Skip to main content
SpringerLink
Log in

Purification and PCR-based cDNA cloning of a plastidial n-6 desaturase

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

Abstract

A plastidial membrane-bound n-6 desaturase from spinach (Spinacia oleracea) was purified from chloroplast envelope membranes by anion exchange, cation exchange and ferredoxin-affinity chromatography. The molecular mass of the protein was estimated by SDS-PAGE to be 40 kDa. The highest specific activity of the desaturase in the final preparation was 196 nmol/min per mg protein with free oleic acid as the substrate. The N-terminal amino acid sequence of the blotted protein was determined and used for the construction of a degenerated and inosine-containing oligonucleotide primer for PCR experiments with cDNA transcribed from leaf mRNA. A 3′-RACE experiment with this primer amplified a single band of 1500 bp that after sequencing showed an open reading frame of 382 amino acids corresponding to a protein of 43 kDa. The 5′ end of the cDNA was amplified by a 5′-RACE experiment and isolated as a 500 bp fragment. Sequencing of this DNA revealed an additional 65 amino acids at the N-terminus of the native protein that are attributed to a plastidial leader peptide. With appropriate primers derived from these sequences a full-length clone was amplified by PCR and sequenced. Comparison of the plastidial oleate desaturase with the homologous enzyme from cyanobacteria showed about 50% amino acid homology. Comparison with other desaturases revealed three histidine boxes with the general sequence HXXXH that are highly conserved in all membrane-bound desaturases. These boxes might be involved in metal ion complexation required for reduction of oxygen.

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. Andrews J, Heinz E: Desaturation of newly synthesized monogalactosyldiacylglycerol in spinach chloroplasts. J Plant Physiol 131: 75–90 (1987).

    Google Scholar 

  2. Andrews J, Schmidt H, Heinz E: Interference of electron transport inhibitors with desaturation of monogalactosyl diacylglycerol in intact chloroplasts. Arch Biochem Biophys 270: 611–622 (1989).

    PubMed  Google Scholar 

  3. Arondel V, Lemieux B, Hwang I, Gibson S, Goodman HM, Somerville CR: Map based cloning of a gene controlling omega-3 fatty acid desaturation in Arabidopsis. Science 258: 1353–1354 (1992).

    PubMed  Google Scholar 

  4. Arthington BA, Bennett LG, Skatrud PL, Guynn CJ, Barbuch RJ, Ulbright CE, Bard M: Cloning, disruption and sequence of the gene encoding yeast C-5 sterol desaturase. Gene 102: 39–44 (1991).

    Article  PubMed  Google Scholar 

  5. Block MA, Joyard J, Douce R: Purification and characterization of E37, a major envelope protein. FEBS Lett 287: 167–170 (1991).

    Article  PubMed  Google Scholar 

  6. 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 

  7. Browse J, Kunst L, Hughly S, Somerville C: Modifications to the two pathway scheme of lipid metabolism based on studies of Arabidopsis mutants. In: Biacs PA, Gruiz K, Kremer T (eds) Biological Role of Plant Lipids, pp. 335–339. Plenum Press, New York (1989).

    Google Scholar 

  8. Bruinsma J: A comment on the spectrophotometric determination of chlorophyll. Biochim Biophys Acta 52: 574–576 (1961).

    Article  PubMed  Google Scholar 

  9. Cahoon EB, Shanklin J, Ohlrogge JB: Expression of a coriander desaturase results in petroselinic acid production in transgenic tobacco. Proc Natl Acad Sci USA 89: 11184–11188 (1992).

    PubMed  Google Scholar 

  10. Devereux J, Haeberli P, Smithies O: A comprehensive set of sequence analysis programs for the VAX. Nucl Acids Res 12: 387–395 (1984).

    PubMed  Google Scholar 

  11. Dreses-Werringloer U, Fischer K, Wachter E, Link TA, Flügge UI: cDNA sequence and deduced amino acid sequence of the precursor of the 37-kDa inner envelope membrane polypeptide from spinach chloroplasts. Eur J Biochem 195: 361–368 (1991).

    PubMed  Google Scholar 

  12. Dresselhaus T, Lörz H, Kranz E: Representative cDNA libraries from few plant cells. Plant J 5: 605–610 (1994).

    Article  PubMed  Google Scholar 

  13. Fischer K, Arbinger B, Kammerer B, Busch C, Brink S, Wallmeier H, Sauer N, Eckerskorn C, Flügge UI: Cloning and in vivo expression of functional triose phosphate/ phosphate translocators from C3- and C4-plants: evidence for the putative participation of specific amino acid residues in the recognition of phosphoenolpyruvate. Plant J 5: 215–226 (1994).

    Article  PubMed  Google Scholar 

  14. Fox BG, Shanklin J, Somerville C, Münck E: Stearoylacyl carrier protein delta-9 desaturase from Ricinus communis is a diiron-oxo protein. Proc Natl Acad Sci USA 90: 2486–2490 (1993).

    PubMed  Google Scholar 

  15. Heinz E: Biosynthesis of polyunsaturated fatty acids. In: MooreJr. TS (ed) Lipid Metabolism in Plants, pp. 34–89: CRC Press, Boca Raton, FL (1993).

    Google Scholar 

  16. Heinz E, Sperling P, Schmidt H: Biosynthesis of polyunsaturated fatty acids in plants: biochemistry, molecular biology and perspectives for gene technology. In: Lifestyle Diseases and the Human Diet, A Challenge to Future Food Production, pp. 43–55. National Institute of Animal Science, Denmark, Research Centre Foulum (1993).

    Google Scholar 

  17. Higgins DG, Sharp PM: Clustal: a package for performing multiple sequence alignment on a microcomputer. Gene 73: 237–244 (1988).

    Article  PubMed  Google Scholar 

  18. Iba K, Gibson S, Nishiuchi t, Fuse T, Nishimura M, Arondel V, Hygly S, Somerville C: A gene encoding a chloroplast omega-3 fatty acid desaturase complements alterations in fatty acid desaturation and chloroplast copy number of the fad7 mutant of Arabidopsis thaliana. J Biol Chem 268: 24099–24105 (1993).

    PubMed  Google Scholar 

  19. Kashan A, Buck F, Haag F, Koch F, thiele HG: A single step purification procedure and partial amino-acid sequence analysis of pmol amounts of the red T cell alloantigen RT6.2. Immunol Lett 23: 133–138 (1990).

    Article  Google Scholar 

  20. Kyhse-Andersen J: Electroblotting of multiple gels: a simple apparatus without buffer tank for rapid transfer of proteins from polyacrylamide to nitrocellulose. J Biochem Biophys Meth 10: 203–209 (1984).

    Article  PubMed  Google Scholar 

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

    PubMed  Google Scholar 

  22. McKeon TA, Stumpf PK: Purification and characterization of the stearoyl-acyl carrier protein desaturase and the acyl-acyl carrier protein thioesterase from maturing seeds of safflower. J Biol Chem 257: 12141–12147 (1982).

    PubMed  Google Scholar 

  23. Murphy DJ (ed) Designer Oil Crops, Breeding, Processing and Biotechnology. VCH, Weinheim (1993).

    Google Scholar 

  24. Ntambi JM, Buhrow SA, Kaestner KH, Christy RJ, Sibley E, KellyJr. TJ, Lane MD: Differentiation-induced gene expression in 3T3-L1 preadipocytes. J Biol Chem 263: 17291–17300 (1988).

    PubMed  Google Scholar 

  25. Ohlrogge JB, Jaworski JG, Post-Beitenmiller D: De novo fatty acid biosynthesis. In: MooreJr. TS (ed) Lipid Metabolism in Plants, pp. 3–32. CRC Press, Boca Raton, FL (1993)

    Google Scholar 

  26. Okayasu T, nagao M, Ishibashi T, Imai Y: Purification and partial characterization of linoleoyl-CoA desaturase from rat liver microsomes. Arch Biochem Biophys 206: 21–28 (1981).

    PubMed  Google Scholar 

  27. Okuley J, Lightner J, Feldmann K, Yadav N, Lark E, Browse J: Arabidopsis FAD2 gene encodes the enzyme that is essential for polyunsaturated lipid synthesis. Plant Cell 6: 147–158 (1994).

    Article  PubMed  Google Scholar 

  28. Reddy AS, Nuccio ML, Gross LM, Thomas TL: Isolation of a delta-6 desaturase from the cyanobacterium Synechocystis sp. strain PCC 6803 by gain-of-function expression in Anabaena sp. strain PCC 7120. Plant Mol Biol 28: 293–300 (1993).

    Google Scholar 

  29. Sanger F, Nicklen S, Coulsen AR: DNA sequencing with chain-termination inhibitors. Proc Natl Acad Sci USA 74: 5463–5467 (1977).

    PubMed  Google Scholar 

  30. Sambrook J, Fritsch T, Maniatis T: Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989).

    Google Scholar 

  31. Schmidt H, Heinz E: Involvement of ferredoxin in desaturation of lipid-bound oleate in chloroplasts. Plant Physiol 94: 214–220 (1990).

    Google Scholar 

  32. Schmidt H, Heinz E: Desaturation of oleoyl groups in envelope membranes from spinach chloroplasts. Proc Natl Acad Sci USA 87: 9477–9480 (1990).

    PubMed  Google Scholar 

  33. Schmidt H, Heinz E: Direct desaturation of intact galactolipids by a desaturase solubilized from spinach (Spinacia oleracea) chloroplast envelopes. Biochem J 289: 777–782 (1993).

    PubMed  Google Scholar 

  34. Schmidt H, Sperling P, Heinz E: New in vivo and in vitro evidence for lipid-linked desaturation in plants, In: Murata N, Somerville C (eds) Biochemistry and Molecular Biology of Membrane and Storage Lipids of Plants. Current Topics Plant Physiol 9: 40–49 (1993).

  35. Shanklin J, Somerville C: Stearoyl-acyl-carrier-protein desaturase from higher plants is structurally unrelated to the animal and fungal homologs. Proc Natl Acad Sci USA 88: 2510–2514 (1991).

    PubMed  Google Scholar 

  36. Stukey JE, McDonough VM, Martin CE: The OLE1 gene of Saccharomyces cerevisiae encodes the delta-9 fatty acid desaturase and can be functionally replaced by the rat stearoyl-CoA desaturase gene. J Biol Chem 265: 20144–20149 (1990).

    PubMed  Google Scholar 

  37. Thies W: Der Einfluss der Chloroplasten auf die Bildung von ungesättigten Fettsüren in reifenden Rapssamen. Fette Seifen Anstrichmittel 73: 710–715 (1971).

    Google Scholar 

  38. Thiede MA, Ozols J, Strittmatter P: Construction and sequence of cDNA for rat liver stearoyl-CoA desaturase. J Biol Chem 261: 13230–13235 (1986).

    PubMed  Google Scholar 

  39. Thompson GA, Scherer DE, Foxall-Van Aken S, Kenny JW, Young HL, Shintani DK, Kridl JC, Knauf VC: Primary structures of the precursor and mature forms of stearoyl-acyl carrier protein desaturase from safflower embryos and requirement of ferredoxin for enzyme activity. Proc Natl Acad Sci USA 88: 2578–2582 (1991).

    PubMed  Google Scholar 

  40. von Heijne G, Nishikawa K: Chloroplast transit peptides. FEBS Lett 278, 1: 1–3 (1991).

    Article  PubMed  Google Scholar 

  41. Wada H, Gombos Z, Murata N: Enhancement of chilling tolerance of a cyanobacterium by genetic manipulation of fatty acid desaturation. Nature 347: 200–203 (1990).

    Article  PubMed  Google Scholar 

  42. Willey DL, Fischer K, Wachter E, Link TA, Flügge UI: Molecular cloning and structural analysis of the phosphate translocator from pea chloroplasts and its comparision to the spinach phosphate translocator. Planta 183: 451–461 (1991).

    Article  Google Scholar 

  43. Yadav NS, Wierzbicki A, Aegerter M, Caster CS, Perez-Grau L, Kinney AJ, Hitz WD, BoothJr. JR, Schweiger B, Stecca KL, Allen SM, Blackwell M, Reiter RS, Carlson TJ, Russel SH, Feldmann KA, Pierce J, Browse J: Cloning of higher plant omega-3 fatty acid desaturases. Plant Physiol 103: 467–476 (1993).

    Article  PubMed  Google Scholar 

  44. Yamamoto K, Mori H, Imaseki H: Novel mRNA sequences induced by indole-3-acetic acid in sections of elongating hypocotyls of mung bean (Vigna radiata). Plant Cell Physiol 33: 13–20 (1992).

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Institut für Allgemeine Botanik, Universität Hamburg, Ohnhorststrasse 18, 22609, Hamburg, Germany

    Hermann Schmidt, Thomas Dresselhaus & Ernst Heinz

  2. Institut für Zellbiochemie und Klinische Neurobiologie, Universitätskrankenhaus Eppendorf, Martinistrasse 52, 20246, Hamburg, Germany

    Friedrich Buck

Authors
  1. Hermann Schmidt
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Thomas Dresselhaus
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Friedrich Buck
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ernst Heinz
    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

Schmidt, H., Dresselhaus, T., Buck, F. et al. Purification and PCR-based cDNA cloning of a plastidial n-6 desaturase. Plant Mol Biol 26, 631–642 (1994). https://doi.org/10.1007/BF00013749

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00013749

Key words

Search

Navigation