Specialized lysophosphatidic acid acyltransferases contribute to unusual fatty acid accumulation in exotic Euphorbiaceae seed oils

Abstract

Main conclusion

In vivo and in vitro analyses of Euphorbiaceae species’ triacylglycerol assembly enzymes substrate selectivity are consistent with the co-evolution of seed-specific unusual fatty acid production and suggest that many of these genes will be useful for biotechnological production of designer oils.

Many exotic Euphorbiaceae species, including tung tree (Vernicia fordii), castor bean (Ricinus communis), Bernardia pulchella, and Euphorbia lagascae, accumulate unusual fatty acids in their seed oils, many of which have valuable properties for the chemical industry. However, various adverse plant characteristics including low seed yields, production of toxic compounds, limited growth range, and poor resistance to abiotic stresses have limited full agronomic exploitation of these plants. Biotechnological production of these unusual fatty acids (UFA) in high yielding non-food oil crops would provide new robust sources for these valuable bio-chemicals. Previous research has shown that expression of the primary UFA biosynthetic gene alone is not enough for high-level accumulation in transgenic seed oils; other genes must be included to drive selective UFA incorporation into oils. Here, we use a series of in planta molecular genetic studies and in vitro biochemical measurements to demonstrate that lysophosphatidic acid acyltransferases from two Euphorbiaceae species have high selectivity for incorporation of their respective unusual fatty acids into the phosphatidic acid intermediate of oil biosynthesis. These results are consistent with the hypothesis that unusual fatty acid accumulation arose in part via co-evolution of multiple oil biosynthesis and assembly enzymes that cooperate to enhance selective fatty acid incorporation into seed oils over that of the common fatty acids found in membrane lipids.

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Abbreviations

DGAT:

Diacylglycerol acyltransferase

ESA:

Eleostearic acid

FADX:

Tung tree fatty acid conjugase X

FAH:

Castor fatty acid hydroxylase

FAME:

Fatty acid methyl ester

FID:

Flame ionization detection

GPAT:

Glycerol-3-phosphate acyltransferase

HFA:

Hydroxy fatty acids

HPLC:

High-performance liquid chromatography

GC:

Gas chromatography

LPA:

Lysophosphatidic acid

LPAT:

Lysophosphatidic acid acyltransferase

PCR:

Polymerase chain reaction

PDAT:

Phospholipid: diacylglycerol acyltransferase

PDCT:

Phosphatidylcholine:diacylglycerol cholinephosphotransferase

TLC:

Thin layer chromatography

UFA:

Unusual fatty acid

References

  1. Adhikari N, Bates PD, Browse J (2016) WRINKLED1 rescues feedback inhibition of fatty acid synthesis in hydroxylase-expressing seeds of Arabidopsis. Plant Physiol 171:179–191

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Arroyo-Caro JM, Chileh T, Kazachkov M, Zou J, Alonso DL, García-Maroto F (2013) The multigene family of lysophosphatidate acyltransferase (LPAT)-related enzymes in Ricinus communis: cloning and molecular characterization of two LPAT genes that are expressed in castor seeds. Plant Sci 199–200:29–40

    PubMed  Google Scholar 

  3. 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  PubMed Central  Google Scholar 

  4. Bansal S, Kim HJ, Na G, Hamilton ME, Cahoon EB, Lu C, Durrett TP (2018) Towards the synthetic design of camelina oil enriched in tailored acetyl-triacylglycerols with medium-chain fatty acids. J Exp Bot 69:4395–4402

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Bates PD (2016) Understanding the control of acyl flux through the lipid metabolic network of plant oil biosynthesis. Biochim Biophys Acta Mol Cell Biol Lipids 1861:1214–1225

    CAS  Google Scholar 

  6. Bates PD, Browse J (2011) The pathway of triacylglycerol synthesis through phosphatidylcholine in Arabidopsis produces a bottleneck for the accumulation of unusual fatty acids in transgenic seeds. Plant J 68:387–399

    CAS  PubMed  Google Scholar 

  7. Bates PD, Browse J (2012) The significance of different diacylgycerol synthesis pathways on plant oil composition and bioengineering. Front Plant Sci 3:147

    PubMed  PubMed Central  Google Scholar 

  8. Bates PD, Fatihi A, Snapp AR, Carlsson AS, Browse J, Lu C (2012) Acyl editing and headgroup exchange are the major mechanisms that direct polyunsaturated fatty acid flux into triacylglycerols. Plant Physiol 160:1530–1539

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Bates PD, Johnson SR, Cao X, Li J, Nam J-W, Jaworski JG, Ohlrogge JB, Browse J (2014) Fatty acid synthesis is inhibited by inefficient utilization of unusual fatty acids for glycerolipid assembly. Proc Nat Acad Sci USA 111:1204–1209

    CAS  PubMed  Google Scholar 

  10. Bourgis F, Kader J-C, Barret P, Renard M, Robinson D, Robinson C, Delseny M, Roscoe TJ (1999) A plastidial lysophosphatidic acid acyltransferase from oilseed rape. Plant Physiol 120:913–921

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Burgal J, Shockey J, Lu CF, 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 Biotech J 6:819–831

    CAS  Google Scholar 

  12. Caldo KMP, Shen W, Xu Y, Hanley-Bowdoin L, Chen G, Weselake RJ, Lemieux MJ (2018) Diacylglycerol acyltransferase 1 is activated by phosphatidate and inhibited by SnRK1-catalyzed phosphorylation. Plant J 96:287–299

    CAS  PubMed  Google Scholar 

  13. Chen GQ, Van Erp H, Martin-Moreno J, Johnson K, Morales E, Eastmond PJ, Lin J-T (2016) Expression of castor LPAT2 enhances ricinoleic acid content at the sn-2 position of triacylglycerols in lesquerella seed. Int J Mol Sci 17:507

    CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  Google Scholar 

  15. Dahlqvist A, Stahl U, Lenman M, Banas A, Lee M, Sandager L, Ronne H, Stymne H (2000) Phospholipid : diacylglycerol acyltransferase: An enzyme that catalyzes the acyl-CoA-independent formation of triacylglycerol in yeast and plants. Proc Natl Acad Sci USA 97(12):6487–6492

    CAS  PubMed  Google Scholar 

  16. Dyer JM, Chapital DC, Kuan J-C, Mullen RT, Turner C, McKeon TA, Pepperman AB (2002) Molecular analysis of a bifunctional fatty acid conjugase/desaturase from tung. Implications for the evolution of plant fatty acid diversity. Plant Physiol 130:2027–2038

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Gunstone FD, Harwood JL, Dijkstra AJ (2007) The lipid handbook with CD-ROM. CRC Press, Boca Raton

    Google Scholar 

  18. Hara A, Radin NS (1978) Lipid extraction of tissues with a low-toxicity solvent. Anal Biochem 90:420–426

    CAS  PubMed  Google Scholar 

  19. Hu Z, Ren Z, Lu C (2012) The phosphatidylcholine diacylglycerol cholinephosphotransferase is required for efficient hydroxy fatty acid accumulation in transgenic Arabidopsis. Plant Physiol 158:1944–1954

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Iskandarov U, Silva JE, Kim HJ, Andersson M, Cahoon RE, Mockaitis K, Cahoon EB (2017) A specialized diacylglycerol acyltransferase contributes to the extreme medium-chain fatty acid content of Cuphea seed oil. Plant Physiol 174:97–109

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Jasieniecka-Gazarkiewicz K, Lager I, Carlsson AS, Gutbrod K, Peisker H, Dörmann P, Stymne S, Banaś A (2017) Acyl-CoA:lysophosphatidylethanolamine acyltransferase activity regulates growth of Arabidopsis. Plant Physiol 174:986–998

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Kanda P, Wells MA (1981) Facile acylation of glycerophosphocholine catalyzed by trifluoroacetic-anhydride. J Lipid Res 22:877–879

    CAS  PubMed  Google Scholar 

  23. Karki N, Bates PD (2018) The effect of light conditions on interpreting oil composition engineering in Arabidopsis seeds. Plant Direct 2:e00067

    PubMed  PubMed Central  Google Scholar 

  24. Katavic V, Reed DW, Taylor DC, Giblin EM, Barton DL, Zou J, Mackenzie SL, Covello PS, Kunst L (1995) Alteration of seed fatty acid composition by an ethyl methanesulfonate-induced mutation in Arabidopsis thaliana affecting diacylglycerol acyltransferase activity. Plant Physiol 108:399–409

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Kennedy EP (1961) Biosynthesis of complex lipids. Fed Proc 20:934–940

    CAS  PubMed  Google Scholar 

  26. Kim HU, Li Y, Huang AH (2005) Ubiquitous and endoplasmic reticulum-located lysophosphatidyl acyltransferase, LPAT2, is essential for female but not male gametophyte development in Arabidopsis thaliana. Plant Cell 17:1073–1089

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Kim HJ, Silva JE, Iskandarov U, Andersson M, Cahoon RE, Mockaitis K, Cahoon EB (2015) Structurally divergent lysophosphatidic acid acyltransferases with high selectivity for saturated medium chain fatty acids from Cuphea seeds. Plant J 84:1021–1033

    CAS  PubMed  Google Scholar 

  28. Knutzon DS, Hayes TR, Wyrick A, Xiong H, Maelor Davies H, Voelker TA (1999) Lysophosphatidic acid acyltransferase from coconut endosperm mediates the insertion of laurate at the sn-2 position of triacylglycerols in lauric rapeseed oil and can increase total laurate levels. Plant Physiol 120:739–746

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Körbes AP, Kulcheski FR, Margis R, Margis-Pinheiro M, Turchetto-Zolet AC (2016) Molecular evolution of the lysophosphatidic acid acyltransferase (LPAAT) gene family. Mol Phylogenet Evol 96:55–69

    PubMed  Google Scholar 

  30. Kotapati HK, Bates PD (2018) A normal phase high performance liquid chromatography method for the separation of hydroxy and non-hydroxy neutral lipid classes compatible with ultraviolet and in-line liquid scintillation detection of radioisotopes. J Chromatogr B 1102–1103:52–59

    Google Scholar 

  31. Kroon JT, Wei W, Simon WJ, Slabas AR (2006) Identification and functional expression of a type 2 acyl-CoA: diacylglycerol acyltransferase (DGAT2) in developing castor bean seeds which has high homology to the major triglyceride biosynthetic enzyme of fungi and animals. Phytochemistry 67:2541–2549

    CAS  PubMed  Google Scholar 

  32. Kumar R, Wallis JG, Skidmore C, Browse J (2006) A mutation in Arabidopsis cytochrome b5 reductase identified by high-throughput screening differentially affects hydroxylation and desaturation. Plant J 48:920–932

    CAS  PubMed  Google Scholar 

  33. Lager I, Yilmaz JL, Zhou XR, Jasieniecka K, Kazachkov M, Wang P, Zou J, Weselake R, Smith MA, Bayon S, Dyer JM, Shockey JM, Heinz E, Green A, Banas A, Stymne S (2013) Plant acyl-CoA:lysophosphatidylcholine acyltransferases (LPCATs) have different specificities in their forward and reverse reactions. J Biol Chem 288:36902–36914

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Lands WEM (1960) Metabolism of glycerolipids. II The enzymatic acylation of lysolecithin. J Biol Chem 235:2233–2237

    CAS  PubMed  Google Scholar 

  35. Li Y, Beisson F, Pollard M, Ohlrogge J (2006) Oil content of Arabidopsis seeds: the influence of seed anatomy, light and plant-to-plant variation. Phytochemistry 67:904–915

    CAS  PubMed  Google Scholar 

  36. Li-Beisson Y, Shorrosh B, Beisson F, Andersson MX, Arondel V, Bates PD, Baud S, Bird D, Debono A, Durrett TP, Franke RB, Graham IA, Katayama K, Kelly AA, Larson T, Markham JE, Miquel M, Molina I, Nishida I, Rowland O, Samuels L, Schmid KM, Wada H, Welti R, Xu C, Zallot R, Ohlrogge J (2013) Acyl-lipid metabolism. The Arabidopsis Book Am Soc Plant Biol 11:e0161

    Google Scholar 

  37. Lin JT, Turner C, Liao LP, McKeon TA (2003) Identification and quantification of the molecular species of acylglycerols in castor oil by HPLC using ELSD. J Liq Chromatogr Relat Technol 26:773–780

    CAS  Google Scholar 

  38. Lu CF, Fulda M, Wallis JG, Browse J (2006) A high-throughput screen for genes from castor that boost hydroxy fatty acid accumulation in seed oils of transgenic Arabidopsis. Plant J 45:847–856

    CAS  PubMed  Google Scholar 

  39. Maisonneuve S, Bessoule JJ, Lessire R, Delseny M, Roscoe TJ (2010) Expression of rapeseed microsomal lysophosphatidic acid acyltransferase isozymes enhances seed oil content in Arabidopsis. Plant Physiol 152:670–684

    CAS  PubMed  PubMed Central  Google Scholar 

  40. McKeon TA (2016) Castor (Ricinus communis, L.). In: McKeon TA, Hayes DG, Hildebrand DF, Weselake RJ (eds) Industrial Oil Crops, 1st edn. AOCS Press, Elsevier, San Diego, pp 75–112

    Google Scholar 

  41. Millar AA, Smith MA, Kunst L (2000) All fatty acids are not equal: discrimination in plant membrane lipids. Trends Plant Sci 5:95–101

    CAS  PubMed  Google Scholar 

  42. Pastor S, Sethumadhavan K, Ullah AH, Gidda S, Cao H, Mason C, Chapital C, Scheffler B, Mullen R, Dyer J, Shockey J (2013) Molecular properties of the class III subfamily of acyl-coenzyme A binding proteins from tung tree (Vernicia fordii). Plant Sci 203–204:79–88

    PubMed  Google Scholar 

  43. Routaboul JM, Benning C, Bechtold N, Caboche M, Lepiniec L (1999) The TAG1 locus of Arabidopsis encodes for a diacylglycerol acyltransferase. Plant Physiol Biochem 37:831–840

    CAS  PubMed  Google Scholar 

  44. Shockey JM, Gidda SK, Chapital DC, Kuan JC, Dhanoa PK, Bland JM, Rothstein SJ, Mullen RT, Dyer JM (2006) Tung tree DGAT1 and DGAT2 have nonredundant functions in triacylglycerol biosynthesis and are localized to different subdomains of the endoplasmic reticulum. Plant Cell 18:2294–2313

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Shockey J, Mason C, Gilbert M, Cao H, Li X, Cahoon E, Dyer J (2015) Development and analysis of a highly flexible multi-gene expression system for metabolic engineering in Arabidopsis seeds and other plant tissues. Plant Mol Biol 89:113–126

    CAS  PubMed  Google Scholar 

  46. Shockey J, Rinehart T, Chen Y, Wang Y, Zhihyong Z, Lisong H (2016) Tung (Vernicia fordii and Vernicia montana). In: McKeon TA, Hayes DG, Hildebrand DF, Weselake RJ (eds) Industrial Oil Crops, 1st edn. AOCS Press, Elsevier, San Diego, pp 243–274

    Google Scholar 

  47. 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

    CAS  PubMed  Google Scholar 

  48. Ståhl U, Stålberg K, Stymne S, Ronne H (2008) A family of eukaryotic lysophospholipid acyltransferases with broad specificity. FEBS Lett 582:305–309

    PubMed  Google Scholar 

  49. Taylor DC, Katavic V, Zou J, MacKenzie SL, Keller WA, An J, Friesen W, Barton DL, Pedersen KK, Giblin EM (2002) Field testing of transgenic rapeseed cv. Hero transformed with a yeast sn-2 acyltransferase results in increased oil content, erucic acid content and seed yield. Mol Breed 8:317–322

    CAS  Google Scholar 

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

    PubMed  Google Scholar 

  51. van Erp H, Bates PD, Burgal J, Shockey J, Browse J (2011) Castor phospholipid:diacylglycerol acyltransferase facilitates efficient metabolism of hydroxy fatty acids in transgenic Arabidopsis. Plant Physiol 155:683–693

    PubMed  Google Scholar 

  52. van Erp H, Shockey J, Zhang M, Adhikari ND, Browse J (2015) Reducing isozyme competition increases target fatty acid accumulation in seed triacylglycerols of transgenic Arabidopsis. Plant Physiol 168:36–46

    PubMed  PubMed Central  Google Scholar 

  53. Wayne LL, Browse J (2013) Homologous electron transport components fail to increase fatty acid hydroxylation in transgenic Arabidopsis thaliana. F1000Res 2:203

    PubMed  PubMed Central  Google Scholar 

  54. Zou J, Katavic V, Giblin EM, Barton DL, MacKenzie SL, Keller WA, Hu X, Taylor DC (1997) Modification of seed oil content and acyl composition in the Brassicaceae by expression of a yeast sn-2 acyltransferase gene. Plant Cell 9:909–923

    CAS  PubMed  PubMed Central  Google Scholar 

  55. Zou J, Wei Y, Jako C, Kumar A, Selvaraj G, Taylor DC (1999) The Arabidopsis thaliana TAG1 mutant has a mutation in a diacylglycerol acyltransferase gene. Plant J 19:645–653

    CAS  PubMed  Google Scholar 

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Acknowledgements

The authors would like to thank Ms. Tien Thuy Vuong for technical assistance. This work was supported by the U.S. Department of Agriculture, Agricultural Research Service Current Research Information System project number 6054-41000-102-00D (to JS and CM) and the National Science Foundation (Directorate for Biological Sciences, Division of Molecular and Cellular Bioscience, award #1613923, to PDB and JS).

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Shockey, J., Lager, I., Stymne, S. et al. Specialized lysophosphatidic acid acyltransferases contribute to unusual fatty acid accumulation in exotic Euphorbiaceae seed oils. Planta 249, 1285–1299 (2019). https://doi.org/10.1007/s00425-018-03086-y

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Keywords

  • Diacylglycerol acyltransferase
  • Eleostearic acid
  • Ricinoleic acid
  • Lysophosphatidic acid acyltransferase
  • Triacylglycerol