Introduction

Phosphatidylcholine (PC), a common phospholipid component of eukaryotic cellular membranes, is not only a building block of lipid bilayers but is also a key regulatory molecule in various biological processes, such as signal transduction and metabolic regulation (Furse and de Kroon 2015). However, this phospholipid has not been detected in some species of green algae, including Chlamydomonas reinhardtii, Acetabularia calyculus, and Closterium acerosum (Sato and Furuya 1985; Giroud et al. 1988). In C. reinhardtii, the betaine lipid diacylglyceryl-N,N,N-trimethylhomoserine (DGTS) is believed to replace PC as an alternative zwitterionic lipid, and this could be advantageous for survival in phosphorus-deficient environments (Giroud et al. 1988). We still do not know how triacylglycerol (TAG), a target of algal biofuel production (Hu et al. 2008), can be biosynthesized without PC, which provides both diacylglycerol (DAG) and acyl groups for the synthesis of TAG in plants (Bates et al. 2013).

The PC synthesis pathway differs among eukaryotes (Lykidis 2007). In the classical Kennedy pathway, free choline is activated to phosphocholine (P-Cho), and then to cytidine 5′-diphosphocholine (CDP-choline), which ultimately provides a choline moiety for PC synthesis (Gibellini and Smith 2010). This pathway, although well-known and important in human nutrition, likely evolved recently, because choline is produced in nature only by the degradation of pre-existing PC. Therefore, this pathway acts to recycle choline. Some bacteria possess a different choline recycling pathway involving the direct addition of choline to CDP-diacylglycerol to synthesize PC (Geiger et al. 2013), but this is beyond the scope of our study.

De novo synthesis of choline or PC requires the methylation of ethanolamine, as either phosphoethanolamine (P-Etn) or phosphatidylethanolamine (PE). PC is synthesized from PE via three methylation steps. Similar three-step methylation provides P-Cho from P-Etn (Fig. 1a). PE methylation is catalyzed by two methyltransferases using S-adenosylmethionine (SAM) as a methyl donor, namely, phosphatidylethanolamine-N-methyltransferase (PEMT) and phospholipid-N-methyltransferase (PLMT), both of which are integral membrane enzymes (Lykidis 2007). Phosphatidyl-N-monomethylethanolamine (PMME) and phosphatidyl-N,N-dimethylethanolamine (PDME) are the intermediates in this reaction series. Some bacteria synthesize PC via the methylation of PE catalyzed by a soluble SAM-dependent methyltransferase called PmtA, which is not found in eukaryotes (Geiger et al. 2013). A soluble methyltransferase called phosphoethanolamine-N-methyltransferase (PEAMT) catalyzes the three methylations of P-Etn, a soluble substrate (Bobenchik et al. 2011). This enzyme was originally discovered in plants, but it has also been found in nematodes and malaria parasites (Lee and Jez 2014). After the complete methylation of P-Etn, the product, P-Cho, is incorporated into PC by the Kennedy pathway.

Fig. 1
figure 1

Methyltransferases involved in PC biosynthesis in eukaryotes. a Universal PC biosynthesis pathways in eukaryotes. b Presence and absence of PC biosynthetic methyltransferases in photosynthetic eukaryotes. The presence of homologs in the Alga-PrAS database is marked by boxes. Phylogenetic relationships are shown as in the Alga-PrAS database

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The PC biosynthesis pathway in plants has been studied using pulse-chase experiments (Hitz et al. 1981; Hanson and Rhodes 1983; Mudd and Datko 1986) and enzymatic assays (Datko and Mudd 1988), and was confirmed by the identification of PEAMT (Nuccio et al. 2000; Smith et al. 2000) and PLMT (Keogh et al. 2009) in plants. In fungi, PEMT catalyzes the first methylation step from PE to PMME, whereas PLMT catalyzes the next two steps from PMME to PC (Kanipes and Henry 1997). In plants, PE is not methylated, because only PLMT is present and not PEMT (Keogh et al. 2009). The first methylation of the ethanolamine moiety is uniquely performed by PEAMT, generating phosphomonomethylethanolamine (P-MMEtn). This reaction also produces phosphodimethylethanolamine (P-DMEtn) and P-Cho, but P-MMEtn can be used to produce PMME. By contrast, all three methylation steps are catalyzed by PLMT in mammals, which lack PEMT and PEAMT (Vance 2014). Red algae possess PEMT and PLMT, but not PEAMT, and are thought to synthesize PC via the methylation of PE, as in fungi (Fig. 1b, Sato and Moriyama 2007; Mori et al. 2016). In green algae, the PC biosynthesis pathway remains unclear and the genes involved are not conserved (Fig. 1b).

The green alga C. reinhardtii lacks PC and all of the PC biosynthesis enzymes (Merchant et al. 2007). However, we previously found PC in several related species: C. applanata NIES-2202, C. asymmetrica NIES-2207, C. debaryana NIES-2212, and C. sphaeroides NIES-2242 (Sakurai et al. 2014). Because the species possessing PC are not monophyletic, PC biosynthesis might have been lost independently in different branches within the genus Chlamydomonas. Alternatively, PC biosynthesis could have been acquired independently in various species after the initial loss of PC biosynthesis at the root of the Chlamydomonas clade. Tracer experiments using radioactive phosphate suggested that the PC biosynthesis pathway differs in C. asymmetrica and C. sphaeroides (Sato et al. 2016). Draft genome sequences of the four species possessing PC showed PEAMT homologs in C. applanata and C. asymmetrica, and PLMT homologs in all four species (Table 1, Hirashima et al. 2016). We succeeded in detecting methyltransferase activity for P-Etn, but not for P-MMEtn, in the recombinant PEAMT homologs from C. applanata and C. asymmetrica (called CapPEAMT and CasPEAMT, respectively). This suggested that the two subsequent methylations could be performed using PE derivatives (PMME and PDME) as substrate, perhaps catalyzed by PLMT (Hirashima et al. 2017).

Table 1 Characteristics of the reference strain and the PC-containing Chlamydomonas species
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To substantiate the presence of putative PLMT enzymes in the green algae and to develop an evolutionary scenario for PC biosynthesis in plants and algae, we determined the cDNA sequences of the four PLMT homologs in C. applanata (CapPLMT), C. asymmetrica (CasPLMT), C. debaryana (CdePLMT), and C. sphaeroides (CspPLMT). Complementation assays of yeast PC biosynthesis mutants showed that the PLMT homologs of C. debaryana and C. sphaeroides have activity for the three successive methylations of PE. A phylogenetic analysis revealed that these homologs are closely related to the homologs found in other green algae and plants, and could have been inherited vertically from the common ancestor of Viridiplantae. Our analysis indicated that parallel losses of PEAMT and PLMT in the genus Chlamydomonas could account for the absence of PC in C. reinhardtii and related species. The ability to synthesize PC with PEAMT and PLMT must have been present in the common ancestor of green algae and land plants, but the combinatorial diversity in the substrate specificities of these enzymes engendered diversity in PC biosynthesis pathways in green algae and land plants.

Materials and Methods

Algal Strains and Culture Conditions

Chlamydomonas applanata NIES-2202, C. asymmetrica NIES-2207, C. debaryana NIES-2212, and C. sphaeroides NIES-2242 were obtained from the Microbial Culture Collection of the National Institution for Environmental Studies, Japan. The cells were grown in modified Bristol’s Medium (Watanabe 1960) at 25 °C under continuous light (45 µmol m−2 s−1) with continuous aeration with 1% CO2 in air.

Cloning and Sequence Analysis

Total RNA was extracted from each algal strain in the exponential growth phase using the RNeasy Plant Mini kit and RNase-Free DNase set (QIAGEN, Venlo, the Netherlands). Complementary DNA was prepared from the RNA using the SMARTer RACE cDNA Amplification Kit (Takara Bio, Kusatsu, Japan). The cDNA sequences of PLMT genes in the algal strains containing 5′- or 3′-ends were amplified by PCR using universal primers and gene-specific primers designed using the results of homology searches of the draft genome sequences. The PCR products were cloned into the pCRII-Blunt-TOPO vector (Thermo Fischer Scientific, Waltham, MA, USA), and the cDNA sequences were determined by the Eurofins Genomics sequencing service (Tokyo, Japan). The nucleotide sequences of CapPLMT, CasPLMT, CdePLMT, and CspPLMT were deposited in the DNA Data Bank of Japan (DDBJ) under accession numbers LC228966 to LC228969, respectively.

The PLMT sequences were aligned using MAFFT software (L-INS-i method, ver. 7.310) (Katoh and Standley 2013). The RefSeq accession numbers of the protein sequences were as follows: Monoraphidium neglectum PLMT, XP_013902006.1; Auxenochlorella protothecoides, XP_011400892.1; Chlorella variabilis, XP_005851476.1; Coccomyxa subellipsoidea C-169, XP_005647515.1; Arabidopsis thaliana, NP_565246.1; and Homo sapiens, NP_680478.1. The Klebsormidium nitens PLMT sequence (kfl00100_g13_v1.1) was obtained from the Klebsormidium genome project (http://www.plantmorphogenesis.bio.titech.ac.jp/~algae_genome_project/klebsormidium/).

Heterologous Complementation of the S. Cerevisiae cho2∆opi3∆ Mutant

A cho2∆opi3∆ deletion mutant of S. cerevisiae (strain YPH499; cho2Δ::KanMX opi3Δ::HphMX4) was generated by homologous recombination (Johnston et al. 2002). The open reading frames of CapPEAMT, CasPEAMT, CapPLMT, CasPLMT, CdePLMT, and CspPLMT were amplified by PCR and cloned into the pORF-CLONE vector (Boca Scientific, Boca Raton, FL, USA) under the control of the copper-inducible CUP1 promoter. The constructs were transformed into the cho2∆opi3∆ mutant, and the transformants were screened on SC-Ura solid medium containing 100 µM choline. To determine growth requirements, 10-fold serial dilutions of yeast cultures were spotted onto SC-Ura solid medium containing 50 µM CuSO4 with or without 100 µM choline and incubated for 3 days at 30 °C.

For the lipid analysis, the lipids were extracted from yeast cells incubated for 1 day in SC-Ura liquid medium containing 50 µM CuSO4 according to the method of Bligh and Dyer (1959), separated by thin layer chromatography using n-propanol/propionate/chloroform/water (3:2:2:1, by volume) as the running solvent, and stained with 10% CuSO4, 8% H3PO4 (w/v) (Churchward et al. 2008). Authentic PE and PC were prepared from wild-type S. cerevisiae cells. Authentic PMME and PDME were prepared from the S. cerevisiae opi3Δ mutant incubated in SC medium containing 1 mM dimethylethanolamine.

Phylogenetic Analysis

Homologs of PLMT were collected using BLAST searches of the Alga-PrAS (Kurotani et al. 2017) and OrthoMCL-DB (Chen 2006) databases. The protein sequences for each homolog group were aligned using Muscle v3.6 software (Edgar 2004). Sequences that were not aligned over their entire length were removed, and the remaining sequences were re-aligned. The alignments were used for phylogenetic analysis as follows. The maximum likelihood tree was constructed using PhyML ver. 20151208 software (the options were: -d aa –m LG –s BEST –b -5) (Guindon et al. 2010). Bayesian Inference analysis was performed using MrBayes ver. 3.2.6 software, using the LG + I + G model with 2,000,000 iterations (Ronquist et al. 2012).

Results and Discussion

Functional Characterization of Chlamydomonas PLMTs

To characterize the Chlamydomonas PLMTs, we determined the full-length cDNA sequences of the PLMT homologs in C. applanata, C. asymmetrica, C. debaryana, and C. sphaeroides. As shown in Fig. 2, the deduced amino acid sequences of the Chlamydomonas PLMTs were similar to those of closely related green algae and plants. Although the detailed structure of PLMT is not known, residues involved in binding to SAM identified by mutagenesis experiments (Shields et al. 2003) are conserved in the Chlamydomonas PLMTs.

Fig. 2
figure 2

Alignment of the PLMT amino acid sequences. Residues corresponding to the transmembrane domains and SAM-binding residues of human PLMT are shown in gray and black, respectively. Abbreviated species names: Monoraphidium, Monoraphidium neglectum; Auxenochlorella, Auxenochlorella protothecoides; Chlorella, Chlorella variabilis; Coccomyxa, Coccomyxa subellipsoidea C-169; Klebsormidium, Klebsormidium nitens; Arabidopsis, Arabidopsis thaliana; Human, Homo sapiens. The accession numbers of the protein sequences used for the alignment are given in the “Materials and Methods”

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To verify the enzymatic activities of CapPLMT, CasPLMT, CdePLMT, CspPLMT, CapPEAMT, and CasPEAMT, we cloned each of the corresponding cDNAs into a yeast expression vector and transformed each construct into the S. cerevisiae cho2Δopi3Δ mutant. As previously reported, the cho2Δopi3Δ mutant lacks PE methylation activity and requires supplementation with exogenous choline or lyso-PC for growth (Riekhof et al. 2007). Heterologous expression of Arabidopsis PEAMT (AT3G18000) complemented the choline auxotrophy of this mutant (Bolognese and McGraw 2000). As shown in Fig. 3, the cho2Δopi3Δ mutant harboring CdePLMT or CspPLMT grew without choline and synthesized PC, which suggested that CdePLMT and CspPLMT are the functional PLMTs possessing methylation activity from PE to PC. The cho2Δopi3Δ mutant harboring CapPEAMT and CasPEAMT did not grow without choline and accumulated P-MMEtn (Fig. 3), which confirmed our previous finding that the recombinant CapPEAMT and CasPEAMT proteins catalyzed the methylation of P-Etn, but not that of P-MMEtn (Hirashima et al. 2017). CapPLMT and CasPLMT failed to complement the mutant: Fig. 3b shows only the lipid analysis in the experiment with CapPLMT because CasPLMT-complemented cells did not grow and died without PC. The CapPLMT-complemented cells could grow without PC supplementation, but the PC level decreased to a very low level by the time of analysis. These results might reflect a lack of PE methylation activity in these enzymes. The results showed that the C. debaryana and C. sphaeroides PLMTs catalyze all three methylations of PE, which is consistent with the absence of PEAMT in these algae (Hirashima et al. 2017). The substrate specificity or substrate range of PLMT could differ between green algae and land plants. A comparable difference in substrate range can be found in yeast and human PLMTs, as described above. These findings suggest that the substrate range of PLMT has been changed in diverse lineages. As the overall structures of the enzymes were highly conserved (Fig. 2), this specificity change could result from subtle mutations.

Fig. 3
figure 3

Heterologous complementation of the S. cerevisiae cho2Δopi3Δ mutant by Chlamydomonas PEAMTs or PLMTs. a Growth of the wild type, cho2Δopi3Δ, and cho2Δopi3Δ harboring Chlamydomonas PEAMT or PLMT on SC-Ura containing 50 µM CuSO4 with (+ Cho) or without (− Cho) 100 µM choline. Ten-fold serial dilutions of the indicated strains from left to right starting at an OD600 of 0.1 were spotted onto solid medium and incubated for 3 days. b The lipid content of the wild type, cho2Δopi3Δ, and cho2Δopi3Δ harboring Chlamydomonas PEAMT or PLMT. Total lipid was extracted from each strain after incubation in SC-Ura liquid medium containing 50 µM CuSO4, and separated by thin layer chromatography, using PE, PMME, PDME, and PC as standards (left lanes). Lipids from cho2Δopi3Δ harboring CasPLMT were not analyzed because the cells died during the incubation without choline

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Phylogenetic Analysis of PLMT

We performed phylogenetic analysis with selected PLMT sequences for algae and plants (Fig. 4). The deduced phylogenetic tree showed that all four Chlamydomonas PLMTs and those of the green algae Monoraphidium, Coccomyxa, Auxenochlorella, and Chlorella were closely related and formed a sister clade with plant PLMTs (Fig. 4). This indicated that Chlamydomonas PLMTs have been transmitted vertically from the common ancestor of Viridiplantae. By contrast, the PLMTs in prasinophytes (Micromonas, Bathycoccus, and Ostreococcus) and other algae (Fragilariopsis, Phaeodactylum, Aureococcus, Bigelowiella, Emiliania, and Guillardia) clustered in a different clade (Fig. 4). These PLMT homologs were relatively large (> 600 amino acid residues) compared with other PLMTs (150–250 amino acid residues). Note that some prasinophyte species, including Ostreococcus tauri, lack PC (Sato and Furuya 1985; Degraeve-Guilbault et al. 2017). The function of the large versions of PLMT in these PC-lacking algae remains to be elucidated. Recently, the phosphosulfolipid, phosphatidyldimethylpropanethiol, was identified as one of the major membrane lipids in O. tauri (Degraeve-Guilbault et al. 2017). The PLMT homologs of prasinophytes could be involved in the biosynthesis of the phosphosulfolipids or betaine lipids in these algae.

Fig. 4
figure 4

sPhylogenetic tree of PLMT. The Bayesian consensus tree is shown and the values on each branch indicate the posterior probability/confidence level of the branch (Bayesian Inference/Maximum Likelihood). For simplicity, 1.00 is shown as “1.” The bar at the bottom is the distance scale

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Evolution of PC Biosynthesis Pathways in Plants and Green Algae

Our results indicated that the putative PLMT is indeed involved in PC biosynthesis and potentially catalyzes all three methylations from PE to PC in C. applanata, C. asymmetrica, C. debaryana, and C. sphaeroides. The phylogenetic analysis showed that Chlamydomonas PLMTs are related to those of land plants, although the biochemical analysis suggested that the substrate ranges differ. Based on the results, we believe that Chlamydomonas and plants have the same sets of enzymes potentially involved in PC biosynthesis, but that the diversity in the substrate ranges of both PEAMT and PLMT results in various PC biosynthesis pathways (Fig. 5). The phylogenetic analysis suggested that PLMTs in Chlamydomonas were the product of a vertical heritage, and not of horizontal gene transfer. Previously, we presented evidence for the vertical heritage of PEAMT in Chlamydomonas (Hirashima et al. 2017). Based on these findings, parallel losses of PEAMT and PLMT within various clades of the genus Chlamydomonas seems to be a plausible explanation for the absence of PC in C. reinhardtii and other species (Fig. 5). An alternative hypothesis, namely, the independent acquisition of these genes in different lineages seems less likely.

Fig. 5
figure 5

Schematic model of the evolution of PC biosynthesis pathways based on the combinatorial diversity of methyltransferases in plants and algae

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We can explain the different PC biosynthesis pathways in various organisms in the following way. We believe that the common ancestor of green algae and land plants had PC, like other eukaryotic organisms. It likely possessed PEAMT and PLMT, which both had a full range of substrate specificity for unmethylated, monomethylated, and dimethylated ethanolamine derivatives. If the specificity for these substrates is indicated by simple numbers, such as 1, 2, and 3, we can call these “all-powerful” enzymes PEAMT_123 and PLMT_123, respectively. During the course of evolution, the substrate range could have been reduced with a loss of activity for the unmethylated derivatives, resulting in PEAMT_23 and PLMT_23, respectively, or by losing the activity for the mono- and dimethylated derivatives, resulting in PEAMT_1 and PLMT_1, respectively. This inference is purely theoretical. According to this diversification of substrate specificities, PEAMT and PLMT are believed to be diversified. We can enumerate possible combinations of these different isozymes that can produce PC in various organisms as follows:

  • PEAMT_123: malaria parasites and nematodes.

  • PLMT_123: C. debaryana and C. sphaeroides, and mammals.

  • PEAMT_1 and PLMT_23: C. applanata and C. asymmetrica.

  • PEAMT_123 and PLMT_23: land plants.

If we also consider PEMT (equivalent to PLMT_1), additional pathways can also be realized:

  • PEMT and PLMT_23: red algae and yeasts.

  • PEMT and PEAMT_123 and/or PLMT_123: yet to be found.

In addition, the loss of PC may be compensated for by the synthesis of DGTS (or phosphosulfolipid as described above); thus, creating more diversity.

The creation of diversity is essential in biological evolution. Mutations in a single enzyme or gene can be compensated for in various different ways in the actual organisms. As explained above, there are several different pathways that produce PC and they differ in eukaryotes and prokaryotes. Even in eukaryotic organisms, various pathways are possible, and many of them are found in different organisms. These findings convinced us that evolution is driven by the trial-and-error of possible combinations of enzymes. Green algae are a good example of evolutionary flexibility.