Skip to main content
SpringerLink
Log in

Combined transgenic expression of Punica granatum conjugase (FADX) and FAD2 desaturase in high linoleic acid Arabidopsis thaliana mutant leads to increased accumulation of punicic acid

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

Abstract

Main conclusion

Arabidopsis was engineered to produce 21.2 % punicic acid in the seed oil. Possible molecular factors limiting further accumulation of the conjugated fatty acid were investigated.

Punicic acid (18:3Δ9cis,11trans,13cis) is a conjugated linolenic acid isomer and is a main component of Punica granatum (pomegranate) seed oil. Medical studies have shown that punicic acid is a nutraceutical with anti-cancer and anti-obesity properties. It has been previously demonstrated that the conjugated double bonds in punicic acid are produced via the catalytic action of fatty acid conjugase (FADX), which is a homolog of the oleate desaturase. This enzyme catalyzes the conversion of the Δ12-double bond of linoleic acid (18:2Δ9cis,12cis) into conjugated Δ11trans and Δ13cis-double bonds. Previous attempts to produce punicic acid in transgenic Arabidopsis thaliana seeds overexpressing P. granatum FADX resulted in a limited accumulation of punicic acid of up to 4.4 %, accompanied by increased accumulation of oleic acid (18:1∆9cis), suggesting that production of punicic acid in some way inhibits the activity of oleate desaturase (Iwabuchi et al. 2003). In the current study, we applied a new strategy to enhance the production of punicic acid in a high linoleic acid A. thaliana fad3/fae1 mutant background using the combined expression of P. granatum FADX and FAD2. This approach led to the accumulation of punicic acid at the level of 21 % of total fatty acids and restored the natural proportion of oleic acid observed in the A. thaliana fad3/fae1 mutant. In addition, we provide new insights into the high oleate phenotype and describe factors limiting the production of punicic acid in genetically engineered plants.

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. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

Abbreviations

CLNAs:

Conjugated linolenic acids

FAD2:

Δ12-oleic acid desaturase

FAD3:

Δ15-linoleic acid desaturase

FADX:

Fatty acid conjugase

ODP:

Oleic acid desaturation proportion

PC:

Phosphatidylcholine

3′ UTR:

Three prime untranslated region

TAG:

Triacylglycerol

References

  • Badami RC, Patil KB (1980) Structure and occurrence of unusual fatty acids in minor seed oils. Prog Lipid Res 19:119–153

    Article  CAS  PubMed  Google Scholar 

  • Baud S, Lepiniec L (2009) Regulation of de novo fatty acid synthesis in maturing oil seeds of Arabidopsis. Plant Physiol Biochem 47:448–455

    Article  CAS  PubMed  Google Scholar 

  • Bevan M (1983) Binary Agrobacterium vectors for plant transformation. Nucleic Acid Res 12:8711–8721

    Article  Google Scholar 

  • Bligh EG, Dyer WJ (1959) A rapid method for total lipid extraction and purification. Can J Biochem Physiol 37:911–917

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Burgal J, Shockey J, Lu C, Dyer J, Larson T, Graham I, Browse J (2008) Metabolic engineering of hydroxy fatty acid production in plants: RcDGAT2 drives dramatic increases in ricinoleate levels in seed oil. Plant Biotechnol J 6:819–831

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Cahoon E, Kinney A (2004) Dimorphecolic acid is synthesized by the coordinate activities of two divergent Δ12-oleic acid desaturases. J Biol Chem 279:12495–12502

    Article  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 Central  PubMed  Google Scholar 

  • Cahoon E, Ripp K, Hall S, Kinney A (2001) Formation of conjugated Δ8, Δ10-double bonds by Δ12-oleic-acid desaturase-related enzymes. Biosynthetic origin of calendic acid. J Biol Chem 276:2637–2643

    Article  CAS  PubMed  Google Scholar 

  • Cahoon EB, Dietrich CR, Meyer K, Damude HG, Dyer JM, Kinney AJ (2006) Conjugated fatty acids accumulate to high levels in phospholipids of metabolically engineered soybean and Arabidopsis seeds. Phytochemistry 67:1166–1176

    Article  CAS  PubMed  Google Scholar 

  • Carlsson AS, Yilmaz JL, Green AG, Stymne S, Hofvander P (2011) Replacing fossil oil with fresh oil—with what and for what? Eur J Lipid Sci Technol 113:812–831

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Chen X, Truksa M, Snyder CL, El-Mezawy A, Shah S, Weselake RJ (2011) Three homologous genes encoding sn-glycerol-3-phosphate acyltransferase 4 exhibit different expression patterns and functional divergence in Brassica napus. Plant Physiol 155:851–865

    Article  CAS  PubMed Central  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  CAS  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 122:383–384

    Article  CAS  PubMed  Google Scholar 

  • Hornung E, Pernstich C, Feussner I (2002) Formation of conjugated Δ11 Δ13-double bonds by Δ12-linoleic acid (1,4)-acyl-lipid-desaturase in pomegranate seeds. Eur J Biochem 269:4852–4859

    Article  CAS  PubMed  Google Scholar 

  • Igarashi M, Miyazawa T (2005) Preparation and fractionation of conjugated trienes from alpa-linolenic acid and their growth-inhibitory effects on human tumor cells and fibroblasts. Lipids 40:109–113

    Article  CAS  PubMed  Google Scholar 

  • Iwabuchi M, Kohno-Murase J, Imamura J (2003) Δ12-Oleate desaturase-related enzymes associated with formation of conjugated trans-Δ11, cis-Δ13 double bonds. J Biol Chem 278:4603–4610

    Article  CAS  PubMed  Google Scholar 

  • Josefsson LG, Lenman M, Ericson ML, Rask L (1987) Structure of a gene encoding the 1.7 S storage protein, napin, from Brassica napus. J Biol Chem 262:12196–12201

    CAS  PubMed  Google Scholar 

  • Kim ND, Mehta R, Yu W, Neeman I, Livney T, Amichay A, Poirier D, Nicholls P, Kirby A, Jiang W, Mansel R, Ramachandran C, Rabi T, Kaplan B, Lansky E (2002) Chemopreventive and adjuvant therapeutic potential of pomegranate (Punica granatum) for human breast cancer. Breast Cancer Res Treat 71:203–217

    Article  CAS  PubMed  Google Scholar 

  • Koba K, Imamura J, Akashoshi A, Kohno-Murase J, Nishizono S, Iwabuchi M, Tanaka K, Sugano M (2007) Genetically modified rapeseed oil containing cis-9, trans-11, cis-13-octadecatrienoic acid affects body fat mass and lipid metabolism in mice. J Agric Food Chem 55:3741–3748

    Article  CAS  PubMed  Google Scholar 

  • Kohno H, Suzuki R, Yasui Y, Hosokawa M, Miyashita K, Tanaka T (2004) Pomegranate seed oil rich in conjugated linolenic acid suppresses chemically induced colon carcinogenesis in rats. Cancer Sci 95:481–486

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  Google Scholar 

  • Lee KN, Pariza MW, Ntambi JM (1998) Conjugated linoleic acid decreases hepatic stearoyl-CoA desaturase mRNA expression. Biochem Biophys Res Commun 248:817–821

    Article  CAS  PubMed  Google Scholar 

  • Li R, Yu K, Hatanaka T, Hildebrand DF (2010) Vernonia DGATs increase accumulation of epoxy fatty acids in oil. Plant Biotechnol J 8:184–195

    Article  PubMed  Google Scholar 

  • Livak K, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2 (-Delta Delta C(T)) method. Methods 25:402–408

    Article  CAS  PubMed  Google Scholar 

  • Mietkiewska E, Brost JM, Giblin EM, Barton DL, Taylor DC (2007) Cloning and functional characterization of the Fatty Acid Elongase 1 (FAE1) gene from high erucic Crambe abyssinica cv. Prophet Plant Biotechnol J 5:636–645

    Article  CAS  Google Scholar 

  • Mietkiewska E, Siloto RM, Dewald J, Shah S, Brindley DN, Weselake RJ (2011) Lipins from plants are phosphatidate phosphatases that restore lipid synthesis in a pah1Δ mutant strain of Saccharomyces cerevisiae. FEBS J 278:764–775

    Article  CAS  PubMed  Google Scholar 

  • Mietkiewska E, Lin Y, Weselake RJ (2014) Engineering production of C18 conjugated fatty acids in developing seeds of oil crops. Biocatal Agric Biotechnol 3:44–48

    Google Scholar 

  • Mirmiran P, Fazeli MR, Asghari G, Shafiee A, Azizi F (2010) Effect of pomegranate seed oil on hyperlipidaemic subjects: a double-blind placebo-controlled clinical trial. Br J Nutr 104:402–406

    Article  CAS  PubMed  Google Scholar 

  • Napier JA (2007) The production of unusual fatty acids in transgenic plants. Annu Rev Plant Biol 58:295–319

    Article  CAS  PubMed  Google Scholar 

  • Rawat R, Yu X-H, Sweet M, Shanklin J (2012) Conjugated fatty acid synthesis: residues 111 and 115 influence product partitioning of Momordica charantia conjugase. J Biol Chem 287:16230–26237

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Shinohara N, Ito J, Tsuduki T, Honma T, Kijima R, Sugawara S, Arai T, Yamasaki M, Ikezaki A, Yokoyama M, Nishiyama K, Nakagawa K, Miyazawa T, Ikeda I (2012) Jacaric acid, a linolenic acid isomer with a conjugated triene system, reduces stearoyl-CoA desaturase expression in liver of mice. J Oleo Sci 61:433–441

    Article  CAS  PubMed  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 CR Jr (1971) Occurrence of unusual fatty acids in plants. Prog Chem Fats Other Lipids 11:137–177

    Article  Google Scholar 

  • Smith MA, Moon H, Chowrira G, Kunst L (2003) Heterologous expression of a fatty acid hydroxylase gene in developing seeds of Arabidopsis thaliana. Planta 217:507–516

    Article  CAS  PubMed  Google Scholar 

  • Sonntag NOV (1979) Composition and characteristics of individual fats and oils. In: Swern D (ed) Bailey’s industrial oil and fat products. Wiley, New York, pp 289–477

    Google Scholar 

  • Stoutjesdijk PA, Singh SP, Liu Q, Hurlstone CJ, Waterhouse PA, Green AG (2002) hpRNA-mediated targeting of the Arabidopsis FAD2 gene gives highly efficient and stable silencing. Plant Physiol 129:1723–1731

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Thomaeus S, Carlsson AS, Lee M, 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 

  • Vanhercke T, Wood CC, Stymne S, Singh SP, Green AG (2013) Metabolic engineering of plant oils and waxes for use as industrial feedstocks. Plant Biotechnol J 11:197–210

    Article  CAS  PubMed  Google Scholar 

  • Vroegrijk IO, van Diepen JA, van den Berg S, Westbroek I, Keizer H, Gambelli L, Hontecillas R, Bassaganya-Riera J, Zondag GC, Romijn JA, Havekes LM, Voshol PJ (2011) Pomegranate seed oil, a rich source of punicic acid, prevents diet-induced obesity and insulin resistance in mice. Food Chem Toxicol 49:1426–1430

    Article  CAS  PubMed  Google Scholar 

  • Zhou X-R, Singh S, Liu Q, Green A (2006) Combined transgenic expression of Δ12-desaturase and Δ12-epoxygenase in high linoleic acid seeds leads to increased accumulation of vernolic acid. Funct Plant Biol 333:585–592

    Article  Google Scholar 

Download references

Acknowledgments

We thank Drs. Ljerka Kunst (University of British Columbia, Vancouver, Canada) and Mark Smith (National Research Council of Canada) for providing seeds of A. thaliana fad3/fae1 mutant. We would like to thank Dr. Joseph Boothe for his critical assessment of the manuscript. We are grateful for the support provided by Alberta Innovates Bio Solutions, Alberta Enterprise and Advanced Education, the Canada Foundation for Innovation and the Canada Research Chairs Program.

Author information

Authors and Affiliations

  1. Alberta Innovates Phytola Centre, Department of Agricultural, Food and Nutritional Science, University of Alberta, 4-10 Agriculture/Forestry Centre, Edmonton, AB, T6G 2P5, Canada

    Elzbieta Mietkiewska, Robin Miles, Aruna Wickramarathna, Ariff Firman Sahibollah, Michael S. Greer, Guanqun Chen & Randall J. Weselake

Authors
  1. Elzbieta Mietkiewska
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Robin Miles
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Aruna Wickramarathna
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ariff Firman Sahibollah
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Michael S. Greer
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Guanqun Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Randall J. Weselake
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Randall J. Weselake.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Mietkiewska, E., Miles, R., Wickramarathna, A. et al. Combined transgenic expression of Punica granatum conjugase (FADX) and FAD2 desaturase in high linoleic acid Arabidopsis thaliana mutant leads to increased accumulation of punicic acid. Planta 240, 575–583 (2014). https://doi.org/10.1007/s00425-014-2109-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00425-014-2109-z

Keywords

Search

Navigation