, Sunchon National University) ;
  • Shin, Ji-San (Dept. of Development in Resources, College of Life Science and Natural Resources, Sunchon National University) ;
  • Yun, Young-Beom (Dept. of Development in Resources, College of Life Science and Natural Resources, Sunchon National University) ;
  • Kwon, Oh-Do (Jeonnam Agricultural Research and Extension Service)
  • 국용인 (순천대학교 생명산업과학대학 자원식물개발학과) ;
  • 신지산 (순천대학교 생명산업과학대학 자원식물개발학과) ;
  • 윤영범 (순천대학교 생명산업과학대학 자원식물개발학과) ;
  • 권오도 (전남농업기술원 작물연구과)
  • Received : 2010.05.28
  • Accepted : 2010.06.23
  • Published : 2010.06.30


We investigated the levels of resistance and accumulation of terapyrroles, reactive oxygen species, lipid peroxidation, and antioxidative enzymes for reasons of growth reduction in herbicide-transgenic rice overexpressing Myxococcus xanthus, Arabidopsis thaliana, and human protoporphyrinogen oxidase (Protox) genes. The transgenic rice overexpressing M. xanthus (MX, MX1, PX), A. thaliana (AP31, AP36, AP37), and human (H45, H48, H49) Protox genes showed 43~65, 41~72 and 17~70-fold more resistance to oxyfluorfen, respectively, than the wild type. Among transgenic rice lines overexpressing Protox genes, several lines showed normal growth compared with the wild type, but several lines showed in reduction of plant height and shoot fresh weight under different light conditions. However, reduction of plant height of AP37 was much higher than other lines for the experimental period. On the other hand, the reduction of plant height and shoot fresh weight in the transgenic rice was higher in high light condition than in low light condition. Enhanced levels of Proto IX were observed in transgenic lines AP31, AP37, and H48 at 7 days after seeding (DAS) and transgenic lines PX, AP37, and H48 at 14 DAS relative to wild type. There were no differences in Mg-Proto IX of transgenic lines except for H41 and H48 and Mg-Proto IX monomethyl ester of transgenic lines except for MX, MX1, and PX. Although accumulation of tetrapyrrole intermediates was observed in transgenic lines, their tetrapyrrole accumulation levels were not enough to inhibit growth of transgenic rice. There were no differences in reactive oxygen species, MDA, ALA synthesizing capacity, and chlorophyll between transgenic lines and wild type indicating that accumulated tetrapyrrole intermediate were apparently not high enough to inhibit growth of transgenic rice. Therefore, the growth reduction in certain transgenic lines may not be caused by a single factor such as Proto IX, but by interaction of many other factors.

본 연구의 목적은 다양한 생물종의 Protox 유전자 과다발현 제초제 저항성 형질전환 벼의 저항성 수준과 생육저해 원인이 생육시기별 tetrapyrrole 중간물질 축적과 대사 경로 물질, 활성산소 발생, 지질과산화 작용 및 항산화 효소 능력과 관련성이 있는지를 조사하는데 있다. Myxococcus xanthus(MX, MX1, PX), Arabidopsis thaliana(AP31, AP36, AP37) 및 인간(H45, H48, H49) Protox 과다발현 형질전환벼는 비형질전환벼에 비해 oxyfluorfen에 각각 43~65, 41~72 및 17~70배 저항성을 보였다. 다양한 생물종의 Protox 유전자 과다발현 제초제 저항성 형질전환 벼 중에 일부 라인은 다른 광조건하에서 정상적인 생육을 보인 반면에 일부 라인은 초장 및 생체중 감소가 야기되었다. 그러나 일관성 있게 파종 후 7일과 14일에 초장의 감소가 야기 되었던 형질전환 라인은 AP37뿐이었다. 또한 이들 형질전환 벼 라인들은 저광 및 암조건보다 고광조건하에서 초장 및 생체중 감소가 뚜렷하였다. 파종 후 7일째 Proto IX 축적은 AP31, AP37 및 H48에서 비형질전환벼에 비해 유의적으로 많았고, 파종 후 14일째에는 PX, AP37 및 H48에서 유의적으로 많았다. 파종 후 7일째 Mg-Proto IX은 일부 라인(H41과 H48)을 제외하고 그리고 Mg-Proto IX monomethyl ester는 MX, MX1, PX를 제외한 형질전환 라인 간에 차이가 없었다. 비록 terapyrrole 중간물질이 축적 되었더라도 이들 축적량은 Protox 과다발현 형질전환벼의 생육을 저해 하는데 충분하지 않았을 것으로 생각된다. 또한 활성산소종 ($H_2O_2$${O_2}^{{\cdot}_-}$), MDA, ALA 합성 및 엽록소 함량에서도 형질전환 라인간에 유의적인 차이가 인정되지 않아 tetrapyrrole 축적량이 형질전환 벼의 생육저해에 충분하지 못했음을 확인할 수 있었다. 따라서 일부 형질전환벼에서 초기 생육저해는 어떤 단일 요인에 기인되는 것보다 복합적인 요인에 의해 기인되는 것으로 생각된다.


Supported by : 연구재단


  1. Bradford, M. M. 1976. A rapid and sensitive method for the quantification of microgram quantities of protein-dye binding. Anal. Biochem. 72:248-254.
  2. Buege, J. A., and S. D. Aust. 1978. Microsomal lipid peroxidation. Methods Enzymol. 52:302-310.
  3. Chen, G. X., and K. Asada. 1989. Ascorbate peroxidase in tea leaves : occurrence of two isozymes and the differences in their enzymatic and molecular propenies. Plant Cell Physiol. 30:987-998.
  4. Duke, S. O., and C. A. Rebeiz. 1994. Porphyrin biosynthesis as a tool in pest management : an overview, in Porphyric pesticides: Chemistry, toxicology, and pharmaceulical applicalions, ed by Duke SO and Rebeiz CA, American Chemical Society, Washington, DC. USA, pp. 1-17.
  5. Duke, S. O., J. Lydon, J. M. Becerril, T. D. Sherman, L. P. Lehnen and H. Matsumoto. 1991. Protoporphyrinogen oxidase-inhibiting herbicides. Weed Sci. 39:465-473.
  6. Egley. G. H, R. N., Paul Jr, K. C. Vaughn and S. O. Duke. 1983. Role of peroxidase in the development of water-impermeable seed coats in Sida spinosa L. Plants. 157:224-232.
  7. Ha, S. B., S. B. Lee, D. E. Lee, J. O. Guh and K. Back. 2003. Transgenic rice plants expressing Bacillus subtilis protoporphyrinogen oxidase gene show low herbicide oxyfluorfen resistance. Biologia Plantarum. 47:277-280.
  8. Ha, S. B., S. B. Lee, Y. Lee, K. Yang, N. Lee, S. M. Jang, J. S. Chung, S. Jung, Y. S. King, S. G. Wi and K. Back. 2004. The plastidic Arabidopsis protoporphyrinogen IX oxidase gene, with or without the transit sequence, confers resistance to the diphenyl ether herbicide in rice. Plant Cell Environ. 27:79-88.
  9. Hiscox, J. D., and G. F. Israetstam. 1979. A method for the extraction of chlorophyll from leaf tissues without maceration. Can. J. Bot. 57:1332-1334.
  10. Jacobs, J. M., N. J. Jacobs, T. D. Sherman and S. O. Duke. 1991. Effect of diphenyl ether herbicides on oxidalion of protoporphyrinogen to protoporphyrin in organella and plasma membrane inriched fractions of barley. Plant Physiol. 97: 197-203.
  11. Jana, S., and M. A. Choudhuri. 1981. Glycolate metabolism of three submerged aquatic angiosperms during aging. Aqual. Bit. 12:345-354.
  12. Jung, H. I., and Y. I. Kuk. 2007. Resistance mechanisms in protoporphyrinogen oxidase (PROTOX) inhibitor-resistant transgenicrice. J. Plant Biol. 50(5):586-594.
  13. Jung. H. I., Y. I. Kuk, K. Back and N. R. Burgos. 2008. Resistance pattern and antioxidant enzyme profiles of protoporphyrinogen oxidase (PROTOX) inhibitor-resistant transgenic rice. Pestic. Biochem. Physiol. 91:53-65.
  14. Jung, S., Y. Lee, K. Yang, S. B. Lee, S. M. Jang, S. B. Ha and K. Back. 2004. Dual targeting of Myxococcus xanthus protoporphyrinogen oxidase into chloroplasts and mitochondria and high level oxytluorfen resistance. Plant Cell Environ. 27: 1436-1446.
  15. Jung, S., H. J. Lee, Y. Lee, K. Kang, Y. S. Kim, B. Grimm and K. Back. 2008. Toxic tetrapyrrole accumulation in proloporphyrinogen IX oxidase-overexpressing transgenic rice plants. Plant Mol. Biol. 67:535-546.
  16. Lee, H. J., S. B. Lee, J. S. Chung. S. U. Han, J. O. Guh, J. S. Jeon, G. An and K. Back. 2000. Transgenic rice plants expressing a Bacillus subtilis protoporphyrinogen oxidase gene are resistant to diphenyl ether herbicide oxytluorfen. Plant Cell Physiol. 41 :743-749.
  17. Lee, Y., S. Jung and K. Back. 2004. Expression of human protoporphyrinogen oxidase in rransgenic rice induces both a phmodynamic response and oxyfluorfen resistance. Pestic. Biochem. Physiol. 80:65-74.
  18. Lee, H. J., M. V. Duke and S. O. Duke. 1993. Cellular localization of protoporphyrinogen-oxidizing activities of etiolated barley (Hordeum vulgare L.) leaves. Plant Physiol. 102:881-889.
  19. Lermontova, I., and B. Grimm. 2000. Overexpression of plastidic proloporphyrinogen IX oxidase leads to resistance to the diphenyl-ether herbicide acifluorfen. Plant Physiol. 122:75-83.
  20. Matringe, M., J. M. Camadro, P. Labbe and R. Scalia. 1989. Protoporphyrinogen oxidase the target for diphenyl ether herbicides. Biochem. J. 260:231-235.
  21. Mishra, N. P., R K. Mishra and G. S. Singhal. 1993. Changes in the activities of antioxidant enzymes during exposure of intact wheat leaves of strong visible light at different temperature in the presence of protein synthesis inhibitors. Plant Physiol. 102:903-910.
  22. Rao. M. V., G. Paliyath and D. P. Onnrod. 1996. Ultraviolet B- and ozone-induced biochemical changes in antioxidant enzymes of Arabidopsis thaliana. Plant Physiol. 110: 125-136.
  23. [SAS] Statistical Analysis System. 2000. SAS/STAT Users Guide, Version 7. Cary, NC : Statistical Analysis Systems Institute, Electronic Version.
  24. Scalia, R., and M. Matringe. 1994. Inhibitors of protoporphyrinogen oxidase as herbicides: diphenyl ethers and related photobleaching molecules. Rev. Weed Sci. 6:103-132.
  25. Spychalla, J. P., and S. L. Desborough. 1990. Superoxide dismutase. catalase, and tocopherol content of stored potato tubers. Plant Physiol. 94:1214-1218.
  26. Wang, A. G., and Luo, G. H. Quantitative relation between the reaction of hydroxylamine and superoxide anion radicals in plants. Plant Physiol. Commun. 6:55-57.

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