Developing Soybean Cotyledons Research Articles

In situ [14C] Glutamate Metabolism by Developing Soybean Cotyledons II. The Importance of Glutamate Decarboxylation

Summary Developing cotyledons of soybean. ( Glycine max . (L.) Merrill) metabolize glutamate in situ via multiple routes including direct decarboxylation, deamination, and transmination reactions. The production of [ 14 C]4-aminobutyrate . (GABA) from [U- 14 C]glutamate, but not from [1- 14 C]glutamate, confirmed that α-decarboxylation is responsible for the production of GABA. [U- 14 C]GABA was rapidly metabolized to [ 14 C]succinate and [ 14 C]malate, consistent with the entry of glutamate carbon into the Krebs cycle via GABA and succinic semialdehyde. Aminooxyacetate, at a concentration of 100 mM, reduced the in situ metabolism of [U- 14 C]glutamate and almost fully inhibited [U- 14 C]GABA synthesis. These data suggested that the GABA shunt . (glutamate → GABA → succinic semialdehyde →succinate) may be important in the glutamate metabolism of a developing soybean seed. Although the GA-BA shunt bypasses the 2-oxoglutarate dehydrogenase reaction of the Krebs cycle, metabolism of [1- 14 C]acetate or [U- 14 C]2-oxoglutarate indicated that this reaction is not restricted under our experimental conditions. The in situ glutamate flux through glutamate decarboxylase, as estimated from the changing specific activity of [ 14 C]GABA during the early metabolism of [U- 14 C]glutamate, indicated that glutamate flux through the GABA shunt is comparable to direct incorporation of glutamate into protein.

Subcellular Compartmentation of the 4-Aminobutyrate Shunt in Protoplasts from Developing Soybean Cotyledons.

The subcellular localization of enzymes involved in the 4-ami-nobutyrate shunt was investigated in protoplasts prepared from developing soybean [Glycine max (L.) Merrill cv Maple Arrow] cotyledons. Protoplast lysate was fractionated by differential and continuous Percoll-gradient centrifugation to separate organelle fractions. Glutamate decarboxylase (EC 4.1.1.15) was found exclusively in the cytosol, whereas 4-aminobutyrate:pyruvate transami-nase (EC 2.6.1.19) and succinic semialdehyde dehydrogenase (EC 1.2.1.16) were associated exclusively with the mitochondrial fractions. Mitochondrial fractions also catabolized [U-14C]4-aminobu-tyrate to labeled succinate.

Use of Photoreactive Substrates for Characterization of Lysophosphatidate Acyltransferases from Developing Soybean Cotyledons

Photoreactive lipid analogs, namely, 1-acyl-2-(12-azidooleoyl)glycero-3-phosphocholine (N3-PC) and 1-acyl-2-(12-axidooleoyl)glycero-3-phosphoethanolamine (N3-PE) have been synthesized as previously described [R. Rajasekharan and J. D. Kemp (1994) J. Lipid Res. 35, 45-51]. Azidophosphatidic acid was produced by hydrolyzing N3-PC with phospholipase D. All of the lysophospholipid analogs, 2-(12-azidooleoyl)glycero-3-phosphate (N3-LPA), 2-(12-azidooleoyl)glycero-3-phosphocholine (N3-LPC), and 2-(12-azidooleoyl)glycero-3-phosphoethanolamine (N3-LPE), were produced from appropriate azidophospholipids by lipase treatment. The photoactive lysophospholipid analogs were recognized as substrates by acyltransferases in the dark and as irreversible inhibitors after photolysis with uv light. The photoinactivation of acyltransferases by azidolysophospholipids was protected by the addition of natural lysophospholipids. Incubation of developing soybean microsomal membranes with N3-LPA followed by photolysis resulted in 69% inhibition of lysophosphatidic acid (LPA) acyltransferase and also had significant inhibitory effects on lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE) acyltransferases, indicating that the LPA analog interacts with all the lysophospholipid acyltransferases. When the membranes were photolyzed with N3-LPC or N3-LPE and assayed, the membranes showed approximately 50% inactivation of LPC and LPE acyltransferase activities, whereas LPA acyltransferase was unaffected, suggesting that a single enzyme might acylate both LPC and LPE. The recognition of these photoreactive lipid analogs by acyltransferases will facilitate the identification and purification of these membrane-bound enzymes.

In situ [ 14C]Glutamate Metabolism by Developing Soybean Cotyledons I. Metabolic Routes

Glutamate metabolism was investigated in developing cotyledons of soybean ( Glycine max (L.) Merrill). Intact excised cotyledons were injected with carrier-free 14 C-glutamate and incubated, in the dark, in sealed vials containing a CO 2 trap. Detection of 14 C-radioactivity as released CO 2 , and ethanol-soluble and -insoluble fractions during 40-minute and 6-hour time courses, indicates that exogenously supplied glutamate was metabolized via CO 2 , 2-oxoglutarate, 4-aminobutyrate and Krebs-cycle intermediates. 14 C-Labeled 4-aminobutyrate was recovered from the metabolism of [U- 14 C]glutamate, but not [1- 14 C]glutamate, demonstrating its production by decarboxylation of the carbon one of glutamate. The rapid metabolism of [ 14 C]4-aminobutyrate and the recovery of 14 C-radioactivity in protein-aspartate suggest that glutamate decarboxylation is not a response to stress, but could contribute Krebs-cycle-derived amino acids for protein synthesis in co-operation with glutamate deamination and transamination. Recovery of 14 C as protein-arginine and -glutamate suggests that glutamate was also incorporated into arginine and protein, possibly without degradation. Multiple cellular amino acid pools were suggested from the rate of glutamate incorporation and the significant, but incomplete utilization of the soluble fraction.

Transient Gene Expression System Using Protoplasts from Developing Soybean Cotyledons.

Transient Gene Expression System Using Protoplasts from Developing Soybean Cotyledons.

Arginine Metabolism in Developing Soybean Cotyledons

Tracerkinetic experiments were performed using l-[guanidino-(14)C]arginine, l-[U-(14)C]arginine, l-[ureido-(14)C]citrulline, and l-[1-(14)C]ornithine to investigate arginine utilization in developing cotyledons of Glycine max (L.) Merrill. Excised cotyledons were injected with carrier-free (14)C compounds and incubated in sealed vials containing a CO(2) trap. The free and protein amino acids were analyzed using high performance liquid chromatography and arginine-specific enzyme-linked assays. After 4 hours, 75% and 90% of the (14)C metabolized from [guanidino-(14)C]arginine and [U-(14)C]arginine, respectively, was in protein arginine. The net protein arginine accumulation rate, calculated from the depletion of nitrogenous solutes in the cotyledon during incubation, was 17 nanomoles per cotyledon per hour. The data indicated that arginine was also catabolized by the arginase-urease reactions at a rate of 5.5 nanomoles per cotyledon per hour. Between 2 and 4 hours (14)CO(2) was also evolved from carbons other than C-6 of arginine at a rate of 11.0 nanomoles per cotyledon per hour. It is suggested that this extra (14)CO(2) was evolved during the catabolism of ornithine-derived glutamate; (14)C-ornithine was a product of the arginase reaction. A model for the estimated fluxes associated with arginine utilization in developing soybean cotyledons is presented.The maximum specific radioactivity ratios between arginine in newly synthesized protein and total free arginine in the (14)C-citrulline and (14)C-ornithine experiments indicated that only 3% of the free arginine was in the protein precursor pool, and that argininosuccinate and citrulline were present in multiple pools.

Arginine Metabolism in Developing Soybean Cotyledons

The free and protein amino acid composition of Glycine max (L.) Merrill cotyledons was determined for the entire developmental period using high performance liquid chromatography. Arginine constituted 18% of the total protein nitrogen throughout development, and there was a linear arginine nitrogen accumulation rate of 1212 nanomoles per cotyledon per day between 16 and 58 days after anthesis. Arginine and asparagine were major constituents of the free amino acid pool, constituting 14 to 62% and 2 to 41% of the total free amino acid nitrogen, respectively. The urea cycle intermediates, citrulline, ornithine, and argininosuccinate were also detected in the free pool. A comparison of the amino acid composition of cotyledonary protein and of seedcoat exudate suggested that 72% of the cotyledon's arginine requirement is satisfied by in situ biosynthesis, and that 20% of the transformed nitrogen is incorporated into arginine. Also, [1-(14)C]glutamate and [U-(14)C]glutamine were fed to excised cotyledons. After 4 hours, (14)C was incorporated into protein and released as (14)CO(2), but none was incorporated into the C-1 and C-6 positions of free and protein arginine, determined using arginine-specific enzyme-linked assays. It is not currently known whether arginine biosynthesis in the cotyledon involves glutamate delivered from the mother plant or glutamate derived in situ.

Arginine Metabolism in Developing Soybean Cotyledons

Tracer kinetic experiments were performed using [ureido-(14)C] citrulline, [1-(14)C]ornithine, and isotope trapping techniques to determine if arginine is synthesized via the urea cycle in developing cotyledons of Glycine max (L.) Merrill. Excised cotyledons were injected with the (14)C-solution and incubated in sealed vials containing a CO(2) trap. The free and protein amino acids were analyzed using high performance liquid chromatography and arginine-specific enzyme-linked assays. In the (14)C-citrulline feeding experiment argininosuccinate was the most highly labeled compound after 5 minutes and it was the first compound to lose (14)C later in the time course. Carbon-14 was also recovered in free arginine, protein arginine, and CO(2) up to 4 hours after introduction of label. All of the (14)C in free and protein arginine could be accounted for in the C-6 position. Metabolism of (14)C-ornithine resulted in (14)C-incorporation into citrulline and free and protein arginine and the evolution of (14)CO(2). Citrulline was the most highly labeled compound after 15 minutes and was the first compound to reach a steady state level of (14)C. With the addition of 800 nanomoles unlabeled citrulline to the (14)C-ornithine feeding solution citrulline was the only compound labeled after 5 minutes and the steady state level of (14)C-citrulline increased 12-fold. The appearance of (14)C in free arginine and protein arginine was also delayed. In both (14)C-ornithine feedings all of the (14)C in free and protein arginine could be accounted for in the C-1 position. Together, the data support the reaction sequence: ornithine --> citrulline --> argininosuccinate --> arginine --> protein arginine.

Identification of Membrane Protein Associated with Sucrose Transport Into Cells of Developing Soybean Cotyledons

The photolyzable sucrose derivative 6'-deoxy-6'-(4-azido-2-hydroxy)-benzamidosucrose (6'-HABS), competitively inhibited the influx of [(14)C] sucrose into protoplasts from developing soybean (Glycine max L. Merr cv Wye) cotyledons. Photolysis of (125)I-labeled 6'-HABS in the presence of 10 millimolar dithiothreitol and microsomal preparations from developing soybean cotyledons led to label incorporation into a moderately abundant membrane protein with an apparent molecular mass of about 62 kilodalton (kD) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The 62 kD protein was partially protected from labeling by the inclusion of 100 millimolar sucrose in the photolysis medium and also by the inclusion of 10 millimolar phenyl alpha-d-thioglucopyranoside. Glucose, raffinose, or phenyl alpha-d-3-deoxy-3-fluoroglucopyranoside did not afford even partial protection from labeling. When the photolyzable moiety of 6'-HABS was attached to 6-deoxy-6-aminoglucose and (125)I labeled, the resulting photoprobe did not label the 62 kD protein above background. The labeled protein at 62 kD is therefore apparently a specific, sucrose binding protein. Sucrose influx into cotlyedons of less than 25 milligrams fresh weight (approximately 10 days after flowering) occurred by passive processes, but metabolically dependent uptake became dominant over the next 5 to 7 days of development. Both the Coomassie staining protein at 62 kD and label incorporation at that position in analysis of membrane proteins appeared concomitant with the onset of active sucrose influx. Polyclonal antibodies to the purified 62 kD protein bound specifically to a protein in the plasmalemma of thin sections prepared from cotyledons and density stained with colloidal gold-protein A. The results suggest that the 62 kD membrane protein is associated with sucrose transport and may be the plasmalemma sucrose transporter.

15N and 13C NMR Determination of Methionine Metabolism in Developing Soybean Cotyledons

The metabolism of d- and l-methionine by immature cotyledons of soybean (Glycine max, L. cv Elf) grown in culture has been investigated using solid-state (13)C and (15)N nuclear magnetic resonance. d-Methionine is taken up by the cotyledons and converted to an amide, most likely by N-malonylation. About 16% of the l-methionine taken up is incorporated intact into protein, and 25% remains as soluble methionine. Almost two-thirds of the l-methionine that enters the cotyledons is degraded. The largest percentage of this is used in transmethylation of the carboxyl groups of pectin. Methionine is not extensively converted to polyamines. We attribute the stimulation of growth of the cotyledons by exogenous methionine to the bypassing of a rate-limiting methyl-transfer step in the synthesis of methionine itself, and subsequently of pectins and proteins.

Regulation by ABA of β-Conglycinin Expression in Cultured Developing Soybean Cotyledons

The regulation of cotyledon-specific gene expression by exogenously applied abscisic acid (ABA) was studied in developing cultured cotyledons of soybean (Glycine max L. Merr. cv Provar). When immature cotyledons were cultured in modified Thompson's medium, the addition of ABA resulted in an increased concentration of the beta-subunit of beta-conglycinin, one of the major storage proteins of soybean seeds. The amount of the alpha'-and alpha-subunits of beta-conglycinin was relatively unaffected by the ABA treatment. When fluridone, an inhibitor of carotenoid biosynthesis that has been shown to decrease ABA levels in plant tissues, was added to the medium the level of ABA and the beta-subunit decreased in the cotyledons. Increasing the concentration of sucrose in the culture medium caused an increase in the concentration of ABA and beta-subunit in the cotyledons. When in vitro translation products from RNA isolated from cotyledons cultured with ABA were immunoprecipitated with antiserum against beta-conglycinin, there was an increased amount of pre-beta-subunit polypetide compared to the translation products from RNA isolated from control cotyledons. The pre-beta-subunit polypeptide was not detected in translation products from RNA isolated from fluridone-treated cotyledons. Nucleic acid hybridization reactions showed that the level of beta-subunit mRNA was higher in ABA-treated cotyledons compared to the control, and was lower in the fluridone-treated cotyledons. We have shown that exogenous ABA is able to modulate the accumulation of the beta-subunit of beta-conglycinin in developing cultured soybean cotyledons.

Linear Sucrose Transport in Protoplasts from Developing Soybean Cotyledons

Previous studies with isolated soybean cotyledon protoplasts revealed the presence of a saturable, simple diffusion, and nonsaturating carrier-mediated uptake of sucrose into soybean cotyledon cells. A proton/sucrose cotransport may be involved in the saturable sucrose uptake (Lin et al. 1984 Plant Physiol 75: 936-940 and Schmitt et al. 1984 Plant Physiol 75: 941-946). In this study, we investigated the linear sucrose uptake mechanism by treating isolated protoplasts with 15 micromolar p-trifluoromethoxy-carbonylcyanide phenylhydrazone (FCCP) or 100 micromolar p-chloromecuribenzenesulfonic acid to eliminate the saturable uptake. We found: (a) increasing external pH decreases the linear sucrose uptake; (b) fusicoccin at 20 micromolar stimulates and FCCP at 15 micromolar inhibits this linear sucrose uptake; and (c) the ratio of the initial influx of proton to sucrose is close to one in both saturable and nondiffusive linear (difference between the total linear and diffusive components) uptakes. The results suggest that a proton/sucrose cotransport is also involved in the nondiffusive linear sucrose uptake into soybean cotyledon cells.

Energetics of Sucrose Transport into Protoplasts from Developing Soybean Cotyledons

The accumulation of tetraphenylphosphonium (TPP(+)), 5,5'-dimethyl-oxazolidine-2,4-dione (DMO), and a micro pH electrode were used to measure membrane potential, intracellular and extracellular pH, respectively, upon the addition of exogenous sucrose to soybean cotyledon protoplasts. Addition of sucrose caused a specific and transient (a) depolarization of the membrane potential (measured by TPP(+) accumulation), (b) acidification of the intracellular pH (measured by DMO accumulation), and (c) alkalization of the external medium (measured by a micro pH electrode). The time course for all these changes was similar (i.e. 5 to 10 minutes). Based on the rate of sucrose uptake and alkalization of the external medium, a stoichiometry of 1.02 to 1.10 for proton to sucrose was estimated. These data strongly support a proton/sucrose cotransporting mechanism in soybean cotyledon cells.

Accumulation and Subcellular Localization of α-Galactosidase-Hemagglutinin in Developing Soybean Cotyledons

We have investigated the accumulation and intracellular localization of soybean (Glycine max [L.] Merr. cv Forrest) alpha-galactosidase-hemagglutinin during seed development. Cotyledon tissue was embedded in Lowicryl K4M and immunocytochemical localization was accomplished through treating thin sections with alpha-galactosidase antisera followed by indirect labeling with protein A coupled to colloidal gold. Gold particles were localized on the Golgi apparatus and protein bodies. We interpret this to indicate that alpha-galactosidase-hemagglutinin is transferred to and transported through the Golgi apparatus and finally deposited within the protein body by a Golgi apparatus-mediated process.

Sugar Transport into Protoplasts Isolated from Developing Soybean Cotyledons

The effects of metabolic inhibitors, pH, and temperature on the kinetics of sucrose uptake protoplasts isolated from developing soybean Glycine max L. cv Wye cotyledons were studied. Structural requirements for substrate recognition by the sucrose carrier were examined by observing the effects of potential alternate substrates for the saturable component on sucrose uptake.Uptake by the three components (saturable, sulfhydryl reagent-sensitive nonsaturable, and diffusive) was calculated over a range of sucrose concentrations. The saturable component dominated uptake at external sucrose concentrations below 12 millimolar and was approximately equal to the nonsaturable and diffusive components at 44 and 22 millimolar external sucrose, respectively. The three uptake components showed different temperature sensitivities.Increasing external pH decreased both the linear component and the V(max) calculated for the saturable component. Conversely, increasing pH increased the calculated K(m) (sucrose) for the saturable component.Sucrose uptake by the saturable component was insensitive to several mono- and divalent cations. Competition for uptake of 0.5 millimolar sucrose by several sugars suggested that the beta-d-fructofuranoside bond and molecular size of sucrose were particularly important in sugar recognition by the saturable component carrier.

Sugar Transport into Protoplasts Isolated from Developing Soybean Cotyledons

A procedure is described to isolated functional protoplasts from developing soybean (Glycine max L. Merr. cv Wye) cotyledons. Studies of sucrose and hexose uptake into these protoplasts show that the plasmalemma of cotyledons during the stage of rapid seed growth contains a sucrose-specific carrier which is energetically and kinetically distinct from the system(s) involved in hexose transport. For example, sucrose, but not hexose uptake: (a) is inhibited by alkaline pH and the nonpermeant SH modifier, p-chloromercuribenzene sulfonic acid; (b) is stimulated by fusicoccin; (c) shows both a saturable and a linear component of uptake in response to substrate concentration; and (d) displays a sharp temperature response (high Q(10) value and high activation energies).

Sucrose Uptake by Developing Soybean Cotyledons

Sucrose uptake by excised developing soybean cotyledons shows a biphasic dependence on sucrose concentration. At concentrations less than about 50 millimolar external sucrose, uptake can be described as a carrier-mediated process, with a K(m) of 8 millimolar. At higher external sucrose concentrations, a linear dependence becomes apparent, which suggests the participation of a nonsaturable component in total uptake. Sucrose absorption is dependent on the presence of an electrochemical potential gradient for protons since agents interfering with the generation or maintenance of this gradient (NaN(3) or carbonylcyanide-m-chlorophenyl hydrazone) decrease sucrose transport to a level at or below that predicted from the operation of the noncarrier-mediated process alone. The saturable component of sucrose uptake is also sensitive to the sulfhydryl-modifying compounds N-ethylmaleimide and p-chloro-mercuribenzenesulfonate. The thiol-reducing agent diethioerythritol reverses fully the p-chloro-mercuri-benzenesulfonate inhibition, but not that of N-ethyl maleim de. Sucrose transport is sensitive to external pH, being decreased at high pH(0). Since sucrose-induced depolarization of the membrane potential and carrier-mediated sucrose influx show similar pH-dependence, inhibitor sensitivity, and values of K(m) for sucrose, a sucrose/proton contransport process appears to operate in developing soybean cotyledon cells. Measurement of free space and intracellular sucrose concentrations in vivo suggests that the carrier-mediated process is fully saturated and that sucrose transport may be limiting for sucrose accumulation by the developing seed.

Aspects of Glycerolipid Metabolism in Developing Soybean Cotyledons1

The metabolism of glycerolipids (GL) labeled with palmitic (16:0), stearic (18:0), oleic (18:1), linoleic (18:2), or linolenic (18:3) acids was studied with unsliced soybean [Glycine max (L.) Merr. cv. Dare] cotyledons harvested at 30 days after flowering. An inhibitor of lipoxygenase, n‐propylgallate, was required in the incubation media to prevent the peroxidation of the exogenous 18:2 and 18:3 substrates by the soybean tissues. Phospholipids (TPL) accounted for greater than 75% of the glycerolipid radioactivity incorporated from each of the acyl‐substrates. The distribution of radioactivity in phospholipid molecular species was determined by assaying the diacylglycerol (DG) molecular species prepared from the phospholipid fraction by phospholipase C hydrolysis. The predominate phospholipid molecular species synthesized from the respective substrates were: SD, [1‐14C] 16:0 or [1‐14C]18:0; SM and MD, [1‐14C]18:1; SD and DD, [1‐14C]18:2; and DT and TT, [1‐14C]18:3; where S= 16:0 or 18:0, M = 18:1, D = 18:2, and T = 18:3. The predominate triacylglycerol (TG) molecular species synthesized from the respective substrates were: S2M, S2D, and SD2, [1‐14C]16:0 or [1‐14C]18:0; M2D and MD2, [1‐14C]18:1; SD2, D3, and D2T, [1‐14C]18:2; and DT2 and T3, [1‐14C]18:3. Stereospecific analysis of the radioactivity present in phospholipid, DG, and TG indicated a preference for unsaturated fatty acid incorporation at the sn‐2 position of each class. The observed acylincorporation patterns supported the precursory interrelation of phospholipids in soybean TG biosynthesis, and also established the nature of soybean TG molecular species biosynthesis from specific acyl‐substrates. The method described has general application for preparing specifically labeled DG molecular species with high specific radioactivity and for determining the biochemical nature of TG biosynthesis in experimental soybean lines selected for altered fatty acid composition.

Electrogenic Sucrose Transport in Developing Soybean Cotyledons

Addition of sucrose to a solution bathing an excised developing soybean cotyledon causes a transient depolarization of the membrane potential, as measured using standard electrophysiological techniques. The magnitude of the depolarization is dependent on the concentration of both sucrose and protons in a manner which suggests carrier mediation; this process has an apparent K(m) for sucrose of about 10 millimolar. Agents interfering with the generation or maintenance of a proton electrochemical gradient eliminate these depolarizations. Electrogenic sugar transport is sensitive to sulfhydryl-modifying reagents; their effect appears to be through a direct interaction with the carrier protein and/or with the process establishing the proton electrochemical gradient across the plasma membrane. p-Chloromercuribenzene sulfonate appears to be a selective inhibitor of the carrier-mediated process itself.

Involvement of Phospholipids in Polyunsaturated Fatty Acid Synthesis in Developing Soybean Cotyledons

Developing soybean (cv. Dare) cotyledons harvested at 30 days after flowering were pulse-labeled with [1-(14)C]oleoyl-CoA. The metabolic interrelation of radiolabeled unsaturated fatty acids between the major glycerolipid classes was determined at various time intervals. At chase time zero, [(14)C]oleic acid accounted for 99.2% of the total glycerolipid radioactivity, and phospholipids contained 92% of the total incorporated radioactivity. With time, phospholipids were metabolized in triacylglycerol biosynthesis and radioactivity was detected in polyunsaturated fatty acids. The hypothesis that phospholipids were metabolic intermediates in polyunsaturated fatty acid biosynthesis was tested by comparing the theoretical and the actual amount of radiolabeled oleic acid that was associated with triacylglycerol as a function of time. The radioactive oleic acid found in triacylglycerol at various intervals was derived from phospholipids via a diacylglycerol intermediate. Assuming no phospholipid desaturation, the potential or theoretical amounts of [(14)C]oleic acid that could be transferred to triacylglycerol from phospholipids was defined by a system of differential equations. The results demonstrated that the decline in [(14)C]oleic acid from phospholipid after long chase intervals was equal to the total amount of radioactive unsaturated fatty acids found in neutral lipids. The difference between the theoretical and actual amounts of [(14)C]oleic acid present in triacylglycerol after long time intervals was equal to the amount of radioactivity present in polyunsaturated fatty acids. Based upon those findings in soybeans, the desaturation of oleic acid associated with phospholipids was highly probable.