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• W2025682709 abstract "In expanding pea leaves, over 95% of fatty acids (FA) synthesized in the plastid are exported for assembly of eukaryotic glycerolipids. It is often assumed that the major products of plastid FA synthesis (18:1 and 16:0) are first incorporated into 16:0/18:1 and 18:1/18:1 molecular species of phosphatidic acid (PA), which are then converted to phosphatidylcholine (PC), the major eukaryotic phospholipid and site of acyl desaturation. However, by labeling lipids of pea leaves with [14C]acetate, [14C]glycerol, and [14C]carbon dioxide, we demonstrate that acyl editing is an integral component of eukaryotic glycerolipid synthesis. First, no precursor-product relationship between PA and PC [14C]acyl chains was observed at very early time points. Second, analysis of PC molecular species at these early time points showed that >90% of newly synthesized [14C]18:1 and [14C]16:0 acyl groups were incorporated into PC alongside a previously synthesized unlabeled acyl group (18:2, 18:3, or 16:0). And third, [14C]glycerol labeling produced PC molecular species highly enriched with 18:2, 18:3, and 16:0 FA, and not 18:1, the major product of plastid fatty acid synthesis. In conclusion, we propose that most newly synthesized acyl groups are not immediately utilized for PA synthesis, but instead are incorporated directly into PC through an acyl editing mechanism that operates at both sn-1 and sn-2 positions. Additionally, the acyl groups removed by acyl editing are largely used for the net synthesis of PC through glycerol 3-phosphate acylation. In expanding pea leaves, over 95% of fatty acids (FA) synthesized in the plastid are exported for assembly of eukaryotic glycerolipids. It is often assumed that the major products of plastid FA synthesis (18:1 and 16:0) are first incorporated into 16:0/18:1 and 18:1/18:1 molecular species of phosphatidic acid (PA), which are then converted to phosphatidylcholine (PC), the major eukaryotic phospholipid and site of acyl desaturation. However, by labeling lipids of pea leaves with [14C]acetate, [14C]glycerol, and [14C]carbon dioxide, we demonstrate that acyl editing is an integral component of eukaryotic glycerolipid synthesis. First, no precursor-product relationship between PA and PC [14C]acyl chains was observed at very early time points. Second, analysis of PC molecular species at these early time points showed that >90% of newly synthesized [14C]18:1 and [14C]16:0 acyl groups were incorporated into PC alongside a previously synthesized unlabeled acyl group (18:2, 18:3, or 16:0). And third, [14C]glycerol labeling produced PC molecular species highly enriched with 18:2, 18:3, and 16:0 FA, and not 18:1, the major product of plastid fatty acid synthesis. In conclusion, we propose that most newly synthesized acyl groups are not immediately utilized for PA synthesis, but instead are incorporated directly into PC through an acyl editing mechanism that operates at both sn-1 and sn-2 positions. Additionally, the acyl groups removed by acyl editing are largely used for the net synthesis of PC through glycerol 3-phosphate acylation. In plants acyl carrier protein (ACP) 2The abbreviations used are: ACP, acyl carrier protein; DAG, diacylglycerol; FA, fatty acid; FAME, fatty acid methyl ester; FAS, fatty acid synthesis; GC, gas chromatography; GPC, glycerophosphorylcholine; LPCAT, lysophosphatidylcholine acyltransferase; PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; MES, 4-morpholineethanesulfonic acid.-dependent de novo fatty acid synthesis (FAS) is restricted to organelles (1Ohlrogge J.B. Kim D.N. Stumpf P.K. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 1194-1198Crossref PubMed Scopus (230) Google Scholar). Essentially all acyl chains for membrane and storage lipid synthesis are produced in the plastid (2Ohlrogge J. Kim J. Plant Cell. 1995; 7: 957-970Crossref PubMed Scopus (1258) Google Scholar, 3Schwender J. Kim J.B. Plant Physiol. 2002; 130: 347-361Crossref PubMed Scopus (160) Google Scholar). The initial products of FAS, 16- and 18-carbon fatty acids (FAs) esterified to ACP, are incorporated into glycerolipids by one of two routes: (i) acyl-ACP is used directly by acyltransferases of the “prokaryotic” pathway within the plastid, or (ii) the acyl-ACP thioester bond is hydrolyzed during acyl export from the plastid prior to FA reactivation and incorporation into glycerolipids by acyltransferases of the “eukaryotic” pathway outside the plastid. This two pathway mechanism for glycerolipid synthesis was first elucidated by radiotracer studies (4Roughan P.G. Kim C.R. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1982; 33: 97-132Crossref Google Scholar), and was confirmed genetically by analysis of Arabidopsis mutants (2Ohlrogge J. Kim J. Plant Cell. 1995; 7: 957-970Crossref PubMed Scopus (1258) Google Scholar, 5Somerville C. Kim J. Science. 1991; 252: 80-87Crossref PubMed Scopus (372) Google Scholar). The proportions of nascent FA (i.e. immediate products of FAS) incorporated into the eukaryotic and prokaryotic pathways vary widely among plants and different tissues within the same plant. The eukaryotic pathway predominates in non-photosynthetic tissues of all higher plants. In 18:3 plants, 3Fatty acids are described by the convention “carbon number:number of double bonds.” For example, oleic acid is represented as 18:1. The molecular species of phospholipids are described by FA/FA. When known, the sn-1 position is represented first, but often the stereochemistry is uncertain. For example, both 1-oleoyl-2-linoleoyl-sn-phosphatidylcholine and 1-linoleoyl-2-oleoyl-sn-phosphatidylcholine are described as 18:1/18:2-PC. so called because they accumulate predominantly 18:3 and not 16:3 in their leaf galactolipids, 95% of the FA produced in the plastid is exported for assembly into glycerolipids by the eukaryotic pathway. Only phosphatidylglycerol (PG) is synthesized directly in the plastid from acyl-ACPs. This is in contrast to leaves of 16:3 plants which can also synthesize glycolipids by the prokaryotic pathway. Consequently, only about 60% of FA produced in the chloroplast by 16:3 plants is exported to the eukaryotic pathway (6Browse J. Kim N. Somerville C.R. Slack C.R. Biochem. J. 1986; 235: 25-31Crossref PubMed Scopus (264) Google Scholar). 18:3 plants, which include pea, make up about 88% of angiosperm species (7Mongrand S. Kim J.J. Cabantous F. Cassagne C. Phytochemistry. 1998; 49: 1049-1064Crossref Scopus (131) Google Scholar). It is generally assumed that the major exported products of chloroplast FAS, namely 18:1 and 16:0 FA, are transferred to the outer envelope of the plastid where they are activated to acyl-CoAs by a long chain acyl-CoA synthetase (8Block M.A. Kim A.J. Joyard J. Douce R. FEBS Lett. 1983; 153: 377-381Crossref Scopus (55) Google Scholar, 9Andrews J. Kim K. Plant Physiol. 1983; 72: 735-740Crossref PubMed Google Scholar). The eukaryotic pathway for de novo PC synthesis then utilizes this pool of newly synthesized acyl-CoAs for sequential sn-1 and sn-2 acylations of glycerol 3-phosphate to produce 18:1/18:1 and 16:0/18:1 molecular species of phosphatidic acid (PA). PA is rapidly converted to phosphatidylcholine (PC) by the action of PA phosphatase and CDP-choline:1,2-diacyl-sn-glycerol choline-phosphotransferase (2Ohlrogge J. Kim J. Plant Cell. 1995; 7: 957-970Crossref PubMed Scopus (1258) Google Scholar). Desaturation of 18:1 to 18:2 and then 18:3 on PC produces the abundant polyunsaturated molecular species of PC (10Sperling P. Kim E. Eur. J. Biochem. 1993; 213: 965-971Crossref PubMed Scopus (33) Google Scholar, 11Harwood J.L. Biochim. Biophys. Acta-Lipids Lipid Metab. 1996; 1301: 7-56Crossref PubMed Scopus (396) Google Scholar). However, several lines of evidence suggest that this model may be inadequate and needs to take account of acyl editing. We define acyl editing, often also termed “remodeling” or “retailoring,” as any process that exchanges acyl groups between polar lipids but which does not by itself result in the net synthesis of the polar lipids. Acyl editing has long been considered an important facet of phospholipid metabolism (12Lands W.E.M. Annu. Rev. Biochem. 1965; 34: 313-346Crossref PubMed Scopus (117) Google Scholar). Acyl editing relevant to this work can occur through two mechanisms. In plants acyl editing via a CoA:PC acyl exchange mechanism was demonstrated in microsomes isolated from developing seeds and was attributed to a reverse reaction of lysophosphatidylcholine acyltransferase (LPCAT) (13Stymne S. Kim A.K. Biochem. J. 1984; 223: 305-314Crossref PubMed Scopus (146) Google Scholar). LPCAT activity has also been described in isolated chloroplasts (14Bertrams M. Kim K. Heinz E. Z. Naturforsch. (C). 1981; 36: 62-70Crossref Scopus (25) Google Scholar, 15Bessoule J.J. Kim E. Cassagne C. Eur. J. Biochem. 1995; 228: 490-497Crossref PubMed Scopus (58) Google Scholar, 16Kjellberg J.M. Kim M. Andersson M. Sandelius A.S. Biochim. Biophys. Acta Mol. Cell. Biol. Lipids. 2000; 1485: 100-110Crossref PubMed Scopus (33) Google Scholar) as well as microsomes (17Stymne S. Kim A.K. Glad G. Biochim. Biophys. Acta. 1983; 752: 198-208Crossref PubMed Scopus (66) Google Scholar, 18Rochester C.P. Kim D.G. Arch. Biochem. Biophys. 1984; 232: 249-258Crossref PubMed Scopus (13) Google Scholar, 19Demandre C. Kim J. Serghini H. Alpha M.J. Mazliak P. Phytochemistry. 1994; 35: 1171-1175Crossref Scopus (9) Google Scholar, 20Serghinicaid H. Kim C. Justin A.M. Mazliak P. Plant Sci. 1988; 54: 93-101Crossref Scopus (13) Google Scholar). Thus LPCAT allows for a mechanism for acyl editing, although the in vitro results do not indicate how prevalent the reaction might be in vivo. In this context, isolated pea chloroplasts incubated with [14C]acetate immediately label PC with newly synthesized FA through a channeled pool of acyl-CoA (21Koo A.J.K. Kim J.B. Pollard M. J. Biol. Chem. 2004; 279: 16101-16110Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). As chloroplasts cannot synthesize PC de novo, this indicated a functional mechanism for PC synthesis with nascent FA through an acyl editing mechanism. A second mechanism for acyl editing involves hydrolysis of the phospholipid, such as PC to lyso-PC or even to glycerolphosphorylcholine (GPC), activation of the released free fatty acid and its reutilization for phospholipid synthesis from lyso-PC or GPC. Based on 18O labeling there is some indication that acyl chains esterified to bulk cellular PC are under a constant flux of acyl editing that proceeds wholly or in part through a hydrolytic deacylation-reacylation cycle (22Pollard M. Kim J. Plant Physiol. 1999; 121: 1217-1226Crossref PubMed Scopus (67) Google Scholar). The most direct line of evidence of acyl editing in plants comes from a careful analysis of the molecular species of monogalactosyldiacylglycerol and PC after labeling leaf disks of the 16:3 plant Brassica napus with carbon dioxide (23Williams J.P. Kim V. Khan M.U. Hodson J.N. Biochem. J. 2000; 349: 127-133Crossref PubMed Scopus (51) Google Scholar). [14C]Carbon dioxide labeling produced initial acyl-labeled species as expected for prokaryotic monogalactosyldiacylglycerol, namely dual labeled 16:0/18:1. By contrast for PC a high degree of scrambling between labeled and unlabeled acyl chains was noted. The authors concluded that there was continuous exchange of acyl groups between all molecular species of PC immediately after labeling and during the prolonged pulse-chase period of 48 h. In this report, we augment and extend these important observations and conclusions in several ways: 1) We perform rapid kinetic studies to more carefully address PC labeling and that of its precursors, PA and 1,2-diacyl-sn-glycerol (DAG). We address whether the initial incorporation of nascent fatty acids occurs via acyl editing, or if there is a rapid incorporation by de novo PC synthesis via glycerol-3-P (G3P) and DAG, which is followed by rapid acyl editing of PC. 2) We perform both total molecular species and stereochemical analyses on acyl-labeled PC using in vivo experiments with expanding pea leaves and seedlings. Pea is an 18:3 plant, so this complements the analysis done previously with the 16:3 plant B. napus (23Williams J.P. Kim V. Khan M.U. Hodson J.N. Biochem. J. 2000; 349: 127-133Crossref PubMed Scopus (51) Google Scholar). 3) We track molecular species of PC labeled in the glycerol backbone. When combined with the analysis of acyl labeling, this allows us to propose that sn-1 acyl editing is as important a component as sn-2 acyl editing. 4) As a control, we perform rapid labeling experiments in planta using carbon dioxide. The results parallel those obtained with excised tissue assays, indicating that there are no wound responses that compromise the metabolic conclusions obtained from excised tissue experiments. From these studies we analyzed possible models by which newly synthesized FA are incorporated into eukaryotic lipids. The data presented in this report and from other studies do not allow us to unambiguously define one particular model, but do narrow down the possible routes in which nascent FA are incorporated into eukaryotic glycerolipids and suggest future directions to reexamine this important yet poorly understood area of plant lipid metabolism. Plant Materials—All experiments were performed with Garden pea (Pisum sativum L. cv Little marvel) grown in soil/perlite/vermiculite (1:1:1) mixture at 22–25 °C under a day/night 8/16-h photoperiod of white light at 185–210 μmol m–2 s–1. Leaves, or in some cases, whole seedlings were harvested 8 days after sowing. Radiochemicals—[1-14C]Acetic acid, sodium salt (specific activity 50 mCi/mmol), [U-14C]glycerol (specific activity 150 mCi/mmol), and [14C]sodium bicarbonate (specific activity 50 mCi/mmol) were from American Radiolabeled Chemicals, Inc. (St. Louis, MO). Leaf [14C]Acetate or [14C]Glycerol Labeling—Leaf-labeling experiments used ∼0.3 g fresh weight of pea leaf strips per assay, incubated in the light (180–220 μmol m–2 s–1) at 22–24 °C with reciprocal shaking in 5 ml of media containing 20 mm MES pH 5.5, 0.1× MS salts, and 0.01% Tween 20. Cut leaves were placed in media without radioisotope and preincubated in ambient light for 5 min. Labeling was started by the addition of radioactive substrate and strong illumination. The reaction was quenched by transfer of leaves into isopropyl alcohol at 80 °C for 10 min. For labeling of acyl groups 250 μCi of [1-14C]acetic acid (1.0 mm) was used per replicate. For [14C]glycerol labeling of PC molecular species each incubation contained 47 μm [U-14C]glycerol (39 μCi). For [14C]glycerol time course the labeling media contained 25 μm [U-14C]glycerol (18 μCi), and the media was reused for consecutive 1, 3, 6, and 9 min time points. In vivo labeling with excised plant tissue can produce considerable variance between samples due to differences in development and in uptake of substrate. To minimize such variance each data point for total incorporation into lipids was normalized against the trend line for all time points to allow improved kinetic plots (Figs. 1 and 3).FIGURE 3Time course for incorporation of label from [14C]glycerol into the backbone and acyl groups of glycerolipids by excised pea leaves. A, amount of radioactivity in the glycerol backbone of each lipid. Results expressed as mean ± S.E. (triplicate determinations): PC, squares; DAG, triangles; PA, inverted triangles. B, % of total lipid [14C]glycerol labeling that is in the acyl chains. The three samples of PC and DAG (Fig. 3A replicates) were each combined for the glycerol to acyl group determination. PC, white bars; DAG, black bars.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Seedling [14C]Carbon Dioxide Labeling—Assays were conducted with 8-day potted pea seedlings in a closed 9.5 L glass desiccator under 250 μmol m–2 s–1 white light. [14C]CO2 was released by injection of 1 ml H2SO4 through the desiccator lid into a vial containing aqueous [14C]NaHCO3. The head space of the vial was quickly flushed with 30 ml of air, and the desiccator sealed for the desired time. Labeling was stopped by removal of the shoots at the base of the first leaves and quenching in 80 °C isopropyl alcohol. The assay used ∼700 μCi substrate and ∼40 seedlings. The seedlings from each labeling were split into two replicate samples (∼20 seedlings each) for analysis. Although each labeling was nominally for 5 min, about 5 min was required to remove all the seedlings and quench, so the assay is reported as of 5–10 min duration. General Methods—Lipids were extracted from hot isopropyl alcohol quenched tissue with hexane/isopropyl alcohol (24Hara A. Kim N.S. Anal. Biochem. 1978; 90: 420-426Crossref PubMed Scopus (2094) Google Scholar) after homogenization with a mortar and pestle. Chlorophyll was determined spectrophotometrically at 652 nm in 20% aqueous acetone (25Arnon D.I. Plant Physiol. 1949; 24: 1-15Crossref PubMed Google Scholar). Radioactivity in the total lipid samples, eluted lipids or organic and aqueous phases recovered from transmethylation was quantified by liquid scintillation counting (Beckman Instrument Inc., Fullerton, CA), while radioactivity on TLC plates was visualized and quantified by electronic radiography (Packard Instrument Co., Meriden, CT). AgNO3-TLC plates were prepared by impregnating Partisil® K6 silica gel 60 Å TLC plates (Whatman, Maidstone, UK) with 10% AgNO3 in acetonitrile (w/v), drying in air and activating at 110 °C for 5 min. Fatty acid methyl esters (FAMEs) were quantified by gas chromatography (GC) using a flame ionization detection and a DB-23 capillary column (30 m length × 0.25 mm inner diameter, 0.25 μ film thickness; J&W). Lipid Class Analysis—For kinetic analyses polar lipids were separated on a K6 silica TLC plates impregnated with 0.15 m ammonium sulfate and heat-activated (110 °C for 3 h) prior to lipid loading. Plates were developed in acetone/toluene/water (91:30:8, v/v/v) (26Khan M.U. Kim J.P. J. Chromatogr. 1977; 140: 179-185Crossref PubMed Scopus (96) Google Scholar). DAG was analyzed by first acetylating an aliquot of the total lipids in acetic anhydride/pyridine (3:2, v/v) and then separating the 1,2-diacyl-3-acetylglycerols by silica TLC developed in hexane/diethyl ether/acetic acid (50:50:1, v/v/v). PA was isolated by a first preparative silica TLC separation using a K6 plate developed in chloroform/methanol/water (65:25:4, v/v/v), and further purified on a second K6 plate developed in chloroform/acetone/methanol/acetic acid/water (10:4: 2:2:1, v/v/v/v/v). For preparative TLC all solvents contained 0.01% butylated hydroxyl-toluene antioxidant. Lipids were eluted from TLC silica with chloroform/methanol/water (5:5:1, v/v/v). Chloroform and 0.88% aq. KCl were added to give chloroform/methanol/water ratios of 2:1:1 (v/v/v), resulting in a phase separation. The aqueous phase was back extracted with chloroform and lipids were recovered from the combined chloroform phases. For [14C]glycerol- and [14C]carbon dioxide-labeled lipids the proportion of label in the acyl groups versus the backbone/head group was determined by transmethylation (27Ichihara K. Kim A. Yamamoto K. Nakayama T. Lipids. 1996; 31: 535-539Crossref PubMed Scopus (350) Google Scholar) and scintillation counting of the separated organic and aqueous phases. Aqueous phase radioactivity from PC was determined to be in the glycerol backbone by silica TLC developed in methanol, water, 28% NH4OH, 3 m NaCl (50:26.6:17.9: 3.4, v/v/v/v), as sample radioactivity co-migrated with glycerol and glycerol 3-phosphate standards, but not choline. Radiolabeled Acyl Group Composition—FAMEs were prepared from purified individual lipids or total lipids by base-catalyzed transmethylation (27Ichihara K. Kim A. Yamamoto K. Nakayama T. Lipids. 1996; 31: 535-539Crossref PubMed Scopus (350) Google Scholar). Recovered FAMEs were separated based on the number of double bonds by AgNO3-TLC, the plates being developed to ¾ height with hexane/diethyl ether (1:1, v/v), then fully with hexane/diethyl ether (9:1, v/v). PC Molecular Species Determination—PC was separated from other lipids by silica TLC (K6 plates developed with chloroform/methanol/acetic acid (75:25:8, v/v/v)). DAG was produced from the purified PC by phospholipase C (B. cereus, Sigma) digestion (28Christie W.W. Lipid Analysis: Isolation, Separation, Identification, and Structural Analysis of Lipids. 2003; (The Oily Press an imprint of PJ Barnes & Associates, Bridgwater, England)Google Scholar). The DAG was acetylated as described above and the 1,2-diacyl-3-acetylglycerols separated into molecular species based on the number of double bonds by argentation-TLC (28Christie W.W. Lipid Analysis: Isolation, Separation, Identification, and Structural Analysis of Lipids. 2003; (The Oily Press an imprint of PJ Barnes & Associates, Bridgwater, England)Google Scholar), using a triple development (½ then ¾ development in chloroform/methanol (96:4, v/v), then fully in chloroform/methanol (99:1, v/v)). The proportion of radioactivity in each band was determined by electronic autoradiography, then each band was eluted, the recovered lipids transmethylated, and the [14C]FAME analyzed by AgNO3-TLC, as described above. To determine endogenous acyl groups from isolated PC molecular species, triheptadecanoin was added as an internal standard to each fraction during elution and prior to transmethylation and GC analysis of FAME. When necessary 1,2-diacyl-3-acetylglycerol fractions recovered from AgNO3 TLC plates were further purified by reverse phase TLC on Partisil® KC18 silica gel 60 Å plates (Whatman) developed with methanol/acetone/water (75:25:2, v/v/v). PC Acyl Group Stereochemistry—PC was isolated as described above and digested with phospholipase A2 (Crotalus atrox, Sigma) (28Christie W.W. Lipid Analysis: Isolation, Separation, Identification, and Structural Analysis of Lipids. 2003; (The Oily Press an imprint of PJ Barnes & Associates, Bridgwater, England)Google Scholar). Briefly, PC was dissolved in 1 ml of diethyl ether and 0.5 unit of PLA2 in 0.1 ml of 50 mm Tris-HCl, pH 8.7, 5 mm CaCl2. The reaction was mixed vigorously for 5 min then the ether was evaporated under N2. To extract lipids 3.8 ml of chloroform/methanol (2:1, v/v) and 1 ml of 0.15 m acetic acid were added, the mixture vortexed, the chloroform phase collected and the aqueous phase back extracted with 2.5 ml of chloroform. Reaction products were separated on silica TLC plates developed with chloroform/methanol/acetic acid/water (50:30: 8/4, v/v/v/v). Radioactivity in the free fatty acid and lyso-PC fractions was quantified by electronic autoradiography, then each product eluted and transmethylated by heating at 50 °C in 5% sulfuric acid in methanol for 30 min. Labeled and unlabeled FAME compositions were determined as described above. Lipid Molecular Species by Mass Spectrometry—Pea leaf samples were analyzed by ESI-MS/MS by the Kansas Lipidomics Research Center. Extraction of lipids was conducted by their standard Arabidopsis leaf protocol. The data set from this analysis is available in supplemental Fig. S1. Net Rate of Fatty Acid Deposition—The net rate of fatty acid synthesis by pea leaves was determined by harvesting 10 leaves from pea seedlings at the start and end of the light cycle for 3 days. After immediate weighing to determine fresh weight, FAME and chlorophyll contents were measured as described above. Triheptadecanoin was added during the lipid extraction as an internal standard, providing methyl heptadecanoate after transmethylation for FAME analysis by GC. Kinetics of Glycerolipid Labeling from [14C]Acetate—Relationships between precursor and product pools in metabolic pathways are revealed by kinetic labeling experiments. The linear accumulation of a product, once reached, coincides with steady state labeling of all precursor pools. In this context [14C]acetate is ideal for the study of acyl lipid metabolism as it is rapidly taken up by excised leaf tissue and utilized for FAS, while the lipids are labeled in their acyl groups, with minimal head group labeling (21Koo A.J.K. Kim J.B. Pollard M. J. Biol. Chem. 2004; 279: 16101-16110Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). The kinetics of [14C]acetate labeling of glycerolipids from rapidly expanding excised pea leaves over a period of 9 min is shown in Fig. 1. PC was the major radiolabeled glycerolipid with over 65% of the label at all time points. Furthermore, steady state labeling was established very rapidly, with no detectable lag (Fig. 1A) and thus suggested that PC was a very early product of nascent FA incorporation into eukaryotic glycerolipids. A second independent 9-min time course gave similar results (data not shown). PE was labeled in a similar manner (Fig. 1C), with no obvious lag phase, but PC labeling was 15-fold greater than PE labeling despite endogenous PC levels only being twice that of PE (supplemental Fig. S1A). Both the PA and the DAG pools were still filling after the onset of PC steady state labeling. The label in PA reached a maximum by 3 min whereas DAG labeling increased throughout the time course (Fig. 1B). Thus, although PA and DAG are intermediates in the de novo synthesis of PC via acylation of glycerol-3-P, these [14C]acyl-labeled intermediates do not show a precursor-product relationship relative to PC labeling, and thus represent different glycerolipid pools. Of course, we cannot rule out that very small labeled PA and DAG pools might contribute to PC labeling in a precursor-product relationship, because experimentally, quenching in these kinetic assays limits the temporal resolution of the method. A statistical analysis suggested about 0.5 min of variance (95% CI) in rate extrapolations to zero time. In conclusion, we were not able to detect a [14C]acyl-labeled glycerolipid precursor pool to the labeling of PC. Acyl Compositions from [14C]Acetate Labeling of Lipids—Fig. 2 shows the composition of labeled fatty acids from the [14C]acetate time course, for total lipids, PC, PA, DAG, PG, and PE. Oleate was the major product in total lipids (Fig. 2A), with saturates, mainly palmitate, decreasing from 32% at 1 min to about 24% for the remainder of the assay period. The reason for this decline is unknown. There was no detectable desaturation of 18:1 at the earliest time point but desaturation to 18:2 was detectable from 3 min onwards. PC had less saturates (11% falling to 8%) whereas PE had more saturates (54% falling to 38%). By the end of the time course desaturation produced 7% linoleate in PC (Fig. 2B). In contrast to PC and PE, PA (Fig. 2C) contained high levels of saturates (>60%) throughout the time course and had an acyl composition with the closest match to PG. PA is also an intermediate of the prokaryotic glycerolipid synthesis pathway, and so the labeled PA pool is likely the precursor for prokaryotic PG labeling. In this context, close inspection of Fig. 1, B and C shows a kinetic precursor-product relationship between PA (label reaching a maximum at 3 min) and PG (lag phase ending at ∼3 min), confirming this conclusion. DAG contained an intermediate level of saturates compared with PC and PE. The origin of the labeled DAG is not certain. It is probably largely eukaryotic in origin, and may arise in part from the reverse action of CDP-choline:DAG cholinephosphotransferase or phospholipase C on labeled PC, or from acyl groups edited from PC re-entering the eukaryotic de novo glycerolipid synthesis pathway. Any of these explanations is supported by the appearance of labeled linoleate in DAG by 9 min (Fig. 2D). Whatever its origin, the difference in labeled acyl group composition of PA and DAG compared with PC supports the conclusion from the kinetic data that they do not represent precursors of initial PC acyl labeling. The Kinetics of Glycerolipid Labeling from [14C]Glycerol—[14C]Glycerol is rapidly taken up by pea leaves and incorporated into the glycerol backbone of glycerolipids. In addition, acyl groups also become labeled because glycerol 3-phosphate equilibrates with glycolytic precursors, leading to plastid acetyl-CoA production (29Slack C.R. Kim P.G. Balasingham N. Biochem. J. 1977; 162: 289-296Crossref PubMed Scopus (61) Google Scholar). To separately analyze the label from the backbone/head-group and the acyl chains, isolated lipid classes were transmethylated. Analysis of the aqueous fraction from PC by TLC indicated that the radioactivity was contained in the glycerol backbone and not the choline headgroup, as noted before (29Slack C.R. Kim P.G. Balasingham N. Biochem. J. 1977; 162: 289-296Crossref PubMed Scopus (61) Google Scholar). In marked contrast to acetate labeling, [14C]glycerol incorporation into lipids exhibited a lag (Fig. 3A). At the earliest time points, radioactivity in DAG was approximately equal to PC. We assume that the labeled PA includes a large contribution from the plastid component. However, chloroplast lipid assembly in 18:3-plants does not require PA conversion to DAG, so we also assume the labeled DAG is largely associated with extraplastidial membranes. Two lines of argument support the notion that the biosynthesis of PC from labeled acetate (Fig. 1) and from labeled glycerol (Fig. 3) report different metabolic processes. First, for de novo PC synthesis the relative movement of label from PA to DAG to PC should be same for both glycerol and acyl group labeling strategies. However, at the earliest time points, it is clear that acetate acyl chain labeling produces PC ≫ DAG (Fig. 1A) whereas glycerol backbone labeling produces DAG ≈ PC (Fig. 3A), suggesting separate metabolic processes. S" @default.
• W2025682709 created "2016-06-24" @default.
• W2025682709 creator A5027619375 @default.
• W2025682709 creator A5045610316 @default.
• W2025682709 creator A5069034028 @default.
• W2025682709 date "2007-10-01" @default.
• W2025682709 modified "2023-10-01" @default.
• W2025682709 title "Incorporation of Newly Synthesized Fatty Acids into Cytosolic Glycerolipids in Pea Leaves Occurs via Acyl Editing" @default.
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