Journal of Plant Biotechnology

The Korean Society of Plant Biotechnology (한국식물생명공학회)
권호 표지
DOI QR코드

DOI QR Code

Proteomic analysis of dehydroascorbate reductase transgenic potato plants

Dehydroascorbate reductase 과발현 형질전환 감자 식물체의 단백질체 분석

  • Han, Eun-Heui (Department of Agronomy & Medicinal Plant Resources, Gyeongnam National University of Science & Technology) ;
  • Goo, Young-Min (Gyeongnam Oriental Medicinal Herb Institute) ;
  • Kim, Yun-Hee (Department of Biology Education, College of Education, IALS, Gyeongsang National University) ;
  • Lee, Shin-Woo (Department of Agronomy & Medicinal Plant Resources, Gyeongnam National University of Science & Technology)
  • 한은희 (국립경남과학기술대학교 생명과학대학 농학.한약자원학부) ;
  • 구영민 (경남한방약초연구원) ;
  • 김윤희 (국립경상대학교 사범대학 생물교육과(농업생명과학연구원)) ;
  • 이신우 (국립경남과학기술대학교 생명과학대학 농학.한약자원학부)
  • Received : 2016.05.11
  • Accepted : 2016.06.14
  • Published : 2016.06.30
Download PDF
⟨ Previous Next ⟩

Abstract

Ascorbic acid (AsA) is a strong antioxidant/reducing agent that can be converted to dehydroascorbate (DHA) by oxidation in plants. DHA, a very short-lived chemical, is recycled to AsA by dehydroascorbate reductase (DHAR). Previously, DHAR cDNA was isolated from the hairy roots of the sesame plant, and DHAR-overexpressing transgenic potato plants were generated under the control of the CaMV35S promoter (CaMV35S::DHAR). An increase in transgene expression and ascorbate levels were observed in the transgenic plants. In the present study, proteomic analysis revealed that transgenic plants not only accumulated DHAR in their cells, but also induced several other antioxidant enzyme-related proteins during plant growth. These results suggest that DHAR is important for stress tolerance via induction of antioxidant proteins, and could improve stress tolerance in transgenic potato plants.

아스코르빈산(ascorbic acid, AsA)는 강력한 항산화 물질 및 환원제로서 식물에서 산화형 AsA인 dehydroascorbate(DHA)를 활성형인 환원형 AsA로 변환시키는 효소인 dehydroascorbate reductase (DHAR)에 의해 생성된다. 선행연구의 결과로서, 본 연구팀은 참깨의 모상근에서 분리한 DHAR 유전자를 이용하여 항시 발현하는 CaMV 35S 프로모터와 괴경 특이적으로 발현하는 patatin 프로모터의 조절하에 과발현 시킨 형질전환 감자를 개발하였다. 형질전환 감자 식물체들은 비형질전환체 보다 증가된 DHAR 활성과 AsA 함량을 보인 바 있다. 본 연구에서는 단백질체 분석을 통해서 형질전환 감자 식물체에서 DHAR 과발현에 의해 조절되는 단백질들을 조사하였다. 단백질체 분석의 결과로서, 형질전환 감자에서 도입된 DHAR 단백질과 다양한 항산화 관련 단백질들이 식물 생장시기 동안 증가 하였다. 본 연구의 결과로서, 도입된 유전자인 DHAR이 항산화 단백질들의 발현 증가를 통해 형질전환 식물체의 스트레스 내성 기작을 향상 시킬 수 있을 것으로 생각된다.

Keywords

References

  1. 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 50:601-639 https://doi.org/10.1146/annurev.arplant.50.1.601
  2. Blum H, Beier H, Gross HJ (1987) Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels. Electrophoresis 8:93-99 https://doi.org/10.1002/elps.1150080203
  3. Chun JA, Seo JY, Han MO, Lee JW, Yi YB, Park GY, Lee SW, Bae SC, Cho KJ, Chung CH (2006) Comparative expression and characterization of dehydroascorbate reductase cDNA from transformed sesame hairy roots using real-time RT-PCR. J Plant Biol 49:507-512 https://doi.org/10.1007/BF03031133
  4. Chun JA, Lee WH, Han MO, Lee JW, Yi YB, Goo YM, Lee SW, Bae SC, Cho KJ, Chung CH (2007) Molecular and biochemical characterizations of dehydroascorbate reductase from sesame (Sesamum indicum L.) hairy root cultures. J Agric Food Chem 55:6067-6073 https://doi.org/10.1021/jf070946t
  5. Conklin PL, Pallanca JE, Last RL, Smirnoff N (1997) L-ascorbic acid metabolism in the ascorbate-deficient arabidopsis mutant vtc1. Plant Physiol 115:1277-1285 https://doi.org/10.1104/pp.115.3.1277
  6. Conklin PL, Williams EH, Last RL (1996) Environmental stress sensitivity of an ascorbate-deficient arabidopsis mutant. Proc Natl Acad USA 93:9970-9974 https://doi.org/10.1073/pnas.93.18.9970
  7. Dixon DP, Cummins I, Cole DJ, Edwards R (1998) Glutathionemediated detoxification systems in plants. Curr Opin Plant Biol 1:258-266 https://doi.org/10.1016/S1369-5266(98)80114-3
  8. Droge W, Broer I, Puhler A (1992) Transgenic plants containing the phosphinothricin-N-acetyltransferase gene metabolize the herbicide l-phosphinothricin (glufosinate) differently from untransformed plants. Planta 187:142-151
  9. Gelhaye E, Rouhier N, Navrot N, Jacquot JP (2005) The plant thioredoxin system. Cell Mol Life Sci 62:24-35 https://doi.org/10.1007/s00018-004-4296-4
  10. Goo YM, Chun HJ, Kim TW, Lee CH, Ahn MJ, Bae SC, Cho KJ, Chun JA, Chung CH, Lee SW (2008) Expressional characterization of dehydroascorbate reductase cDNA in transgenic potato plants. J Plant Biol 51:35-41 https://doi.org/10.1007/BF03030738
  11. Green LS, Yee BC, Buchanan BB, Kamide K, Sanada Y, Wada K (1991) Ferredoxin and ferredoxin-NADP reductase from photosynthetic and nonphotosynthetic tissues of tomato. Plant Physiol 96:1207-1213 https://doi.org/10.1104/pp.96.4.1207
  12. Kim ST, Cho KS, Jang YS, Kang KY (2001) Two-dimensional electrophoresis analysis of rice proteins by polyethylene glycol fractionation for protein arrays. Electrophoresis. 22: 2103-2109 https://doi.org/10.1002/1522-2683(200106)22:10<2103::AID-ELPS2103>3.0.CO;2-W
  13. Kwon SY, Ahn YO, Lee HS, Kwak SS (2001) Biochemical characterization of transgenic tobacco plants expressing a human dehydroascorbate reductase gene. J Biochem Mol Biol 34:316-321
  14. Kwon SY, Choi SM, Ahn YO, Lee HS, Lee HB, Park YB, Kwak SS (2003) Enhanced stress-tolerance of transgenic tobacco plants expressing a human dehydroascorbate reductase gene. J Plant Physiol 160:347-353 https://doi.org/10.1078/0176-1617-00926
  15. Lee DG, Ahsan N, Lee SH, Kang KY, Bahk JD, Lee IJ, Lee BH (2007) A proteomic approach in analyzing heat-responsive proteins in rice leaves. Proteomics 7:3369-3383 https://doi.org/10.1002/pmic.200700266
  16. Lowry OH, Rosebrough JN, Farr AI, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193: 265-275
  17. Meyer Y, Siala W, Bashandy T, Riondet C, Vignols F, Reichheld JP (2008) Glutaredoxins and thioredoxins in plants. Biochim Biophysic Acta 1783: 589-600 https://doi.org/10.1016/j.bbamcr.2007.10.017
  18. Mittler R, Vanderauwera S, Gollery M, Breusegem FV (2004) Reactive oxygen gene network of plants. Trend Plant Sci 9: 490-498 https://doi.org/10.1016/j.tplants.2004.08.009
  19. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15: 473-97 https://doi.org/10.1111/j.1399-3054.1962.tb08052.x
  20. Neuhoff V, Arold N, Taube D, Ehrhardt W (1988) Improved staining of proteins in polyacrylamide gels include isoelectric focusing gels with clear background at nonogram sensitivity using Coomassie Brilliant Blue G-250 and R-250. Electrophoresis 9:255-262 https://doi.org/10.1002/elps.1150090603
  21. Noctor G, Foyer CH (1998) Ascorbate and glutathione: keeping active oxygen under control. Annu Rev Plant Physiol Plant Mol Biol 49:249-279 https://doi.org/10.1146/annurev.arplant.49.1.249
  22. Shigeoka S, Ishikawa T, Tamoi M, Miyakawa Y, Takeda T, Yabuta Y, Yoshimura K (2002) Regulation and function of ascorbate peroxidase isoenzymes. J Exp Bot 53:1305-1319 https://doi.org/10.1093/jexbot/53.372.1305
  23. Stitt M, Gerbardt R, Wilke I, Hddt HW (1987) The contribution of fructose 2,6-bisphosphate to the regulation of sucrose synthesis during photosynthesis. Physiol Plant 69:377-386 https://doi.org/10.1111/j.1399-3054.1987.tb04304.x
  24. Toledano MB, Delaunay A, Monceau L, Tacnet F (2004) Microbial H2O2 sensors as archetypical redox signaling modules. Trends Biochem Sci 29:351-357 https://doi.org/10.1016/j.tibs.2004.05.005
  25. Wheeler GL, Grant CM (2004) Regulation of redox homeostasis in the yeast Saccharomyces cerevisiae. Physiol Plant 120:12-20 https://doi.org/10.1111/j.0031-9317.2004.0193.x
  26. Wheeler GL, Jones MA, Smirnoff N (1998) The biosynthetic pathway of vitamin C in higher plants. Nature 393:365-369 https://doi.org/10.1038/30728
  27. Wohlleben W, Arnold W, Broer I, Hillemann D, Strauch E, Punier A (1988) Nucleotide sequence of the phosphinothricin N-acetyltransferase gene from Streptomyces viridochromogenes Tu494 and its expression in Nicotiana tabacum. Gene 70:25-37 https://doi.org/10.1016/0378-1119(88)90101-1