Castor patatin-like phospholipase A IIIβ facilitates removal of hydroxy fatty acids from phosphatidylcholine in transgenic Arabidopsis seeds
- 169 Downloads
Castor patatin-like phospholipase A IIIβ facilitates the exclusion of hydroxy fatty acids from phosphatidylcholine in developing transgenic Arabidopsis seeds.
Hydroxy fatty acids (HFAs) are industrial useful, but their major natural source castor contains toxic components. Although expressing a castor OLEATE 12-HYDROXYLASE in Arabidopsis thaliana leads to the synthesis of HFAs in seeds, a high proportion of the HFAs are retained in phosphatidylcholine (PC). Thus, the liberation of HFA from PC seems to be critical for obtaining HFA-enriched seed oils. Plant phospholipase A (PLA) catalyzes the hydrolysis of PC to release fatty acyl chains that can be subsequently channeled into triacylglycerol (TAG) synthesis or other metabolic pathways. To further our knowledge regarding the function of PLAs from HFA-producing plant species, two class III patatin-like PLA cDNAs (pPLAIIIβ or pPLAIIIδ) from castor or Physaria fendleri were overexpressed in a transgenic line of A. thaliana producing C18-HFA, respectively. Only the overexpression of RcpPLAIIIβ resulted in a significant reduction in seed HFA content with concomitant changes in fatty acid composition. Reductions in HFA content occurred in both PC and TAG indicating that HFAs released from PC were not incorporated into TAG. These results suggest that RcpPLAIIIβ may catalyze the removal of HFAs from PC in the developing seeds synthesizing these unusual fatty acids.
KeywordsCastor Hydroxy fatty acid Phosphatidylcholine Phospholipase A Physaria fendleri Ricinus communis
The authors thank Dr. John Browse of Washington State University for kindly providing Arabidopsis CL7 line. This work was supported by the Canada Research Chairs (R.J.W. and G.C.), Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grants (RGPIN-2016-05926 to G.C. and RGPIN-2014-04585 to R.J.W.), and Alberta Innovates Bio Solutions (R.J.W.). The infrastructure used in this work was funded by the Canadian Foundation for Innovation and Research Capacity Program of Alberta Enterprise and Advanced Education.
R.J.W. oversaw the project; R.J.W. and G.C. conceived the project; Y.L., G.C., E.M. and R.J.W. designed the experiments; R.J.W., G.C. and S.D.S. supervised the experiments; Y.L. performed most of the experiments and data analysis; G.C. conducted some of the experiments and data analysis; Z.S. performed qRT-PCR, K.C. and Y.L. made constructs for gene transformation; J.D., M.S., and T.M. generated important plant materials, genes, and gene libraries. Y.L. and G.C. wrote the initial draft of the article. All authors participated in interpretation of the data and were instrumental in the preparation of the final article.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
- 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(2):507–514. https://doi.org/10.1042/bj2800507 PubMedPubMedCentralCrossRefGoogle Scholar
- Bates PD, Fatihi A, Snappp 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(3):1530–1539. https://doi.org/10.1104/pp.112.204438 PubMedPubMedCentralCrossRefGoogle Scholar
- 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 Natl Acad Sci USA 111(3):1204–1209. https://doi.org/10.1073/pnas.1318511111 PubMedCrossRefGoogle 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 bean. Plant Physiol 113(3):933–942. https://doi.org/10.1104/pp.113.3.933 PubMedPubMedCentralCrossRefGoogle Scholar
- Brown AP, Kroon JT, Swarbreck D, Febrer M, Larson TR, Graham IA, Caccamo M, Slabas AR (2012) Tissue-specific whole transcriptome sequencing in castor, directed at understanding triacylglycerol lipid biosynthetic pathways. PLoS ONE 7(2):e30100. https://doi.org/10.1371/journal.pone.0030100 PubMedPubMedCentralCrossRefGoogle 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(8):819–831. https://doi.org/10.1111/j.1467-7652.2008.00361.x PubMedPubMedCentralCrossRefGoogle Scholar
- Chen G (2016) Lesquerella (Physaria spp.). In: McKeon T, Hayes DH, Hildebrand DF, Weselake RJ (eds) Industrial oil crops. Elsevier/AOCS Press, New York/Urbana, pp 313–316Google Scholar
- Chen G, Greer MS, Lager I, Yilmaz JL, Mietkiewska E, Carlsson AS, Stymne S, Weselake RJ (2012) Identification and characterization of an LCAT-like Arabidopsis thaliana gene encoding a novel phospholipase A. FEBS Lett 586(4):373–377. https://doi.org/10.1016/j.febslet.2011.12.034 PubMedCrossRefGoogle Scholar
- 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(4):507. https://doi.org/10.3390/ijms17040507 PubMedPubMedCentralCrossRefGoogle Scholar
- Dahlqvist A, Stahl U, Lenman M, Banas A, Lee M, Sandager L, Ronne H, Stymne S (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. https://doi.org/10.1073/pnas.120067297 PubMedCrossRefGoogle Scholar
- de Marcos Lousa C, van Roermund CW, Postis VL, Dietrich D, Kerr ID, Wanders RJ, Baldwin SA, Baker A, Theodoulou FL (2013) Intrinsic acyl-CoA thioesterase activity of a peroxisomal ATP binding cassette transporter is required for transport and metabolism of fatty acids. Proc Natl Acad Sci USA 110(4):1279–1284. https://doi.org/10.1073/pnas.1218034110 CrossRefGoogle Scholar
- Horn PJ, Liu J, Cocuron J-C, McGlew K, Thrower NA, Larson M, Lu C, Alonso AP, Ohlrogge J (2016) Identification of multiple lipid genes with modifications in expression and sequence associated with the evolution of hydroxyl fatty acid accumulation in Physaria fendleri. Plant J 86(4):322–348. https://doi.org/10.1111/tpj.13163 PubMedCrossRefGoogle Scholar
- 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(23):2541–2549. https://doi.org/10.1016/j.phytochem.2006.09.020 PubMedCrossRefGoogle Scholar
- Kunst L, Taylor DC, Underhill EW (1992) Fatty acid elongation in developing seeds of Arabidopsis thaliana. Plant Physiol Biochem 30(4):425–434Google Scholar
- Li M, Bahn SC, Guo L, Musgrave W, Berg H, Welti R, Wang X (2011) Patatin-related phospholipase pPLAIIIβ-induced changes in lipid metabolism alter cellulose content and cell elongation in Arabidopsis. Plant Cell 23(3):1107–1123. https://doi.org/10.1105/tpc.110.081240 PubMedPubMedCentralCrossRefGoogle Scholar
- Li M, Bahn SC, Fan C, Li J, Phan T, Ortiz M, Roth MR, Welti R, Jaworski J, Wang X (2013) Patatin-related phospholipase pPLAIII δ increases seed oil content with long-chain fatty acids in Arabidopsis. Plant Physiol 162(1):39–51. https://doi.org/10.1104/pp.113.216994 PubMedPubMedCentralCrossRefGoogle Scholar
- Lin J, Woodruff CL, Lagouche OJ, McKeon TA, Stafford AE, Goodrich-Tanrikulu M, Singleton JA, Haney CA (1998) Biosynthesis of triacylglycerols containing ricinoleate in castor microsomes using 1-acyl-2-oleoyl-sn-glycero-3-phosphocholine as the substrate of oleoyl-12-hydroxylase. Lipids 33(1):59–69PubMedCrossRefGoogle Scholar
- Mavraganis I, Meesapyodsuk D, Vrinten P, Smith M, Qiu X (2010) Type II diacylglycerol acyltransferase from Claviceps purpurea with ricinoleic acid, a hydroxyl fatty acid of industrial importance, as preferred substrate. Appl Environ Microbiol 76(4):1135–1142. https://doi.org/10.1128/aem.02297-09 PubMedCrossRefGoogle Scholar
- Mietkiewska E, Miles R, Wickramarathna A, Sahibollah AF, Greer MS, Chen G, Weselake RJ (2014) 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(3):575–583. https://doi.org/10.1007/s00425-014-2109-z PubMedCrossRefGoogle Scholar
- Shockey J, Lager I, Stymne S, Kotapati HK, Sheffield J, Mason C, Bates PD (2019) Specialized lysophosphatidic acid acyltransferases contribute to unusual fatty acid accumulation in exotic Euphorbiaceae seed oils. Planta 249(5):1285–1299. https://doi.org/10.1007/s00425-018-03086-y PubMedCrossRefGoogle Scholar
- Thomæus 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(5):997–1003. https://doi.org/10.1016/S0168-9452(01)00500-3 CrossRefGoogle Scholar
- Wang L, Shen W, Kazachkov M, Chen G, Chen Q, Carlsson AS, Stymne S, Weselake RJ, Zou J (2012) Metabolic interactions between the lands cycle and the Kennedy pathway of glycerolipid synthesis in Arabidopsis developing seeds. Plant Cell 24(11):4652–4669. https://doi.org/10.1105/tpc.112.104604 PubMedPubMedCentralCrossRefGoogle Scholar
- Waschburger E, Kulcheski FR, Veto NM, Margis R, Margis-Pinheiro M, Turchetto-Zolet AC (2018) Genome-wide analysis of the glycerol-3-phosphate acyltransferase (GPAT) gene family reveals the evolution and diversification of plant GPATs. Genet Mol Biol 41(1 suppl 1):355–370. https://doi.org/10.1590/1678-4685-GMB-2017-0076 PubMedPubMedCentralCrossRefGoogle Scholar
- Xu Y, Caldo KMP, Pal-Nath D, Ozga J, Lemieux MJ, Weselake RJ, Chen G (2018) Properties and biotechnological applications of acyl-CoA: diacylglycerol acyltransferases and phospholipid: diacylglycerol acyltransferases from terrestrial plants and microalgae. Lipids 53(7):663–688. https://doi.org/10.1002/lipd.12081 PubMedCrossRefGoogle Scholar
- Yurchenko OP, Nykiforuk CL, Moloney MM, Ståhl U, Banaś A, Stymne S, Weselake RJ (2009) A 10-kDa acyl-CoA-binding protein (ACBP) from Brassica napus enhances acyl exchange between acyl-CoA and phosphatidylcholine. Plant Biotechnol J 7(7):602–610. https://doi.org/10.1111/j.1467-7652.2009.00427.x PubMedCrossRefGoogle Scholar