Skip to main content
SpringerLink
Log in

Accumulation of type I fish antifreeze protein in transgenic tobacco is cold-specific

  • Research Articles
  • Published:
Plant Molecular Biology Aims and scope Submit manuscript

Abstract

Expression of fish antifreeze protein (AFP) genes in plants is a possible means of increasing their frost resistance and freeze tolerance. Initial work involved transfer into tobacco of an AFP gene from winter flounder which codes for the alanine-rich, α-helical Type I AFP. Plants were transformed with a gene construct in which the preproAFP cDNA was inserted between the cauliflower mosaic virus 19S RNA promoter and the nopaline synthetase polyadenylation site. Although transgenic plants produced AFP mRNA, no AFP was detected on western blots. Re-evaluation of AFP expression in these transgenic plants showed that AFP accumulated to detectable levels only after exposure of the plant to cold. Extracts of plants incubated at 4°C for 24 h contained a protein which co-migrated with winter flounder proAFP and was cross-reactive to Type I AFP antisera. Two other minor protein bands of slightly higher apparent M r also cross-reacted with the antisera and are thought to represent processing intermediates. The proAFP was unique to the transgenic plants and was absent in extracts taken prior to cold exposure. AFP levels increased over the first 48 h of cold incubation then remained stable. Since the α-helix content of Type I AFP has been shown to decrease markedly at warmer temperatures, we postulate that Type I AFP stability in transgenic plants is dependent on its secondary structure.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Ananthanarayanan VS, Hew CL: Structural studies on the freezing point-depressing protein of the winter flounder Pseudopleuronectes americanus. Biochem Biophys Res Commun 74: 685–689 (1977).

    Article  PubMed  Google Scholar 

  2. Bernatzky R, Tanksley SD: Genetics of actin-related sequences in tomato. Theor Appl Genet 72: 314–321 (1986).

    Article  Google Scholar 

  3. Bradford MM: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal Biochem 72: 248–254 (1976).

    Article  PubMed  Google Scholar 

  4. Burnette WH: Western blotting: electophoretic transfer of proteins from SDS-polyacrylamide gels to unmodified nitrocellulose and ratio-graphic detection with antibody and radioiodinated protein A. Anal Biochem 112: 195–203 (1981).

    PubMed  Google Scholar 

  5. Cutler AJ, Saleem M, Kendall E, Gusta LV, Georges F, Fletcher GL: Winter flounder antifreeze proteins improves the cold hardiness of plant tissues. J Plant Physiol 135: 351–354 (1989).

    Google Scholar 

  6. Davies PL, Hew CL; Fletcher GL: Fish antifreeze proteins: physiology and evolutionary biology. Can J Zool 66: 2611–2617 (1988).

    Google Scholar 

  7. Davies PL: Antifreeze protein: prospects for transferring freeze resistance. New Biotechnol 1: 11–16.

  8. Davies PL, Roach AH, Hew CL: DNA sequence coding for an antifreeze protein precursor form winter flounder. Proc Natl Acad Sci USA 79: 335–339 (1982).

    PubMed  Google Scholar 

  9. Davies PL: Conservation of antifreeze protein-encoding genes in tandem repeats. Gene 112: 163–170 (1992).

    Article  PubMed  Google Scholar 

  10. Denecke J, Botterman J, Deblaere R: Protein secretion in plant cells can occur via a default pathway. Plant Cell 2: 51–59 (1990).

    Article  PubMed  Google Scholar 

  11. DeVries AL: Antifreeze peptides and glycopeptides in cold-water fishes. Annu Rev Physiol 45: 245–260 (1983).

    Article  PubMed  Google Scholar 

  12. Ditta G, Stanfield S, Corbin D, Helinski DR: Broad host range DNA cloning system for gram-negative bacteria: construction of a gene bank or Rhizobium meliloti. Proc Natl Acad Sci USA 77: 7347–7351 (1982).

    Google Scholar 

  13. Duman JG, DeVries AL: Isolation and characterization and physical properties of protein antifreezes from the winter flounder, Pseudopleuronectes americanus. Comp Biochem Physiol 54B: 375–380 (1976).

    Google Scholar 

  14. Fletcher GL, Idler DR, Vaisius A, Hew CL: Hormonal regulation of antifreeze protein gene expression in winter flounder. Fish Physiol Biochem 7: 387–393 (1989).

    Google Scholar 

  15. Gengenheimer P: Preparation of extracts from plants. Meth Enzymol 182: 184–185 (1990).

    Google Scholar 

  16. Georges G, Saleem M, Cutler AJ: Design and cloning of a synthetic gene for the flounder antifreeze protein and its expression in plant cells. Gene 91: 159–165 (1990).

    Article  PubMed  Google Scholar 

  17. Hew CL, Liunardo N, Fletcher GL: In vivo biosynthesis of the antifreeze protein in the winter flounder-evidence for a larger precursor. Biochem Biophys Res Commun 85: 421–427 (1978).

    PubMed  Google Scholar 

  18. Hew CL, Wang NC, Yan S, Cai H, Sclater A, Fletcher GL: Biosynthesis of antifreeze polypeptides in the winter flounder. Eur J Biochem 160: 267–272 (1986).

    PubMed  Google Scholar 

  19. Hightower R, Baden C, Penzes E, Lund P, Dunsmuir P: Expression of antifreeze proteins in transgenic plants. Plant Mol Biol 17: 1013–1021 (1991).

    PubMed  Google Scholar 

  20. Horsch RD, Fry JE, Hoffmann NL, Eichholtz D, Rogers SG, Fraley RT: A simple and general method for transferring genes into plants. Science 227: 1229–1231 (1985).

    Google Scholar 

  21. Kenward KD, Davies PL, Downing W, MacHutchon M, McPherson JC: Expression of a type III fish antifreeze protein gene in transgenic tobacco improves plant freeze tolerance. Proceedings of the 1992 Miami Bio/Technology Winter Symposium, p. 59 (1992).

  22. Knight CA, Hallett J, De Vries AL: Solute effects on ice recrystallization: an assessment technique. Cryobiology 25: 55–60 (1988).

    PubMed  Google Scholar 

  23. Kriel G, Haiml L, Suchanek G: Stepwise cleavage of the pro part of promelittin by dipeptidylpeptidase IV. Eur J Biochem 111: 49–58 (1980).

    PubMed  Google Scholar 

  24. Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685 (1970).

    PubMed  Google Scholar 

  25. Maniatis T, Fritsch EF, Sambrook J: Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982).

    Google Scholar 

  26. Mazur BJ, Chui CF, Smith JK: Isolation and characterization of plant genes coding for acetolactate synthase, the target enzyme for two classes of herbicides. Plant Physiol 85: 1110–1117 (1987).

    Google Scholar 

  27. Palmiter RD: Magnesium precipitation of ribonucleoprotein complexes: Expedient techniques for the isolation of undegraded polysomes and messenger ribonucleic acid. Biochemistry 13: 3606–3615 (1974).

    PubMed  Google Scholar 

  28. Parody-Morreale P, Murphy KP, Di Cera E, Fall R, De Vries AL, Gill SJ: Inhibition of bacterial ice nucleators by fish antifreeze glycoproteins. Nature 333: 782–783 (1988).

    Article  PubMed  Google Scholar 

  29. Peters ID, Hew CL, Davies PL: Biosynthesis of winter flounder antifreeze proprotein in E. coli. Protein Engng 3: 145–151 (1989).

    Google Scholar 

  30. Peters ID, Rancourt DE, Davies PL, Walker VK: Isolation and characterization of an antifreeze protein precursor from transgenic Drosophila: evidence for partial processing. Biochem Biophys Acta 1171: 247–254 (1993).

    PubMed  Google Scholar 

  31. Pickett M, Scott G, Davies P, Wang N, Joshi S, Hew C: Sequence of an antifreeze precursor. Eur J Biochem 143: 35–38 (1984).

    PubMed  Google Scholar 

  32. Pickett MH: Antifreeze protein gene expression in the liver of the winter flounder, Pseudopleuronectes americanus. Ph. D. thesis, Queen's University, Kingston, Ontario (1984).

  33. Rancourt DE, Walker VK, Davies PL: Flounder antifreeze protein synthesis under heat shock control in transgenic Drosophila melanogaster. Mol Cell Biol 7: 2188–2195 (1987).

    PubMed  Google Scholar 

  34. Rubinsky B, Arav A, Fletcher GL: Hypothermic Protection — A Fundamental Property of ‘Antifreeze’ Proteins. Biochem Biophys Res Commun 180: 566–571 (1991).

    PubMed  Google Scholar 

  35. Shears MA, Fletcher GL, Hew CL, Gauthier S, Davies PL: Transfer, expression and stable inheritance of antifreeze protein in atlantic salmon (Salmo salar). Mol Mar Biol Biotechnol 1: 58–63 (1991).

    Google Scholar 

  36. Sijmons PC, Dekker BMM, Schrammeijer B, Verwoerd TC, van den Elzen PJM, Hoekema A: Production of correctly processed human serum albumin in transgenic plants. Bio/Technology 8: 217–221 (1990).

    Article  PubMed  Google Scholar 

  37. Slaughter D, Hew CL: Radioimmunoassay for the antifreeze polypeptides of the winter flounder: seasonal profile and immunological cross-reactivity with other fish antifreezes. Can J Biochem 60: 824–829 (1982).

    PubMed  Google Scholar 

  38. Thomas PS: Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Natl Acad Sci USA 77: 5201–5205 (1980).

    PubMed  Google Scholar 

  39. von Heijne G: A new method for predicting signal sequence cleavage sites. Nucl Acids Res 14: 4683–4690 (1986).

    PubMed  Google Scholar 

  40. Yang DSC, Sax M, Chakrabartty A, Hew CL: Crystal structure of an antifreeze polypeptide and its mechanistic implications. Nature 333: 232–237 (1988).

    Article  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Biochemistry, Queen's University, K7L 3N6, Kingston, Ontario, Canada

    Kimberly D. Kenward & Peter L. Davies

  2. Plant Molecular Biology Center, Northern Illinois University, 60115, DeKalb, IL, USA

    Mitchell Altschuler

  3. Department of Agronomy, University of Kentucky, 40546, Lexington, KY, USA

    David Hildebrand

Authors
  1. Kimberly D. Kenward
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Mitchell Altschuler
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. David Hildebrand
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Peter L. Davies
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Kenward, K.D., Altschuler, M., Hildebrand, D. et al. Accumulation of type I fish antifreeze protein in transgenic tobacco is cold-specific. Plant Mol Biol 23, 377–385 (1993). https://doi.org/10.1007/BF00029012

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00029012

Key words

Search

Navigation