Modified Suppression Subtractive Hybridization Identifies an AP2-containing Protein Involved in Metal Responses in Physcomitrella patens

  • Cho, Sung Hyun (Department of Biology, Washington University) ;
  • Hoang, Quoc Truong (School of Life Sciences and Biotechnology, Korea University) ;
  • Phee, Jeong Won (School of Life Sciences and Biotechnology, Korea University) ;
  • Kim, Yun Young (School of Life Sciences and Biotechnology, Korea University) ;
  • Shin, Hyun Young (School of Life Sciences and Biotechnology, Korea University) ;
  • Shin, Jeong Sheop (School of Life Sciences and Biotechnology, Korea University)
  • Received : 2006.10.09
  • Accepted : 2006.11.23
  • Published : 2007.02.28

Abstract

The moss Physcomitrella patens has two life cycles, filamentous protonema and leafy gametophore. A modified from of suppression subtractive hybridization (SSH), mirror orientation selection (MOS), was applied to screen genes differentially expressed in the P. patens protonema. Using reverse Northern blot analysis, differentially expressed clones were identified. The identified genes were involved mainly in metal binding and detoxification. One of these genes was an AP2 (APETALA2) domain-containing protein (PpACP1), which was highly up-regulated in the protonema. Alignment with other AP2/EREBPs (Ethylene Responsive Element Binding Proteins) revealed significant sequence homology of the deduced amino acid sequence in the AP2/EREBP DNA binding domain. Northern analysis under various stress conditions showed that PpACP1 was induced by ethephon, cadmium, copper, ABA, IAA, and cold. In addition, it was highly expressed in suspension-cultured protonema. We suggest that PpACP1 is involved in responses to metals, and that suspension culture enhance the expression of genes responding to metals.

Keywords

AP2-Containing Protein;Physcomitrella patens;Protonema;Stress-induced;Suppression Subtractive Hybridization;Suspension Culture

Acknowledgement

Supported by : Rural Development Administration

References

  1. Ashton, N. W. and Cove, D. J. (1977) The isolation and preliminary characterisation of auxotrophic and analogue-resistant mutants of the moss, Physcomitrella patens. Mol. Gen. Genet. 154, 87−95
  2. Cove, D. (2000) The Moss, Physcomitrella patens. J. Plant Growth Regul. 19, 275−283
  3. Diatchenko, L., Lukyanov, S., Lau, Y. F., and Siebert, P. D. (1999) Suppression subtractive hybridization: a versatile method for identifying differentially expressed genes. Methods Enzymol. 303, 349−380 https://doi.org/10.1016/S0076-6879(99)03023-2
  4. Faraco, V., Giardina, P., and Sannia, G. (2003) Metal-responsive elements in Pleurotus ostreatus laccase gene promoters. Microbiology 149, 2155−2162
  5. Coupe, S. A., Taylor, J. E., and Roberts, J. A. (1995) Characterization of am mRNA encoding a metallothionein-like protein that accumulates during ethylene-promoted abscission of Sambucus nigra L. leaflets. Planta 197, 442−447
  6. Ohme-Takagi, M. and Shinshi, H. (1995) Ethylene-inducible DNA binding proteins that interact with an ethyleneresponsive element. Plant Cell 7, 173−182
  7. Cho, S. K., Jeung, J. U., Kang, K. H., Shim, K. S., Jung, K. W., et al. (2004) Identification of Genes Induced in Woundtreated Wild Rice (Oryza minuta). Mol. Cells 17, 230−236
  8. Jofuku, K. D., den Boer, B. G., Van Montagu, M., and Okamuro, J. K. (1994) Control of Arabidopsis flower and seed development by the homeotic gene APETALA2. Plant Cell 6, 1211−1225
  9. Singh, P. K. and Tewari, R. K. (2003) Cadmium toxicity induced changes in plant water relations and oxidative metabolism of Brassica juncea L. plants. J. Environ. Biol. 24, 107−112
  10. Tang, W., Charles, T. M., and Newton, R. J. (2005) Overexpression of the pepper transcription factor CaPF1 in transgenic Virginia pine (Pinus Virginia Mill.) confers multiple stress tolerance and enhances organ growth. Plant Mol. Biol. 59, 603−617
  11. Cho, S. H., Hoang, Q. T., Kim, Y. Y., Shin, H. Y., Ok, S. H., et al. (2006) Proteome analysis of gametophores identified a metallothionein involved in various abiotic stress responses in Physcomitrella patens. Plant Cell Rep. 25, 475−488
  12. Mlotshwa, S., Yang, Z., Kim, Y., and Chen, X. (2006) Floral patterning defects induced by Arabidopsis APETALA2 and microRNA172 expression in Nicotiana benthamiana. Plant Mol. Biol. 61, 781−793
  13. Weigel, D. (1995) The APETALA2 domain is related to a novel type of DNA binding domain. Plant Cell 7, 388−389
  14. O'Donnell, P. J., Calvert, C., Atzorn, R., Wasternack, C., Leyser, H. M. O., et al. (1996) Ethylene as a signal mediating the wound response of tomato plants. Science 274, 1914−1917 https://doi.org/10.1126/science.274.5294.1917
  15. Sponsel, V. M. (2001) The deoxyxylulose phosphate pathway for the biosynthesis of plastidic isoprenoids: early days in our understanding of the early stages of gibberellin biosynthesis. J. Plant Growth Regul. 20, 332−345
  16. Allen, M. D., Yamasaki, K., Ohme-Takagi, M., Tateno, M., and Suzuki, M. (1998) A novel mode of DNA recognition by a beta-sheet revealed by the solution structure of the GCC-box binding domain in complex with DNA. EMBO J. 17, 5484−5496
  17. Johnson, P. R. and Ecker, J. R. (1998) The ethylene gas signal transduction pathway: a molecular perspective. Annu. Rev. Genet. 32, 227−254
  18. Reski, R. (1999) Molecular genetics of Physcomitrella. Planta 208, 301−309
  19. Sambrook, J. and Russell, D. W. (2001) Molecular Cloning, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York
  20. Kagaya, Y., Ohmiya, K., and Hattori, T. (1999) RAV1, a novel DNA-binding protein, binds to bipartite recognition sequence through two distinct DNA-binding domains uniquely found in higher plants. Nucleic Acids Res. 27, 470−478
  21. Cobbett, C. S., May, M. J., Howden, R., and Rolls, B. (1998) The glutathione-deficient, cadmium-sensitive mutant, cad2-1, of Arabidopsis thaliana is deficient in gamma-glutamylcysteine synthetase. Plant J. 16, 73−78
  22. Dushenkov, V., Kumar, P. B., Motto, H., and Raskin, I. (1995) Rhizofiltration: the use of plants to remove heavy metals from aqueous stream. Environ. Sci. Technol. 29, 1239−1245
  23. Hunter, T. C. and Mehra, R. K. (1998) A role for HEM2 in cadmium tolerance. J. Inorg. Biochem. 69, 293−303
  24. Cobbett, C. and Goldsbrough, P. (2002) Phytochelatins and metallothioneins: roles in heavy metal detoxification and homeostasis. Annu. Rev. Plant Biol. 53, 159−182
  25. Rebrikov, D. V., Britanova, O. V., Gurskaya, N. G., Lukyanov, K. A., Tarabykin, V. S., et al. (2000) Mirror orientation selection (MOS): a method for eliminating false positive clones from libraries generated by suppression subtractive hybridization. Nucleic Acids Res. 28, E90 https://doi.org/10.1093/nar/28.20.e90
  26. Mine, T., Hiyoshi, T., Kasaoka, K., and Ohyama, A. (2003) CIP35 encodes an AP2/ERF-domain protein in potato (Solanum tuberosum L.) and responds slowly to cold stress. Plant Cell Physiol, 44, 10−15
  27. Han, C. U., Lee, C. H., Jang, K. S., Choi, G. J., Lim, H. K., et al. (2004) Identification of rice genes induced in a rice blastresistant mutant. Mol. Cells 17, 462−468
  28. Wang, K. L., Li, H., and Ecker, J. R. (2002) Ethylene biosynthesis and signaling networks, Plant Cell, 14 (Suppl), S131−151