Journal of Life Science (생명과학회지)

Korean Society of Life Science (한국생명과학회)
권호 표지
DOI QR코드

DOI QR Code

Overexpression of NtHSP70-1 Protects Chlorophyll from High Temperature in Plants

NtHSP70-1에 의한 클로로필의 고온 내성 효과

  • Cho, Eun-Kyung (Department of Bio-Food Materials, College of Medical Life Sciences, Silla University) ;
  • Hong, Choo-Bong (Institute of Molecular Biology and Genetics and School of Biological Sciences, Seoul National University)
  • 조은경 (신라대학교 바이오식품소재학과) ;
  • 홍주봉 (서울대학교 생명과학부 유전공학연구소)
  • Published : 2008.03.31
Download PDF
⟨ Previous Next ⟩

Abstract

Heat shock protein 70 (HSP70) is known as molecular chaperone, the fundamental protein participating in various processes, from nascent protein synthesis to protection of proteins during abiotic stresses and developmental programs. However, their biological functions in plants are not yet well known. Here, NtHSP70-1 (AY372069), HSP70 of Nicotiana tabacum induced by heat stress was investigated. To analyze the protective role of NtHSP70-1, transgenic tobacco plants, which constitutively overexpressed NtHSP70-1 as well as contained either the vector alone or having NtHSP70-1 in the antisense orientation, were constructed. The altered NtHSP70-1 levels in plants were confirmed by western blotting and transgenic sense lines exhibited tolerance to heat stress. Seedlings with the constitutively expressed NtHSP70-1 grew as green or healthy plants after heat stress. In contrast, transgenic vector or antisense lines exhibited yellowing of leaves or some delay in growth, which finally led to death. Evaluation of chlorophyll contents of heat-shocked transgenic tobacco seedlings indicated that NtHSP70-1 contributes to thermotolerance by preventing chlorophyll synthesis in plants.

고온 단백질 heat shcok protein 70 (HSP70)은 분자샤페론으로써 환경스트레스와 발달단계 동안 단백질을 보호하고 합성하는 다양한 과정에 관여하는 기본적인 단백질이다. 하지만 그 생물학적 기능이 식물에서 아직 정확하게 밝혀지지 않았다. 이에 본 연구에서는 담배에서 고온에 의해 유도된 HSP70인 NtHSP70-1 (AY372069)를 분리하여 그 기능을 연구하였다. NtHSP70-1의 고온 내성 기능을 분석하기 위해 NtHSP70-1이 식물 형질전환용 벡터인 pBKS1-1에 sense 또는 antisense 방향으로 도입되어 형질전환된 식물체와 pBKS1-1만 도입된 형질전환 식물체들을 제조하였다. 형질전환체에 있어서 NtHSP70-1의 발현량은 western blot 분석법을 사용하여 수행하였고 확인된 형질전환체들은 고온 내성 기능분석에 이용되었다. 그 결과 고온 환경에 있어서 NtHSP70-1이 과다발현된 형질전환체들은 그 클로로필의 함량과 생존율이 정상환경 일 때와 유사하였고 반대로 벡터 또는 벡터인 pBKS1-1에 antisense 방향으로 도입되어 형질전환된 식물체들은 클로로필의 파괴로 인한 감소된 생존율을 나타내었다. 고온 처리된 형질전환 식물체에서 클로로필의 함량비교 결과로 NtHSP70-1이 클로로필을 보호함으로써 식물의 고온내성에 기여함을 알 수 있었다.

Keywords

References

  1. Andersson, B. and J. Barber. 1994. Molecular processes in photosynthesis. Adv. Mol. Cell Biol. 10, 1-53. https://doi.org/10.1016/S1569-2558(08)60394-X
  2. Aro, E. M., I. Virgin and B. Andersson. 1993. Photoinhibition of photosystem II. Inactivation, protein damage and turnover. Biochim. Biophys. Acta. 1143, 113-134. https://doi.org/10.1016/0005-2728(93)90134-2
  3. Beale, S. I. 1999. Enzymes of chlorophyll biosynthesis. Photosyn. Res. 60, 43-73. https://doi.org/10.1023/A:1006297731456
  4. Buchanan, M., L. Starrs, S. U. Egelhaaf and M. E. Cates. 2000. Kinetic pathways of multiphase surfactant systems. Phys. Rev. E 62, 6895-6905.
  5. Canjura, F. L., S. J. Schwartz and R. V. Nunes. 1991. Degradation kinetics of chlorophylls and chlorophyllides. J. Food Sci. 56, 1639-1643. https://doi.org/10.1111/j.1365-2621.1991.tb08660.x
  6. Cho, E. K. and C. B. Hong. 2006. Over-expression of tobacco NtHSP70-1 contributes to drought-stress tolerance in plants. Plant Cell Rep. 25, 349-358 https://doi.org/10.1007/s00299-005-0093-2
  7. Dix, D. J. 1997. Hsp70 expression and function during gametogenesis. Cell Stress Chaperon 2, 73-77. https://doi.org/10.1379/1466-1268(1997)002<0073:HEAFDG>2.3.CO;2
  8. Eckhardt, U., B. Grimm and S. Hortensteiner. 2004. Recent advances in chlorophyll biosynthesis and breakdown in higher plants. Plant Mol. Biol. 56, 1-14. https://doi.org/10.1007/s11103-004-2331-3
  9. Grossman, A. R., M. Lohr and C. S. Im. 2004. Chlamydomonas reinhardtii in the landscape of pigments. Annu. Rev. Genet. 38, 119-173. https://doi.org/10.1146/annurev.genet.38.072902.092328
  10. Heaton, J. W. and A. G. Marangoni. 1996. Chlorophyll degradation in processed foods and senescent plant tissues. Trends Food Sci. Tech. 7, 8-15. https://doi.org/10.1016/0924-2244(96)81352-5
  11. Hortensteiner, S., K. L. Wuthrich, P. Matile, K. H. Ongania and B. Krautler. 1998. The key step in chlorophyll breakdown in higher plants: cleavage of pheophorbide a macrocycle by a monooxygenase. J. Biol. Chem. 273, 15335-15339. https://doi.org/10.1074/jbc.273.25.15335
  12. Ko, K., O. Bornemisza, L. Kourtz, Z. W. Ko, W. C. Plaxton and A. R. Cashmore. 1992. Isolation and characterization of a cDNA clone encoding a cognate 70 kDa heat shock protein of the chloroplast envelope. J. Biol. Chem. 267, 2986-2993.
  13. Koca, N., F. Karadeniz and H. S. Burdurlu. 2007. Effect of pH on chlorophyll degradation and colour loss in blanched green peas. Food Chem. 100, 609-615. https://doi.org/10.1016/j.foodchem.2005.09.079
  14. Krebs, R. A. and M. E. Feder. 1997. Negative consequences of Hsp70 overexpression in Drosophila melanogaster larvae. Cell Stress Chaperon 2, 60-71. https://doi.org/10.1379/1466-1268(1997)002<0060:DCOHOI>2.3.CO;2
  15. Kropat, J., U. Oster, W. Rue diger and C. F. Beck. 1997. Chlorophyll precursors are signals of chloroplast origin involved in light induction of nuclear heat-shock genes. Proc. Natl. Acad. Sci. USA. 94, 14168-14172. https://doi.org/10.1073/pnas.94.25.14168
  16. Kropat, J., U. Oster, W. Rue digger and C. F. Beck. 2000. Chloroplast signalling in the light induction of nuclear HSP70 genes requires the accumulation of chlorophyll precursors and the export of these compounds to the cytoplasm/ nucleus. Plant J. 24, 523-531. https://doi.org/10.1046/j.1365-313x.2000.00898.x
  17. Li, Q. B., D. Haskell, C. Zhang, D. Y. Sung and C. Guy. 2000. Diurnal regulation of Hsp70s in leaf tissue. Plant J. 21, 373-378. https://doi.org/10.1046/j.1365-313x.2000.00673.x
  18. Luft, J. C. and D. J. Dix. 1999. Hsp70 expression and function during embryogenesis. Cell Stress Chaperon 4, 162-170. https://doi.org/10.1379/1466-1268(1999)004<0162:HEAFDE>2.3.CO;2
  19. Melis, A. 1991. Dynamics of photosynthetic membrane composition and function. Biochim. Biophys. Acta. 1058, 87-106. https://doi.org/10.1016/S0005-2728(05)80225-7
  20. Oh, S. A., J. H. Park, G. I. Lee, K. H. Paek, S. K. Park andH. G. Nam. 1997. Identification of three genetic loci controlling leaf senescence in Arabidopsis thaliana. Plant J. 12, 527-535. https://doi.org/10.1046/j.1365-313X.1997.00527.x
  21. Ohad, I., N. Keren, H. Zer, H. Gong, T. S. Mor, A. Gal, S. Tal and Y. Domovich. 1994. Light-induced degradation of the Photosystem II reaction centre D1 protein in vivo: an integrated approach, pp. 161-178, In Baker, N. R. and J. R. Bowyer (eds.), Photoinhibition Photosynth.: From Mol. Mechanisms to Field, Oxford: BIOS Scientific Publishers.
  22. Ohad, I., D. J. Kyle and J. Arntzen. 1984. Membrane protein damage and repair: removal and replacement of inactivated 32-kilodalton polypeptides in chloroplast membranes. J. Cell Biol. 99, 481-485. https://doi.org/10.1083/jcb.99.2.481
  23. Papenbrock, J., H. P. Mock, R., Tanaka, E. Kruse and B. Grimm. 2000. Role of magnesium chelatase activity in the early steps of the tetrapyrrole biosynthetic pathway. Plant Physiol. 122, 1161-1169. https://doi.org/10.1104/pp.122.4.1161
  24. Sambrook, J., E. F. Fritsch and T. Maniatis. 1989. Molecular cloning: a laboratory manual 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor., New York.
  25. Schnell, D. J., F. Kessler and G. Blobel. 1994. Isolation of components of the chloroplast protein import machinery. Science 266, 1007-1012. https://doi.org/10.1126/science.7973649
  26. Schroda, M., J. Kropat, U. Oster, W. Rdiger, O. Vallon, F. A. Wollman, C. F. Beck. 2001. A role for molecular chaperones in assembly and repair of photosystem II. Biochem. Soc. Trans. 29, 413-418. https://doi.org/10.1042/BST0290413
  27. Schroda, M., O. Vallon, F. A. Wollman, and C. F. Beck. 1999. A chloroplast-targeted heat shock protein 70 (HSP70) contributes to the photoprotection and repair of photosystem II during and after photoinhibition. Plant Cell 11, 1165-1178. https://doi.org/10.1105/tpc.11.6.1165
  28. Schuster, G., D. Even, K. Kloppstech, and I. Ohad. 1988. Evidence for protection by heat-shock proteins against photoinhibition during heat-shock. J. Eur. Mol. Biol. Organ. 7, 1-6.
  29. Schuster, G., R. Timberg and I. Ohad. 1988. Turnover of thylakoid photosystem II proteins during photoinhibition of Chlamydomonas reinhardtii. Eur. J. Biochem. 177, 403-410. https://doi.org/10.1111/j.1432-1033.1988.tb14389.x
  30. Schwartz, S. J. and T. V. Lorenzo. 1991. Chlorophyll stability during continuous aseptic processing and storage. J. Food Sci. 56, 1059-1062. https://doi.org/10.1111/j.1365-2621.1991.tb14641.x
  31. Schwartz, S. J. and J. H. von Elbe, 1983. Kinetics of chlorophyll degradation to pyropheophytin in vegetables. J. Food Sci. 48, 1303-1306 https://doi.org/10.1111/j.1365-2621.1983.tb09216.x
  32. Suh, M. C., C. B. Hong, S. S. Kim and W. S. Sim. 1994. Transgenic tobacco plants with Bacillus thuringiensis delta- toxin gene resistant to Korean born tobacco budworms. Mol. Cells 4, 211-219.
  33. Sweeney, J. P., and M. E. Martin. 1961. Stability of chlorophyll in vegetables as affected by pH. Food Tech. 15, 263-266.
  34. Tanaka, A. and R. Tanaka. 2006. Chlorophyll metabolism. Curr. Opin. Plant Biol. 9, 248-255. https://doi.org/10.1016/j.pbi.2006.03.011
  35. Tijkens, L. M. M., S. A. Barringer and E. S. A. Biekman. 2001. Modelling the effect of pH on the colour degradation of blanched broccoli. Innov. Food Sci. Emer. Tech. 2, 315-322. https://doi.org/10.1016/S1466-8564(01)00048-0
  36. Vavilin, D. V. and W. F. Vermaas. 2002. Regulation of the tetrapyrrole biosynthetic pathway leading to heme and chlorophyll in plants and cyanobacteria. Physiol. Plant 115, 9-24. https://doi.org/10.1034/j.1399-3054.2002.1150102.x
  37. von Elbe, J. H. and S. J. Schwartz. 1996. Colorants. pp. 651-722, In Fennema, O. R. (ed.), Food Chem. Marcel Dekker Inc., New York.
  38. Willows, R. D. 2003. Biosynthesis of chlorophylls from protoporphyrin IX. Nat. Prod. Rep. 20, 327-341. https://doi.org/10.1039/b110549n
  39. Zer, H., O. Prasil and I. Ohad. 1994. Role of plastoquinol oxidoreduction in regulation of photochemical reaction center II D1 protein turnover in vivo. J. Biol. Chem. 269, 17670- 17676.
  40. Zer, H. and I. Ohad. 1995. Photoinactivation of photosystem II induces changes in the photochemical reaction center II abolishing the regulatory role of the QB site in the D1 protein degradation. Eur. J. Biochem. 231, 448-453. https://doi.org/10.1111/j.1432-1033.1995.tb20718.x