DOI QR코드

DOI QR Code

Protoporphyrinogen oxidase (Protox) 저해제 저항성 형질전환 벼의 적응성과 Porphyrin 생합성 경로물질과 관련성

Relationship of Fitness and Substance of Porphyrin Biosynthesis Pathway in Resistant Transgenic Rice to Protoporphyrinogen Oxidase (Protox) Inhibitor

  • 윤영범 (순천대학교 생명산업과학대학 자원식물개발학과) ;
  • 권오도 (전남농업기술원 쌀연구소) ;
  • 백경환 (전남대학교 농업생명과학대학 분자생물공학) ;
  • 이도진 (순천대학교 사범대학 농업교육과) ;
  • 정하일 (코넬대학교 작물토양학과) ;
  • 국용인 (순천대학교 생명산업과학대학 자원식물개발학과)
  • Yun, Young-Beom (Department of Development in Resources, College of Life Science and Natural Resources, Sunchon National University) ;
  • Kwon, Oh-Do (Jeonnam Agricultural Research and Extension Service) ;
  • Back, Kyoung-Whan (Major in Molecular Biotechnology, College of Agriculture and Life Sciences, Chonnam National University) ;
  • Lee, Do-Jin (Department of Agricultural Education, Sunchon National University) ;
  • Jung, Ha-Il (Department of Crop and Soil Sciences, Cornell University) ;
  • Kuk, Yong-In (Department of Development in Resources, College of Life Science and Natural Resources, Sunchon National University)
  • 투고 : 2011.05.20
  • 심사 : 2011.06.10
  • 발행 : 2011.06.30

초록

Myxococcus xanthus (MX, PX)와 Arabidopsis thaliana (AP37)의 protoporphyrinogen oxidase (Protox)유전자 과다발현 제초제 저항성 형질전환 벼와 비형질전환벼의 생육 및 수량에 관한 적응성 차이와 벼생육시기별 ALA 합성능력, tetrapyrrole 중간물질, 활성산소 발생, 지질과산화 작용 및 항산화효소 능력의 연관성이 조사되었다. Protox 과다발현 형질전환 벼 MX와 AP37의 초장은 이앙 후 43, 50, 65일에 비형질전환벼 비형질전환벼에 비해 유의적으로 적었고, 분얼수는 이앙 후 50일과 65일에 MX와 AP37 뿐만 아니라 PX에서도 비형질전환벼에 비해 유의적으로 적었다. 수확기의 간장과 수량은 MX, PX 및 AP37에서 그리고 고건중은 MX와 AP37에서 비형질전환벼에 비해 유의적으로 적었다. 형질전환벼 계통 MX, PX 및 AP37의 수량감소는 MX의 경우 영화수와 천립중에 의해, PX는 등숙율에 의해, AP37은 영화수, 등숙율 및 천립중에 의해서 기인되었다. 한편, 형질전환벼 계통 MX, PX 및 AP37의 수량감소는 또 다른 년차변이 연구에서도 관찰되었다. 이러한 형질전환벼계통의 생육 감소는 이앙 후 생육기간 뿐만 아니라 이앙 전 육묘기간 동안에서도 발생하여 결과적으로 수량이 감소되는 것으로 생각된다. Tetrapyrrole 중간물질 Proto IX, Mg-Proto IX 및 Mg-Proto IX monomethyl ester의 축적량, 활성산소종($H_2O_2$${O_2}^-$), MDA, 및 항산화효소(superoxide dismutase, caltalase, peroxidase, ascorbate peroxidase, glutathione reductase) 및 엽록소 함량은 Protox 유전자 과다발현형질전환벼 계통과 비형질전환벼간에 유의적인 차이가 없어 이들 porphyrin 대사 경로 물질과 벼 생육 및 수량감소의 연관성은 적은 것으로 사료된다. 그러나 MX, PX, AP37의 ALA 합성능력은 광노출 후 1일과 이앙 후 52일에 비형질전환벼에 비해 유의적으로 감소하여 이 부분에 대한 상세한 연구가 추후에 수행되어야 할 것으로 보인다.

The objectives of this study were to investigate fitness difference in growth and rice yield in herbicide-transgenic rice overexpressing Myxococcus xanthus and Arabidopsis thaliana protoporphyrinogen oxidase (Protox) genes and non-transgenic rice. We also aimed to determine whether these fitness differences are related to ALA synthesizing capacity, accumulation of terapyrroles, reactive oxygen species, lipid peroxidation, and antioxidative enzymes at different growth stages of rice. Plant height of the transgenic rice overexpressing M. xanthus (MX) and A. thaliana (AP37) Protox genes at 43, 50, and 65 days after transplanting (DAT) was significantly lower than that of WT. Number of tiller of PX as well as MX and AP37 at 50 and 65 DAT was significantly lower than that of WT. At harvest time, culm length and yield of MX, PX and AP37 and rice straw weight of MX and AP37 were significantly low compared with WT. The reduction of yield in MX, PX, and AP37 was caused by spikelets per panicle and 1000 grain weight, ripened grain, spikelets per panicle, 1000 grain weight, and ripened grain, respectively. On the other hand, 135 the reduction of yield in MX, PX, and AP37 was also observed in another yearly variation experiment. The reduction of rice growth in MX, PX, and AP37 was observed in seedling stage as well as growth duration in field. There were no differences in tetrapyrrole intermediate Proto IX, Mg-Proto IX and Mg-Proto IX monomethyl ester, reactive oxygen species ($H_2O_2$ and ${O_2}^-$), MDA, antioxidative enzymes (SOD, CAT, POX, APX, and GR) and chlorophyll between transgenic lines and wild type, indicating that accumulated tetrapyrrole intermediate and other parameters were not related to growth reduction in transgenic rice. However, ALA synthesizing capacity in MX, PX, and AP37 at one day after exposure to light and 52 DAT was significantly lower than that of WT. Further study is required to elucidate the mechanisms underlying the growth and yield difference between transgenic and WT lines.

키워드

참고문헌

  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. https://doi.org/10.1016/0003-2697(76)90527-3
  2. Buege, J. A., and S. D. Aust. 1978. Microsomal lipid peroxidation. Methods Enzymol. 52:302-310. https://doi.org/10.1016/S0076-6879(78)52032-6
  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 properties. Plant Cell Physiol. 30:987-998.
  4. 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.
  5. 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. https://doi.org/10.1007/BF00405186
  6. 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. https://doi.org/10.1023/B:BIOP.0000022265.66891.73
  7. 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. https://doi.org/10.1046/j.0016-8025.2003.01127.x
  8. Hiscox, J. D., and G. F. Israelstam. 1979. A method for the extraction of chlorophyll from leaf tissues without maceration. Can. J. Bot. 57:1332-1334. https://doi.org/10.1139/b79-163
  9. Jacobs, J. M., N. J. Jacobs, T. D. Sherman and S. O. Duke. 1991. Effect of diphenyl ether herbicides on oxidation of protoporphyrinogen to protoporphyrin in organella and plasma membrane inriched fractions of barley. Plant Physiol. 97:197-203. https://doi.org/10.1104/pp.97.1.197
  10. Jana, S., and M. A. Choudhuri. 1981. Glycolate metabolism of three submerged aquatic angiosperms during aging. Aquat. Bit. 12:345-354.
  11. Jeong, E. G., G. W. Yi, Y. J. Won, H. M. Park, N. S. Cheon, J. H. Choi, Y. C. Ku, C. D. Han, M. Y. Eun, T. S. Kim and M. H. Nam. 2005. Agronomic characteristics of transgenic japonica rice "Milyang 204" with herbicide resistance gene (bar). Kor. J. Plant Biotechnol. 32:85-90. https://doi.org/10.5010/JPB.2005.32.2.085
  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. https://doi.org/10.1007/BF03030713
  13. Jung, H. I., Y. I. Kuk, K. Back and N. R. Burgos. 2008a. Resistance pattern and antioxidant enzyme profiles of protoporphyrinogen oxidase (PROTOX) inhibitor-resistant transgenic rice. Pestic. Biochem. Physiol. 91:53-65. https://doi.org/10.1016/j.pestbp.2008.01.005
  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 oxyfluorfen resistance. Plant Cell Environ. 27:1436-1446. https://doi.org/10.1111/j.1365-3040.2004.01247.x
  15. Jung, S., H. J. Lee, Y. Lee, K. Kang, Y. S. Kim, B. Grimm and K. Back. 2008b. Toxic tetrapyrrole accumulation in protoporphyrinogen IX oxidase-overexpressing transgenic rice plants. Plant Mol. Biol. 67:535-546. https://doi.org/10.1007/s11103-008-9338-0
  16. Jung, H. I., Y. I. Kuk, H. Y. Kim, K. Back, D. J. Lee, S. Lee and N. R. Burgos. 2010. Resistance levels and fitness of protoporphyrinogen oxidase (PROTOX) inhibitor-resistant transgenic rice in paddy fields. Field Crops Research 115:125-131. https://doi.org/10.1016/j.fcr.2009.10.010
  17. Kuk, Y. I., J. S. Shin, Y. B. Yun and O. D. Kwon. 2010. Mechanism of growth inhibition in herbicide-resistant transgenic rice overexpressing protoporphyrinogen oxidase (Protox) gene. Kor. J. Weed Sci. 30:122-134. https://doi.org/10.5660/KJWS.2010.30.2.122
  18. 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 oxyfluorfen. Plant Cell Physiol. 41:743-749. https://doi.org/10.1093/pcp/41.6.743
  19. Lee, Y., S. Jung and K. Back. 2004. Expression of human protoporphyrinogen oxidase in transgenic rice induces both a photodynamic response and oxyfluorfen resistance. Pestic. Biochem. Physiol. 80:65-74. https://doi.org/10.1016/j.pestbp.2004.06.008
  20. Lermontova, I., and B. Grimm. 2000. Overexpression of plastidic protoporphyrinogen IX oxidase leads to resistance to the diphenyl-ether herbicide acifluorfen. Plant Physiol. 122:75-83. https://doi.org/10.1104/pp.122.1.75
  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. https://doi.org/10.1104/pp.102.3.903
  22. Rao, M. V., G. Paliyath and D. P. Ormrod. 1996. Ultraviolet B- and ozone-induced biochemical changes in antioxidant enzymes of Arabidopsis thaliana. Plant Physiol. 110:125-136. https://doi.org/10.1104/pp.110.1.125
  23. [SAS] Statistical Analysis System. 2000. SAS/ STAT Users Guide, Version 7. Cary, NC : Statistical Analysis Systems Institute, Electronic Version.
  24. Scalla, 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. https://doi.org/10.1104/pp.94.3.1214
  26. Schuh, W., M. R. Nelson, D. M. Bigelow, T. V. Orum, C. E. Orth, P. T. Lynch, P.S. Eyles, N. W. Blackhall, J. Jones, E. C. Cocking and M. R. Davey. 1993. The phenotypic characterrization of R2 generation transgenic rice plants under field conditions. Plant Sci. 89:69-79. https://doi.org/10.1016/0168-9452(93)90171-U
  27. Tanaka, R., and A. Tanaka. 2007. Tetrapyrrole biosynthesis in higher plants. Annu. Rev. Plant Biol. 58:321-346. https://doi.org/10.1146/annurev.arplant.57.032905.105448
  28. Wang, A. G., and G. H. Luo. 1990. Quantitative relation between the reaction of hydroxylamine and superoxide anion radicals in plants. Plant Physiol. Commun. 6:55-57.