Skip to main content
SpringerLink
Log in

The linin promoter is highly effective in enhancing punicic acid production in Arabidopsis

  • Original Article
  • Published:
Plant Cell Reports Aims and scope Submit manuscript

Abstract

Key message

Enhanced levels of punicic acid were produced in the seed oil of Arabidopsis over-expressing pomegranate FATTY ACID CONJUGASE driven by heterologous promoters, among which the linin promoter was the most efficient.

Abstract

Fatty acids with conjugated double bonds play a special role in determining both the nutritional and industrial uses of plant oils. Punicic acid (18:3Δ9cis,11trans,13cis), a conjugated fatty acid naturally enriched in the pomegranate (Punica granatum) seeds, has gained increasing attention from the biotechnology community toward its production in metabolically engineered oilseed crops because of its significant health benefits. The present study focused on selecting the best heterologous promoter to drive the expression of the P. granatum FATTY ACID CONJUGASE (PgFADX) cDNA as a means of producing punicic acid in Arabidopsis seed oil. Among the four promoters of genes encoding seed storage proteins from different crop species, the linin promoter led to the highest accumulation of punicic acid (13.2% of total fatty acids in the best homozygous line). Analysis of the relative expression level of PgFADX in developing seeds further confirmed that the linin promoter was most efficient in Arabidopsis. In addition, a conserved profile of cis-regulatory elements were identified in four heterologous promoters by bioinformatic analysis, and their possible roles in regulating gene expression during plant development were also discussed based on the results of this study in combination with the literature. This study contributes to metabolic engineering strategies aimed at enhancing the production of bioactive fatty acids in oilseed crops.

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

ANOVA:

One-way analysis of variance

CLNA:

Conjugated linolenic acid

EV:

Empty vector

FA:

Fatty acid

FAD:

Fatty acid desaturase

FAE:

Fatty acid elongase

FAMEs:

Fatty acid methyl esters

HSD:

Honestly significant difference

ORF:

Open reading frame

PgFADX:

Punica granatum fatty acid conjugase

References

  • Aruna P, Venkataramanamma D, Singh AK, Singh RP (2016) Health benefits of punicic acid: a review. Compr Rev Food Sci Food Saf 15:16–27

    Article  CAS  Google Scholar 

  • Banihani S, Swedan S, Alguraan Z (2013) Pomegranate and type 2 diabetes. Nutr Res 33:341–348

    Article  CAS  PubMed  Google Scholar 

  • Boothe J, Nykiforuk C, Shen Y, Zaplachinski S, Szarka S, Kuhlman P, Murray E, Morck D, Moloney MM (2010) Seed-based expression systems for plant molecular farming. Plant Biotechnol J 8:588–606

    Article  CAS  PubMed  Google Scholar 

  • Bustos MM, Guiltinan MJ, Jordano J, Begum D, Kalkan FA, Hall TC (1989) Regulation of beta-glucuronidase expression in transgenic tobacco plants by an A/T-rich, cis-acting sequence found upstream of a French bean beta-phaseolin gene. Plant Cell 1:839–853

    CAS  PubMed  PubMed Central  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  PubMed Central  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 

  • Chandrasekharan MB, Bishop KJ, Hall TC (2003) Module-specific regulation of the beta-phaseolin promoter during embryogenesis. Plant J 33:853–866

    Article  CAS  PubMed  Google Scholar 

  • Chaudhary S, van Rooijen G, Moloney MM, Singh S (2010) Flax seed specific promoters. Patent US 7642346 B2

  • Chen G, Woodfield HK, Pan X, Harwood JL, Weselake RJ (2015) Acyl-trafficking during plant oil accumulation. Lipids 50:1057–1068

    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  CAS  PubMed  Google Scholar 

  • Edwards K, Johnstone C, Thompson C (1991) A simple and rapid method for the preparation of plant genomic DNA for PCR analysis. Nucleic Acids Res 19:1349

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ellerstrom M, Stalberg K, Ezcurra I, Rask L (1996) Functional dissection of a napin gene promoter: identification of promoter elements required for embryo and endosperm-specific transcription. Plant Mol Biol 32:1019–1027

    Article  CAS  PubMed  Google Scholar 

  • Ezcurra I, Ellerström M, Wycliffe P, Stålberg K, Rask L (1999) Interaction between composite elements in the napA promoter: both the B-box ABA-responsive complex and the RY/G complex are necessary for seed-specific expression. Plant Mol Biol 40:699–709

    Article  CAS  PubMed  Google Scholar 

  • Gasmi J, Sanderson JT (2010) Growth inhibitory, antiandrogenic, and pro-apoptotic effects of punicic acid in LNCaP human prostate cancer cells. J Agric Food Chem 58:12149–12156

    Article  CAS  PubMed  Google Scholar 

  • Goderis IJ, De Bolle MF, Francois IE, Wouters PF, Broekaert WF, Cammue BP (2002) A set of modular plant transformation vectors allowing flexible insertion of up to six expression units. Plant Mol Biol 50:17–27

    Article  CAS  PubMed  Google Scholar 

  • Grossmann ME, Mizuno NK, Schuster T, Cleary MP (2010) Punicic acid is an omega-5 fatty acid capable of inhibiting breast cancer proliferation. Int J Oncol 36:421–426

    CAS  PubMed  Google Scholar 

  • Hernandez-Garcia CM, Finer JJ (2014) Identification and validation of promoters and cis-acting regulatory elements. Plant Sci 217–218:109–119

    Article  PubMed  Google Scholar 

  • Hontecillas R, O’Shea M, Einerhand A, Diguardo M, Bassaganya-Riera J (2009) Activation of PPAR gamma and alpha by punicic acid ameliorates glucose tolerance and suppresses obesity-related inflammation. J Am Coll Nutr 28:184–195

    Article  CAS  PubMed  Google Scholar 

  • Iwabuchi M, Kohno-Murase J, Imamura J (2003) Delta 12-oleate desaturase-related enzymes associated with formation of conjugated trans-delta 11, cis-delta 13 double bonds. J Biol Chem 278:4603–4610

    Article  CAS  PubMed  Google Scholar 

  • Jaworski J, Cahoon EB (2003) Industrial oils from transgenic plants. Curr Opin Plant Biol 6:178–184

    Article  CAS  PubMed  Google Scholar 

  • Johanningsmeier SD, Harris GK (2011) Pomegranate as a functional food and nutraceutical source. Annu Rev Food Sci Technol 2:181–201

    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 carried by a novel type of Agrobacterium binary vector. Mol Gen Genet 204:383–396

    Article  CAS  Google Scholar 

  • Lescot M, Dehais P, Thijs G, Marchal K, Moreau Y, Van de Peer Y, Rouze P, Rombauts S (2002) PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucleic Acids Res 30:325–327

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Google Scholar 

  • Mietkiewska E, Miles R, Wickramarathna A, Sahibollah AF, Greer MS, Chen G, Weselake RJ (2014b) 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

    Article  CAS  PubMed  Google Scholar 

  • Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473–497

    Article  CAS  Google Scholar 

  • Napier JA, Haslam RP, Beaudoin F, Cahoon EB (2014) Understanding and manipulating plant lipid composition: metabolic engineering leads the way. Curr Opin Plant Biol 19:68–75

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Qiu X, Truksa M, Hu Z (2008) Flax (Linum usitatissimum L.) seed-specific promoters. Patent EP 1925671 A1

  • Rogalski M, Carrer H (2011) Engineering plastid fatty acid biosynthesis to improve food quality and biofuel production in higher plants. Plant Biotechnol J 9:554–564

    Article  CAS  PubMed  Google Scholar 

  • Rossak M, Smith M, Kunst L (2001) Expression of the FAE1 gene and FAE1 promoter activity in developing seeds of Arabidopsis thaliana. Plant Mol Biol 46:717–725

    Article  CAS  PubMed  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 

  • Spilmont M, Leotoing L, Davicco MJ, Lebecque P, Mercier S, Miot-Noirault E, Pilet P, Rios L, Wittrant Y, Coxam V (2013) Pomegranate seed oil prevents bone loss in a mice model of osteoporosis, through osteoblastic stimulation, osteoclastic inhibition and decreased inflammatory status. J Nutr Biochem 24:1840–1848

    Article  CAS  PubMed  Google Scholar 

  • Truksa M, MacKenzie Samuel L, Qiu X (2003) Molecular analysis of flax 2S storage protein conlinin and seed specific activity of its promoter. Plant Physiol Biochem 41:141–147

    Article  CAS  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 

  • Wang L, Martins-Green M (2014) Pomegranate and its components as alternative treatment for prostate cancer. Int J Mol Sci 15:14949–14966

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Wang L, Li W, Lin M, Garcia M, Mulholland D, Lilly M, Martins-Green M (2014) Luteolin, ellagic acid and punicic acid are natural products that inhibit prostate cancer metastasis. Carcinogenesis 35:2321–2330

    Article  CAS  PubMed  Google Scholar 

  • Yuan GF, Chen XE, Li D (2014) Conjugated linolenic acids and their bioactivities: a review. Food Funct 5:1360–1368

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank Dr. Xiao Qiu (University of Saskatchewan, Saskatoon, Canada) for kindly providing the plasmid carrying the conlinin promoter used as a PCR template. Financial support provided by Alberta Innovates Bio Solutions and the Natural Sciences and Engineering Research Council (NSERC) of Canada is gratefully acknowledged.

Author information

Authors and Affiliations

  1. Agricultural Lipid Biotechnology Program, Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, AB, T6G 2P5, Canada

    Ziliang Song, Elzbieta Mietkiewska & Randall J. Weselake

Authors
  1. Ziliang Song
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Elzbieta Mietkiewska
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Randall J. Weselake
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Randall J. Weselake.

Additional information

Communicated by M. C. Jordan.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOCX 25 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Song, Z., Mietkiewska, E. & Weselake, R.J. The linin promoter is highly effective in enhancing punicic acid production in Arabidopsis. Plant Cell Rep 36, 447–457 (2017). https://doi.org/10.1007/s00299-016-2094-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00299-016-2094-8

Keywords

Search

Navigation