Two plastidial lysophosphatidic acid acyltransferases differentially mediate the biosynthesis of membrane lipids and triacylglycerols in Phaeodactylum tricornutum.

Publisher: Wiley on behalf of New Phytologist Trust Country of Publication: England NLM ID: 9882884 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1469-8137 (Electronic) Linking ISSN: 0028646X NLM ISO Abbreviation: New Phytol Subsets: MEDLINE

Publication: Oxford : Wiley on behalf of New Phytologist Trust
Original Publication: London, New York [etc.] Academic Press.

Membrane Lipids* ; Acyltransferases*/metabolism; Triglycerides ; Plastids/metabolism ; Phosphatidic Acids ; Nitrogen

Lysophosphatidic acid acyltransferases (LPAATs) catalyze the formation of phosphatidic acid (PA), a central metabolite in both prokaryotic and eukaryotic organisms for glycerolipid biosynthesis. Phaeodactylum tricornutum contains at least two plastid-localized LPAATs (ptATS2a and ptATS2b), but their roles in lipid synthesis remain unknown. Both ptATS2a and ptATS2b could complement the high temperature sensitivity of the bacterial plsC mutant deficient in LPAAT. In vitro enzyme assays showed that they prefer lysophosphatidic acid over other lysophospholipids. ptATS2a is localized in the plastid inner envelope membrane and CRISPR/Cas9-generated ptATS2a mutants showed compromised cell growth, significantly changed plastid and extra-plastidial membrane lipids at nitrogen-replete condition and reduced triacylglycerols (TAGs) under nitrogen-depleted condition. ptATS2b is localized in thylakoid membranes and its knockout led to reduced growth rate and TAG content but slightly altered molecular composition of membrane lipids. The changes in glycerolipid profiles are consistent with the role of both LPAATs in the sn-2 acylation of sn-1-acyl-glycerol-3-phosphate substrates harboring 20:5 at the sn-1 position. Our findings suggest that both LPAATs are important for membrane lipids and TAG biosynthesis in P. tricornutum and further highlight that 20:5-Lyso-PA is likely involved in the massive import of 20:5 back to the plastid to feed plastid glycerolipid syntheses.
(© 2023 The Authors New Phytologist © 2023 New Phytologist Foundation.)

Abida H, Dolch LJ, Mei C, Villanova V, Conte M, Block MA, Finazzi G, Bastien O, Tirichine L, Bowler C et al. 2015. Membrane glycerolipid remodeling triggered by nitrogen and phosphorus starvation in Phaeodactylum tricornutum. Plant Physiology 167: 118-136.
Apt KE, Zaslavkaia L, Lippmeier JC, Lang M, Kilian O, Wetherbee R, Grossman AR, Kroth PG. 2002. In vivo characterization of diatom multipartite plastid targeting signals. Journal of Cell Science 115: 4061-4069.
Awai K, Xu C, Lu B, Benning C. 2006. Lipid trafficking between the endoplasmic reticulum and the chloroplast. Biochemical Society Transactions 34: 395-398.
Balamurugan S, Wang X, Wang HL, An CJ, Li H, Li DW, Yang WD, Liu JS, Li HY. 2017. Occurrence of plastidial triacylglycerol synthesis and the potential regulatory role of AGPAT in the model diatom Phaeodactylum tricornutum. Biotechnology for Biofuels 10: 97.
Benghezal M, Roubaty C, Veepuri V, Knudsen J, Conzelmann A. 2007. SLC1 and SLC4 encode partially redundant acyl-coenzyme A 1-acylglycerol-3-phosphate O-acyltransferases of budding yeast. The Journal of Biological Chemistry 282: 30845-30855.
Benning C. 2008. A role for lipid trafficking in chloroplast biogenesis. Progress in Lipid Research 47: 381-389.
Cabantous S, Terwilliger TC, Waldo GS. 2005. Protein tagging and detection with engineered self-assembling fragments of green fluorescent protein. Nature Biotechnology 23: 102-107.
Chen Q, Kazachkov M, Zheng Z, Zou J. 2007. The yeast acylglycerol acyltransferase LCA1 is a key component of Lands cycle for phosphatidylcholine turnover. FEBS Letters 581: 5511-5516.
Coleman J. 1990. Characterization of Escherichia coli cells deficient in 1-acyl-sn-glycerol-3-phosphate acyltransferase activity. The Journal of Biological Chemistry 265: 17215-17221.
Collos Y, Mornet F, Sciandra A, Waser N, Larson A, Harrison PJ. 1999. An optical method for the rapid measurement of micromolar concentrations of nitrate in marine phytoplankton cultures. Journal of Applied Phycology 11: 179-184.
Cullinane M, Baysse C, Morrissey JP, O'Gara F. 2005. Identification of two lysophosphatidic acid acyltransferase genes with overlapping function in Pseudomonas fluorescens. Microbiology 151: 3071-3080.
Dolch LJ, Marechal E. 2015. Inventory of fatty acid desaturases in the pennate diatom Phaeodactylum tricornutum. Marine Drugs 13: 1317-1339.
Dolch LJ, Rak C, Perin G, Tourcier G, Broughton R, Leterrier M, Morosinotto T, Tellier F, Faure JD, Falconet D et al. 2017. A palmitic acid elongase affects eicosapentaenoic acid and plastidial monogalactosyldiacylglycerol levels in Nannochloropsis. Plant Physiology 173: 742-759.
Frentzen M, Heinz E, McKeon TA, Stumpf PK. 1983. Specificities and selectivities of glycerol-3-phosphate acyltransferase and monoacylglycerol-3-phosphate acyltransferase from pea and spinach chloroplasts. European Journal of Biochemistry 129: 629-636.
Gietz RD, Woods RA. 2002. Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method. Methods in Enzymology 350: 87-96.
Grosche C, Hempel F, Bolte K, Zauner S, Maier UG. 2014. The periplastidal compartment: a naturally minimized eukaryotic cytoplasm. Current Opinion in Microbiology 22: 88-93.
Gruber A, Vugrinec S, Hempel F, Gould SB, Maier UG, Kroth PG. 2007. Protein targeting into complex diatom plastids: functional characterisation of a specific targeting motif. Plant Molecular Biology 64: 519-530.
Guillard RRL. 1975. Culture of phytoplankton for feeding marine invertebrates. In: Smith W, Chanley M, eds. Culture of marine invertebrate animals. New York, NY, USA: Springer, 29-60.
Haslam RP, Hamilton ML, Economou CK, Smith R, Hassall KL, Napier JA, Sayanova O. 2020. Overexpression of an endogenous type 2 diacylglycerol acyltransferase in the marine diatom Phaeodactylum tricornutum enhances lipid production and omega-3 long-chain polyunsaturated fatty acid content. Biotechnology for Biofuels 13: 87.
Heinz E, Roughan G. 1983. Similarities and differences in lipid metabolism of chloroplasts isolated from 18:3 and 16:3 plants. Plant Physiology 72: 273-279.
Huang T, Pan Y, Maréchal E, Hu H. 2023. Proteomes reveal the lipid metabolic network in the complex plastid of Phaeodactylum tricornutum. The Plant Journal: 1-19.
Karas BJ, Diner RE, Lefebvre SC, McQuaid J, Phillips AP, Noddings C, Brunson JK, Valas RE, Deerinck TJ, Jablanovic J. 2015. Designer diatom episomes delivered by bacterial conjugation. Nature Communications 6: 6925.
Karki N, Johnson BS, Bates PD. 2019. Metabolically distinct pools of phosphatidylcholine are involved in trafficking of fatty acids out of and into the chloroplast for membrane production. Plant Cell 31: 2768-2788.
Kilian O, Kroth PG. 2005. Identification and characterization of a new conserved motif within the presequence of proteins targeted into complex diatom plastids. The Plant Journal 41: 175-183.
Kim HU, Huang AH. 2004. Plastid lysophosphatidylacyltransferase is essential for embryo development in Arabidopsis. Plant Physiology 134: 1206-1216.
Kim Y, Terng EL, Riekhof WR, Cahoon EB, Cerutti H. 2018. Endoplasmic reticulum acyltransferase with prokaryotic substrate preference contributes to triacylglycerol assembly in Chlamydomonas. Proceedings of the National Academy of Sciences, USA 115: 1652-1657.
Körbes AP, Kulcheski FR, Margis R, Margis-Pinheiro M, Turchetto-Zolet AC. 2016. Molecular evolution of the lysophosphatidic acid acyltransferase (LPAAT) gene family. Molecular Phylogenetics and Evolution 96: 55-69.
Kunst L, Browse J, Somerville C. 1988. Altered regulation of lipid biosynthesis in a mutant of Arabidopsis deficient in chloroplast glycerol-3-phosphate acyltransferase activity. Proceedings of the National Academy of Sciences, USA 85: 4143-4147.
Lewin TM, Wang P, Coleman RA. 1999. Analysis of amino acid motifs diagnostic for the sn-glycerol-3-phosphate acyltransferase reaction. Biochemistry 38: 5764-5771.
Leyland B, Zarka A, Didi-Cohen S, Boussiba S, Khozin-Goldberg I. 2020. High resolution proteome of lipid droplets isolated from the pennate diatom Phaeodactylum tricornutum (Bacillariophyceae) strain pt4 provides mechanistic insights into complex intracellular coordination during nitrogen deprivation. Journal of Phycology 56: 1642-1663.
Lu B, Benning C. 2009. A 25-amino acid sequence of the Arabidopsis TGD2 protein is sufficient for specific binding of phosphatidic acid. The Journal of Biological Chemistry 284: 17420-17427.
Lupette J, Jaussaud A, Seddiki K, Morabito C, Brugière S, Schaller H, Kuntz M, Putaux JL, Jouneau PH, Rébeillé F et al. 2019. The architecture of lipid droplets in the diatom Phaeodactylum tricornutum. Algal Research 38: 101415.
Miège C, Maréchal É. 1999. 1,2-sn-Diacylglycerol in plant cells: product, substrate and regulator. Plant Physiology and Biochemistry 37: 795-808.
Moog D, Rensing SA, Archibald JM, Maier UG, Ullrich KK. 2015. Localization and evolution of putative triose phosphate translocators in the diatom Phaeodactylum tricornutum. Genome Biology Evolution 7: 2955-2969.
Moosburner MA, Gholami P, McCarthy JK, Tan M, Bielinski VA, Allen AE. 2020. Multiplexed knockouts in the model diatom Phaeodactylum by episomal delivery of a selectable Cas9. Frontiers in Microbiology 11: 5.
Niu YF, Wang X, Hu DX, Balamurugan S, Li DW, Yang WD, Liu JS, Li HY. 2016. Molecular characterization of a glycerol-3-phosphate acyltransferase reveals key features essential for triacylglycerol production in Phaeodactylum tricornutum. Biotechnology for Biofuels 9: 60.
Okazaki K, Sato N, Tsuji N, Tsuzuki M, Nishida I. 2006. The significance of C16 fatty acids in the sn-2 positions of glycerolipids in the photosynthetic growth of Synechocystis sp. PCC6803. Plant Physiology 141: 546-556.
Petroutsos D, Amiar S, Abida H, Dolch LJ, Bastien O, Rebeille F, Jouhet J, Falconet D, Block MA, McFadden GI et al. 2014. Evolution of galactoglycerolipid biosynthetic pathways-from cyanobacteria to primary plastids and from primary to secondary plastids. Progress in Lipid Research 54: 68-85.
Riekhof WR, Wu J, Jones JL, Voelker DR. 2007. Identification and characterization of the major lysophosphatidylethanolamine acyltransferase in Saccharomyces cerevisiae. The Journal of Biological Chemistry 282: 28344-28352.
Roughan PG, Slack CR. 1982. Cellular organization of glycerolipid metabolism. Annual Review of Plant Physiology 33: 97-132.
Schujman GE, de Mendoza D. 2008. Regulation of type II fatty acid synthase in gram-positive bacteria. Current Opinion in Microbiology 11: 148-152.
Slack CR, Roughan PG, Balasingham N. 1977. Labelling studies in vivo on the metabolism of the acyl and glycerol moieties of the glycerolipids in the developing maize leaf. The Biochemical Journal 162: 289-296.
Slattery SS, Diamond A, Wang H, Therrien JA, Lant JT, Jazey T, Lee K, Klassen Z, Desgagné-Penix I, Karas BJ et al. 2018. An expanded plasmid-based genetic toolbox enables Cas9 genome editing and stable maintenance of synthetic pathways in Phaeodactylum tricornutum. ACS Synthetic Biology 7: 328-338.
Wang X, Dong HP, Wei W, Balamurugan S, Yang WD, Liu JS, Li HY. 2018. Dual expression of plastidial GPAT1 and LPAT1 regulates triacylglycerol production and the fatty acid profile in Phaeodactylum tricornutum. Biotechnology for Biofuels 11: 318.
Wang X, Liu SF, Li RY, Yang WD, Liu JS, Lin CSK, Balamurugan S, Li HY. 2020. TAG pathway engineering via GPAT2 concurrently potentiates abiotic stress tolerance and oleaginicity in Phaeodactylum tricornutum. Biotechnology for Biofuels 13: 160.
Xu C, Cornish AJ, Froehlich JE, Benning C. 2006a. Phosphatidylglycerol biosynthesis in chloroplasts of Arabidopsis mutants deficient in acyl-ACP glycerol-3-phosphate acyltransferase. The Plant Journal 47: 296-309.
Xu C, Fan J, Froehlich JE, Awai K, Benning C. 2005. Mutation of the TGD1 chloroplast envelope protein affects phosphatidate metabolism in Arabidopsis. Plant Cell 17: 3094-3110.
Xu C, Yu B, Cornish AJ, Froehlich JE, Benning C. 2006b. Phosphatidylglycerol biosynthesis in chloroplasts of Arabidopsis mutants deficient in acyl-ACP glycerol-3- phosphate acyltransferase. Plant Journal 47: 296-309.
You L, Jouhet J, Maréchal E, Amato A, Hao X, Zhang D, Banaś A, Gong Y. 2023. Acyl-CoA:lysophosphatidylcholine acyltransferase from the unicellular diatom Phaeodactylum tricornutum (PtLPCAT1) is involved in triacylglycerol and galactoglycerolipid synthesis and enhances eicosapentaenoic acid accumulation in recombinant oleaginous yeast. Plant Biotechnology Journal 21: 238-240.
Zaslavskaia LA, Lippmeier JC, Kroth PG, Grossman AR, Apt KE. 2000. Transformation of the diatom Phaeodactylum tricornutum (Bacillariophyceae) with a variety of selectable marker and reporter genes. Journal of Phycology 36: 379-386.

31961133008 National Natural Science Foundation of China

Keywords: Phaeodactylum tricornutum; chloroplast membrane lipids; lysophosphatidic acid acyltransferase; multiplexed CRISPR/Cas9; triacylglycerol

EC 2.3.1.52 (2-acylglycerophosphate acyltransferase)
0 (Triglycerides)
0 (Membrane Lipids)
EC 2.3.- (Acyltransferases)
0 (Phosphatidic Acids)
N762921K75 (Nitrogen)

Date Created: 20231130 Date Completed: 20240126 Latest Revision: 20240126

20240126

10.1111/nph.19434

38031462