Acyl coenzyme a preference of the glycerol phosphate pathway in the microsomes from the maturing seeds of palm, maize, and rapeseed

The acyl coenzyme A (CoA) preference of the glycerol phosphate pathway in the microsomes from the maturing seeds of palm (Butia capitata Becc.), maize (Zea mays L.), and rapeseed (Brassica napus L.) was tested. Each microsomal preparation was incubated with [¹⁴C-U]-glycerol-3-phosphate and either lauroyl CoA, oleoyl CoA, or erucoyl CoA, and the ¹⁴C-lipid products were separated and quantitated. In the presence of oleoyl CoA, the microsomes from each of the three species produced lysophosphatidic acid, phosphatidic acid, diacylglycerol, and triacylglycerol with kinetics consistent with the operation of the glycerol phosphate pathway. In the presence of erucoyl CoA, the microsomes from all the three species did not produce di- or tri-acyl lipids. In the presence of lauroyl CoA, only the microsomes from palm, but not those from maize or rapeseed, synthesized di- and tri-acyl lipids. This lack of reactivity of lauroyl CoA was also observed in the microsomes from maturing castor bean, peanut, and soybean. In maize seed and rapeseed, but not palm seed, the kinetics of labeling suggest that lauroyl and erucoyl moieties of the acyl CoAs were incorporated into lysophosphatidic acid but failed to enter into phosphatidic acid and thus the subsequent lipid products. We propose that the high degree of acyl specificity of lysophosphatidyl acyltransferase is the blocking step in the synthesis of triacylglycerols using lauroyl CoA or erucoyl CoA. The significance of the findings in seed oil biotechnology is discussed.     

Lysophosphatidate Acyltransferase in the Microsomes from Maturing Seeds of Meadowfoam (Limnanthes alba)

Lysophosphatidate (LPA) acyltransferase (EC 2.3. 1.51) in the microsomes from the maturing seeds of meadowfoam (Limnanthes alba), nasturtium (Tropaeolum majus), palm (Syagrus cocoides), castor bean (Ricinus communis), soybean (Glycine max), maize (Zea mays), and rapeseed (Brassica napus) were tested for their specificities toward 1-oleoyl-LPA or 1-erucoyl-LPA, and oleoyl coenzyme A (CoA) or erucoyl CoA. All the enzymes could use either of the two acyl acceptors and oleoyl CoA, but only the meadowfoam enzyme could use erucoyl CoA as the acyl donor to produce dierucoyl phosphatidic acid (PA). The meadowfoam enzyme was studied further. It had an optimal activity at pH 7 to 8, and its activity was inhibited by 1 millimolar MnCl₂, ZnCl₂, or p-chloromercuribenzoate. In a test of substrate specificity using increasing concentrations of either 1-oleoyl-LPA or 1-erucoyl-LPA, and either oleoyl CoA or erucoyl CoA, the enzyme activity in producing PA was highest for dioleoyl-PA, followed successively by 1-oleoyl-2-erucoyl-PA, dierucoyl-PA, and 1-erucoyl-2-oleoyl-PA. In a test of substrate selectivity using a fixed combined concentration, but varying proportions, of 1-oleoyl-LPA and 1-erucoyl-LPA, and of oleoyl CoA and erucoyl CoA, the enzyme showed a pattern of acyl preference similar to that observed in the test of substrate specificity, but the preference toward oleoyl moiety in the substrates was slightly stronger. The meadowfoam microsomes could convert [¹⁴C]glycerol-3-phosphate to diacylglycerols and triacylglycerols in the presence of erucoyl CoA. The meadowfoam LPA acyltransferase is unique in its ability to produce dierucoyl-PA, and should be a prime candidate for use in the production of trierucin oils in rapeseed via genetic engineering.     

Lysophosphatidate acyltransferase activities in the microsomes from palm endosperm, maize scutellum, and rapeseed cotyledon of maturing seeds

Lysophosphatidate (LPA) acyltransferase (EC 2.3.1.51) in the microsomes from palm endosperm (Syagrus cocoides Martius), maize scutellum (Zea mays L.), and rapeseed cotyledon (Brassica napus L.) of maturing seeds were studied for their specificities toward the acyl moiety of the substrates lysophosphatidate and acyl coenzyme A (CoA). The LPA acceptor greatly influenced the acyl CoA specificity of the enzyme and vice versa. With 1-oleoyl-lysophosphatidate (LPA-18:1), the palm enzyme was equally active on oleoyl CoA and lauroyl CoA, whereas the maize and rapeseed enzymes were more active on oleoyl CoA than on lauroyl CoA. With 1-lauroyl-lysophosphatidate (LPA-12), which generated less activity than LPA-18:1, the palm enzyme was three times more active on lauroyl CoA than on oleoyl CoA. LPA-12 was an inactive substrate for the maize and rapeseed enzymes. The selectivity of the enzymes was also studied using a mixture of LPA-18:1 and LPA-12, as well as lauroyl CoA and oleoyl CoA. Under this selectivity condition and compared to the specificity condition, the enzymes from all the three seeds exerted stronger preference for oleoyl moiety in either the LPA or acyl CoA, and again, only the palm enzyme could act on LPA-12. Similar studies, although in lesser detail, showed that the enzymes from soybean and castor bean were similar to the maize and rapeseed enzymes in having little activity on substrates containing lauroyl moiety. The results demonstrate the importance of the acyl group in the sn-1 position of LPA in determining the acyl preference in the sn-2 position in phosphatidate synthesis. The palm enzyme appears to be the only one capable of synthesizing phosphatidates containing high amounts of lauric moieties.     

A Comparison of Oleic Acid Metabolism in the Soybean (Glycine max [L.] Merr.) Genotypes Williams and A5, a Mutant with Decreased Linoleic Acid in the Seed

The metabolism of oleoyl coenzyme A (CoA) was examined in developing seed from two soybean (Glycine max [L.] Merr.) genotypes: Williams, a standard cultivar and A5, a mutant containing nearly twice the oleic acid (18:1) content of Williams. The in vitro rates of esterification of oleoyl-CoA to lysophosphatides by acyl-CoA: lysophosphatidylcholine acyltransferase was similar in both genotypes and lysophosphatidyl-ethanolamine was a poor substrate. Crude extracts desaturated exogenous [1-¹⁴C]dioleoyl phosphatidylcholine at 14% of the rate achieved with [1-¹⁴C]oleoyl-CoA, and 50 micromolar lysophosphatidylcholine. The desaturase enzyme also required NADH for full activity. Extracts from Williams contained 1.5-fold more oleoyl phosphatidylcholine desaturase activity, on a fresh weight basis, than did A5 and appeared to have a similar affinity for oleoyl-CoA. There was 1.2- to 1.9-fold more linoleic acid (18:2) in phosphatidylcholine from Williams than from A5, measured at two stages of development, but both genotypes had a similar distribution of fatty acids in the one and two positions. Phosphatidylethanolamine in A5 contained relatively more linoleic acid (18:2) in the one position than did Williams. The increased oleic acid (18:1) content in A5 appeared to be a result of decreased rates of 18:1 desaturation of oleoyl-phosphatidylcholine in this genotype.     

Glycerolipid synthesis by homogenate and oil bodies from developing mustard (Sinapis alba L.) seed

[1-¹⁴C]Oleic acid and [14-¹⁴C]erucic acid were converted to their acyl-CoA derivatives and incorporated into acyl lipids by a homogenate from developing mustard (Sinapis alba L.) seed and oil bodies, as well as supernatant isolated by centrifugation at 20000 g. In both homogenate and oil bodies, the oleoyl moieties from exogenous [1-¹⁴C]oleoyl-CoA were most extensively incorporated into phosphatidic acids, but very little into phosphatidylcholines. The pattern of labelling of acyl lipids by oleoyl versus erucoyl moieties from either of the corresponding fatty acids, added individually or as a mixed substrate, indicates that oleoyl-CoA directly acylates sn-glycerol-3-phosphate to yield lysophosphatidic acids and phosphatidic acids that are subsequently converted to mono- and diacylglycerols. In contrast, erucoyl-CoA predominantly acylates preformed mono-and diacylglycerols containing oleoyl moieties to yield triacylglycerols containing erucoyl moieties.     

Lipids in Cruciferae IX. The Effect of Growth Temperature and Stage of Development on the Fatty Acid Composition of Leaves, Siliques and Seeds of "Zero-erucic-acid" Breeding Lines of Brassica napus

Two breeding lines of “zero-erucic-acid” rapeseed (Brassica napus) were grown in climate chambers at a constant night temperature (12°C) and constant photoperiod (16 hours) but with different day temperatures (15, 20 and 25°C). Samples of leaves, siliques and immature seeds were analysed for total fatty acid pattern. The content of different acyl lipids and the fatty acid pattern of these lipids were also determined in some of the samples by use of preparative TLC followed by GLC of the fatty acids. The mature seeds produced by ten plants of each selection in each climate were analysed separately for total fatty acid composition. Mono- and digalactosyl diglycerides (MGDG, DGDG) were the predominant acyl lipids in leaves and siliques. In developing seeds they also were more abundant than the phospholipids, but in this case the neutral lipids, mainly triacylglycerols, contained about 95% of the total fatty acids. Large variations were found in the fatty acid composition of monogalactosyl diglyceride and digalactosyl diglyceride, isolated from leaves, siliques and immature seeds. The palmitic acid content of leaf MGDG was about 15 %, atypically high for MGDG from photosynthetic tissue. The linolenic acid content of the MGDG was about 45 %, 30 % and 10 % in the leaf, silique and seed tissues respectively. A hexadecatrienoic acid (16: 3) was found almost exclusively in the MGDG samples of leaves, siliques and immature seeds (about 25 %, 10 % and 3 % 16:3 respectively). The lipids of siliques — mainly photosynthetising tissue — were different from those of leaves and had especially high contents of stearic acid (6–12 % in the different lipids). For all lipid classes studied, leaves grown at the lowest day temperature had a slightly lower oleic and higher linolenic acid content than those grown at the highest temperature. On the other hand, increasing the day temperature caused a decreased level of oleic, an increased level of linoleic and an essentially unchanged level of linolenic acids in the mature seeds from both selections.     

Triacylglycerol Bioassembly in Microspore-Derived Embryos of Brassica napus L. cv Reston

Erucic acid (22:1) was chosen as a marker to study triacylglycerol (TAG) biosynthesis in a Brassica napus L. cv Reston microspore-derived (MD) embryo culture system. TAGs accumulating during embryo development exhibited changes in acyl composition similar to those observed in developing zygotic embryos of the same cv, particularly with respect to erucic and eicosenoic acids. However, MD embryos showed a much higher rate of incorporation of ¹⁴C-erucoyl moieties into TAGs in vitro than zygotic embryos. Homogenates of early-late cotyledonary stage MD embryos (14-29 days in culture) were assessed for the ability to incorporate 22:1 and 18:1 (oleoyl) moieties into glycerolipids. In the presence of [1-¹⁴C]22:1-coenzyme A (CoA) and various acyl acceptors, including glycerol-3-phosphate (G-3-P), radiolabeled erucoyl moieties were rapidly incorporated into the TAG fraction, but virtually excluded from other Kennedy Pathway intermediates as well as complex polar lipids. This pattern of erucoyl incorporation was unchanged during time course experiments or upon incubation of homogenates with chemicals known to inhibit Kennedy Pathway enzymes. In marked contrast, parallel experiments conducted using [1-¹⁴C]18:1-CoA and G-3-P indicated that ¹⁴C oleoyl moieties were incorporated into lyso-phosphatidic acids, phosphatidic acids, diacylglycerols, and TAGs of the Kennedy Pathway, as well as other complex polar lipids, such as phosphatidylcholines and phosphatidylethanolamines. When supplied with l-[2-³H(N)]G-3-P and [1-¹⁴C]22:1-CoA, the radiolabeled TAG pool contained both isotopes, indicating G-3-P to be a true acceptor of erucoyl moieties. Radio-high-performance liquid chromatography, argentation thin-layer chromatography/gas chromatography-mass spectrometry, and stereospecific analyses of radiolabeled TAGs indicated that 22:1 was selectively incorporated into the sn-3 position by a highly active diacylglycerol acyltransferase (DGAT; EC 2.3.1.20), while oleoyl moieties were inserted into the sn-1 and sn-2 positions. In the presence of sn-1,2-dierucin and [1-¹⁴C]22:1-CoA, homogenates and microsomal preparations were able to produce radiolabeled trierucin, a TAG not found endogenously in this species. A 105,000g pellet fraction contained 22:1-CoA:DGAT exhibiting the highest specific activity. The rate of 22:1-CoA:DGAT activity in vitro could more than account for the maximal rate of TAG biosynthesis observed in vivo during embryo development. In double label experiments, G-3-P was shown to stimulate the conversion of [³H]phosphatidylcholines to [³H]diacylglycerols, which subsequently acted as acceptors for ¹⁴C erucoyl moieties. In vitro, 22:1 moieties did not enter the sn-1 position of TAGs by a postsynthetic modification or transacylation of preformed TAGs.     

Acylation of lysolecithin in the intestinal mucosa of rats

1. The presence of an active acyl-CoA-lysolecithin (1-acylglycerophosphorylcholine) acyltransferase was demonstrated in rat intestinal mucosa. 2. ATP and CoA were necessary for the incorporation of free [1-(14)C]oleic acid into lecithin (phosphatidylcholine). 3. The reaction was about 20 times as fast with [1-(14)C]oleoyl-CoA as with free oleic acid, CoA and ATP. 4. With 1-acylglycerophosphorylcholine as the acceptor, both oleic acid and palmitic acid were incorporated into the beta-position of lecithin; the incorporation of palmitic acid was 60% of that of oleic acid. 5. Of the various analogues of lysolecithin tested as acyl acceptors from [1-(14)C]oleoyl CoA, a lysolecithin with a long-chain fatty acid at the 1-position was most efficient. 6. The enzyme was mostly present in the brush-border-free particulate fraction of the intestinal mucosa. 7. Of the various tissues of rats tested for the activity, intestinal mucosa was found to be the most active, with testes, liver, kidneys and spleen following it in decreasing order.     

Effect of Growth Temperature on the Biosynthesis of Chloroplastic Galactosyldiacylglycerol Molecular Species in Brassica napus Leaves

Brassica napus leaves developed at low temperature display rapid in situ desaturation of monogalactosyldiacylglycerol (MGDG) fatty acids leading to the production of hexadecatrienoic/linolenic acid. This was shown by radioactivity-tracer experiments to occur via a sequence of desaturations proceeding from the initially synthesized palmitic/oleic acid molecular species to palmitic/linoleic acid, palmitoleic/linoleic acid, hexadecadienoic/linoleic acid, hexadecadienoic/linolenic acid, and finally to hexadecatrienoic/linolenic acid. The results suggest that there is increased activity in all five desaturation steps in leaves developed at low temperatures. Labeling data also indicate that there is another pool of MGDG which is more slowly desaturated before galactosylation to digalactosyldiacylglycerol (DGDG). Our data further suggest that relative rates of galactosylation of chloroplastic and cytosolic MGDG molecular species may regulate the final amounts of chloroplastic and cytosolic MGDG and DGDG in the leaf. We have proposed a model for chloroplastic biosynthesis and desaturation of galactosyldiacylglycerols in the leaves of Brassica napus, a 16:3 plant.     

Functional analysis and tissue-differential expression of four FAD2 genes in amphidiploid Brassica napus derived from Brassica rapa and Brassica oleracea

Fatty acid desaturase 2 (FAD2), which resides in the endoplasmic reticulum (ER), plays a crucial role in producing linoleic acid (18:2) through catalyzing the desaturation of oleic acid (18:1) by double bond formation at the delta 12 position. FAD2 catalyzes the first step needed for the production of polyunsaturated fatty acids found in the glycerolipids of cell membranes and the triacylglycerols in seeds. In this study, four FAD2 genes from amphidiploid Brassica napus genome were isolated by PCR amplification, with their enzymatic functions predicted by sequence analysis of the cDNAs. Fatty acid analysis of budding yeast transformed with each of the FAD2 genes showed that whereas BnFAD2-1, BnFAD2-2, and BnFAD2-4 are functional enzymes, and BnFAD2-3 is nonfunctional. The four FAD2 genes of B. napus originated from synthetic hybridization of its diploid progenitors Brassica rapa and Brassica oleracea, each of which has two FAD2 genes identical to those of B. napus. The BnFAD2-3 gene of B. napus, a nonfunctional pseudogene mutated by multiple nucleotide deletions and insertions, was inherited from B. rapa. All BnFAD2 isozymes except BnFAD2-3 localized to the ER. Nonfunctional BnFAD2-3 localized to the nucleus and chloroplasts. Four BnFAD2 genes can be classified on the basis of their expression patterns.     

Inhibition of hormone-sensitive lipase by intermediary lipid metabolites.

Hormone-sensitive lipase (HSL) is inhibited in a non-competitive manner by oleoyl CoA, oleic acid and 2-monopalmitoylglycerol, 50% inhibition being observed at concentrations of approx. 0.1 microM, 0.5 microM and 500 microM, respectively. HSL is a key enzyme in lipid metabolism, mobilising triacylglycerol and cholesterol ester stores in several tissues. Feedback inhibition of HSL by oleoyl CoA and oleic acid may therefore prevent accumulation of free fatty acids and cholesterol in the cell, whereas 2-monoacylglycerol may act as a feedback inhibitor if the capacity of monoacylglycerol lipase is exceeded.     

ACYLATION OF LYSOPHOSPHATIDYLSERINE BY RAT BRAIN MICROSOMES

Abstract— The acylation of lysophosphatidylserine, prepared by snake venom digestion of phosphatidylserine, by rat brain microsomes is described. Acylation was monitored by spectrophotometric assay and by measuring the incorporation of radioactively labelled acyl CoA thioesters. Acylation was time dependent, showed an approximately linear response to enzyme concentration and had a pH optimum of 9.0. Maximum acylation was attained at a concentration of about 100 μM for lysophosphatidylserine and about 40μM for acyl CoA thioesters. Positional distribution studies with [¹⁴C]oleoyl CoA and [¹⁴C]arachidonoyl CoA showed incorporation was predominantly at position -2, but with significant labelling at position–1, particularly with oleoyl CoA, possibly as a result of isomerization of the 1–acyl isomer of lysophosphatidylserine. Both saturated and unsaturated thioesters could serve as acyl group donors. Myristoyl CoA was considerably superior to palmitoyl CoA and stearoyl CoA, which were poor acyl group donors. Some selectivity was shown among the long chain unsaturated thioesters, linoleoyl, linolenoyl and arachidonoyl CoA being the most effective acylating agents. Although docosahexaenoic acid is a major unsaturated fatty acid in brain phosphatidylserine, its CoA ester was a relatively poor acyl group donor. Relative acylation rates remained essentially constant over a wide range of lysophosphatidylserine concentrations. It is concluded that acyl transfer mechanisms are active in brain for the regulation of the fatty acid profile of phosphatidylserine.     

Mapping of QTLs controlling content of fatty acid composition in rapeseed (Brassica napus)

The improvement of fatty acid composition is one of the major goals of breeding in rapeseed (Brassica napus). The aim of this study was to provide more information on the genetic determination of fatty acid composition by investigating quantitative trait loci (QTLs). The study was based on two-year of field trials (in 2006 and 2007) with a population of recombinant inbred lines (RILs), which originated from a cross between GH06 and P174. The level of erucic acid (C22:1) was significantly negatively correlated with those of palmitic acid (C16:0), oleic acid (C18:1), linoleic acid (C18:2), linolenic acid (C18:3), and eicosenoic acid (C20:1) in both years. A total of 40 QTLs for six fatty acids were detected and most of them were clustered on linkage groups N8, N9, and N13. These results suggested strongly that there were significant correlations between the levels of fatty acid components and would be useful for the future improvement of breeding programs focused on fatty acids in rapeseed.     

Biosynthesis of triacylglycerols containing very long chain monounsaturated acyl moieties in developing seeds

Particulate (15,000g) fractions from developing seeds of honesty (Lunaria annua L.) and mustard (Sinapis alba L.) synthesize radioactive very long chain monounsaturated fatty acids (gadoleic, erucic, and nervonic) from [1-¹⁴C]oleoyl-CoA and malonyl-CoA or from oleoyl-CoA and [2-¹⁴C]malonyl-CoA. The very long chain monounsaturated fatty acids are rapidly channeled to triacylglycerois and other acyl lipids without intermediate accumulation of their CoA thioesters. When [1-¹⁴C]oleoyl-CoA is used as the radioactive substrate, phosphatidylcholines and other phospholipids are most extensively radiolabeled by oleoyl moieties rather than by very long chain monounsaturated acyl moieties. When [2-¹⁴C]malonyl-CoA is used as the radioactive substrate, no radioactive oleic acid is formed and the newly synthesized very long chain monounsaturated fatty acids are extensively incorporated into phosphatidylcholines and other phospholipids as well as triacylglycerols. The pattern of labeling of the key intermediates of the Kennedy pathway, e.g. lysophosphatidic acids, phosphatidic acids, and diacylglycerols by the newly synthesized very long chain monounsaturated fatty acids is consistent with the operation of this pathway in the biosynthesis of triacylglycerols.     

Effects of jasmonic Acid on embryo-specific processes in brassica and linum oilseeds

A number of effects on embryogenesis of the putative phytohormone jasmonic acid (JA), and its methyl ester (MeJA), were investigated in two oilseed plants, repeseed (Brassica napus) and flax (Linum usitatissimum). Results from treatments with JA and MeJA were compared with those of a known effector of several aspects of embryogenesis, abscisic acid (ABA). Jasmonic acid was identified by gas chromatography-mass spectrometry as a naturally occurring substance in both plant species during embryo development. Both JA and MeJA can prevent precocious germination of B. napus microspore embryos and of cultured zygotic embryos of both species at an exogenous concentration of >1 micromolar. This dose-response was comparable with results obtained with ABA. Inhibitory effects were also observed on seed germination with all three growth regulators in rapeseed and flax. A number of molecular aspects of embryogenesis were also investigated. Expression of the B. napus storage protein genes (napin and cruciferin) was induced in both microspore embryos and zygotic embryos by the addition of 10 micromolar JA. The level of napin and cruciferin mRNA detected was similar to that observed when 10 micromolar ABA was applied to these embryos. For MeJA only slight increases in napin or cruciferin mRNA were observed at concentrations of 30 micromolar. Several oilbody-associated proteins were found to accumulate when the embryos were incubated with either JA or ABA in both species. The MeJA had little effect on oilbody protein synthesis. The implications of JA acting as a natural regulator of gene expression in zygotic embryogenesis are discussed.     

The microspore-derived embryo ofBrassica napus L. as a tool for studying embryo-specific lipid biogenesis and regulation of oil quality

Summary A time-course study of lipid accumulation in microspore-derived embryos and developing zygotic embryos of rapeseed (Brassica napus L. ssp.oleifera) is presented. Rapid storage fat (triacylglycerol) biosynthesis was induced in microspore-derived embryos of oilseed rape (cv Topas) when the embryos were transferred from standing cultures (10 ml) to fresh medium (75 ml) and shake cultured. Triacylglycerols accumulated, after a lag period of 7 days, at a linear rate of approximately twice that of the developing zygotic embryo. The fatty acid composition of triacylglycerols in microspore-derived embryos closely parallelled that of the developing zygotic embryos. In the microspore-derived embryos, the amount of phosphatidylcholine, the major substrate for the production of polyunsaturated fatty acids in oilseeds, remained constant during the linear phase of triacylglycerol production, whereas it increased steadily in the zygotic embryos. The fatty acid composition of individual cotyledons from microspore embryos shake cultured for 15 days was compared with that of individual mature seeds. Relative amounts of the major fatty acids, i.e. palmitic, oleic and linoleic acids, were essentially the same, whereas the microspore-derived embryos had about 35% less stearic acid and 35% more linolenic acid than the mature seeds. Variation in the amounts of oleic, linoleic and linolenic acids between seeds was similar to that found between cotyledons of microspore-derived embryos, whereas variation in palmitic and stearic acid levels was significantly lower between microsporederived cotyledons than between the seeds. The results indicate that microspore-derived embryos from shake cultures should be convenient for use in studying the regulation of oil biosynthesis and for rapidly screening for oil quality in genetically altered rapeseed.     

6-d-glucopyranosyl fatty acid esters from Brassica napus pollen

6-d-Glucopyranosyl esters of palmitic, oleic, linoleic and linolenic acids were identified in Brassica napus (rape) pollen. These esters are inactive as plant growth promoters in the bean second-internode bioassay.     

Inhibition of Neutral Lipase from Castor Bean Lipid Bodies by Coenzyme A (CoA) and Oleoyl-CoA

The neutral lipase (EC 3.1.1.3) in lipid body membranes isolated from the endosperm of 4 day old castor (Ricinus communis L.) seedlings catalyzes the hydrolysis of [¹⁴C]trioleoylglycerol, releasing [¹⁴C]oleic acid for up to 4 hours. However, the addition of Mg-ATP and coenzyme A (CoA), which are present in the cytoplasm of plant cells, caused a progressive inhibition of the neutral lipase such that after 15 minutes, release of [¹⁴C]oleic acid was almost undetectable. A fatty acyl CoA synthetase was found in the lipid body membrane which converts [¹⁴C]oleic acid produced from the lipase reaction to [¹⁴C]oleoyl-CoA under these conditions. The concentration of free oleoyl-CoA in the reaction mixture when the lipase was inhibited by 50% was calculated to be about 21 micromolar. It was found that a mixture of exogenously added oleoyl-CoA and CoA was most effective in causing lipase inhibition. Little inhibition of lipase was detected in the presence of CoA alone. It is possible that this effect is important In vivo in coordinating lipase activity with fatty acid oxidation.     

Lipid biosynthesis in developing mustard seed: formation of triacylglycerols from endogenous and exogenous Fatty acids

Cotyledons of developing mustard (Sinapis alba L.) seed have been found to synthesize lipids containing the common plant fatty acids and very long-chain monounsaturated (icosenoic, erucic, and tetracosenic) and saturated (icosanoic, docosanoic, and tetracosanoic) fatty acids from various radioactive precursors. The in vivo pattern of labeling of acyl lipids, either from fatty acids synthesized `endogenously' from radioactive acetate or malonate, or from radioactive fatty acids added `exogenously', indicates the involvement of the following pathways in the biosynthesis of triacylglycerols. Palmitic, stearic, and oleic acid, synthesized in the acyl carrier protein-track, are channeled to the Coenzyme A (CoA)-track and converted to triacylglycerols via the glycerol-3-phosphate pathway. Pools of stearoyl-CoA and oleoyl-CoA are elongated to very long-chain saturated and monounsaturated acyl-CoA, respectively. Most of the very long-chain saturated acyl-CoAs acylate preformed diacylglycerols. Very long-chain monounsaturated acyl-CoAs are converted to triacylglycerols, partly via phosphatidic acids and diacylglycerols, and partly by acylation of preformed diacylglycerols.     

Lipase in lipid bodies of cotyledons of rape and mustard seedlings

Lipolytic activity was absent in the crude cotyledon extract of ungerminated rapeseed (Brassica napm L. var. Dwarf Essex), and increased to a peak at day 4 in seedling growth, concomitant with the decrease in total lipids. About 50% of the lipase activity was recovered in the lipid bodies isolated from the cotyledon extract by flotation centrifugation. Isolated lipid bodies underwent autolysis of internal triacylglycerols resulting in the release of fatty acids. After the triacylglycerols in isolated lipid bodies had been extracted with diethyl ether, the lipase was recovered in the remaining membrane fraction. The lipase had a maximal activity at pH 6.5 on trierucin, trilinolein, or endogenous triacylglycerols, and at pH 8.0 on N-methylindoxylmyristate. The lipase was most active on trierucin and trilinolein, and hydrolyzed the related di- and monoacylglycerols at lower rates. There was little enhancement of the lipase activity in the presence of NaCl, CaCl₂, or detergents, and detergents in general reduced the activity. The hydrolysis of trierucin was linear until about 50% of the trierucin had been converted to erucic acid, and there was little accumulation of dierucin and monoerucin. Lipase extracted from lipid bodies isolated from germinated rapeseed of the variety Tower, which contains little or no erucic acids in the storage triacylglycerols, also had the highest activities on trierucin and trilinolein. A comparative study on mustard seed (Brassica juncea) revealed that the mustard lipase possessed characteristics very similar to those of the rapeseed lipase.