Production of complex ether glycerolipids from exogenous alkylglycerols by cell suspension cultures of rape

Summary Neutral and ionic ether glycerolipids, especially alkylacylglycerophosphocholines and 1-alkenylacylglycerophosphocholines, are formed from exogenous 1-O-alkylglycerols, 1-O-(1-alkenyl)glycerols or 2-O-alkylglycerols by photomixotrophic cell suspension cultures of rape (Brassica napus). Best yields of ether glycerolipids were obtained by incubating rape cells with optically active 1-O-alkyl-sn-glycerols. Racemic or symmetric alkylglycerols are also utilized by rape cell suspension cultures for the biosynthesis of optically active ionic ether glycerolipids. In contrast, 3-O-hexadecyl-sn-glycerol is not incorporated into ether glycerophospholipids of rape cells. Incorporation of the substrates into ionic ether lipids is dependent on chain length (C₁₄>C₁₆>C₁₈) and degree of unsaturation (C_18:1C_18:0) of alkyl chains.Stereochemically uniform 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholines and 2-O-alkyl-1-acyl-sn-glycero-3-phosphocholines with defined alkyl moieties can be prepared from exogenous alkylglycerols. This method recommends itself especially for the preparation of 1-O-(1-alkenyl)-2-acyl-sn-glycero-3-phosphocholines (choline plasmalogens) from 1-O-(1-alkenyl)-sn-glycerols. Ether glycerophospholipids with physiological activity, such as 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholines (platelet activating factor, PAF) and 1-O-alkyl-sn-glycero-3-phosphocholines (lyso PAF), were synthesized from 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholines formed by cell suspension cultures of rape.     

Ether glycerolipids: novel substrates for studying specificity of enzymes involved in glycerolipid biosynthesis in higher plants

Ether glycerolipids, predominantly alkylacylglycerols and alkylacylglycerophosphocholines, are synthesized in photomixotrophic rape (Brassica napus) suspension cells from various exogenous monoalkylglycerols. The stereospecific distribution of acyl moieties was studied in these ether glycerolipids with regard to chain-length and degree of unsaturation of alkyl moieties and compared with the distribution of acyl moieties in the corresponding endogenous acyl glycerolipids. The results show the following: (1) Alkylacylglycerophosphocholines replaced up to one-half of the corresponding physiological membrane lipids, i.e. diacylglycerophosphocholines, without changing the total amount of cholineglycerophospholipids as compared to untreated cells. (2) The composition of acyl moieties in total lipids of rape cells was practically unaltered by fatty acids derived via oxidative cleavage from the various alkyl moieties of either glycerolipids. (3) In 1-O-alkyl-2-acylglycerols derived from exogenous alkylglycerols and in endogenous 1,2-diacylglycerols compositions of acyl moieties were found to be different indicating that different pathways were operative in the biosynthesis of these two neutral glycerolipids. (4) Enzymes involved in synthesizing molecular species of 1-O-alkyl-2-acylglycerophosphocholines or 2-O-alkyl-1-acylglycerophosphocholines as well as 1,2-diacylglycerophosphocholines showed similar specificities with regard to chain-length and degree of unsaturation of both alkyl and corresponding acyl moieties. Thus, ether glycerolipids formed by plant cells from exogenous alkylglycerols are suitable metabolites for studying the specificity of enzymes involved in the biosynthesis of glyerolipids.     

Phase behavior of ethanolamine plasmalogen

In the present study the phase behavior of multilamellar dispersions of 1-O-(1′-alkenyl)-2-oleoyl-glycerophosphoethanolamine (ethanolamine plasmalogen), 1-O-alkyl-2-oleoyl-glycerophosphoethanolamine and 1-acyl-2-oleoyl-glycerophosphoethanolamine was compared using differential scanning calorimetry (DSC) and ³¹P-NMR. The three compounds differed only in the type of bonding (vinyl ether, alkyl ether or acyl ester) linking the aliphatic moiety to position 1 of sn-glycerol.The gel to liquid-crystalline phase transition temperature as determined by DSC was lowest for ethanolamine plasmalogen (26°C) and was similar for the alkylacyl and diacyl analogs (29.5° and 30°C, respectively). Enthalpies of the G → L phase transition were not significantly different for the three phospholipids tested.Ethanolamine plasmalogen undergoes the lamellar to hexagonal phase transition at 30°C, the analogous alkylacyl-glycerophosphoethanolamine(alkylacyl-GPE) and diacyl-GPE at 53°C and 69°C, respectively. Thus, an alkenyl ether bond in position 1 of sn-glycerol, the structural characteristic of plasmalogens, effectively stabilizes the hexagonal H_II arrangement of ethanolamine glycerophospholipids, while it has relatively little effect on destabilization of the lamellar gel state.     

Formation of optically active ether lipids from race-mic-1-O-tetradecylglycerol in plant cell cultures

Photomixotrophic rape cells in culture specifically incorporate 1-O-tetradecyl-sn-glycerol from a racemic mixture into complex alkyl glycerolipids. Thus, both neutral and ionic 1-O- alkyl-2-O-acyl-sn-glycerolipids with defined alkyl moieties can be prepared from racemic mixtures of alkylglycerols.     

Comparison of linoleate,palmitate and acetate metabolism in rat ventral prostate

Rat ventral prostate incorporated (1-¹⁴C)acetate, (1-¹⁴C)palmitate and (1-¹⁴C)linoleate into different phospholipids in a time-dependent process. The rate of incorporation into total phospholipids was higher with linoleate (10.0 nmol/g) than with either palmitate (5.8 nmol/g) or acetate (4.7 nmol/g). Predominant labelling with all the radioactive substrates assayed was found in choline glycerophospholipids (PC). The radioactive profiles for linoleate in the other ventral prostate phospholipids differed from those obtained with palmitate and acetate. Specifically linoleate was incorporated into inositol glycerophospholipids plus lysoethanolamine glycerophospholipids (PI+LPE) and not into sphingomyelin (SM), while palmitate and acetate incorporated into SM but not into PI+LPE. Acetate showed the highest oxidation to CO₂ whereas no differences were observed in the radioactivity incorporated into CO₂ from a saturated (palmitate) or an essential unsaturated fatty acid (linoleate). These studies also show zinc-dependence by the acetate to CO₂ oxidation.Abbreviations PL total phospholipids - PC choline glycerophospholipids - PE ethanolamine glycerophospholipids - PI+LPE inositol glycerophospholipids plus lysoethanolamine glycerophospholipids - PS serine glycerophospholipids - SM sphingomyelin     

The synthesis of choline and ethanolamine phosphoglycerides in neuronal and glial cells of rabbit in vitro

Abstract— The de novo synthesis of phosphatidylcholine and phosphatidylethanolamine in isolated neuronal and glial cells from adult rabbit brain cortex was investigated in vitro, using labelled phosphorylcholine (phosphorylethanolamine) or cytidine-5′-phosphate choline (cytidine-5′-phosphate ethanolamine), as lipid precursors. Synthesis of phospholipid from phosphorylcholine and phosphorylethanolamine in both fractions was extremely low when compared to that derived from the corresponding cytidine nucleotides. The neuronal cell-enriched fraction was found to possess a much higher rate of synthesis of both lipids from all precursors. Neuronal/glial ratios of about 5–9 were found for the synthesis of phosphatidylcholine and phosphatidylethanolamine from cytidine-5′-phosphate choline and cytidine-5′-phosphate ethanolamine, respectively. Several kinetic properties of the choline-phosphotransferase (EC 2.7.8.2) and ethanolaminephosphotransferase (EC 2.7.8.1) were found to be similar both in neurons and in glia (e.g. K_m of cytidine-5′-phosphate ethanolamine, K_m of diacyl glycerol, pH optimum, need for divalent cations), but the K_m value for cytidine-5′-phosphate choline in glial cells was much lower (2.3 × 10^?4m ) than in neurons (1 × 10^?3m ). The K_mfor cytidine-5′-phosphate ethanolamine in both cells was much lower than in whole brain microsomes. It is concluded that the cytidine-dependent enzymic system for phosphatidylcholine and phosphatidylethanolamine synthesis is concentrated mostly in the neuronal cells, as compared to glia.     

Posttranslational Regulation of Fatty Acyl-CoA Reductase 1, Far1, Controls Ether Glycerophospholipid Synthesis

Plasmalogens are a major subclass of ethanolamine and choline glycerophospholipids in which a long chain fatty alcohol is attached at the sn-1 position through a vinyl ether bond. This ether-linked alkyl bond is formed in peroxisomes by replacement of a fatty acyl chain in the intermediate 1-acyl-dihydroxyacetone phosphate with a fatty alcohol in a reaction catalyzed by alkyl dihydroxyacetone phosphate synthase. Here, we demonstrate that the enzyme fatty acyl-CoA reductase 1 (Far1) supplies the fatty alcohols used in the formation of ether-linked alkyl bonds. Far1 activity is elevated in plasmalogen-deficient cells, and conversely, the levels of this enzyme are restored to normal upon plasmalogen supplementation. Down-regulation of Far1 activity in response to plasmalogens is achieved by increasing the rate of degradation of peroxisomal Far1 protein. Supplementation of normal cells with ethanolamine and 1-O-hexadecylglycerol, which are intermediates in plasmalogen biosynthesis, accelerates degradation of Far1. Taken together, our results indicate that ether lipid biosynthesis in mammalian cells is regulated by a negative feedback mechanism that senses cellular plasmalogen levels and appropriately increases or decreases Far1.     

Relative degradation of different arachidonoyl molecular species of choline glycerophospholipids in opsonized zymosan-stimulated rabbit alveolar macrophages

The relative degradation of arachidonoyl molecular species of glycerophospholipids prelabeled with [3H]20:4 caused by opsonized zymosan was studied in rabbit alveolar macrophages using a recently developed high-performance liquid chromatographic method. The opsonized zymosan caused the release of [3H]20:4 only from choline glycerophospholipids, no significant changes being observed in the radioactivities of other glycerophospholipids and triacylglycerol. Choline glycerophospholipids were resolved into seven arachidonoyl molecular species, which differed as to the alkyl ether or acyl residue bound at the 1-position, by high-performance liquid chromatography. Arachidonate was predominantly located in the alkyl type having 16:0 at the 1-position which comprised more than half of the total arachidonoyl molecular species of choline glycerophospholipids. The radioactivities of all arachidonoyl molecular species of choline glycerophospholipids, except for the 18:2-20:4 and 18:1-20:4 species of diacylglycerophosphocholine, decreased to 80-85% of the control values as a result of the challenge with opsonized zymosan for 1 h. However, 50% of the released 20:4 came from the 16:0-20:4 species of alkylacylglycerophospholipids, which were the most predominant species of choline glycerophospholipids. The present results indicate that the 16:0-20:4 species of alkylacylglycerophosphocholine is a significant source of arachidonate and 1-O-alkyl-2-lysoglycerophosphocholine, the precursor of the platelet-activating factor, relative to other arachidonoyl species in activated alveolar macrophages.     

Choline and ethanolamine glycerophospholipid synthesis in isolated synaptosomes of rat brain.

Substantial activities of cholinephosphotransferase (EC 2.7.8.2) and ethanolaminephosphotransferase (EC 2.7.8.1) were found with lysed synaptosomes but not with intact synaptosomes isolated from adult rat brains. Synaptosomal and non-synaptosomal microsomal transferases were similar in kinetic properties. Substantial activities of synaptosomal transferases have not been described previously. Part of the glycerophospholipids in synaptosomal membranes may be synthesized in the nerve ending in addition to the glycerophospholipids supplied by axonal transport. The synthesis of the alkylacyl type of choline and ethanolamine glycerophospholipids was moderately inhibited by 1 mM ATP and 1 microM cyclic AMP. This synthesis was also inhibited by more than 50% by 1 mM norepinephrine and to a lesser extent by 5 mM hydroxytryptamine and 1 mM acetylcholine. Cyclic AMP may mediate the effects of biogenic amines. The relative synthesis of different glycerophospholipid classes and the relative proportion of alkylacyl type (plasmalogen precursors) and diacyl type of glycerophospholipids may be influenced by the levels of adenine nucleotides and/or biogenic amines. Elevated cyclic AMP levels will decrease the synthesis of plasmalogen precursors.     

Crystal structures and thermal analyses of alkyl 2-deoxy-α-d-arabino-hexopyranosides

The crystal structures of alkyl 2-deoxy-α-d-arabino-hexopyranosides, with the alkyl chain lengths from C₈ to C₁₈, are established by the single crystal X-ray structural determination. The even-alkyl chain length derivatives crystallized orthorhombic, with space group P2₁2₁2₁, whereas the odd-alkyl chain length derivatives crystallized monoclinic, with space group P2₁. The sugar moieties retained a ⁴C₁ chair conformation and the conformation of the alkyl chains was all-trans. The molecules formed a bilayer structure, in which alkyl chains were interdigitated. The hydrogen bonds, originating from the sugar moieties, were observed in adjacent layers and also within the same layer, resulting in the formation of infinite chains. The alkyl chains arranged parallel to each other and formed planar structures. The thermal properties of the alkyl 2-deoxy glucosides were analyzed further. It was observed that none of the derivatives exhibited mesomorphism. This study establishes that the absence of the hydroxyl group at C-2 of the sugar moiety results in a non-mesogenic nature of the alkyl 2-deoxy-α-d-glycosides, as opposed to the profound mesogenic nature of the normal alkyl glycosides.     

Semi-synthetic preparation of 1-O-[1'-14C]hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (platelet activating factor) using plant cell cultures

Incubation of photomixotrophic cell suspension cultures of rape (Brassica napus) and heterotrophic cell suspension cultures of soya (Glycine max) with 1-O-[1'-14C]hexadecyl-sn-glycerol or rac-1-O-[1'-14C]hexadecylglycerol leads in high yield (up to 78%) to labeled 1-O-hexadecyl-2-acyl-sn-glycero-3-phosphocholines. Alkaline hydrolysis of the choline glycerophospholipids yields pure 1-O-[1'-14C]hexadecyl-sn-glycero-3-phosphocholine. 1-O-[1'-14C]Hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (platelet activating factor) is obtained by acetylating the lyso compound. The semi-synthetic preparation described leads to labeled platelet activating factor in an overall yield of 50-60% without loss of specific activity.     

Synthesis of Ethanolamine and Its Regulation in Lemna paucicostata

The metabolism of ethanolamine and its derivatives in Lemna paucicostata has been investigated, with emphasis on the path-way for synthesis of phosphoethanolamine, a precursor of phosphatidylcholine in higher plants. In experiments involving labeling of intact plants with radioactive serine, ambiguities of interpretation due to entry of radioactivity into methyl groups of methylated ethanolamine derivatives were mitigated by pregrowth of plants with methionine. Difficulties due to labeling of diacylglyceryl moieties of phospholipids were avoided by acid hydrolysis of crucial samples and determination of radioactivity in isolated serine or ethanolamine moieties. The results obtained from such experiments are most readily reconciled with the biosynthetic sequence: serine → ethanolamine → phosphoethanolamine → phosphatidylethanolamine. A possible alternative is: serine → phosphatidylserine → phosphatidylethanolamine → ethanolamine → phosphoethanolamine. Cell-free extracts of L. paucicostata were shown to produce CO₂ from the carbon originating as C-1 of serine at a rate sufficient to satisfy the demand for ethanolamine moieties. A number of experiments produced no support for a hypothetical role for phosphoserine in phosphoethanolamine formation. Uptake of exogenous ethanolamine commensurately down-regulates the synthesis of ethanolamine moieties (considered as a whole, and regardless of their state of derivatization at the time of their formation). In agreement with previous observations, uptake of exogenous choline down-regulates the methylation of phosphoethanolamine, without being accompanied by secondary accumulation of a marked excess of ethanolamine derivatives.     

The base-catalyzed isomerization of trans-RCo(H2O)L (L=5,7,7,12,14,14-hexamethyl-1,4,8,11-tetraazacyclotetradeca-4,11-diene)

The sec, rac-CH₃Co(H₂O)L²⁺ (L=5,7,7,12,14,14-hexamethyl-1,4,8,11-tetraazacyclotetradeca-4,11-diene) was prepared successfully via meso-CH₃Co(H₂O)L²⁺ in aqueous solution. The isomerizations from meso-RCo(H₂O)L²⁺ (R=CH₃, C₂H₅ and C₃H₇) and sec, rac-CH₃Co(H₂O)L²⁺ to pri, rac-RCo(H₂O)L²⁺ were both base catalyzed in aqueous solution. The kinetic results showed the reaction to be first order in both organocobalt complex and hydroxide ion with the reactivity order for the alkyl group being C₃H₇ ∼ C₂H₅ ? CH₃. However, the conversion from the most steric hindered isomer form of sec, rac- was slow. The ratio of the isomerization rate constants between meso-CH₃Co(H₂O)L²⁺ and sec, rac-CH₃Co(H₂O)L²⁺ to pri, rac-CH₃Co(H₂O)L²⁺ is almost a factor of 100. The thermodynamic activation parameters for these isomerization reactions were investigated.     

Organogold(I) complexes of a C2-symmetric diacetylide ligand derived from 1,1′-bi-2-naphthol

Alkynylgold(I) complexes incorporating a chiral binaphthyl group have been prepared. Bis(alkyne) reagents [rac-1,1′-C₂₀H₁₂-2,2′-(OCH₂CCH)₂] (1) and [rac-1,1′-C₂₀H₁₂-2,2′-(OC(O)CH₂CCH)₂] (2), react with [AuCl(SMe₂)] and base to give insoluble oligomeric alkynylgold(I) complexes [rac-1,1′-C₂₀H₁₂-2,2′-(OCH₂CCAu)₂]_n (3) and [rac-1,1′-C₂₀H₁₂-2,2′-(OC(O)CH₂CCAu)₂]_n (4), which react with phosphine or diphosphine ligands to give soluble complexes [rac-1,1′-C₂₀H₁₂-2,2′-(OCH₂CCAuPR₃)₂] (5), R = Ph or Cy, [rac-1,1′-C₂₀H₁₂-2,2′-(OCH₂CCAu)₂(Ph₂P(CH₂)_nPPh₂)] (6), or [rac-1,1′-C₂₀H₁₂-2,2′-(OC(O)CH₂CCAu)₂(Ph₂P(CH₂)_nPPh₂)] (7), with n = 3–5. Several of the complexes 6 and 7 are shown to exist as mixtures of isomeric forms in solution.     

Influence of lindane on the fluidity of the rat ventral prostate membranes

The influence of lindane upon the dynamic properties of plasma membranes from rat ventral prostate has been investigated using a fluorescence polarization technique. Preincubation with lindane decreased the fluorescence polarization in a dose dependent manner. This effect, which is associated with an increased membrane fluidity, occurred in a very short period of time.Lindane also provoked a number of changes in lipid biosynthesis from acetate in the membrane. Less [1-¹⁴C]acetate was incorporated into cholesterol and more into phospholipids when this liposoluble toxicant was added to the preincubation medium. However, not all phospholipid classes were equally increased, because while the rate of acetate incorporation was greater into choline glycerophospholipids than into ethanolamine glycerophospholipids, both were higher than the rates of acetate incorporation into serine glycerophospholipids and sphingomyelin.     

Androgenic control of acetate incorporation into membrane lipids in rat ventral prostate

The rat ventral prostate plasma membranes incorporated acetate into total lipids, which was a time-dependent process. The acetate incorporation was mainly into phospholipids followed by cholesterol. The main phospholipids subclasses were choline and ethanolamine glycerophospholipids. Castration modified drastically both cholesterol-phospholipids and choline glycerophospholipids-ethanolamine glycerophospholipids ratios. These effects of castration were reversed after testosterone treatment, which could suggest an influence of this hormone in the modification of some lipid classes into cellular membrane.     

A comparison of the reversibility of phosphoethanolamine transferase and phosphocholine transferase in rat brain microsomes

The reversibility of phosphoethanolamine transferase (EC 2.7.8.1) in rat brain is demonstrated in this paper. Microsomal ethanolamine glycerophospholipids were prelabeled with an intracerebral injection of [3H]ethanolamine 4 h before killing young rats. Labeled CDPethanolamine was produced by incubation of the microsomes with CMP, although to a lesser extent than for the previously observed release of CDPcholine. Ethanolamine and choline glycerophospholipids were labeled with [2-3H]glycerol by incubation with primary cultures of rat brain. Microsomes from rat brains, with diisopropyl phosphofluoridate for inhibition of lipases, were incubated with the labeled glycerophospholipids separately, and labeled diacylglycerols were produced. The kinetic parameters of phosphoethanolamine transferase and phosphocholine transferase (EC 2.7.8.2) were compared by incubating rat brain microsomes with [3H]CMP. Inclusion of AMP in the reaction mixture was necessary in order to inhibit the hydrolysis of CMP by an enzyme with the properties of 5'-nucleotidase (EC 3.1.3.5). For phosphoethanolamine transferase and phosphocholine transferase respectively, the Km values for CMP were 40 and 125 microM and the V values were 2.3 and 21.6 nmol/h per mg protein. The reversibility of both enzymes permits the interconversion of the diacylglycerol moieties of choline and ethanolamine glycerophospholipids. During brain ischemia, a principal pathway for degradation of ethanolamine glycerophospholipids may be by reversal of phosphoethanolamine transferase followed by hydrolysis of diacylglycerols by the lipase.     

Synthetic pathways of glycerophospholipids in adult Brugia pahangi and Brugia patei

The pathways of glycerophospholipid syntheses in adult Brugia pahangi and Brugia patei were examined by radioisotopic incorporation and demonstration of the enzymatic steps. Radiolabelling studies showed that l-U-¹⁴C-glycerol-3-phosphate was rapidly incorporated into glycerophospholipids of B. pahangi and B. patei, respectively, with the label distributed in phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylglycerol (PG) and cardiolipin (CL) fractions. Crude extracts of these worms were found to contain significant activities of sn-glycerol-3-phosphate acyl-transferase (EC 2.3.1.15), phosphatidic acid phosphatase (EC 3.1.3.4), choline phosphotransferase (EC 2.7.8.2), ethanolamine phosphotransferase (EC 2.7.8.1), PE methyltransferase (EC 2.1.1.17), PS decarboxylase (EC 4.1.1.65), phosphatidylglycerolphosphate synthetase (EC 2.7.8.5), phosphatidylinositol synthetase (EC 2.7.8.11), and base exchange enzymes of ethanolamine, serine and inositol. These findings suggest that filarial worms can synthesize PC by two pathways, PE by three pathways, and PI by two pathways and fabricate PS, PG and CL.     

Diacyl,Alkylacyl, and Alkenylacyl Phospholipids of Meloidogyne javanica Females

The phospholipid composition and acyl, alkyl, and alkenyl group compositions of diacyl, alkylacyl, and alkenylacyl phosphoglycerides of M. javanica were investigated. Phospholipid was comprised of 61.7% choline phosphoglyceride, 22.0% ethanolamine phosphoglyceride, and smaller quantities of six other lipids. Phospholipid fatty acid was more unsaturated than neutral lipid fatty acid and contained 61.3% octadecenoic (18:1) acid. Fatty acid at the 1-position of diacyl phospholipids was shorter and more saturated than that at the 2-position. Compared to choline phosphoglyceride, ethanolantine phosphoglyceride contained less 18:1 and 20:5 and more 18:0 and 20:0 acid. Alkenylacyl and alkylacyl compounds comprised 34.6% and 9.3%, respectively, of the ethanolamine phosphoglyceride but only 0.5% and 0.6% of the choline phosphoglyceride. Alkenylacyl and alkylacyl ethanolamine phosphoglycerides contained a smaller percentage of 20-carbon polyunsaturated acid at their 2-positions than did their diacyl analogue. At least 95% of the alkenyl and alkyl groups were 18:0 compounds. Tomato roots did not contain alkenylacyl or alkylacyl phosphoglycerides; their occurrence in M. javanica is a significant biochemical difference between the nematode and its host.     

Synthesis of methylated ethanolamine moieties: regulation by choline in soybean and carrot

The results of experiments in which intact plants of Lemna paucicostata were labeled with either l-[³H₃C]methionine, l-[¹⁴CH₃]methionine, or [1,2-¹⁴C]ethanolamine support the conclusion that growth in concentrations of choline of 3.0 micromolar or above brings about marked decreases in the rate of biosynthesis of methylated forms of ethanolamine (normally present chiefly as phosphatidylcholine, with lesser amounts of choline and phosphocholine). The in vivo locus of the block is at the committing step in the biosynthetic sequence at which phosphoethanolamine is methylated by S-adenosylmethionine to form phosphomethylethanolamine. The block is highly specific: flow of methyl groups originating in methionine continues into S-adenosylmethionine, S-methylmethionine, the methyl moieties of pectin methyl ester, and other methylated metabolites. When choline uptake is less than the total that would be synthesized by control plants, phosphoethanolamine methylation is down-regulated to balance the uptake; total plant content of choline and its derivatives remains essentially constant. At maximum down-regulation, phosphoethanolamine methylation continues at 5 to 10% of normal. A specific decrease in the total available activity of AdoMet: phosphoethanolamine N-methyltransferase, as well as feedback inhibition of this enzyme by phosphocholine, and prevention of accumulation of phosphoethanolamine by down-regulation of ethanolamine synthesis may each contribute to effective control of phosphoethanolamine methylation. This down-regulation may necessitate major changes in S-adenosylmethionine metabolism. Such changes are discussed.