BMB Reports

Korean Society for Biochemistry and Molecular Biology (생화학분자생물학회)
권호 표지
DOI QR코드

DOI QR Code

PDAT1 genome editing reduces hydroxy fatty acid production in transgenic Arabidopsis

  • Mid-Eum Park (Department of Molecular Biology, Sejong University) ;
  • Hyun Uk Kim (Department of Molecular Biology, Sejong University)
  • Received : 2023.03.21
  • Accepted : 2023.08.18
  • Published : 2024.02.29
Download PDF
⟨ Previous Next ⟩

Abstract

The fatty acids content of castor (Ricinus communis L.) seed oil is 80-90% ricinoleic acid, which is a hydroxy fatty acid (HFA). The structures and functional groups of HFAs are different from those of common fatty acids and are useful for various industrial applications. However, castor seeds contain the toxin ricin and an allergenic protein, which limit their cultivation. Accordingly, many researchers are conducting studies to enhance the production of HFAs in Arabidopsis thaliana, a model plant for oil crops. Oleate 12-hydroxylase from castor (RcFAH12), which synthesizes HFA (18:1-OH), was transformed into an Arabidopsis fae1 mutant, resulting in the CL37 line producing a maximum of 17% HFA content. In addition, castor phospholipid:diacylglycerol acyltransferase 1-2 (RcPDAT1-2), which catalyzes the production of triacylglycerol by transferring HFA from phosphatidylcholine to diacylglycerol, was transformed into the CL37 line to develop a P327 line that produces 25% HFA. In this study, we investigated changes in HFA content when endogenous Arabidopsis PDAT1 (AtPDAT1) of the P327 line was edited using the CRISPR/Cas9 technique. The successful mutation resulted in three independent lines with different mutation patterns, which were transmitted until the T4 generation. Fatty acid analysis of the seeds showed that HFA content decreased in all three mutant lines. These findings indicate that AtPDAT1 as well as RcPDAT1-2 in the P327 line are involved in transferring and increasing HFAs to triacylglycerol.

Keywords

Acknowledgement

This work was supported by grants from the Mid-Career Researcher Program of the National Research Foundation of Korea (NRF-2020R1A2C2008175) and New Breeding Technologies Development Program (RS-2022-RD009977), and Program of the Rural Development Administration, Republic of Korea.

References

  1. Binder RG, Kohler GO, Goldblatt LA and Applewhite TH (1962) Chromatographic analysis of seed oils - fatty acid composition of castor oil. J Am Oil Chem Soc 39, 513-517  https://doi.org/10.1007/BF02672540
  2. Prasad RBN and Rao BVSK (2017) Chapter 8 - chemical derivatization of castor oil and their industrial utilization; in fatty acids, Ahmad MU (ed.), 279-303, AOCS Press, USA
  3. Patel VR, Dumancas GG, Kasi Viswanath LC, Maples R and Subong BJ (2016) Castor oil: properties, uses, and optimization of processing parameters in commercial production. Lipid Insights 9, 1-12  https://doi.org/10.4137/LPI.S40233
  4. Severino LS, Auld DL, Baldanzi M et al (2012) A review on the challenges for increased production of castor. Agron J 104, 853-880  https://doi.org/10.2134/agronj2011.0210
  5. Park ME, Lee KR, Chen GQ and Kim HU (2022) Enhanced production of hydroxy fatty acids in Arabidopsis seed through modification of multiple gene expression. Biotechnol Biofuels Bioprod 15, 66 
  6. Lunn D, Wallis JG and Browse J (2019) Tri-hydroxy-triacylglycerol is efficiently produced by position-specific castor acyltransferases. Plant Physiol 179, 1050-1063  https://doi.org/10.1104/pp.18.01409
  7. Shockey J, Lager I, Stymne S et al (2019) Specialized lysophosphatidic acid acyltransferases contribute to unusual fatty acid accumulation in exotic Euphorbiaceae seed oils. Planta 249, 1285-1299  https://doi.org/10.1007/s00425-018-03086-y
  8. Aryal N and Lu CF (2018) A phospholipase C-like protein from ricinus communis increases hydroxy fatty acids accumulation in transgenic seeds of Camelina sativa. Front Plant Sci 9, 1576 
  9. Snapp AR, Kang J, Qi X and Lu C (2014) A fatty acid condensing enzyme from Physaria fendleri increases hydroxy fatty acid accumulation in transgenic oilseeds of Camelina sativa. Planta 240, 599-610  https://doi.org/10.1007/s00425-014-2122-2
  10. Lu C and Kang J (2008) Generation of transgenic plants of a potential oilseed crop Camelina sativa by Agrobacterium-mediated transformation. Plant Cell Rep 27, 273-278  https://doi.org/10.1007/s00299-007-0454-0
  11. Smith MA, Moon H, Chowrira G and Kunst L (2003) Heterologous expression of a fatty acid hydroxylase gene in developing seeds of Arabidopsis thaliana. Planta 217, 507-516  https://doi.org/10.1007/s00425-003-1015-6
  12. Lu C, Fulda M, Wallis JG and 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  https://doi.org/10.1111/j.1365-313X.2005.02636.x
  13. Broun P and 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, 933-942  https://doi.org/10.1104/pp.113.3.933
  14. van de Loo FJ, Broun P, Turner S and 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  https://doi.org/10.1073/pnas.92.15.6743
  15. Lunn D, Le A, Wallis JG and Browse J (2020) Castor LPCAT and PDAT1A Act in concert to promote transacylation of hydroxy-fatty acid onto triacylglycerol. Plant Physiol 184, 709-719  https://doi.org/10.1104/pp.20.00691
  16. Kim HU, Lee KR, Go YS, Jung JH, Suh MC and Kim JB (2011) Endoplasmic reticulum-located PDAT1-2 from castor bean enhances hydroxy fatty acid accumulation in transgenic plants. Plant Cell Physiol 52, 983-993  https://doi.org/10.1093/pcp/pcr051
  17. van Erp H, Bates PD, Burgal J, Shockey J and Browse J (2011) Castor phospholipid:diacylglycerol acyltransferase facilitates efficient metabolism of hydroxy fatty acids in transgenic Arabidopsis. Plant Physiol 155, 683-693  https://doi.org/10.1104/pp.110.167239
  18. Kunst L, Taylor DC and Underhill EW (1992) Fatty acid elongation in developing seeds of Arabidopsis thaliana. Plant Physiol Biochem 30, 425-434 
  19. Chapman KD and Ohlrogge JB (2012) Compartmentation of triacylglycerol accumulation in plants. J Biol Chem 287, 2288-2294  https://doi.org/10.1074/jbc.R111.290072
  20. Kim HU, Li Y and Huang AH (2005) Ubiquitous and endoplasmic reticulum-located lysophosphatidyl acyltransferase, LPAT2, is essential for female but not male gametophyte development in Arabidopsis. Plant Cell 17, 1073-1089  https://doi.org/10.1105/tpc.104.030403
  21. Shockey J, Regmi A, Cotton K, Adhikari N, Browse J and Bates PD (2016) Identification of Arabidopsis GPAT9 (At5g60620) as an essential gene involved in triacylglycerol biosynthesis. Plant Physiol 170, 163-179  https://doi.org/10.1104/pp.15.01563
  22. Zou J, Wei Y, Jako C, Kumar A, Selvaraj G and Taylor DC (1999) The Arabidopsis thaliana TAG1 mutant has a mutation in a diacylglycerol acyltransferase gene. Plant J 19, 645-653  https://doi.org/10.1046/j.1365-313x.1999.00555.x
  23. Burgal J, Shockey J, Lu C et al (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  https://doi.org/10.1111/j.1467-7652.2008.00361.x
  24. Kim HU, Park ME, Lee KR, Suh MC and Chen GQ (2020) Variant castor lysophosphatidic acid acyltransferases acylate ricinoleic acid in seed oil. Ind Crops Prod 150, 112245 
  25. Dahlqvist A, Stahl U, Lenman M et al (2000) Phospholipid:diacylglycerol acyltransferase: an enzyme that catalyzes the acyl-CoA-independent formation of triacylglycerol in yeast and plants. Proc Natl Acad Sci U S A 97, 6487-6492  https://doi.org/10.1073/pnas.120067297
  26. Behera J, Rahman MM, Shockey J and Kilaru A (2022) Acyl-CoA-dependent and acyl-CoA-independent avocado acyltransferases positively influence oleic acid content in nonseed triacylglycerols. Front Plant Sci 13, 1056582 
  27. Falarz LJ, Xu Y, Caldo KMP, Garroway CJ, Singer SD and Chen G (2020) Characterization of the diversification of phospholipid:diacylglycerol acyltransferases in the green lineage. Plant J 103, 2025-2038  https://doi.org/10.1111/tpj.14880
  28. Hernandez ML, Moretti S, Sicardo MD et al (2021) Distinct physiological roles of three phospholipid:diacylglycerol acyltransferase genes in olive fruit with respect to oil accumulation and the response to abiotic stress. Front Plant Sci 12, 751959 
  29. Bates PD and 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  https://doi.org/10.1111/j.1365-313X.2011.04693.x
  30. Dauk M, Lam P and Smith MA (2009) The role of diacylglycerol acyltransferase-1 and phospholipid:diacylglycerol acyltransferase-1 and -2 in the incorporation of hydroxy fatty acids into triacylglycerol in Arabidopsis thaliana expressing a castor bean oleate 12-hydroxylase gene. Botany 87, 552-560  https://doi.org/10.1139/B09-011
  31. van Erp H, Shockey J, Zhang M and Adhikari ND (2015) Reducing isozyme competition increases target fatty acid accumulation in seed triacylglycerols of transgenic Arabidopsis. Plant Physiol 168, 36-46  https://doi.org/10.1104/pp.114.254110
  32. Zhang M, Fan J, Taylor DC and Ohlrogge JB (2009) DGAT1 and PDAT1 acyltransferases have overlapping functions in Arabidopsis triacylglycerol biosynthesis and are essential for normal pollen and seed development. Plant Cell 21, 3885-3901  https://doi.org/10.1105/tpc.109.071795
  33. Lei Y, Lu L, Liu HY, Li S, Xing F and Chen LL (2014) CRISPR-P: a web tool for synthetic single-guide RNA design of CRISPR-system in plants. Mol Plant 7, 1494-1496  https://doi.org/10.1093/mp/ssu044
  34. Weber E, Engler C, Gruetzner R, Werner S and Marillonnet S (2011) A modular cloning system for standardized assembly of multigene constructs. PLoS One 6, e16765 
  35. Clough SJ and Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16, 735-743 https://doi.org/10.1046/j.1365-313x.1998.00343.x