Oil bodies isolated from the mature seeds of rape (Brassica napus L.), mustard (Brassica juncea l.), cotton (Gossypium hirsutum L.), flax (Linus usifafis simum), maize (Zea mays L.), peanut (Arachis hypogaea L.), and sesame (Sesamum indicum L.) had average diameters that were different but within a narrow range (0.6-2.0 fim), as measured from electron micrographs of seria1 sections. Their contents of triacylglycerols (TAC), phospholipids, and proteins (oleosins) were correlated with their sizes. The correlation fits a formula that describes a spherical particle surrounded by a shell of a monolayer of phospholipids embedded with oleosins. Oil bodies from the various species contained substantial amounts of the uncommon negatively charged phosphatidylserine and phosphatidylinositol, as well as small amounts of free fatty acids. These acidic lipids are assumed to interact with the basic amino acid residues of the oleosins on the surface of the phospholipid layer. lsoelectrofocusing revealed that the oil bodies from the various species had an isoelectric point of 5.7 to 6.6 and thus possessed a negatively charged surface at neutra1 pH. We conclude that seed oil bodies from diverse species are very similar in structure. In rapeseed during maturation, TAC and oleosins accumulated concomitantly. TAC-synthesizing acyltransferase activities appeared at an earlier stage and peaked during the active period of TAC accumulation. l h e concomitant accumulation of TAC and oleosins is similar to that reported earlier for maize and soybean, and the finding has an implication for the mode of oil body synthesis during seed maturation.Seeds store TAG as food reserves for germination and postgerminative growth of the seedlings. The TAG are present in small, discrete intracellular organelles called oil bodies (Yatsu and Jacks, 1972; Appelquist, 1975; Stymne and Stobart, 1987; Huang, 1992). Isolated oil bodies have a spherical shape and possess diameters ranging from about 0.5 to 2.0 pm. They contain mostly TAG and small amounts of PL and proteins called oleosins. It is generally agreed that the oil body has a matrix of TAG surrounded by a layer of PL embedded with oleosins. The PL form a monolayer such that the acyl moieties of the molecules face inward to interact with the hydrophobic TAG in the matrix, and the hydrophilic PL head groups are exposed to the cytosol. The embedded oleosin molecule is composed of three structural domains: an N-terminal amphipathic domain, a central hydrophobic domain, and a C-terminal amphipathic a-helical domain (Vance and Huang, 1987; Qu and Huang, 1990; Murphy et al., 1991; Tzen et al., 1992). It is predicted that the hydrophobic portion Supported by U.S. Department of Agriculture grant 91-01430 (A.H.C.H.).* Corresponding author; fax 1-714-787-4437. 267 of the oleosin molecule penetrates the PL layer into the TAG matrix, and its amphipathic portion resides on the PL layer or protrudes to the exterior. The structure of an oil body as described in the preceding paragraph implies that the relative...

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 MnCI2, ZnCI2, 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 pattem 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.In oil seeds, TAG3 is synthesized from fatty acid via the Kennedy pathway which consists of four major enzymatic reactions (14). Glycerol-3-P is first acylated at the sn

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 1[4C-UIglycerol-3-phosphate and either lauroyl CoA, oleoyl CoA, or erucoyl CoA, and the '4C-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.In oil seeds, the FA3 composition of the storage TG is speciesand variety-specific, and it can be modified by environmental factors such as temperature (14,18). Within a seed species, the FA composition in each of the three positions of a TG is also largely inherited (18).In seeds as well as in mammalian tissues, TG is synthesized from acyl CoA and glycerol-P via at least four enzyme reactions in the glycerol-P pathway (Kennedy pathway). The first two enzymes are glycerol-P acyltransferase and LPA acyltransferase. These two enzymes possess some acyl CoA preference, especially the second enzyme, such that their specificities as well as the in vivo pool sizes of acyl CoAs produce the observed positional acyl specificity in the TG (1,14,18,19 is concerned is unknown (18). The last enzyme, DG acyltransferase, is supposed to be relatively less specific for acyl CoAs, and it is assumed that the in vivo pool sizes of the acyl CoAs largely determine the acyl moiety in the sn-3 position of the TG. In seed oil biotechnology, one major goal is to alter the chain length of the fatty acyl moiety of the TG (7, 13, 18). Researchers are using different approaches to manipulate the genes controlling enzymes for the elongation of FA. This manipulation appears to be theoretically workable. However, what is not known is whether the newly designed FA can be accommodated by the other components of the TG synthesis machinery. The acyl preference of all the three glycerol acyltransferases in maturing seeds ofdifferent species had bee...

Diacylglycerol acyltransferase (EC 2.3.1.20) activity was detected in the microsomal fractions of maturing maize scutellum, soybean cotyledon, peanut cotyledon, and castor bean endosperm. The activity detected was high enough to account for the in vivo rate of triacylglycerol synthesis. The activity of the maize enzyme was characterized using diolein micelles prepared by sonication in Tween 20 as the substrate. The activity was highest at pH values of 6 to 7. The activity was proportional to the amount of enzyme added, and the reaction rate was linear for about 2 minutes. The enzyme was not inactivated by Tween 20, Zwitterion 3-08, Triton-X 100, and cholate, but was inactivated completely by sodium dodecyl sulfate. The enzyme was active on linoleoyl coenzyme A (CoA), palmitoyl CoA, and oleoyl CoA, although the activity was highest on linoleoyl CoA. Endogenous diacylglycerol was present in the microsomes, and the enzyme activity was only partially dependent on the addition of external diolein. Subcellular fractionation of the total scutellum extract in sucrose density gradients was performed. By comparing the migration of the enzyme between rate and equilibrium centrifugation, and between equilibrium centrifugation in the presence and absence of magnesium ions in the preparative media, the enzyme was shown to be associated with the rough endoplasmic reticulum. Some of the above findings on the maize enzyme were extended to the enzymes from castor bean, soybean, and peanuts.Diacylglycerol acyltransferase (EC 2.3.1.20) catalyzes the final step in the synthesis of triacylglycerols in oil seeds (18,22). It is also the only known enzyme unique to the long biosynthetic pathway of triacylglycerols, since the diacylglycerol produced could also be used to produce phospholipids or galactolipids. In spite of the importance of this enzyme in triacylglycerol biosynthesis in oil seeds, its properties have not been well studied.Diacylglycerol acyltransferase in the microsomal fraction of developing seeds has been assayed directly or together with other enzymes (6,10,20,21). In a recent, and so far the most detailed, study, the general properties of the enzyme in a safflower microsomal fraction were characterized (10). However, the detected level ofactivity was almost a magnitude lower than that required to catalyze the sequence of reaction from glycerol phosphate to triacylglycerol (21, 22). Using an enzyme assay which had been used successfully to study the enzyme in spinach leaves, Martin and Wilson (13) failed to detect enzyme activity in the cotyledon extract of developing soybean.An important but unknown aspect of diacylglycerol acyltransferase is its subcellular location. In general, the microsomal fractions were used to study the enzyme activity, and they 'Supported by National Science Foundation grant DMB 85-15556.presumably contained vesicles of the ER as well as membranes of other subcellular particles, including broken plastids. In a recent detailed analysis of the subcellular location ofthe enzyme in spinach...

ABSTRACrIn their seed triacylglycerols, Cuphea carthagenensis contains 62% lauric acid; maize possesses 50% linoleic acid and 30% oleic acid; rapeseed (Brassica napus L. var Dwarf Essex) has 40% erucic acid; and Canola (Brassica napus L. var Tower) holds 60% oleic acid and 23% linoleic acid. Diacylglycerol acyltransferase (EC 2.3.1.20) in the microsomal preparations fron maturing seeds of the above species were tested for their preference in using different forms of acyl coenzyme A (CoA). Lauroyl CoA, oleoyl CoA, and erucoyl CoA individually or in equimolar mixtures at increasing concentrations were added to the assay mixture containing diolein, and the formation of trinacylglycerols from the acyl groups at 24, 32, and 40°C was analyzed. The Cuphea enzyme preferred lauroyl CoA to oleoyl CoA, and was inactive on erucoyl CoA. The maize enzyme had about equal activities on oleoyl CoA and lauroyl CoA, and was inactive on erucoyl CoA. Enzymes from both rapeseed and Canola had the same pattern of acyl CoA preference, with highest activities on lauroyl CoA. The two enzymes were more active on oleoyl CoA than on erucoyl CoA at high acyl CoA concentrations (10 and 20 micromolar) at 24°C, but were more active on erucoyl CoA than on oleoyl CoA at low acyl CoA concentrations (1.36 micromolar or less) at 32 and 40°C. These findings are discussed in terms of the contribution of the enzyme to the acyl specificity in storage triacylglycerols and the implication in seed oil biotechnology.it is assumed that the in vivo pool sizes ofthe acyl CoAs determine the acyl specificity in the 3-position of the triacylglycerol.In seed oil biotechnology, one major goal is to alter the chain length of the fatty acyl moiety of the triacylglycerols (5,12,15 In an attempt to resolve part of the above unknown, we have studied the acyl CoA preference of seed diacylglycerol acyltransferase. We also would like to see if the enzyme exerts preference on specific acyl CoA, such that this preference reflects the acyl moiety in the triacylglycerol. The enzyme was obtained from four selected oil seeds which have unique and very contrasting fatty acyl moieties in the triacylglycerols. MATERIALS AND METHODSIn oil seeds, the fatty acid composition of the storage triacylglycerols is species-and variety-specific, and environmental factors such as temperature exert some modifying effects (13, 16). Within a seed species, the fatty acid composition in each of the three positions of a triacylglycerol is also largely inherited (16).In oil seeds as well as in mammalian tissues, triacylglycerols are synthesized from acyl CoA and glycerol-P via three different acyltransferases (1,13,16,17). The first two acyltransferases (glycerol-P acyltransferase and lysophosphatidic acid acyltransferase) possess some acyl CoA preference, such that their specificities as well as the in vivo pool sizes of acyl CoAs produce the observed positional acyl specificity in the triacylglycerol (1,17