Skip to main content
SpringerLink
Log in

Heterologous expression of a fatty acid hydroxylase gene in developing seeds of Arabidopsis thaliana

  • Original Article
  • Published:
Planta Aims and scope Submit manuscript

Abstract

Expression of a cDNA encoding the castor bean (Ricinus communis L.) oleate Δ12-hydroxylase in the developing seeds of Arabidopsis thaliana (L.) Heynh. results in the synthesis of four novel hydroxy fatty acids. These have been previously identified as ricinoleic acid (12-hydroxy-octadec-cis-9-enoic acid: 18:1-OH), densipolic acid (12-hydroxy-octadec-cis-9,15-enoic acid: 18:2-OH), lesquerolic acid (14-hydroxy-eicos-cis-11-enoic acid: 20:1-OH) and auricolic acid (14-hydroxy-eicos-cis-11,17-enoic acid: 20:2-OH). Using mutant lines of Arabidopsis that lack the activity of the FAE1 condensing enzyme or FAD3 ER Δ-15-desaturase, we have shown that these enzymes are required for the synthesis of C20 hydroxy fatty acids and polyunsaturated hydroxy fatty acids, respectively. Analysis of the seed fatty acid composition of transformed plants demonstrated a dramatic increase in oleic acid (18:1) levels and a decrease in linoleic acid (18:2) content correlating to the levels of hydroxy fatty acid present in the seed. Plants in which FAD2 (ER Δ12-desaturase) activity was absent showed a decrease in 18:1 content and a slight increase in 18:2 levels corresponding to hydroxy fatty acid content. Expression of the castor hydroxylase protein in yeast indicates that this enzyme has a low level of fatty acid Δ12-desaturase activity. Lipase catalysed 1,3-specific lipolysis of triacylglycerol from transformed plants demonstrated that ricinoleic acid is not excluded from the sn-2 position of triacylglycerol, but is the only hydroxy fatty acid present at this position.

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

Fig. 1.
Fig. 2a–e.
Fig. 3a, b.
Fig. 4a, b.
Fig. 5. a
Fig. 6a–c.
Fig. 7.
Fig. 8. a

Similar content being viewed by others

Abbreviations

DAG:

diacylglycerol

ER:

endoplasmic reticulum

FAE1:

Fatty Acid Elongation 1

FAMES:

fatty acid methyl esters

MAG:

monoacylglycerol

ODP:

Oleate Derivative Proportion

PC:

phosphatidylcholine

TAG:

triacylglycerol

VLCFA:

very long chain fatty acid (C20 and longer)

X:Y:

a fatty acid containing X carbons with Y double bonds

References

  • Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K, Albright LM, Coen DM, Varki A (eds) (1995) Current protocols in molecular biology. Wiley, New York

  • Bafor M, Jonsson L, Stobart AK, Stymne S (1990) Regulation of triacylglycerol biosynthesis in embryos and microsomal preparations from the developing seeds of Cuphea lanceolata. Biochem J 272:31–38

    CAS  PubMed  Google Scholar 

  • Bafor M, Smith MA, Jonsson L, Stobart K, Stymne S (1991) Ricinoleic acid biosynthesis and triacylglycerol assembly in microsomal preparations from developing castor-bean (Ricinus communis) endosperm. Biochem J 280:507–514

    CAS  PubMed  Google Scholar 

  • Bafor M, Smith MA, Jonsson L, Stobart K, Stymne S (1993) Biosynthesis of vernoleate (cis-12-epoxyoctadeca-cis-9-enoate) in microsomal preparations from developing endosperm of Euphorbia lagascae. Arch Biochem Biophys 303:141–151

    Article  Google Scholar 

  • Broadwater JA, Whittle E, Shanklin J (2002) Desaturation and hydroxylation: Residues 148 and 324 of Arabidopsis FAD2, in addition to substrate chain length, exert a major influence in partitioning of catalytic specificity. J Biol Chem 277:15613–15620

    Article  CAS  PubMed  Google Scholar 

  • Broun P, Somerville C (1997) Accumulation of ricinolic, lesquerolic and densipolic acids in seeds of transgenic Arabidopsis plants that express a fatty acyl hydroxylase cDNA from castor bean. Plant Physiol 113:933–942

    CAS  PubMed  Google Scholar 

  • Broun P, Boddupalli S, Somerville C (1998a) A bifunctional oleate 12-hydroxylase: desaturase from Lesquerella fendleri. Plant J 13:201–210

    CAS  PubMed  Google Scholar 

  • Broun P, Shanklin J, Whittle E, Somerville C (1998b) Catalytic plasticity of fatty acid modification enzymes underlying chemical diversity of plant lipids. Science 282:1315–1317

    CAS  PubMed  Google Scholar 

  • Cahoon EB, Carlson TJ, Ripp KG, Schweiger BJ, Cook GA, Hall SE, Kinney AJ (1999) Biosynthetic origin of conjugated double bonds: production of fatty acid components of high-value drying oils in transgenic soybean embryos. Proc Natl Acad Sci USA 96:12935–12940

    Article  CAS  PubMed  Google Scholar 

  • Cahoon EB, Ripp KG, Hall SE, McGonigle BM (2002) Transgenic production of epoxy fatty acids by expression of a cytochrome P450 enzyme from Euphorbia lagascae seed. Plant Physiol 128:615–624

    Article  CAS  PubMed  Google Scholar 

  • Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16:735–743

    Article  PubMed  Google Scholar 

  • Datla RSS, Hammerlindl JK, Panchuk B, Pelcher LE, Keller W (1992) Modified binary plant transformation vectors with the wild-type gene encoding NPTII. Gene 211:383–384

    Google Scholar 

  • Engeseth N, Stymne S (1996) Desaturation of oxygenated fatty acids in Lesquerella and other oil seeds. Planta 198:238–245

    CAS  Google Scholar 

  • Fehling E, Mukherjee KD (1991) Acyl-CoA elongase from a higher plant (Lunaria annua): metabolic intermediates of very long chain acyl-CoA products and substrate specificity. Biochim Biophys Acta 1082:239–246

    CAS  PubMed  Google Scholar 

  • Hayes DG, Kleiman R, Phillips BS (1995) The triglyceride composition, structure and presence of estolides in the oils of Lesquerella and related species. J Am Oil Chem Soc 72:559–569

    CAS  Google Scholar 

  • Katavic V, Haughn GW, Reed D, Martin M, Kunst L (1994) In planta transformation of Arabidopsis thaliana. Mol Gen Genet 245:363–370

    CAS  PubMed  Google Scholar 

  • Koncz C, Schell J (1986) The promoter of TL-DNA gene 5 controls the tissue specific expression of chimeric genes by a novel type of Agrobacterium binary vector. Mol Gen Genet 204:383–396

    CAS  Google Scholar 

  • Kunst L, Taylor DC, Underhill EW (1992) Fatty acid elongation in developing seeds of Arabidopsis thaliana. Plant Physiol Biochem 30:425–434

    CAS  Google Scholar 

  • Miquel M, Browse J (1992) Arabidopsis mutants deficient in polyunsaturated fatty acid synthesis. Biochemical and genetic characterization of a plant oleoyl-phosphatidylcholine desaturase. J Biol Chem 267:1502–1509

    CAS  PubMed  Google Scholar 

  • Moon H, Smith MA, Kunst L (2001) A condensing enzyme from the seeds of Lesquerella fendleri that specifically elongates hydroxy fatty acids. Plant Physiol 127:1635–1643

    Article  CAS  PubMed  Google Scholar 

  • Moreau RA, Stumpf PK (1981) Recent studies of the enzymatic synthesis of ricinoleic acid by developing castor bean. Plant Physiol 67:672–676

    CAS  Google Scholar 

  • Norton G, Harris, JF (1983) Triacylglycerols in oilseed rape during seed development. Phytochemistry 22:2703–2707

    Article  CAS  Google Scholar 

  • Reed DW, Taylor DC, Covello PS (1997) Metabolism of hydroxy fatty acids in developing seeds in the genera Lesquerella (Brassicaceae) and Linum (Linaceae). Plant Physiol 114:63–68

    CAS  PubMed  Google Scholar 

  • Reed DW, Schafer UA, Covello PS (2000) Characterization of the Brassica napus extraplastidial linoleate desaturase by expression in Saccharomyces cerevisiae. Plant Physiol 122:715–720

    CAS  PubMed  Google Scholar 

  • Shanklin J, Cahoon EB (1998) Desaturation and related modifications of fatty acids. Annu Rev Plant Physiol Plant Mol Biol 49:611–641

    Article  CAS  Google Scholar 

  • Shaul O, Galili G (1992) Increased lysine synthesis in tobacco plants that express high levels of bacterial dihydrodipicolinate synthase in their chloroplasts. Plant J 2:203–209

    CAS  Google Scholar 

  • Singh S, Thomaeus S, Lee M, Stymne S, Green A (2001) Transgenic expression of a Δ12-epoxygenase gene in Arabidopsis seeds inhibits accumulation of linoleic acid. Planta 212:872–879

    Article  CAS  PubMed  Google Scholar 

  • Smith MA, Jonsson L, Stymne S, Stobart K (1992) Evidence for cytochrome b5 as an electron donor in ricinoleic acid biosynthesis in microsomal preparations from developing castor bean (Ricinus communis L.). Biochem J 287:141–144

    CAS  PubMed  Google Scholar 

  • Smith MA, Stobart AK, Shewry PR, Napier JA (1994) Tobacco cytochrome b5: cDNA isolation, expression analysis and in vitro protein targeting. Plant Mol Biol 25:527–537

    CAS  PubMed  Google Scholar 

  • Stymne S, Griffiths G, Stobart K (1987) Desaturation of fatty acids on complex lipid substrates. In: Stumpf PK et al (eds) The metabolism, structure and function of plant lipids. Plenum, New York, pp 405–412

  • Thomaeus S, Carlsson AS, Stymne S (2001) Distribution of fatty acids in polar and neutral lipids during seed development in Arabidopsis thaliana genetically engineered to produce acetylenic, epoxy and hydroxy fatty acids. Plant Sci 161:997–1003

    Article  CAS  Google Scholar 

  • van de Loo FJ, Broun P, Turner S, Somerville C (1995). An oleate 12-hydroxylase from castor (Ricinus communis) is a fatty acyl desaturase homologue. Proc Natl Acad Sci USA 92:6743–6747

    PubMed  Google Scholar 

Download references

Acknowledgements

The authors acknowledge the financial support of Linnaeus Plant Sciences Inc. and The Science Council of British Columbia.

Author information

Authors and Affiliations

  1. Department of Botany, University of British Columbia, 6270 University Boulevard, Vancouver, British Columbia, V6T 1Z4, Canada

    Mark A. Smith, Gangamma Chowrira & Ljerka Kunst

  2. Department of Botany, Iowa State University, 353 Bessey Hall, Ames, IA 50011, USA

    Hangsik Moon

Authors
  1. Mark A. Smith
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Hangsik Moon
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Gangamma Chowrira
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ljerka Kunst
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Mark A. Smith.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Smith, M.A., Moon, H., Chowrira, G. et al. Heterologous expression of a fatty acid hydroxylase gene in developing seeds of Arabidopsis thaliana . Planta 217, 507–516 (2003). https://doi.org/10.1007/s00425-003-1015-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00425-003-1015-6

Keywords

Search

Navigation