Fatty Acid Specificity and Selectivity of the Chloroplast sn-Glycerol 3-Phosphate Acyltransferase of the Chilling Sensitive Plant, Amaranthus lividus

Chilling sensitivity of plants is strongly correlated with the presence of high levels of a species of chloroplast phosphatidylglycerol that contains two saturated fatty acids. The most straightforward synthetic pathway for this lipid would require the primary acylation of sn-glycerol 3-phosphate (G3P) with a saturated fatty acid (palmitic acid) rather than with oleic acid, an unsaturated acid. This selective incorporation would differ markedly from the reported properties of the chloroplast G3P acyltransferases of pea and spinach, two chilling resistant plants and thus we have studied the chloroplast G3P acyltransferase of Amaranthus lividus, a chilling sensitive plant. In contrast to our results and those of others (M. Frentzen et al. 1983 Eur J Biochem 129: 629-636 and previous work) with the pea and spinach enzymes, the amaranthus chloroplast G3P acyltranferase did not select oleic acid donors from a mixture of oleic and palmitic acid donors (either coenzyme A or acyl carrier protein thioesters). Instead the fatty acid composition of the synthesized 1-acyl G3P faithfully reflected the composition of the acyl donor mixture. However, the amaranthus enzyme did strongly select against incorporation of stearic acid. The properties of the amaranthus G3P acyltransferase are consistent with this enzyme having the major role in synthesis of the disaturated phosphatidylglycerol species.     

On the export of fatty acids from the chloroplast

The model for export of fatty acids from plastids proposes that the acyl-ACP (acyl carrier protein) product of de novo fatty acid synthesis is hydrolyzed in the stroma by acyl-ACP thioesterases and the free fatty acid (FFA) released is then transferred to the outer envelope of the plastid where it is reactivated to acyl-CoA for utilization in cytosolic glycerolipid synthesis. Experiments were performed to assess whether the delivery of nascent FFA from the stroma for long chain acyl-CoA synthesis (LACS) occurs via simple diffusion or a more complex mechanism. The flux through the in vivo FFA pool was estimated using kinetic labeling experiments with spinach and pea leaves. The maximum half-life for FFA in the export pool was < or =1 s. Isolated pea chloroplasts incubated in the light with [14C]acetate gave a linear accumulation of FFA. When CoASH and ATP were present there was also a linear accumulation of acyl-CoA thioesters (plus derived polar lipids), with no measurable lag phase (<30 s), indicating that the FFA pool supplying LACS rapidly reached steady state. The LACS reaction was also measured independently in the dark after in situ generated FFA had accumulated yielding estimates of LACS substrate-velocity relationships. Based on these experiments the LACS reaction with in situ generated FFA as substrate is only about 3% of the LACS activity required in vivo at the very low concentrations of the FFA export pool calculated from the in vivo experiment. Furthermore, bovine serum albumin rapidly removed in situ generated FFA from chloroplasts, but could not compete effectively for "nascent" FFA substrates of LACS. Together the data suggest a locally channeled pool of exported FFA that is closely linked to LACS.     

Comparison of glycerolipid biosynthesis in non-green plastids from sycamore (Acer pseudoplatanus) cells and cauliflower (Brassica oleracea) buds.

The availability of methods to fractionate non-green plastids and to prepare their limiting envelope membranes [Alban, Joyard & Douce (1988) Plant Physiol. 88, 709-717] allowed a detailed analysis of the biosynthesis of lysophosphatidic acid, phosphatidic acid, diacylglycerol and monogalactosyl-diacylglycerol (MGDG) in two different types of non-green starch-containing plastids: plastids isolated from cauliflower buds and amyloplasts isolated from sycamore cells. An enzyme [acyl-ACP (acyl carrier protein):sn-glycerol 3-phosphate acyltransferase) recovered in the soluble fraction of non-green plastids transfers oleic acid from oleoyl-ACP to the sn-1 position of sn-glycerol 3-phosphate to form lysophosphatidic acid. Then a membrane-bound enzyme (acyl-ACP:monoacyl-sn-glycerol 3-phosphate acyltransferase), localized in the envelope membrane, catalyses the acylation of the available sn-2 position of 1-oleoyl-sn-glycerol 3-phosphate by palmitic acid from palmitoyl-ACP. Therefore both the soluble phase and the envelope membranes are necessary for acylation of sn-glycerol 3-phosphate. The major difference between cauliflower (Brassica oleracea) and sycamore (Acer pseudoplatanus) membranes is the very low level of phosphatidate phosphatase activity in sycamore envelope membrane. Therefore, very little diacylglycerol is available for MGDG synthesis in sycamore, compared with cauliflower. These findings are consistent with the similarities and differences described in lipid metabolism of mature chloroplasts from 'C18:3' and 'C16:3' plants (those with MGDG containing C18:3 and C16:3 fatty acids). Sycamore contains only C18 fatty acids in MGDG, and the envelope membranes from sycamore amyloplasts have a low phosphatidate phosphatase activity and therefore the enzymes of the Kornberg-Pricer pathway have a low efficiency of incorporation of sn-glycerol 3-phosphate into MGDG. By contrast, cauliflower contains MGDG with C16:3 fatty acid, and the incorporation of sn-glycerol 3-phosphate into MGDG by the enzymes associated with envelope membranes is not limited by the phosphatidate phosphatase. These results demonstrate that: (1) non-green plastids employ the same biosynthetic pathway as that previously established for chloroplasts (the formation of glycerolipids is a general property of all plastids, chloroplasts as well as non-green plastids), (2) the envelope membranes are the major structure responsible for the biosynthesis of phosphatidic acid, diacylglycerol and MGDG, and (3) the enzymes of the envelope Kornberg-Pricer pathway have the same properties in non-green starch-containing plastids as in mature chloroplasts from C16:3 and C18:3 plants.     

Isoforms of acyl carrier protein involved in seed-specific fatty acid synthesis

Seeds of coriandrum sativum (coriander) and Thunbergia alata (black-eyed Susan vine) produce unusual monoenoic fatty acids which constitute over 80% of the total fatty acids of the seed oil. The initial step in the formation of these fatty acids is the desaturation of palmitoyl-ACP (acyl carrier protein) at the delta(4) or delta(6) positions to produce delta(4)-hexadecenoic acid (16:1(delta(4)) or delta(6)-hexadecenoic acid (16:1(delta(6)), respectively. The involvement of specific forms of ACP in the production of these novel monoenoic fatty acids was studied. ACPs were partially purified from endosperm of coriander and T. alata and used to generate 3H- and 14C-labelled palmitoyl-ACP substrates. In competition assays with labelled palmitoyl-ACP prepared from spinach (Spinacia oleracea), delta(4)-acyl-ACP desaturase activity was two- to threefold higher with coriander ACP than with spinach ACP. Similarly, the T. alata delta(6) desaturase favoured T. alata ACP over spinach ACP. A cDNA clone, Cs-ACP-1, encoding ACP was isolated from a coriander endosperm cDNA library. Cs-ACP-1 mRNA was predominantly expressed in endosperm rather than leaves. The Cs-ACP-1 mature protein was expressed in E. coli and comigrated on SDS-PAGE with the most abundant ACP expressed in endosperm tissues. In in vitro delta(4)-palmitoyl-ACP desaturase assays, the Cs-ACP-1 expressed from E. coli was four- and 10-fold more active than spinach ACP or E. coli ACP, respectively, in the synthesis of delta(4)-hexadecenoic acid from palmitoyl-ACP. In contrast, delta(9)-stearoyl-ACP desaturase activity from coriander endosperm did not discriminate strongly between different ACP species. These results indicate that individual ACP isoforms are specifically involved in the biosynthesis of unusual seed fatty acids and further suggest that expression of multiple ACP isoforms may participate in determining the products of fatty acid biosynthesis.     

L-acetylcarnitine, a substrate for chloroplast fatty acid synthesis

Abstract. H¹⁴CO₃ was not incorporated into fatty acids by isolated pea leaf chloroplasts, which, therefore, do not possess a self-contained pathway for the synthesis of fatty acids from early intermediates of the Calvin cycle. Citrate, pyruvate, acetate and L-acetylcarnitine were all shown to act as sources of acetyl groups for fatty acid synthesis by pea leaf chloroplasts. L-acetylcarnitine was the best substrate, being incorporated into fatty acids at rates that were at least five-fold higher than those achieved with the other substrates. Citrate was incorporated into fatty acids at the lowest rate, followed by pyruvate, with acetate being incorporated at the second highest rate of all. When the isolated chloroplasts were ruptured, an inhibition of L-acetylcarnitine incorporation into fatty acids was noted, whilst acetate incorporation remained unaffected. L-acetylcarnitine also increased the ratio of monoenoic: saturated fatty acids synthesized, compared with a 1:1 ratio observed when citrate, pyruvate and acetate were supplied as substrates. It is suggested that L-carnitine and carnitine acyltransferases play a central role in plant acyl CoA metabolism by facilitating the transfer of activated acyl groups across membranes (acyl CoA barriers).     

Positional Specificity and Fatty Acid Selectivity of Purified sn-Glycerol 3-Phosphate Acyltransferases from Chloroplasts

Soluble acyl-CoA:sn-glycerol 3-phosphate acyltransferases (EC 2.3.1.15) which are localized in chloroplasts were purified from leaves of Pisum sativum and Spinacia oleracea and obtained free from interfering activities. The purification raised the specific activities by factors of about 1,000 for pea and 200 for spinach preparations. In pea chloroplasts, acyltransferase activity occurs in two soluble forms with apparent isoelectric points of 6.3 and 6.6. For both forms, the same molecular weight of about 42,000 was determined. The enzyme from spinach chloroplasts showed a slightly higher molecular weight and a lower isoelectric point of 5.2.     

Thematic review series: Glycerolipids. Acyltransferases in bacterial glycerophospholipid synthesis

Phospholipid biosynthesis is a vital facet of bacterial physiology that begins with the synthesis of the fatty acids by a soluble type II fatty acid synthase. The bacterial glycerol-phosphate acyltransferases utilize the completed fatty acid chains to form the first membrane phospholipid and thus play a critical role in the regulation of membrane biogenesis. The first bacterial acyltransferase described was PlsB, a glycerol-phosphate acyltransferase. PlsB is a key regulatory point that coordinates membrane phospholipid formation with cell growth and macromolecular synthesis. Phosphatidic acid is then produced by PlsC, a 1-acylglycerol-phosphate acyltransferase. These two acyltransferases use thioesters of either CoA or acyl carrier protein (ACP) as the acyl donors and have homologs that perform the same reactions in higher organisms. However, the most prevalent glycerol-phosphate acyltransferase in the bacterial world is PlsY, which uses a recently discovered acyl-phosphate fatty acid intermediate as an acyl donor. This unique activated fatty acid is formed from the acyl-ACP end products of the fatty acid biosynthetic pathway by PlsX, an acyl-ACP:phosphate transacylase.     

Metabolic Evidence for the Involvement of a [delta]4-Palmitoyl-Acyl Carrier Protein Desaturase in Petroselinic Acid Synthesis in Coriander Endosperm and Transgenic Tobacco Cells

We have previously demonstrated that the double bond of petroselinic acid (18:1[delta]6cis) in coriander (Coriandrum sativum L.) seed results from the activity of a 36-kD desaturase that is structurally related to the [delta]9-stearoyl-acyl carrier protein (ACP) desaturase (E.B. Cahoon, J. Shanklin, J.B. Ohlrogge [1992] Proc Natl Acad Sci USA 89: 11184-11188). To further characterize the biosynthetic pathway of this unusual fatty acid, 14C-labeling experiments were conducted using developing endosperm of coriander. Studies were also performed using suspension cultures of transgenic tobacco (Nicotiana tabacum L.) that express the coriander 36-kD desaturase, and as a result produce petroselinic acid and [delta]4-hexadecenoic acid. When supplied exogenously to coriander endosperm slices, [1-14C]palmitic acid and stearic acid were incorporated into glycerolipids but were not converted to petroselinic acid. This suggested that petroselinic acid is not formed by the desaturation of a fatty acid bound to a glycerolipid or by reactions involving acyl-coenzyme As (CoA). Instead, evidence was most consistent with an acyl-ACP route of petroselinic acid synthesis. For example, the exogenous feeding of [1-14C]lauric acid and myristic acid to coriander endosperm slices resulted in the incorporation of the radiolabels into long-chain fatty acids, including primarily petroselinic acid, presumably through acyl-ACP-associated reactions. In addition, using an in vitro fatty acid biosynthetic system, homogenates of coriander endosperm incorporated [2-14C]malonyl-CoA into petroselinic acid, of which a portion was detected in a putative acyl-ACP fraction. Furthermore, analysis of transgenic tobacco suspension cultures expressing the coriander 36-kD desaturase revealed significant amounts of petroselinic acid and [delta]4-hexadecenoic acid in the acyl-ACP pool of these cells. Also presented is evidence derived from [U-14C]nonanoic acid labeling of coriander endosperm, which demonstrates that the coriander 36-kD desaturase positions double bonds relative to the carboxyl end of acyl-ACP substrates. The data obtained in these studies are rationalized in terms of a biosynthetic pathway of petroselinic acid involving the [delta]4 desaturation of palmitoyl-ACP by the 36-kD desaturase followed by two-carbon elongation of the resulting [delta]4-hexadecenoyl-ACP.     

Intraorganelle localization and substrate specificities of the mitochondrial acyl-CoA: sn-glycerol-3-phosphate O-acyltransferase and acyl-CoA: 1-acyl-sn-glycerol-3-phosphate O-acyltransferase from potato tubers and pea leaves

The mitochondrial sn-glycerol-3-phosphate and 1-acyl-sn-glycerol-3-phosphate O-acyltransferases from potato tubers and pea leaves were investigated with respect to their intraorganelle localization, their positional and substrate specificities, and their fatty acid selectivities. In mitochondria from potato tubers both enzymes were found to be located in the outer membrane. The 1-acyl-sn-glycerol-3-phosphate O-acyltransferase of pea mitochondria showed the same intraorganelle localization whereas the sn-glycerol-3-phosphate O-acyltransferase behaved like a soluble protein of the intermembrane space. The sn-glycerol-3-phosphate O-acyltransferase of both potato and pea mitochondria used sn-glycerol-3-phosphate but not dihydroxyacetone phosphate as acyl acceptor and exclusively catalyzed the formation of 1-acyl-sn-glycerol-3-phosphate which subsequently served as substrate for the second acylation reaction at its C-2 position. Both acyltransferases of potato as well as pea mitochondria showed higher activities with acyl-CoA than with the corresponding acyl-(acyl carrier protein) thioesters. When different acyl-CoA thioesters were offered separately, the sn-glycerol-3-phosphate O-acyltransferase of potato mitochondria displayed no fatty acid specificity whereas the enzyme of pea mitochondria revealed one for saturated acyl groups. On the other hand, the mitochondrial 1-acyl-sn-glycerol-3-phosphate O-acyltransferases from both potato tubers and pea leaves were more active on unsaturated than on saturated acyl-CoA thioesters. Furthermore, these enzymes preferentially used oleoyl- and linoleoyl-CoA when they were offered in a mixture with saturated ones, although the fatty acid selectivity of the pea enzyme was less pronounced than that of the potato enzyme. The sn-glycerol-3-phosphate O-acyltransferase of potato mitochondria displayed a slight preference for saturated acyl groups.     

Inhibition of plant fatty acid synthesis by nitroimidazoles.

1. The effect of the addition of a number of nitroimidazoles was tested on fatty acid synthesis by germinating pea seeds, isolated lettuce chloroplasts and a soluble fraction from pea seeds. 2. All the compounds tested had a marked inhibition on stearate desaturation by lettuce chloroplasts and on the synthesis of very-long-chain fatty acids by pea seeds. 3. In contrast, the effect of the drugs on total fatty acid synthesis from [14C]acetate in chloroplasts was related to the compound's electron reduction potentials. 4. Of the compounds used, only metronidazole had a marked inhibition on palmitate elongation in the systems tested. 5. The mechanism of inhibition of plant fatty acid synthesis by nitroimidazoles is discussed and the possible relevance of these findings to their neurotoxicity is suggested.     

Characterisation of acyltransferases from Synechocystis sp. PCC6803

As phylogenetic ancestors of plant chloroplasts cyanobacteria resemble plastids with respect to lipid and fatty acid composition. These membrane lipids show the typical prokaryotic fatty acid pattern in which the sn-2 position is exclusively esterified by C(16) acyl groups. In the course of de novo glycerolipid biosynthesis this prokaryotic fatty acid pattern is established by the sequential acylation of glycerol-3-phosphate with acyl-ACPs by the activity of different acyltransferases. In silico approaches allowed the identification of putative Synechocystis acyltransferases involved in glycerolipid metabolism. Functional expression studies in Escherichia coli showed that sll1848 codes for a lysophosphatidic acid acyltransferase with a high specificity for 16:0-ACP, whereas slr2060 encodes a lysophospholipid acyltransferase, with a broad acyl-ACP specificity but a strong preference for lysophosphatidyglycerol especially its sn-2 acyl isomer as acyl-acceptor. The generation and analysis of the corresponding Synechocystis knockout mutants revealed that lysophosphatidic acid acyltransferase unlike the lysophospholipid acyltransferase is essential for the vital functions of the cells.     

On the control of long-chain-fatty acid synthesis in isolated intact spinach (Spinacia oleracea) chloroplasts

1. Chloroplasts isolated from spinach leaves by using the low-ionic-strength buffers of Nakatani & Barber [(1977) Biochim. Biophys. Acta.461, 510-512] had higher rates of HCO(3) (-)-dependent oxygen evolution (up to 369mumol/h per mg of chlorophyll) and higher rates of [1-(14)C]acetate incorporation into long-chain fatty acids (up to 1500nmol/h per mg of chlorophyll) than chloroplasts isolated by using alternative procedures. 2. Acetate appeared to be the preferred substrate for fatty acid synthesis by isolated chloroplasts, although high rates of synthesis were also measured from H(14)CO(3) (-) in assays permitting high rats of photosynthesis. Incorporation of H(14)CO(3) (-) into fatty acids was decreased by relatively low concentrations of unlabelled acetate. Acetyl-CoA synthetase activity was present 3-4 times in excess of that required to account for rates of [1-(14)C]acetate incorporation into fatty acids, but pyruvate dehydrogenase was either absent or present in very low activity in spinach chloroplasts. 3. Rates of long-chain-fatty acid synthesis from [1-(14)C]acetate in the highly active chloroplast preparations, compared with those used previously, were less dependent on added cofactors, but showed a greater response to light. The effects of added CoA plus ATP, Triton X-100 and sn-glycerol 3-phosphate on the products of [1-(14)C]acetate incorporation were similar to those reported for less active chloroplast preparations. 4. Endogenous [(14)C]acetyl-CoA plus [(14)C]malonyl-CoA was maintained at a constant low level even when fatty acid synthesis was limited by low HCO(3) (-) concentrations. Endogenous [(14)C]acyl-(acyl-carrier protein) concentrations increased with increasing HCO(3) (-) concentration and higher rates of fatty acid synthesis, but were slightly lower in the presence of Triton X-100. It is proposed that rates of long-chain-fatty acid synthesis in isolated chloroplasts at saturating [1-(14)C]acetate concentrations and optimal HCO(3) (-) concentrations may be primarily controlled by rates of removal of the products of the fatty acid synthetase.     

A novel, general radioimmunoassay for acyl carrier proteins

A radioimmunoassay (RIA) for acyl carrier proteins (ACP) is described that is based on the competitive binding between [3H]acyl-ACP and unlabeled ACP of the same species. The radiolabeled antigen, [3H]palmitoyl-ACP, is enzymatically synthesized by Escherichia coli acyl-ACP synthetase. Because acyl-ACP synthetase can specifically radiolabel ACP in crude extracts from several plant sources, the use of this enzyme to prepare [3H]acyl-ACP obviates the need for pure preparations of each ACP. Preparation of [3H]acyl-ACP with a specific activity of 15 Ci/mmol allows RIA detection of total ACP in crude plant extracts at the nanogram level. Because antibodies against spinach ACP partially crossreact with ACP from many plant sources, RIAs for other plant species can be constructed using only one preparation of antibody. ACP preparations from safflower, soybean, avocado, corn, and E. coli show a decreasing order of partial immunocross-reactivity with spinach ACP-specific antiserum, as examined by RIA using spinach [3H]palmitoyl-ACP.     

Acyl-ACP thioesterases from castor (Ricinus communis L.): An enzymatic system appropriate for high rates of oil synthesis and accumulation

Acyl–acyl carrier protein (ACP) thioesterases are enzymes that terminate the intraplastidial fatty acid synthesis in plants by hydrolyzing the acyl-ACP intermediates and releasing free fatty acids to be incorporated into glycerolipids. These enzymes are classified in two families, FatA and FatB, which differ in amino acid sequence and substrate specificity. In the present work, both FatA and FatB thioesterases were cloned, sequenced and characterized from castor (Ricinus communis) seeds, a crop of high interest in oleochemistry. Single copies of FatA and FatB were found in castor resulting to be closely related with those of Jatropha curcas. The corresponding mature proteins were heterologously expressed in Escherichia coli for biochemical characterization after purification, resulting in high catalytic efficiency of RcFatA on oleoyl-ACP and palmitoleoyl-ACP and high efficiencies of RcFatB for oleoyl-ACP and palmitoyl-ACP. The expression profile of these genes displayed the highest levels in expanding tissues that typically are very active in lipid biosynthesis such as developing seed endosperm and young expanding leaves. The contribution of these two enzymes to the synthesis of castor oil is discussed.     

Metabolism of exogenous long-chain fatty acids by spinach leaves

When applied in liquid paraffin to the upper surface of expanding spinach leaves, [1-14C]palmitic acid was efficiently and exclusively incorporated into the sn-1 position of cellular glycerolipids, principally phosphatidylcholine and triacylglycerol. A slow transfer of fatty acids from phosphatidylcholine to chloroplast glycolipids subsequently occurred with the positional specificity of the label remaining intact. Labeled palmitate at the sn-1 position of monogalactosyldiacylglycerol was desaturated to hexadecatrienoate so that 1-[14C]hexadecatrienoyl-2-linolenoyl-3-galactosoylglycerol became the major labeled species of the lipid between 8 and 24 h. There was no evidence of deacylation/reacylation reactions modifying the acyl compositions of spinach leaf glycerolipids for at least 48 h after labeling with [1-14C]palmitic acid; even the partially prokaryotic glycerolipids remained firmly labeled at the sn-1 position. Exogenous [1-14C]stearic acid was also incorporated into the sn-1 position of MGD, presumably by the same mechanism, and was there desaturated to [14C]linolenate. Exogenous [1-14C]oleic acid was initially incorporated equally into both sn-1 and sn-2 positions of phosphatidylcholine, and was desaturated to linoleate at both positions before the label was rapidly transferred to monogalactosyldiacylglycerol. There, desaturation of linoleate to linolenate took place. Galactolipids remained equally labeled at both positions throughout the 6 days of the experiment, but label was concentrated in the 1-saturated-2-[14C]linolenoyl molecular species of phosphatidylcholine as those species with two [14C]linoleoyl residues were drawn off for monogalactolipid synthesis. Glycerolipids synthesised from exogenous [1-14C]acetate by spinach leaves were labeled equally at both the sn-1 and the sn-2 positions. These results are interpreted as providing strong support for the two-pathway scheme of glycerolipid synthesis in plants.     

Purification and characterization of beta-ketoacyl-ACP synthetase I from Spinacia oleracea leaves

beta-Ketoacyl-acyl carrier protein (ACP) synthetase I was purified 180-fold from crude extracts of spinach leaves. The purified preparation was completely free from other component enzymes of the de novo fatty acid synthetase (FAS) system. Its molecular weight was estimated to be 56,000 by gel filtration. The apparent Km value for malonyl-CoA in the presence of ACP and malonyl-CoA:ACP transacylase was 4 microM. Purified synthetase I was highly active with acyl-ACP having chain lengths from C2 to C14, with hexanoyl-ACP being the most effective substrate, but palmitoyl-ACP was far less effective and stearoyl-ACP almost inactive. The antibiotic, cerulenin, strongly inhibited synthetase I activity. The inhibition by cerulenin was protected by prior incubation with hexanoyl-ACP, decanoyl-ACP, and myristoyl-ACP. The synthetase was inhibited by 1 mM p-CMB and 5 mM NEM, but not by 1 mM arsenite.     

Phosphatidylglycerol synthesis in pea chloroplasts: pathway and localization

Isolated intact pea chloroplasts synthesized phosphatidylglycerol from either [¹⁴C]acetate or [¹⁴C]glycerol 3-phosphate. Both time-course and pulse-chase labeling studies demonstrated a precursor-product relationship between newly synthesized phosphatidic acid and newly synthesized phosphatidylglycerol.

The synthesis both of CDP-diacylglycerol from exogenous phosphatidic acid and CTP, and of phosphatidylglycerol from exogenous CDP-diacylglycerol and glycerol 3-phosphate, could be assayed in fractions obtained from disrupted chloroplasts. Moreover, the enzymes catalyzing these reactions were localized in the inner envelope membrane. Exogenous phosphatidic acid was incorporated into phosphatidylglycerol, but only following its incorporation into CDP-diacylglycerol. Finally, radio-active phosphatidic acid synthesized in the envelope membranes from [¹⁴C]palmitoyl-ACP and 1-oleoyl-glycerol 3-phosphate was sequentially incorporated into labeled CDP-diacylglycerol and phosphatidylglycerol upon the addition of appropriate substrates and cofactors. Thus, we have demonstrated that (a) the synthesis of phosphatidylglycerol in chloroplasts occurs by the pathway: phosphatidic acid → CDP-diacylglycerol →→ phosphatidylglycerol, and (b) phosphatidylglycerol synthesis is located in the inner envelope membrane.

     

Regulation of plant Fatty Acid biosynthesis : analysis of acyl-coenzyme a and acyl-acyl carrier protein substrate pools in spinach and pea chloroplasts

In previous work (D. Post-Beittenmiller, J.G. Jaworski, J.B. Ohlrogge [1991] J Biol Chem 266: 1858-1865), the in vivo acyl-acyl carrier protein (ACP) pools were measured in spinach (Spinacia oleracea) leaves and changes in their levels were compared to changes in the rates of fatty acid biosynthesis. To further examine the pools of substrates and cofactors for fatty acid biosynthesis and to evaluate metabolic regulation of this pathway, we have now examined the coenzyme A (CoA) and short chain acyl-CoA pools, including acetyl- and malonyl-CoA, in isolated spinach and pea (Pisum sativum) chloroplasts. In addition, the relationships of the acetyl- and malonyl-CoA pools to the acetyl- and malonyl-ACP pools have been evaluated. These studies have led to the following conclusions: (a) Essentially all of the CoA (31-54 μm) in chloroplasts freshly isolated from light-grown spinach leaves or pea seedling was in the form of acetyl-CoA. (b) Chloroplasts contain at least 77% of the total leaf acetyl-CoA, based on comparison of acetyl-CoA levels in chloroplasts and total leaf. (c) CoA-SH was not detected either in freshly isolated chloroplasts or in incubated chloroplasts and is, therefore, less than 2 μm in the stroma. (d) The malonyl-CoA:ACP transacylase reaction is near equilibrium in both light- and dark-incubated chloroplasts, whereas the acetyl-CoA:ACP transacylase reaction is far from equilibrium in light-incubated chloroplasts. However, the acetyl-CoA:ACP transacylase reaction comes nearer to equilibrium when chloroplasts are incubated in the dark. (e) Malonyl-CoA and -ACP could be detected in isolated chloroplasts only during light incubations, and increased with increased rates of fatty acid biosynthesis. In contrast, both acetyl-CoA and acetyl-ACP were detectable in the absence of fatty acid biosynthesis, and acetyl-ACP decreased with increased rates of fatty acid biosynthesis. Together these data have provided direct in situ evidence that acetyl-CoA carboxylase plays a regulatory role in chloroplast fatty acid biosynthesis.     

Comparison of acetate- and pyruvate-dependent fatty-acid synthesis by spinach chloroplasts

In recent studies using intact chloroplasts of spinach (Spinacia oleracea L.) to investigate the accumulation of acetyl-CoA produced by the activity of either acetyl-CoA synthetase (EC 6.2.1.1) or the pyruvate-dehydrogenase complex, this product was not detectable. These results in combination with new information on the physiological levels of acetate and pyruvate in spinach chloroplasts (H.-J. Treede et al. 1986, Z. Naturforsch. 41 C, 733–740) prompted a reinvestigation of the incorporation of [1-¹⁴C] acetate and [2-¹⁴C] pyruvate into fatty acids at physiological concentrations.The K _m for the incorporation into fatty acids was about 0.1 mM for both metabolites and thus agreed with the values obtained by H.-J. Treede et al. (1986) for acetyl-CoA synthetase and the pyruvate dehydrogenase complex. However, acetate was incorporated with a threefold higher V _max. Saturation for pyruvate incorporation into the fattyacid fraction was achieved only at physiological pyruvate concentrations (<1.0 mM). The diffusion kinetics observed at higher concentrations may be the result of contamination with derivates of the labeled substrate. Competition as well as double-labeling experiments with [³H]acetate and [2-¹⁴C]pyruvate support the notion that, at least in spinach, chloroplastic acetate is the preferred substrate for fatty-acid synthesis when both substrates are supplied concurrently (P.G. Roughan et al., 1979 b, Biochem. J. 184, 565–569).Experiments with spinach leaf discs confirmed the predominance of fatty-acid incorporation from acetate. Radioactivity from [1-¹⁴C]acetate appeared to accumulate in glycerolipids while that from [2-¹⁴C]pyruvate was apparently shifted in favor of the products of prenyl metabolism.Abbreviations Chl chlorophyll - TLC thin-layer chromatography     

Control of Lipid Synthesis during Soybean Seed Development: Enzymic and Immunochemical Assay of Acyl Carrier Protein

During soybean seed (Glycine max, var Am Soy 71) development, the rate of lipid biosynthesis per seed increases greatly. As the seed reaches maturity, lipid synthesis declines. To study the controls over the oil synthesis and storage process, we have chosen acyl carrier protein (ACP) as a representative marker for the fatty acid synthetase pathway. We have quantitated soybean ACP levels by both enzymic and immunochemical methods. Escherichia coli acyl-ACP synthetase was used as an assay for enzymically active ACP. Total ACP protein was determined by immunoassay using antibodies prepared in rabbits against spinach ACP. These antibody preparations also bind ACP isolated from soybeans, allowing development of a radioimmunoassay based on competition with [³H]palmitoyl-ACP. The enzymic and immunochemical measurement of ACP at various stages of seed development have indicated that ACP activity and ACP antigen increase markedly in correlation with the in vivo increase in lipid synthesis. These results indicate that a major control over the increase in lipid synthesis arises through regulation of the levels of the fatty acid biosynthetic proteins. However, as the seed reaches maturity and lipid biosynthesis declines, ACP per seed remains relatively high. In the mature seed, we found that more than 95% of the ACP is localized in the cotyledons, less than 5% is in the axis, and less than 1% is in the seed coat.