Functional Characterization of Phospholipid N-Methyltransferases from Arabidopsis and Soybean

Abstract

Phospholipid N-methyltransferase (PLMT) enzymes catalyze the S-adenosylmethionine-dependent methylation of ethanolamine-containing phospholipids to produce the abundant membrane lipid phosphatidylcholine (PtdCho). In mammals and yeast, PLMT activities are required for the de novo synthesis of the choline headgroup found in PtdCho. PLMT enzyme activities have also been reported in plants, yet their roles in PtdCho biosynthesis are less clear because most plants can produce the choline headgroup entirely via soluble substrates, initiated by the methylation of free ethanolamine-phosphate. To gain further insights into the function of PLMT enzymes in plants, we isolated PLMT cDNAs from Arabidopsis and soybean (Glycine max) based upon primary amino acid sequence homology to the rat PLMT, phosphatidylethanolamine N-methyltransferase. Using a heterologous yeast expression system, it was shown that plant PLMTs methylate phosphatidylmonomethylethanolamine and phosphatidyldimethylethanolamine but cannot utilize phosphatidylethanolamine as a substrate. Identification of an Arabidopsis line containing a knock-out dissociator transposon insertion within the single copy AtPLMT gene allowed us to investigate the consequences of loss of PLMT function. Although the accumulation of the PLMT substrates phosphatidylmonomethylethanolamine and phosphatidyldimethylethanolamine was considerably elevated in the atplmt knock-out line, PtdCho levels remained normal, and no obvious differences were observed in plant morphology or development under standard growth conditions. However, because the metabolic routes through which PtdCho is synthesized in plants vary greatly among differing species, it is predicted that the degree with which PtdCho synthesis is dependent upon PLMT activities will also vary widely throughout the plant kingdom. Phospholipid N-methyltransferase (PLMT) enzymes catalyze the S-adenosylmethionine-dependent methylation of ethanolamine-containing phospholipids to produce the abundant membrane lipid phosphatidylcholine (PtdCho). In mammals and yeast, PLMT activities are required for the de novo synthesis of the choline headgroup found in PtdCho. PLMT enzyme activities have also been reported in plants, yet their roles in PtdCho biosynthesis are less clear because most plants can produce the choline headgroup entirely via soluble substrates, initiated by the methylation of free ethanolamine-phosphate. To gain further insights into the function of PLMT enzymes in plants, we isolated PLMT cDNAs from Arabidopsis and soybean (Glycine max) based upon primary amino acid sequence homology to the rat PLMT, phosphatidylethanolamine N-methyltransferase. Using a heterologous yeast expression system, it was shown that plant PLMTs methylate phosphatidylmonomethylethanolamine and phosphatidyldimethylethanolamine but cannot utilize phosphatidylethanolamine as a substrate. Identification of an Arabidopsis line containing a knock-out dissociator transposon insertion within the single copy AtPLMT gene allowed us to investigate the consequences of loss of PLMT function. Although the accumulation of the PLMT substrates phosphatidylmonomethylethanolamine and phosphatidyldimethylethanolamine was considerably elevated in the atplmt knock-out line, PtdCho levels remained normal, and no obvious differences were observed in plant morphology or development under standard growth conditions. However, because the metabolic routes through which PtdCho is synthesized in plants vary greatly among differing species, it is predicted that the degree with which PtdCho synthesis is dependent upon PLMT activities will also vary widely throughout the plant kingdom. Phosphatidylcholine (PtdCho) 2The abbreviations used are:PtdChophosphatidylcholinePLMTphospholipid N-methyltransferasePtdMMEtnphosphatidylmonomethylethanolaminePtdDMEtnphosphatidyldimethylethanolaminePtdEtnphosphatidylethanolamineMMEtn-Pmonomethylethanolamine-phosphatePEAMTphosphoethanolamine N-methyltransferasePEMTphosphatidylethanolamine N-methyltransferaseAdoMetS-adenosylmethionineESI/MS/MSelectrospray ionization/tandem mass spectrometrydH2Odistilled H2OMES4-morpholineethanesulfonic acidDsdissociator. is the most abundant phospholipid in most non-plastid membranes of eukaryotes. PtdCho biosynthesis has been studied intensively in plants not only because of its importance as a structural membrane lipid, but also because of its role as a precursor to important lipid-based signaling molecules, such as phosphatidic acid, and phospholipase A2-derived free fatty acids (1.Meijer H.J. Munnik T. Annu. Rev. Plant Biol. 2003; 54: 265-306Crossref PubMed Scopus (486) Google Scholar). The choline headgroup of PtdCho serves multiple functions as well. In addition to being an essential human nutrient (2.Zeisel S.H. IUBMB Life. 2007; 59: 380-387Crossref PubMed Scopus (60) Google Scholar), in many plant species choline can be oxidized to produce the potent osmoprotectant glycine betaine (3.Hitz W.D. Rhodes D. Hanson A.D. Plant Physiol. 1981; 68: 814-822Crossref PubMed Google Scholar, 4.Rontein D. Basset G. Hanson A.D. Metab. Eng. 2002; 4: 49-56Crossref PubMed Scopus (320) Google Scholar). phosphatidylcholine phospholipid N-methyltransferase phosphatidylmonomethylethanolamine phosphatidyldimethylethanolamine phosphatidylethanolamine monomethylethanolamine-phosphate phosphoethanolamine N-methyltransferase phosphatidylethanolamine N-methyltransferase S-adenosylmethionine electrospray ionization/tandem mass spectrometry distilled H2O 4-morpholineethanesulfonic acid dissociator. For over 2 decades it has been apparent that there are fundamental differences between the manner in which PtdCho is produced in plants versus how it is synthesized in mammals and fungi. In the latter two systems, PtdCho can be formed through two distinct pathways as follows: (a) the “nucleotide pathway” in which free choline is incorporated in PtdCho using CDP-choline as an intermediate, and (b) the “methylation pathway” whereby PtdCho is produced directly from phosphatidylethanolamine (PtdEtn) via three sequential methylation reactions using S-adenosylmethionine (AdoMet) as the methyl donor (5.Carman G.M. Henry S.A. Annu. Rev. Biochem. 1989; 58: 635-669Crossref PubMed Google Scholar, 6.Vance D.E. Biochemistry of Lipids, Lipoproteins, and Membranes.in: Vance D.E. Vance J.E. Elsevier Science Publishers B.V., Amsterdam, The Netherlands1996: 153-180Google Scholar). In contrast, PtdCho biosynthesis in plants occurs through a branched pathway that utilizes components of both the nucleotide and methylation pathways (7.Kinney A.J. Lipid Metabolism in Plants.in: Moore T.S. CRC Press, Inc., Boca Raton, FL1993: 259-284Google Scholar). The greatest distinction between the contrasting mechanisms of PtdCho biosynthesis can be attributed to the presence of plant enzymes that are capable of converting ethanolamine headgroups to choline at the phospho-base level, activities that are absent in mammals and yeast. Conversely, mammals and fungi possess methylation enzymes that act directly on PtdEtn, a reaction that cannot be detected in most plant systems investigated (reviewed in Ref. 7.Kinney A.J. Lipid Metabolism in Plants.in: Moore T.S. CRC Press, Inc., Boca Raton, FL1993: 259-284Google Scholar). A diagram of the most widely accepted model of phosphoamino alcohol biosynthesis in plants is shown in Fig. 1. Similar to animals and yeast, free choline can be directly incorporated into PtdCho via nucleotide pathway enzymes in plants. In the absence of choline, however, the methylation of Etn-phosphate represents the first committed step in PtdCho biosynthesis. The resulting monomethylethanolamine-phosphate (MMEtn-P) metabolite can be further methylated at the phospho-base level to produce Cho-P. Alternatively, MMEtn-P can be incorporated into phosphatidylmonomethylethanolamine (PtdMMEtn) via the cytidylyltransferase and amino alcohol phosphotransferase activities of the nucleotide pathway and then methylated at the phosphatidyl-base level to complete the synthesis of PtdCho (Fig. 1). The extent with which PtdCho is formed by the flow of metabolites through phospho-bases as opposed to phosphatidyl-bases varies greatly among different plant species. In most higher plants, it is likely that the methylation of the phosphoamino alcohol headgroups involves the flow of metabolites through both branches of the pathway, as has been shown in species such as barley, carrot, and tobacco (3.Hitz W.D. Rhodes D. Hanson A.D. Plant Physiol. 1981; 68: 814-822Crossref PubMed Google Scholar, 8.Datko A.H. Mudd S.H. Plant Physiol. 1988; 88: 1338-1348Crossref PubMed Google Scholar, 9.McNeil S.D. Nuccio M.L. Rhodes D. Shachar-Hill Y. Hanson A.D. Plant Physiol. 2000; 123: 371-380Crossref PubMed Scopus (58) Google Scholar). Nevertheless, examples have also been reported where only one of the branches appears to be utilized. In Lemna paucicostata, for example, the methylation steps in PtdCho biosynthesis were shown to occur almost exclusively at the phospho-base level (10.Mudd S.H. Datko A.H. Plant Physiol. 1986; 82: 126-135Crossref PubMed Google Scholar). At the other end of the spectrum is soybean, where all methylations beyond the initial formation of MMEtn-P were reported to occur on phosphatidyl-bases (8.Datko A.H. Mudd S.H. Plant Physiol. 1988; 88: 1338-1348Crossref PubMed Google Scholar, 11.Datko A.H. Mudd S.H. Plant Physiol. 1988; 88: 854-861Crossref PubMed Google Scholar). The tremendous variability observed among plants with regard to PtdCho formation is also exemplified by a study conducted by Williams and Harwood (12.Williams M. Harwood J.L. Biochem. J. 1994; 304: 463-468Crossref PubMed Scopus (24) Google Scholar) where it was shown that the predominant route of PtdCho synthesis in olive culture cells involved the first two methylation reactions taking place at the phospho-base level (producing dimethylethanolamine phosphate) and the final methylation occurring on a phosphatidyldimethylethanolamine (PtdDMEtn) substrate. Our understanding on the mechanisms by which plants synthesize PtdCho and regulate its accumulation has been further enhanced as the the steps of the phosphoamino alcohol pathway have been isolated and For example, to the that all of the amino alcohol phosphotransferase reactions in Fig. can be by the of a single gene that a Plant 1994; Google Scholar, Plant Physiol. Biochem. Scopus Google Scholar). it was the of the phosphoethanolamine from Arabidopsis and that to the that all three phospho-base methylation reactions be by a single enzyme Plant Physiol. 2000; PubMed Scopus Google Scholar, M.L. Henry S.A. Hanson A.D. J. Biol. 2000; PubMed Scopus Google Scholar). of gene function in Arabidopsis through insertion or between the phosphoamino alcohol pathway and and Y. J. Y. J. Plant 2002; PubMed Scopus Google Scholar, J. G. Plant PubMed Scopus Google Scholar). Although most of the reactions in Fig. have been at the level, absent is on the plant for the methylation reactions conducted at the phosphatidyl-base In contrast, reactions are among the most in animals and yeast, by enzymes to as phospholipid In the membrane phosphatidylethanolamine N-methyltransferase is a PLMT that is in the Vance D.E. J. Biol. PubMed Google Scholar). all three of the methylation reactions to to PtdCho. two distinct PLMT enzymes to catalyze the three methylation reactions as follows: that the methylation of to produce T. J. Biol. PubMed Google Scholar, Henry S.A. 1988; PubMed Google Scholar), and an enzyme to the that the methylation of to and to the final two steps of the methylation pathway T. J. Biol. PubMed Google Scholar, Henry S.A. 1989; PubMed Google Scholar). PLMT activities are in both of knock-out are dependent on choline for and levels of choline metabolites within the and with choline J. S.H. Biochem. J. 2003; PubMed Scopus Google Scholar). PLMT activities are choline to synthesize PtdCho de novo in the absence of To gain a understanding of the function of PLMT reactions in higher plants, and their PtdCho we and PLMT from Arabidopsis and the cDNAs in yeast, we were to that PLMT activities as as to their also a Arabidopsis line containing a knock-out in the single copy PLMT gene found in the Arabidopsis us to the consequences of loss of gene function in model species. were in yeast for or yeast amino acids for as with amino was on with choline used a and was by and using the by and to and Press, Scholar). To place the plant PLMT cDNAs under the of a yeast were into both the and of the AtPLMT and cDNAs via with the insertion of the plant cDNAs between the and of the yeast gene in yeast expression 88: PubMed Scopus Google Scholar). A of the resulting a containing the yeast were required for AtPLMT to an which was and into yeast expression 1989; PubMed Google Scholar). a that the of the expression into and was conducted using the by and 2007; PubMed Scopus Google Scholar). were to and were isolated as by Plant J. PubMed Scopus Google Scholar). In methylation were the of and T. J. Biol. PubMed Google Scholar). of and of A reaction of was by the addition of were at for and by the addition of of The was to a and the was using of The were using final and the was to and in of were on and using acid to and PtdCho were by with Lipids, in and using plants used in study were under at was from to PubMed Scopus Google Scholar). were by using the three and to AtPLMT the predicted insertion and a to the The were as follows: for by of for for and for and a final at For of line was isolated with to the was synthesized using the synthesis The used to AtPLMT expression were and To to the gene and were used as and The were as follows: for by of for for and for and a final at was isolated from to and PubMed Scopus Google Scholar). was using the as acid were conducted using the by and J. A Press, Scholar) or using the to the were using the the by the with were as follows: two with at for two using at for two with at for and a final using at for were by the to The for Arabidopsis with was from Hanson and A.D. Plant Physiol. PubMed Google Scholar). Arabidopsis were on with and and to were to of containing and in the by growth for on a using a of was to the and the were in the for to was by the addition of acid by a on were with then with a in of at by at for of was to and at To of was and at 2 at The was under a and in of of were on a and in a containing acid to with were from the and was by Arabidopsis were on containing and were from plants as M. Y. Williams J. Biol. 2002; PubMed Scopus Google Scholar). Plant lipid was in a for at and then to for to was conducted using by the Using the rat and yeast as a of the sequence of an Arabidopsis predicted homology with the rat and yeast Arabidopsis with homology to the yeast sequence were A of its sequence was from the Arabidopsis and a was using of A sequence from soybean was by a soybean using the Arabidopsis as a shown in Fig. the predicted Arabidopsis and soybean are and amino acids in The plant sequence to but only to the yeast or rat The presence of three predicted that the plant function as membrane of the complete Arabidopsis sequence that the sequence as a single copy within the of the soybean two distinct not the gene that to the soybean isolated from the The other soybean sequence on is likely to be a as were with the that soybean two with homology to sequence Fig. 1). The of the Arabidopsis and soybean are shown in Fig. a single between two of Arabidopsis with the sequence that two from A within the of the gene is in of the Arabidopsis to not of the soybean gene on with that on with their that a of and a in the in from the latter gene that not be likely to produce a not is that the gene on is the only copy within the soybean of the primary sequence between the rat and yeast PLMT and the predicted of the Arabidopsis and soybean it was to the plant were of the and yeast to the most widely accepted model of PtdCho biosynthesis in plants (Fig. and be the most likely substrates for a plant PLMT of the membrane of we of yeast to the function and of the plant an knock-out and methylation activities and have been shown to levels of the Henry S.A. 1989; PubMed Google Scholar). cDNAs of both the Arabidopsis and soybean were into a yeast and into the knock-out were with and for their to methylate the phospholipids within membrane The of the in methylation are shown in Fig. and were observed using membranes from a yeast was the only observed from of with the In cells the Arabidopsis or soybean however, and PtdCho species were observed in addition to (Fig. that the plant enzymes were capable of the final two steps in the methylation pathway of PtdCho synthesis in yeast. of their to alcohol substrates in the Arabidopsis and soybean were AtPLMT and Although both plant enzymes the reaction under from yeast AtPLMT observed from yeast have that plants are different from animals or yeast by not being to produce PtdCho directly via methylation of (7.Kinney A.J. Lipid Metabolism in Plants.in: Moore T.S. CRC Press, Inc., Boca Raton, FL1993: 259-284Google Scholar, 8.Datko A.H. Mudd S.H. Plant Physiol. 1988; 88: 1338-1348Crossref PubMed Google Scholar, M. PubMed Scopus Google Scholar). is further by the that using Arabidopsis and to yeast in the PtdCho methylation pathway only enzymes capable of phospho-base substrates Plant Physiol. 2000; PubMed Scopus Google Scholar, M.L. Henry S.A. Hanson A.D. J. Biol. 2000; PubMed Scopus Google Scholar). the is that plant cells cannot synthesize PtdCho directly from PtdEtn, then plant PLMT their and yeast be of as a substrate. by the Arabidopsis or soybean PLMT a yeast with knock-out in both the and In yeast, choline is not by the because the enzyme is capable of a a level of methylation Henry S.A. PubMed Scopus Google Scholar). however, have an for was through the of with AtPLMT and cDNAs were of the alcohol of yeast and into shown in Fig. the expression of plant PLMTs to the growth of the on The to choline in that AtPLMT can as To that the plant enzymes are in was on with a metabolite that can be incorporated into via enzymes of the nucleotide Although with the remained on expression of both plant PLMT growth (Fig. with the from the in enzyme expression of AtPLMT a growth membrane of the plant PLMTs in with were also in the in methylation of were detected using not the expression in yeast the that plant PLMT enzymes are to the final two steps of the to PtdCho methylation pathway, but cannot catalyze the initial methylation of of the expression that AtPLMT is at levels in most plant or where a in accumulation is observed not AtPLMT expression levels to be by to or In PLMT activities are at the level of in to soluble phospholipid Henry S.A. PubMed Scopus Google Scholar, T. Henry S.A. G.M. PubMed Scopus Google Scholar). and accumulation is in containing To AtPLMT expression is in a Arabidopsis were on with levels of choline or no obvious on AtPLMT levels in to Fig. with the of two of or as shown in Fig. AtPLMT on as a In animals and yeast, PLMTs the route for de novo choline synthesis in The of the phospho-base pathway for de novo choline synthesis in plants, however, the of the PLMT enzymes in plants. To a was to the consequences of the loss of AtPLMT gene function in A insertion line, was in the PubMed Scopus Google Scholar). sequence that the was at the first nucleotide of the Fig. of that the line was for the insertion not To the of the insertion on AtPLMT was isolated from Arabidopsis and the line and by Using and to and were detected in but not be observed using Fig. The to AtPLMT in that the a The insertion in at the AtPLMT was also by Fig. under standard growth in or on nutrient choline, atplmt no obvious or not To gain insights into the of the atplmt at the level, were conducted to the synthesis and accumulation of phospholipids with methylated amino alcohol headgroups and in versus plants. Arabidopsis were in the presence of for to is incorporated into the methyl donor of via the pathway (3.Hitz W.D. Rhodes D. Hanson A.D. Plant Physiol. 1981; 68: 814-822Crossref PubMed Google Scholar). was in the to the of that been oxidized to which then be into other of phospholipids through S.D. Nuccio M.L. Rhodes D. Shachar-Hill Y. Hanson A.D. Plant Physiol. 2000; 123: 371-380Crossref PubMed Scopus (58) Google Scholar). shown in Fig. incorporated into were in the line at all The was at the where in that found in PtdCho in the Arabidopsis plants. levels in study were to To the of AtPLMT in an in the levels of and in plants, plant lipid were using at the and levels were by and in atplmt plants with Arabidopsis (Fig. however, was observed in the of PtdCho (Fig. that of PtdCho through the phospho-base pathway of PtdCho levels via phospholipase activities can for in PtdCho synthesis by the PLMT Similar to no differences were observed in of the other phospholipids or in plants (Fig. and In cells it has been shown that PtdCho synthesized through the nucleotide pathway is not with PtdCho produced via the methylation in between the two of PtdCho is to be one of the for of J. Biol. PubMed Scopus Google Scholar, S.H. J. 2003; PubMed Scopus Google Scholar). In yeast as of the and enzymes in the formation of PtdCho from that through the enzymes of the nucleotide pathway A.J. J. Biol. PubMed Scopus Google Scholar). To the of metabolites through the PLMT enzyme has the of PtdCho species that from that produced through the route (Fig. the fatty acid of the was in both and Arabidopsis by differences in PtdCho fatty acid were observed between and plants (Fig. Although it cannot be from that AtPLMT based on it is clear that AtPLMT is not for PtdCho species, as has been shown for in the there were no differences observed in the fatty acid of PtdEtn, and (Fig. or of the other phospholipid and species not between and atplmt Arabidopsis plants. no differences were observed in the fatty acid of the in versus plants, one from was the of fatty acid species in and Although fatty acids of for of the and PtdCho species in both is in and represents less of (Fig. study represents the first of the PLMT of the PtdCho pathway in higher plants. Similar to the rat and yeast the predicted soybean and Arabidopsis PLMT enzymes are of and The of multiple predicted is with the of PLMT activities within membrane of plant cells A.H. Mudd S.H. Plant Physiol. 1988; 88: 854-861Crossref PubMed Google Scholar, M. PubMed Scopus Google Scholar). the Arabidopsis and soybean PLMT enzymes only primary amino acid sequence with their and yeast is the of the in the plant PLMTs in not that are of most Vance D.E. J. Biol. 2003; PubMed Scopus Google Scholar) conducted a study to the involved in for human were in human a amino in the of the and an the (Fig. and in the human were the glycine in the or the first acid of the sequence was with amino acid The soybean and Arabidopsis however, A in the plant PLMTs to the rat and yeast occurs at the to the of the and a is found in place of the in the (Fig. that plant PLMTs utilize different to and the substrate. the that plant PLMTs as a in to the enzyme that as yeast to a also that there are differences in the of plant versus and yeast The of plant PLMTs to utilize as a with that the methylation of not occur in plants (7.Kinney A.J. Lipid Metabolism in Plants.in: Moore T.S. CRC Press, Inc., Boca Raton, FL1993: 259-284Google Scholar, 8.Datko A.H. Mudd S.H. Plant Physiol. 1988; 88: 1338-1348Crossref PubMed Google Scholar, M. PubMed Scopus Google Scholar). that the de novo synthesis of a choline via the methylation at the phospho-base level by the MMEtn-P and can as substrates for the nucleotide pathway, which in the synthesis of or lipid and Moore Moore T.S. Plant Physiol. PubMed Scopus Google Scholar) that the cytidylyltransferase of has a level of using MMEtn-P or as a substrate. for most plants species it appears that the flow of metabolites through phospho-base versus phosphatidyl-base is by the of the cytidylyltransferase with the enzymes as for MMEtn-P and The of AtPLMT gene function in Arabidopsis not to obvious in plant growth and but it in and lipid species that are found in only in membrane The and in and in atplmt plants elevated the accumulation of species to levels to that observed for (Fig. In contrast, the levels of the end of the PLMT remained that the phospho-base route appears to be for of PtdCho within atplmt plants (Fig. it is on PLMT function has been Arabidopsis the most be that PLMT functions to metabolites of the PtdCho pathway to the end and the accumulation of the and Although the enhanced accumulation of appears to be atplmt plants are in an be growth at or the plant is to or atplmt plants to an of be a for to gene function on plant of the variability observed among different plant species with regard to the methylation of amino alcohol in the PtdCho pathway, the from study of the Arabidopsis atplmt not the importance of gene in other higher plants. In soybean, for example, in conducted both in and and in enzyme using clear that the final two methylation reactions occur exclusively at the phosphatidyl-base level in species (8.Datko A.H. Mudd S.H. Plant Physiol. 1988; 88: 1338-1348Crossref PubMed Google Scholar, 11.Datko A.H. Mudd S.H. Plant Physiol. 1988; 88: 854-861Crossref PubMed Google Scholar). the soybean gene be predicted to be essential for PtdCho and its likely in a In contrast, the of PLMT in likely a to that observed in metabolite was shown to occur through phosphatidyl-base in system, the that PLMT activities were in (8.Datko A.H. Mudd S.H. Plant Physiol. 1988; 88: 1338-1348Crossref PubMed Google Scholar, 11.Datko A.H. Mudd S.H. Plant Physiol. 1988; 88: 854-861Crossref PubMed Google Scholar). Although no differences were observed in the fatty acid of the alcohol from versus atplmt Arabidopsis plants, the of species within and of both was in of the that is in and PtdCho (Fig. of the absence of species within all phospholipids of Arabidopsis J. Biochem. J. 1986; PubMed Scopus Google Scholar), it is to that the species detected in were of The absence of fatty acid in and its in be by the of the species by the amino alcohol phosphotransferase enzyme with a or substrate. Alternatively, of be and the of fatty acid in PtdCho and be a of PtdCho and species as substrates for the and the species of and substrates for the of and be by an enhanced of species to phospholipase or through an as by M. J. Biol. 2007; PubMed Scopus Google Scholar). In through the heterologous expression of cDNAs in yeast, we have that the AtPLMT and in study PLMT enzymes involved in PtdCho biosynthesis. The enhanced accumulation of and in an Arabidopsis atplmt line that the AtPLMT gene functions within the PtdCho pathway in The and of the PLMT in plants not only understanding of the PtdCho pathway but also an important for further the variability observed among different plants species the route by which PtdCho is synthesized within the plant and how is the of for in the yeast that also and the for phospholipid and fatty acid of the Arabidopsis plants. was by Science and and Science with from the of through and The is also by of from the of the for with