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Functional Characterization of Flax Fatty Acid Desaturase FAD2 and FAD3 Isoforms Expressed in Yeast Reveals a Broad Diversity in Activity

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With 45 % or more oil content that contains more than 55 % alpha linolenic (LIN) acid, linseed (Linum usitatissimum L.) is one of the richest plant sources of this essential fatty acid. Fatty acid desaturases 2 (FAD2) and 3 (FAD3) are the main enzymes responsible for the Δ12 and Δ15 desaturation in planta. In linseed, the oilseed morphotype of flax, two paralogous copies, and several alleles exist for each gene. Here, we cloned three alleles of FAD2A, four of FAD2B, six of FAD3A, and seven of FAD3B into a pYES vector and transformed all 20 constructs and an empty construct in yeast. The transformants were induced in the presence of oleic (OLE) acid substrate for FAD2 constructs and linoleic (LIO) acid for FAD3. Conversion rates of OLE acid into LIO acid and LIO acid into LIN acid were measured by gas chromatography. Conversion rate of FAD2 exceeded that of FAD3 enzymes with FAD2B having a conversion rate approximately 10 % higher than FAD2A. All FAD2 isoforms were active, but significant differences existed between isoforms of both FAD2 enzymes. Two FAD3A and three FAD3B isoforms were not functional. Some nonfunctional enzymes resulted from the presence of nonsense mutations causing premature stop codons, but FAD3B-C and FAD3B-F seem to be associated with single amino acid changes. The activity of FAD3A-C was more than fivefold greater than the most common isoform FAD3A-A, while FAD3A-F was fourfold greater. Such isoforms could be incorporated into breeding lines to possibly further increase the proportion of LIN acid in linseed.

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References

  1. Warude, D., Joshi, K., & Harsulkar, A. (2006). Polyunsaturated fatty acids: Biotechnology. Critical Review of Biotechnology, 26, 83–93.

    Article  CAS  Google Scholar 

  2. Damude, H. G., & Kinney, A. J. (2008). Engineering oilseeds to produce nutritional fatty acids. Physiologia Plantarum, 132, 1–10.

    CAS  Google Scholar 

  3. Simopoulos, A. P. (1999). Essential fatty acids in health and chronic diseases. American Journal of Clinical Nutrition, 70, 560s–569s.

    CAS  Google Scholar 

  4. Simopoulos, A. P. (2002). Genetic variation and dietary response: Nutrigenetics/nutrigenomics. Asia Pacific Journal of Clinical Nutrition, 11, S117–S128.

    Article  CAS  Google Scholar 

  5. Dyer, J. M., Stymne, S., Green, A. G., & Carlsson, A. S. (2008). High-value oils from plants. Plant Journal, 54, 640–656.

    Article  CAS  Google Scholar 

  6. Lopez Alonso, D., & Garcia Maroto, F. (2000). Plants as ‘chemical factories’ for the production of polyunsaturated fatty acids. Biotechnology Advances, 18, 481–497.

    Article  Google Scholar 

  7. Simopoulos, A. P. (2008). The omega-6/omega-3 fatty acid ratio, genetic variation, and cardiovascular disease. Asia Pacific Journal of Clinical Nutrition, 7, 131–134.

    Google Scholar 

  8. Fofana, B., Ragupathy, R., & Cloutier, S. (2010). Flax Lipids: Classes, biosynthesis, genetics and the promise of applied genomics for understanding and altering of fatty acids (pp. 4–15). New York: Nova Science Publishers Inc.

    Google Scholar 

  9. Ayerza (h), R. (1995). Oil content and fatty acid composition of chia (Salvia hispanica L.) from five northwestern locations in Argentina. Journal of the American Oil Chemists’ Society, 72, 1079–1081.

    Article  Google Scholar 

  10. Budin, J. T., Breene, W. M., & Putnam, D. H. (1995). Some compositional properties of Camelina (Camelina sativa L. Crantz) seeds and oils. Journal of the American Oil Chemists’ Society, 72, 309–315.

    Article  CAS  Google Scholar 

  11. Dubois, V., Breton, S., Linder, M., Fanni, J., & Parmentier, M. (2007). Fatty acid profiles of 80 vegetable oils with regard to their nutritional potential. European Journal of Lipid Science and Technology, 109, 710–732.

    Article  CAS  Google Scholar 

  12. Silvia Taga, M., Miller, E. E., & Pratt, D. E. (1984). Chia seeds as a source of natural lipid antioxidants. Journal of the American Oil Chemists’ Society, 61, 928–931.

    Article  Google Scholar 

  13. Earle, F. R., & Jones, Q. (1962). Analyses of seed samples from 113 plant families. Economic Botany, 16, 221–250.

    Article  CAS  Google Scholar 

  14. Duguid, S. D., Kenaschuk, E. O., & Rashid, K. Y. (2004). Prairie blue flax. Canadian Journal of Plant Science, 84, 801–803.

    Article  Google Scholar 

  15. Duguid, S. D., Kenaschuk, E. O., & Rashid, K. Y. (2004). Macbeth flax. Canadian Journal of Plant Science, 84, 803–805.

    Article  Google Scholar 

  16. Diederichsen, A., & Raney, J. P. (2006). Seed colour, seed weight and seed oil content in Linum usitatissimum accessions held by plant gene resources of Canada. Plant Breeding, 125, 372–377.

    Article  Google Scholar 

  17. Rowland, G. G., Hormis, Y. A., & Rashid, K. Y. (2002). CDC bethune flax. Canadian Journal of Plant Science, 82, 101–102.

    Article  Google Scholar 

  18. Ohlrogge, J., & Browse, J. (1995). Lipid biosynthesis. Plant Cell, 7, 957–970.

    Article  CAS  Google Scholar 

  19. Baud, S., & Lepiniec, L. (2010). Physiological and developmental regulation of seed oil production. Progress in Lipid Research, 49, 235–249.

    Article  CAS  Google Scholar 

  20. McConn, M., & Browse, J. (1998). Polyunsaturated membranes are required for photosynthetic competence in a mutant of Arabidopsis. Plant Journal, 15, 521–530.

    Article  CAS  Google Scholar 

  21. Weber, H. (2002). Fatty acid-derived signals in plants. Trends in Plant Science, 7, 217–224.

    Article  CAS  Google Scholar 

  22. Khadake, R., Khonde, V., Mhaske, V., Ranjekar, P., & Harsulkar, A. (2011). Functional and bioinformatic characterisation of sequence variants of FAD3 gene from flax. Journal of Science of Food and Agriculture, 91, 2689–2696.

    Article  CAS  Google Scholar 

  23. Arondel, V., Lemieux, B., Hwang, I., Gibson, S., Goodman, H. M., & Somerville, C. R. (1992). Map-based cloning of a gene controlling omega-3 fatty acid desaturation in Arabidopsis. Science, 20, 1353–1355.

    Article  Google Scholar 

  24. Yadav, N. S., Wierzbicki, A., Aererter, M., Caster, C. S., Perez-Grau, L., Kinney, A. J., et al. (1993). Cloning of higher plant omega-3 fatty acid desaturases. Plant Physiology, 103, 467–476.

    Article  CAS  Google Scholar 

  25. Bilyeu, K., Palavalli, L., Sleper, D. A., & Beuselincka, P. R. (2003). Three microsomal omega-3 fatty-acid desaturase genes contribute to soybean linolenic acid levels. Crop Science, 43, 1833–1838.

    Article  CAS  Google Scholar 

  26. Heppard, E. P., Kinney, A. J., Stecca, K. L., & Miao, G. H. (1996). Developmental and growth temperature regulation of two different microsomal omega-6 desaturase genes in soybeans. Plant Physiology, 110, 311–319.

    Article  CAS  Google Scholar 

  27. Reed, D. W., Schafer, U. A., & Covello, P. S. (2000). Characterization of the Brassica napus extraplastidial linoleate desaturase by expression in Saccharomyces cerevisiae. Plant Physiology, 122, 715–720.

    Article  CAS  Google Scholar 

  28. Hernandez, M. L., Mancha, M., & Martinez-Rivas, J. M. (2005). Molecular cloning and characterization of genes encoding two microsomal oleate desaturases (FAD2) from olive. Phytochemistry, 66, 1417–1426.

    Article  CAS  Google Scholar 

  29. Fofana, B., Duguid, S., & Cloutier, S. (2004). Cloning of fatty acid biosynthetic genes B-ketoacyl CoA synthase, fatty acid elongase, stearoyl-ACP desaturase, and fatty acid desaturase and analysis of expression in the early developmental stages of flax (Linum usitatissimum L.) seeds. Plant Science, 166, 1487–1496.

    Article  CAS  Google Scholar 

  30. Khadake, R. M., Ranjekar, P. K., & Harsulkar, A. M. (2009). Cloning of a novel omega-6 desaturase from flax (Linum usitatissimum L.) and its functional analysis in Saccharomyces cerevisiae. Molecular Biotechnology, 42, 168–174.

    Article  CAS  Google Scholar 

  31. Krasowska, A., Dziakowiec, D., Polinceusz, A., Plonka, A., & Lukaszewicz, M. (2007). Cloning of flax oleic fatty acid desaturase and its expression in yeast. Journal of the American Oil Chemists’ Society, 84, 809–816.

    Article  CAS  Google Scholar 

  32. Vrinten, P., Hu, Z., Munchinsky, M. A., Rowland, G., & Qiu, X. (2005). Two FAD3 desaturase genes control the level of linolenic acid in flax seed. Plant Physiology, 139, 79–87.

    Article  CAS  Google Scholar 

  33. Banik, M., Duguid, S., & Cloutier, S. (2011). Transcript profiling and gene characterization of three fatty acid desaturase genes in high, moderate, and low linolenic acid genotypes of flax (Linum usitatissimum L.) and their role in linolenic acid accumulation. Genome, 54, 471–483.

    Article  CAS  Google Scholar 

  34. Avelange-Macherel, M. H., Macherel, D., Wada, H., & Murata, N. (1995). Site-directed mutagenesis of histidine residues in the Δ12 acyl-lipid desaturase of Synechocystis. FEBS Letters, 361, 111–114.

    Article  CAS  Google Scholar 

  35. Thambugala, D., Duguid, S., Loewen, E., Rowland, G., Booker, H., You, F. M., et al. (2013). Genetic variation of six desaturase genes in flax and their impact on fatty acid composition. Theoretical and Applied Genetics, 126, 2627–2641.

    Article  CAS  Google Scholar 

  36. Huang, X. Q., & Cloutier, S. (2007). Hemi-nested touchdown PCR combined with primer-template mismatch PCR for rapid isolation and sequencing of gene families from BAC libraries. BMC Genetics, 8, 18.

    Article  CAS  Google Scholar 

  37. Cloutier, S., Ragupathy, R., Niu, Z., & Duguid, S. (2010). SSR-based linkage map of flax (Linum usitatissimum L.) and mapping of QTLs underlying fatty acid composition traits. Molecular Breeding, 28, 437–451.

    Article  Google Scholar 

  38. Green, A. G. (1986). Genetic control of polyunsaturated fatty acid biosynthesis in flax (Linum usitatissimum) seed oil. Theoretical and Applied Genetics, 72, 654–661.

    Article  CAS  Google Scholar 

  39. Li, L. Y., Wang, X. L., Gai, J. Y., & Yu, D. Y. (2007). Molecular cloning and characterization of a novel microsomal oleate desaturase gene from soybean. Journal of Plant Physiology, 164, 1516–1526.

    Article  CAS  Google Scholar 

  40. Mikkilineni, V., & Rocheford, T. R. (2003). Sequence variation and genomic organization of fatty acid desaturase-2 (fad2) and fatty acid desaturase-6 (fad6) cDNAs in maize. Theoretical and Applied Genetics, 106, 1326–1332.

    CAS  Google Scholar 

  41. Los, D. A., & Murata, N. (1998). Structure and expression of fatty acid desaturases. Biochimica et Biophysica Acta, 1394, 3–15.

    Article  CAS  Google Scholar 

  42. Shanklin, J., & Cahoon, E. B. (1998). Desaturation and related modifications of fatty acids. Annual Review of Plant Physiology and Plant Molecular Biology, 49, 611–641.

    Article  CAS  Google Scholar 

  43. Luo, T., Deng, W. Y., Zeng, J., & Zhang, F. L. (2009). Cloning and characterization of a stearoyl-acyl carrier protein desaturase gene from Cinnamomum longepaniculatum. Plant Molecular Biology Reporter, 27, 13–19.

    Article  CAS  Google Scholar 

  44. Shanklin, J., Whittle, E., & Fox, B. G. (1994). Eight histidine residues are catalytically essential in a membrane associate diron enzyme, stearoyl-CoA desaturase, and are conserved in alkane hydroxylase and xylene monooxygenase. Biochemistry, 33, 12787–12794.

    Article  CAS  Google Scholar 

  45. Shilman, F., Brand, Y., Brand, A., Hedvat, I., & Hovav, R. (2011). Identification and molecular characterization of homologous Δ9-stearoyl acyl carrier protein desaturase 3 genes from the allotetraploid peanut (Arachis hypogaea). Plant Molecular Biology Reporter, 29, 232–241.

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  47. Scheffler, J. A., Sharpe, A. G., Schimdt, H., Sperling, P., Parkin, I. A. P., Luhs, W., et al. (1997). Desaturase multigene families of Brassica napus arose through genome duplication. Theoretical and Applied Genetics, 94, 583–591.

    Article  CAS  Google Scholar 

  48. Pirtle, I. L., Wisatre, K., Mongkol, N., John, E. K., Kent, D. C., & Robert, M. P. (2001). Molecular cloning and functional expression of the gene for a cotton Delta-12 fatty acid desaturase (FAD2). Biochimica et Biophysica Acta, 1522, 122–129.

    Article  CAS  Google Scholar 

  49. McCartney, A. W., Dyer, J. M., Dhanoa, P. K., Kim, P. K., Andrews, D. W., McNew, J. A., et al. (2004). Membrane-bound fatty acid desaturases are inserted co-translationally into the ER and contain different ER retrieval motifs at their carboxy termini. Plant Journal, 37, 156–173.

    Article  CAS  Google Scholar 

  50. Covello, P. S., & Reed, D. W. (1996). Functional expression of the extraplastidial Arabidopsis thaliana oleate desaturase gene (FAD2) in Saccharomyces cerevisiae. Plant Physiology, 111, 223–226.

    Article  CAS  Google Scholar 

  51. Fofana, B., Cloutier, S., Duguid, S., Ching, J., & Rampitsch, C. (2006). Gene expression of stearoyl-ACP desaturase and Δ12 fatty acid desaturase 2 is modulated during seed development of flax (Linum usitatissimum). Lipids, 41, 705–712.

    Article  CAS  Google Scholar 

  52. Pham, A.-T., Lee, J.-D., Shannon, J. G., & Bilyeu, K. D. (2011). A novel FAD2-1A allele in a soybean plant introduction offers an alternate means to produce soybean seed oil with 85% oleic acid content. Theoretical and Applied Genetics, 123, 793–802.

    Article  CAS  Google Scholar 

  53. Wang, M. L., Barkley, N. A., Chen, Z., & Pittman, R. N. (2011). FAD2 gene mutations significantly alter fatty acid profiles in cultivated peanuts (Arachis hypogaea). Biochemistry Genetics, 49, 748–759.

    Article  CAS  Google Scholar 

  54. Chapman, M. A., & Burke, J. M. (2012). Evidence of selection on fatty acid biosynthetic genes during the evolution of cultivated sunflower. Theoretical and Applied Genetics, 125, 897–907.

    Article  CAS  Google Scholar 

  55. Tang, G. Q., Novitzky, W. P., Griffin, H. C., Huber, S. C., & Dewey, R. E. (2005). Oleate desaturase enzymes of soybean: evidence of regulation through differential stability and phosphorylation. Plant Journal, 44, 433–446.

    Article  CAS  Google Scholar 

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Acknowledgments

This work was conducted as part of the Total Utilization Flax Genomics (TUFGEN) project funded by Genome Canada and co-funded by the Government of Manitoba, the Flax Council of Canada, the Saskatchewan Flax Development Commission, Agricultural Development Fund and the Manitoba Flax Growers Association. Project management and support by Genome Prairie are also gratefully acknowledged.

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Authors and Affiliations

  1. Cereal Research Centre, Agriculture and Agri-Food Canada, 195 Dafoe Road, Winnipeg, MB, R3T 2M9, Canada

    Natasa Radovanovic & Sylvie Cloutier

  2. Department of Plant Science, University of Manitoba, 66 Dafoe Road, Winnipeg, MB, R3T 2N2, Canada

    Dinushika Thambugala & Sylvie Cloutier

  3. Morden Research Station, Agriculture and Agri-Food Canada, 101 Route 100, Unit 100, Morden, MB, R6M 1Y5, Canada

    Scott Duguid & Evelyn Loewen

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  1. Natasa Radovanovic
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  2. Dinushika Thambugala
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  4. Evelyn Loewen
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Correspondence to Sylvie Cloutier.

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12033_2014_9737_MOESM1_ESM.docx

Supplementary Table S1 List of isoforms for each of the six desaturases associated with the accessions, molecule type, tissue, and developmental stage used for cloning (DOCX 15 kb)

12033_2014_9737_MOESM2_ESM.docx

Supplementary Table S2 List of primers used for PCR amplification and sequencing of cDNA and genomic DNA of the six fatty acid desaturase genes (DOCX 14 kb)

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Radovanovic, N., Thambugala, D., Duguid, S. et al. Functional Characterization of Flax Fatty Acid Desaturase FAD2 and FAD3 Isoforms Expressed in Yeast Reveals a Broad Diversity in Activity. Mol Biotechnol 56, 609–620 (2014). https://doi.org/10.1007/s12033-014-9737-1

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