Abstract
Phosphatidylcholine (PC) is one of the most common phospholipids in eukaryotes, although some green algae such as Chlamydomonas reinhardtii are known to lack PC. Recently, we detected PC in four species in the genus Chlamydomonas: C. applanata NIES-2202, C. asymmetrica NIES-2207, C. debaryana NIES-2212, and C. sphaeroides NIES-2242. To reveal the PC biosynthesis pathways in green algae and the evolutionary scenario involved in their diversity, we analyzed the PC biosynthesis genes in these four algae using draft genome sequences. Homology searches suggested that PC in these species is synthesized by phosphoethanolamine-N-methyltransferase (PEAMT) and/or phosphatidylethanolamine-N-methyltransferase (PEMT), both of which are absent in C. reinhardtii. Recombinant PEAMTs from these algae showed methyltransferase activity for phosphoethanolamine but not for monomethyl phosphoethanolamine in vitro, in contrast to land plant PEAMT, which catalyzes the three methylations from phosphoethanolamine to phosphocholine. This suggested an involvement of other methyltransferases in PC biosynthesis. Here, we characterized the putative phospholipid-N-methyltransferase (PLMT) genes of these species by genetic and phylogenetic analysis. Complementation assays using a PC biosynthesis-deficient yeast suggested that the PLMTs of these algae can synthesize PC from phosphatidylethanolamine. These results indicated that the PC biosynthesis pathways in green algae differ from those of land plants, although the enzymes involved are homologous. Phylogenetic analysis suggested that the PEAMTs and PLMTs in these algae were inherited from the common ancestor of green algae. The absence of PC biosynthesis in many Chlamydomonas species is likely a result of parallel losses of PEAMT and PLMT in this genus.
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References
Bates PD, Stymne S, Ohlrogge J (2013) Biochemical pathways in seed oil synthesis. Curr Opin Plant Biol 16:358–364. https://doi.org/10.1016/j.pbi.2013.02.015
Bligh EG, Dyer WJ (1959) A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37:911–917. https://doi.org/10.1139/o59-099
Bobenchik AM, Augagneur Y, Hao B et al (2011) Phosphoethanolamine methyltransferases in phosphocholine biosynthesis: functions and potential for antiparasite therapy. FEMS Microbiol Rev 35:609–619. https://doi.org/10.1111/j.1574-6976.2011.00267.x
Bolognese CP, McGraw P (2000) The isolation and characterization in yeast of a gene for Arabidopsis S-adenosylmethionine:phospho-ethanolamine N-methyltransferase. Plant Physiol 124:1800–1813. https://doi.org/10.1104/pp.124.4.1800
Chen F (2006) OrthoMCL-DB: querying a comprehensive multi-species collection of ortholog groups. Nucleic Acids Res 34:D363–D368. https://doi.org/10.1093/nar/gkj123
Churchward MA, Brandman DM, Rogasevskaia T, Coorssen JR (2008) Copper (II) sulfate charring for high sensitivity on-plate fluorescent detection of lipids and sterols: quantitative analyses of the composition of functional secretory vesicles. J Chem Biol 1:79–87. https://doi.org/10.1007/s12154-008-0007-1
Datko AH, Mudd SH (1988) Enzymes of phosphatidylcholine synthesis in Lemna, soybean, and carrot. Plant Physiol 88:1338–1348. https://doi.org/10.1104/pp.88.4.1338
Degraeve-Guilbault C, Bréhélin C, Haslam R et al (2017) Glycerolipid characterization and nutrient deprivation-associated changes in the green picoalga Ostreococcus tauri. Plant Physiol 173:2060–2080. https://doi.org/10.1104/pp.16.01467
Edgar RC (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32:1792–1797. https://doi.org/10.1093/nar/gkh340
Furse S, de Kroon AIPM. (2015) Phosphatidylcholine’s functions beyond that of a membrane brick. Mol Membr Biol 32:117–119. https://doi.org/10.3109/09687688.2015.1066894
Geiger O, López-Lara IM, Sohlenkamp C (2013) Phosphatidylcholine biosynthesis and function in bacteria. Biochim Biophys Acta Mol Cell Biol Lipids 1831:503–513. https://doi.org/10.1016/j.bbalip.2012.08.009
Gibellini F, Smith TK (2010) The Kennedy pathway—De novo synthesis of phosphatidylethanolamine and phosphatidylcholine. IUBMB Life 62:414–428. https://doi.org/10.1002/iub.337
Giroud C, Gerber A, Eichenberger W (1988) Lipids of Chlamydomonas reinhardtii. Analysis of molecular species and intracellular site(s) of biosynthesis. Plant Cell Physiol 29:587–595
Guindon S, Dufayard JF, Lefort V et al (2010) New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Syst Biol 59:307–321. https://doi.org/10.1093/sysbio/syq010
Hanson AD, Rhodes D (1983) 14C tracer evidence for synthesis of choline and betaine via phosphoryl base intermediates in salinized sugarbeet leaves. Plant Physiol 71:692–700. https://doi.org/10.1104/pp.71.3.692
Hirashima T, Tajima N, Sato N (2016) Draft genome sequences of four species of Chlamydomonas containing phosphatidylcholine. Genome Announc 4:e01070–e01016. https://doi.org/10.1128/genomeA.01070-16
Hirashima T, Toyoshima M, Moriyama T et al (2017) Characterization of phosphoethanolamine-N-methyltransferases in green algae. Biochem Biophys Res Commun 488:141–146. https://doi.org/10.1016/j.bbrc.2017.05.026
Hitz WD, Rhodes D, Hanson AD (1981) Radiotracer evidence implicating phosphoryl and phosphatidyl bases as intermediates in betaine synthesis by water-stressed barley leaves. Plant Physiol 68:814–822. https://doi.org/10.1104/pp.68.4.814
Hu Q, Sommerfeld M, Jarvis E et al (2008) Microalgal triacylglycerols as feedstocks for biofuel production: perspectives and advances. Plant J 54:621–639. https://doi.org/10.1111/j.1365-313X.2008.03492.x
Johnston M, Riles L, Hegemann JH (2002) Gene disruption. Methods Enzym 350:290–315. https://doi.org/10.1016/S0076-6879(02)50970-8
Kanipes MI, Henry SA (1997) The phospholipid methyltransferases in yeast. Biochim Biophys Acta 1348:134–141. https://doi.org/10.1016/S0005-2760(97)00121-5
Katoh K, Standley DM (2013) MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol 30:772–780. https://doi.org/10.1093/molbev/mst010
Keogh MR, Courtney PD, Kinney AJ, Dewey RE (2009) Functional characterization of phospholipid N-methyltransferases from Arabidopsis and soybean. J Biol Chem 284:15439–15447. https://doi.org/10.1074/jbc.M109.005991
Kurotani A, Yamada Y, Sakurai T (2017) Alga-PrAS (Algal Protein Annotation Suite): a database of comprehensive annotation in algal proteomes. Plant Cell Physiol 58:e6. https://doi.org/10.1093/pcp/pcw212
Lee SG, Jez JM (2014) Nematode phospholipid metabolism: an example of closing the genome-structure-function circle. Trends Parasitol 30:241–250. https://doi.org/10.1016/j.pt.2014.03.001
Lykidis A (2007) Comparative genomics and evolution of eukaryotic phospholipid biosynthesis. Prog Lipid Res 46:171–199. https://doi.org/10.1016/j.plipres.2007.03.003
Merchant SS, Prochnik SE, Vallon O et al (2007) The Chlamydomonas genome reveals the evolution of key animal and plant functions. Science 318:245–250. https://doi.org/10.1126/science.1143609
Mori N, Moriyama T, Toyoshima M, Sato N (2016) Construction of global acyl lipid metabolic map by comparative genomics and subcellular localization analysis in the red alga Cyanidioschyzon merolae. Front Plant Sci 7:958. https://doi.org/10.3389/fpls.2016.00958
Mudd SH, Datko AH (1986) Phosphoethanolamine bases as intermediates in phosphatidylcholine synthesis by Lemna. Plant Physiol 82:126–135. https://doi.org/10.1104/pp.82.1.126
Nuccio ML, Ziemak MJ, Henry SA et al (2000) cDNA cloning of phosphoethanolamine N-methyltransferase from spinach by complementation in Schizosaccharomyces pombe and characterization of the recombinant enzyme. J Biol Chem 275:14095–14101. https://doi.org/10.1074/jbc.275.19.14095
Riekhof WR, Wu J, Gijón MA et al (2007) Lysophosphatidylcholine metabolism in Saccharomyces cerevisiae: the role of P-type ATPases in transport and a broad specificity acyltransferase in acylation. J Biol Chem 282:36853–36861. https://doi.org/10.1074/jbc.M706718200
Ronquist F, Teslenko M, Van Der Mark P et al (2012) MrBayes 3.2: efficient bayesian phylogenetic inference and model choice across a large model space. Syst Biol 61:539–542. https://doi.org/10.1093/sysbio/sys029
Sakurai K, Mori N, Sato N (2014) Detection and characterization of phosphatidylcholine in various strains of the genus Chlamydomonas (Volvocales, Chlorophyceae). J Plant Res 127:641–650. https://doi.org/10.1007/s10265-014-0644-0
Sato N, Furuya M (1985) Distribution of diacylglyceryltrimethylhomoserine and phosphatidylcholine in non-vascular green plants. Plant Sci 38:81–85. https://doi.org/10.1016/0168-9452(85)90134-7
Sato N, Moriyama T (2007) Genomic and biochemical analysis of lipid biosynthesis in the unicellular rhodophyte Cyanidioschyzon merolae: lack of a plastidic desaturation pathway results in the coupled pathway of galactolipid synthesis. Eukaryot Cell 6:1006–1017. https://doi.org/10.1128/EC.00393-06
Sato N, Mori N, Hirashima T, Moriyama T (2016) Diverse pathways of phosphatidylcholine biosynthesis in algae as estimated by labeling studies and genomic sequence analysis. Plant J 87:281–292. https://doi.org/10.1111/tpj.13199
Shields DJ, Altarejos JY, Wang X et al (2003) Molecular dissection of the S-adenosylmethionine-binding site of phosphatidylethanolamine N-methyltransferase. J Biol Chem 278:35826–35836. https://doi.org/10.1074/jbc.M306308200
Smith DD, Summers PS, Weretilnyk EA (2000) Phosphocholine synthesis in spinach: characterization of phosphoethanolamine N-methyltransferase. Physiol Plant 108:286–294. https://doi.org/10.1034/j.1399-3054.2000.108003286.x
Vance DE (2014) Phospholipid methylation in mammals: from biochemistry to physiological function. Biochim Biophys Acta Biomembr 1838:1477–1487. https://doi.org/10.1016/j.bbamem.2013.10.018
Watanabe A (1960) List of algal strains in collection at the Institute of Applied Microbiology, University of Tokyo. J Gen Appl Microbiol 6:283–292. https://doi.org/10.2323/jgam.6.283
Yumoto K, Kasai F, Kawachi M (2013) Taxonomic re-examination of Chlamydomonas strains maintained in the NIES-Collection. Microbiol Cult Collect 29:1–12
Acknowledgements
The authors thank Dr. Ikuo Nishida, Saitama University, for helpful advice in the initial phase of the work, Drs. Takahiro Nakamura and Kunihiro Ohta for technical advice, and Dr. Hajime Wada for valuable discussion and encouragement. This work was supported, in part, by Core Research for Evolutional Science and Technology, Japan Science and Technology Agency.
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Hirashima, T., Toyoshima, M., Moriyama, T. et al. Evolution of the Phosphatidylcholine Biosynthesis Pathways in Green Algae: Combinatorial Diversity of Methyltransferases. J Mol Evol 86, 68–76 (2018). https://doi.org/10.1007/s00239-017-9826-4
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DOI: https://doi.org/10.1007/s00239-017-9826-4