Transgenic expression of a delta 12-epoxygenase gene in Arabidopsis seeds inhibits accumulation of linoleic acid.

The Crepis palaestina cDNA Cpal2 encodes a delta 12-epoxygenase that can catalyse the synthesis of 12,13-epoxy-cis-9-octadecenoic acid (18:1E) from linoleic acid (18:2). When the Cpal2 gene was expressed under the control of the napin seed-specific promoter in Arabidopsis thaliana (L.) Heynh., the seed lipids accumulated only low levels of 18:1E and also 12,13-epoxy-cis-9,15-octadec-2-enoic acid (18:2E). Despite the fact that the levels of these epoxy fatty acids comprised only up to 6.2% of the total fatty acids, there was a very marked increase in oleic acid (18:1) and decrease in linoleic (18:2) and alpha-linolenic (18:3) acids in these plants, indicating that endogenous delta 12-desaturation was greatly reduced in these plants. Significant between-line differences in the levels of Cpal2 mRNA were observed during seed development, but were not associated with any major variation in mRNA levels for the endogenous Arabidopsis delta 12-desaturase (Fad2). This suggests that if an unfavourable interaction occurs between the transgenic delta 12-epoxygenase and the endogenous delta 12-desaturase, which decreases the level of desaturation, it occurs at either the translational or post-translational level. We further show that the co-expression of a delta 12-desaturase gene from C. palaestina in Cpal2 transgenic Arabidopsis returns the relative proportions of the C18 seed fatty acids to normal levels and results in an almost twofold increase in total epoxy fatty acids.

Phospholipid:diacylglycerol acyltransferase: an enzyme that catalyzes the acyl-CoA-independent formation of triacylglycerol in yeast and plants.

Triacylglycerol (TAG) is known to be synthesized in a reaction that uses acyl-CoA as acyl donor and diacylglycerol (DAG) as acceptor, and which is catalyzed by the enzyme acyl-CoA:diacylglycerol acyltransferase. We have found that some plants and yeast also have an acyl-CoA-independent mechanism for TAG synthesis, which uses phospholipids as acyl donors and DAG as acceptor. This reaction is catalyzed by an enzyme that we call phospholipid:diacylglycerol acyltransferase, or PDAT. PDAT was characterized in microsomal preparations from three different oil seeds: sunflower, castor bean, and Crepis palaestina. We found that the specificity of the enzyme for the acyl group in the phospholipid varies between these species. Thus, C. palaestina PDAT preferentially incorporates vernoloyl groups into TAG, whereas PDAT from castor bean incorporates both ricinoleoyl and vernoloyl groups. We further found that PDAT activity also is present in yeast microsomes. The substrate specificity of this PDAT depends on the head group of the acyl donor, the acyl group transferred, and the acyl chains of the acceptor DAG. The gene encoding the enzyme was identified. The encoded PDAT protein is related to lecithin:cholesterol acyltransferase, which catalyzes the acyl-CoA-independent synthesis of cholesterol esters. However, budding yeast PDAT and its relatives in fission yeast and Arabidopsis form a distinct branch within this protein superfamily, indicating that a separate PDAT enzyme arose at an early point in evolution.

A bifunctional delta-fatty acyl acetylenase/desaturase from the moss Ceratodon purpureus. A new member of the cytochrome b5 superfamily.

Many plant genes have been cloned that encode regioselective desaturases catalyzing the formation of cis-unsaturated fatty acids. However, very few genes have been cloned that encode enzymes catalyzing the formation of the functional groups found in unusual fatty acids (e.g. hydroxy, epoxy or acetylenic fatty acids). Here, we describe the characterization of an acetylenase from the moss Ceratodon purpureus with a regioselectivity differing from the previously described Delta12-acetylenase. The gene encoding this protein, together with a Delta6-desaturase, was cloned by a PCR-based approach with primers derived from conserved regions in Delta5-, Delta6-fatty-acid desaturases and Delta8-sphingolipid desaturases. The proteins that are encoded by the two cloned cDNAs are likely to consist of a N-terminal extension of unknown function, a cytochrome b5-domain, and a C-terminal domain that is similar to acyl lipid desaturases with characteristic histidine boxes. The proteins were highly homologous in sequence to the Delta6-desaturase from the moss Physcomitrella patens. When these two cDNAs were expressed in Saccharomyces cerevisiae, both transgenic yeast cultures desaturated Delta9-unsaturated C16- and C18-fatty acids by inserting an additional Delta6cis-double bond. One of these transgenic yeast clones was also able to introduce a Delta6-triple bond into gamma-linolenic and stearidonic acid. This resulted in the formation of 9,12,15-(Z,Z,Z)-octadecatrien-6-ynoic acid, the main fatty acid found in C. pupureus. These results demonstrate that the Delta6-acetylenase from C. pupureus is a bifunctional enzyme, which can introduce a Delta6cis-double bond into 9,12,(15)-C18-polyenoic acids as well as converting a Delta6cis-double bond to a Delta6-triple bond.

Characterisation of acyl-ACP desaturases from Macadamia integrifolia Maiden & Betche and Nerium oleander L.

The seed oil in Macadamia integrifolia contains about 30% palmitoleic acid (16:1(Delta9)) and Nerium oleander about 12% isoricinoleic acid (Delta9-hydroxy-18:1(Delta12)). It has been shown that palmitoleic acid can be produced by acyl-acyl carrier protein (ACP) desaturases and it has also been shown that fatty acid hydroxylation can occur via direct substitution of a hydrogen atom. Therefore it seemed possible that the enzymes responsible for the making of these unusual fatty acids in M. integrifolia and N. oleander were of acyl-ACP desaturase type. Extracts from developing M. integrifolia developing seeds showed a relative ratio of 16:0-ACP to 18:0-ACP desaturation that was about 13 times higher than in sunflower seeds. N. oleander seed extracts catalysed conversion of 18:0-ACP to 18:1(Delta9) but only trace amounts of Delta9-hydroxy fatty acids were formed. A total of four cDNAs were isolated from developing seeds, of both species, using a fragment isolated with PCR amplification. The M. integrifolia acyl-ACP desaturase cDNA was expressed in Escherichia coli. A partly purified fraction of the enzyme showed a 16:0-ACP to 18:0-ACP desaturation ratio about 90-fold less than that in the Macadamia extracts. Expressed N. oleander acyl-ACP desaturase cDNAs showed predominantly 18:0-ACP desaturase activity and no hydroxylase activity. Thus it is not likely that any of the four acyl-ACP desaturases cloned from M. integrifolia or N. oleander is involved in the production of unusual fatty acids.

The distribution of caprylate, caprate and laurate in lipids from developing and mature seeds of transgenic Brassica napus L.

The composition and positional distribution of lipids in developing and mature transgenic Brassica napus seeds accumulating up to 7 mol% of caprylate (8:0), 29 mol% caprate (10:0) or 63 mol% of laurate (12:0) were examined. The accumulation of 8:0 and 10:0 resulted from over-expression of the medium-chain-specific thioesterase (Ch FatB2) alone or together with the respective chain-length-specific condensing enzyme (Ch KASIV). Seeds containing high levels of 12:0 were obtained from plants expressing bay thioesterase (BTE) alone or crossed with a line over-expressing the coconut lysophosphatidic acid acyltransferase (LPAAT), an enzyme responsible for the increase in acylation of 12:0 at the sn-2 position. In all instances, 10:0 and 12:0 fatty acids were present in substantial amounts in phosphatidylcholine during seed development with a drastic decrease of 80-90% in mature seeds. At all stages of seed development however, 8:0 was barely detectable in this membrane lipid. Altogether, these results indicate that these transgenic seeds exclude and/or remove the medium-chain fatty acids from their membrane and that this mechanism(s) is more effective with the shorter-chain fatty acids. Furthermore, seeds of 8:0- and 10:0-producing lines had only negligible levels of these fatty acids present in the sn-2 position of the triacylglycerols. In contrast, all 12:0-producing seeds had a substantial amount of this fatty acid in the sn-2 position of the triacylglycerols, suggesting that the endogenous LPAAT is able to acylate 12:0 if no other acyl-CoA species are available.

The involvement of phospholipid:diacylglycerol acyltransferases in triacylglycerol production.

We have characterized three CoA-independent types of enzyme, phospholipases, phospholipid:diacylglycerol acyltransferases (PDATs) and cholinephosphotransferases, responsible for the removal of unusual fatty acids from phosphatidylcholine (PC) in microsomal preparations from developing oil seeds. The metabolism of sn-2-[(14)C]acyl-PC was monitored in microsomal preparations from various oilseeds having either medium-chain, acetylenic, epoxy or hydroxy fatty acids as their major fatty acids in the oil. The results indicate that PDAT plays a major role in removing ricinoleic acid and vernolic acid from phospholipids in Ricinus communis and Crepis palaestina seeds, respectively. However, vernolic, crepenynic and capric acids are primarily removed from phospholipids by phospholipases in Euphorbia lagascae, Crepis rubra and elm seeds, respectively. Further, we show that significant PDAT activity is also present in vegetative tissues of Arabidopsis thaliana.

An acyl-CoA:cholesterol acyltransferase (ACAT)-related gene is involved in the accumulation of triacylglycerols in Saccharomyces cerevisiae.

The major route for the synthesis of triacylglycerol (TAG) in yeast as well as in all TAG-accumulating organisms has been suggested to occur via the acylation of diacylglycerol (DAG) by acyl-CoA:diacylglycerol acyltransferase (DAGAT). Genes encoding DAGAT have been identified in both plant and animal tissues. These genes show strong sequence similarities to genes encoding acyl-CoA:cholesterol acyltransferase (ACAT). So far no Saccharomyces cerevisiae DAGAT gene has been published; however, two ACAT-like genes, ARE1 and ARE2, are present in the yeast genome. Both these genes have been suggested to be involved in the synthesis of sterol esters. We have now shown that the ARE1 gene in yeast also is involved in the synthesis of TAG, whereas the ARE2 gene is more specifically involved in the synthesis of sterol esters.

Accumulation of storage products in oat during kernel development.

Lipids, proteins and starch are the main storage products in oat seeds. As a first step in elucidating the regulatory mechanisms behind the deposition of these compounds, two different oat varieties, 'Freja' and 'Matilda', were analysed during kernel development. In both cultivars, the majority of the lipids accumulated at very early stage of development but Matilda accumulated about twice the amount of lipids compared to Freja. Accumulation of proteins and starch started also in the early stage of kernel development but, in contrast to lipids, continued over a considerably longer period. The high-oil variety Matilda also accumulated higher amounts of proteins than Freja. The starch content in Freja kernels was higher than in Matilda kernels and the difference was most pronounced during the early stage of development when oil synthesis was most active. Oleosin accumulation continued during the whole period of kernel development.

Selective increase in acyl hydrolase activity by graminicides in wheat.

Seedlings of wheat were grown for 24 h in control nutrient solution and in solutions containing haloxyfop, alloxydim, diquat or paraquat, and thereafter the roots were used for microsomal preparations. Phosphatidylcholine or diacylglycerol with various 1-(14)C-labelled fatty acids (oleic, linoleic, linolenic or ricinoleic acids) in position sn-2 were added to the prepared microsomes. After incubation for 2 h at 30 degrees C, the lipids were extracted and the distribution of radioactivity among lipid classes was determined. In the microsomal preparations of plants treated with diquat and paraquat, the amounts of fatty acids released were similar to the control, whereas they were 1.4-2 times higher in the microsomal preparation of plants treated with haloxyfop and alloxydim. Thus, the data indicate that graminicides could increase lipid catabolism in sensitive plants and that this is not a general phenomenon connected with inhibition of growth.

Inhibition of polyunsaturated fatty acid accumulation in plants expressing a fatty acid epoxygenase.

Earlier, we described the isolation of a Crepis palaestina cDNA (Cpal2) which encoded a Delta12-epoxygenase that could catalyse the synthesis of 12,13-epoxy-cis-9-octadecenoic acid (18:1E) from linoleic acid (18:2). When the Cpal2 gene was expressed under the control of a seed-specific promoter in Arabidopsis, plants were able to accumulate small amounts 18:1E and 12,13-epoxy-cis-9,15-octadec-2-enoic acid in their seed lipids. In this report we give results obtained from a detailed analysis of transgenic Arabidopsis plants containing the Cpal2 gene. The seeds from these plants accumulate varying levels of 18:1E, but show a marked increase in 18:1 and equivalent decrease in 18:2 and 18:3. We further observed that the co-expression of a C. palaestina Delta12-desaturase in Arabidopsis appears to return the relative proportions of the C(18) seed fatty acids to normal levels and results in a 2-fold increase in total epoxy fatty acids.

Purification and characterization of a low-molecular-weight phospholipase A2 from developing seeds of elm.

Phospholipase A2 (PLA2) was purified about 180,000 times compared with the starting soluble-protein extract from developing elm (Ulmus glabra) seeds. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis the purified fraction showed a single protein band with a mobility that corresponded to 15 kD, from which activity could be recovered. When analyzed by matrix-assisted laser-desorption ionization-time-of-flight mass spectrometry, the enzyme had a deduced mass of 13,900 D. A 53-amino acid-long N-terminal sequence was determined and aligned with other sequences, giving 62% identity to the deduced amino acid sequence of some rice (Oryza sativa) expressed sequence tag clones. The purified enzyme had an alkaline pH optimum and required Ca2+ for activity. It was unusually stable with regard to heat, acidity, and organic solvents but was sensitive to disulfide bond-reducing agents. The enzyme is a true PLA2, neither hydrolyzing the sn-1 position of phosphatidylcholine nor having any activity toward lysophosphatidylcholine or diacylglycerol. The biochemical data and amino acid sequence alignments indicate that the enzyme is related to the well-characterized family of animal secretory PLA2s and, to our knowledge, is the first plant enzyme of this type to be described.

Identification of non-heme diiron proteins that catalyze triple bond and epoxy group formation.

Acetylenic bonds are present in more than 600 naturally occurring compounds. Plant enzymes that catalyze the formation of the Delta12 acetylenic bond in 9-octadecen-12-ynoic acid and the Delta12 epoxy group in 12,13-epoxy-9-octadecenoic acid were characterized, and two genes, similar in sequence, were cloned. When these complementary DNAs were expressed in Arabidopsis thaliana, the content of acetylenic or epoxidated fatty acids in the seeds increased from 0 to 25 or 15 percent, respectively. Both enzymes have characteristics similar to the membrane proteins containing non-heme iron that have histidine-rich motifs.

Fatty acid distribution and lipid metabolism in developing seeds of laurate-producing rape (Brassica napus L.).

The fatty acid composition and content of membrane and storage lipids of two transgenic laurate-producing rape (Brassica napus L.) lines were monitored during seed development. The two lines, the medium-laurate (ML) line and the high-laurate (HL) line, accumulated 34 mol% and 55 mol% of laurate in their seed triacylglycerols, respectively. The diacylglycerols contained about 17 and 33 mol% of laurate in the ML- and HL-lines, respectively, from the mid-stage of seed development up to seed maturity. The ML-line showed a maximal relative laurate content in phosphatidylcholine (17 mol%) at the mid-stage of seed development whereafter the content decreased to 2.7 mol% with seed maturity. The laurate content in phosphatidylcholine was observed to remain high (26 mol%) in the HL-line from the mid-stage to the end of triacylglycerol deposition. Thereafter, the relative content decreased and reached 6.6 mol% in the mature seeds. There was an enhanced activity of lauroyl-phosphatidylcholine-metabolizing enzymes in the seed membranes from laurate-producing lines compared with control lines, which might explain the decrease seen in laurate content in phosphatidylcholine during seed maturation. A comparison of the laurate distribution in the lipids from developing laurate-producing rape seeds and developing seeds from three species naturally accumulating laurate at similar levels revealed differences in laurate metabolism compared with these species. The results suggest that phospholipids and triacylglycerols are synthesized from the same diacylglycerol pool in rape seeds and that rape lysophosphatidic acid acyltransferase and diacylglycerol acyltransferase do not have the same preference for laurate substrates as the corresponding enzymes in seed tissues naturally accumulating this acyl group. In addition, the mechanisms that specifically remove or exclude laurate from membrane lipids appear less effective in rape seed than in tissues naturally evolved to synthesize laurate-rich oils.

Plant Microsomal Phospholipid Acyl Hydrolases Have Selectivities for Uncommon Fatty Acids.

Developing endosperms and embryos accumulating triacylglycerols rich in caproyl (decanoyl) groups (i.e. developing embryos of Cuphea procumbens and Ulmus glabra) had microsomal acyl hydrolases with high selectivities toward phosphatidylcholine with this acyl group. Similarly, membranes from Euphorbia lagascae and Ricinus communis endosperms, which accumulate triacylglycerols with vernoleate (12-epoxy-octadeca-9-enoate) and ricinoleate (12-hydroxy-octadeca-9-enoate), respectively, had acyl hydrolases that selectively removed their respective oxygenated acyl group from the phospholipids. The activities toward phospholipid substrates with epoxy, hydroxy, and medium-chain acyl groups varied greatly between microsomal preparations from different plant species. Epoxidated and hydroxylated acyl groups in sn-1 and sn-2 positions of phosphatidylcholine and in sn-1-lysophosphatidylcholine were hydrolyzed to a similar extent, whereas the hydrolysis of caproyl groups was highly dependent on the positional localization.

Expression of a biologically active plant cytochrome b5 in Escherichia coli.

Cytochrome b5 from tobacco (Nicotiana tabacum) was expressed in Escherichia coli using a T7 polymerase/promoter system as described by Studier, Rosenberg, Dunn and Dubendorff (1990) (Methods Enzymol. 185, 60-89). Transformed cells were red in colour and accumulated cytochrome b5 to a level of around 30% of the total cell protein. The purified cytochrome had oxidized, reduced and low-temperature absorbance spectra characteristic of plant microsomal cytochrome b5, and exhibited a c.d. spectrum resembling that of a mammalian cytochrome b5. The recombinant protein appeared to be correctly assembled and biologically active, being reduced by NADH in the presence of microsomal membranes prepared from the developing seeds of sunflower (Helianthus annuus). Inhibition of haem synthesis in the transformed E. coli cells expressing cytochrome b5, by the use of gabaculin or succinylacetone, prevented the assembly of the cytochrome b5 holoprotein but had little effect on the accumulation of cytochrome apoprotein. The recombinant protein expressed in E. coli therefore has the biochemical features of the higher-plant cytochrome b5 and can be used in studies of plant microsomal oxidation/reduction reactions.

Intertribal somatic hybrids between Brassica napus and Thlaspi perfoliatum with high content of the T. perfoliatum-specific nervonic acid.

Protoplast fusions were performed between hypocotyl protoplasts of Brassica napus and mesophyll protoplasts of Thlaspi perfoliatum. The two species are members of the Lepidieae and Brassiceae tribes, respectively, in the family of Brassicaceae. Seeds of T. perfoliatum are rich in the fatty acid C24∶1 (nervonic acid), an oil valuable for technical purposes. In the search for renewable oils to replace the mineral oils, plant breeders have been trying to develop oil crops with a high content of long-chain fatty acids. After fusion of B. napus protoplasts with non-irradiated as well as irradiated protoplasts of T. perfoliatum selection was carried out by flow cytometry and cell sorting. Of the shoots regenerated from different calli 27 were verified as hybrids or partial hybrids using the isoenzyme phosphoglucose isomerase (PGI) as a marker. Another 6 plants were identified as partial hybrids using a T. perfoliatum-specific repetitive DNA sequence. Slot blot experiments were performed to estimate the copy number of the repetitive DNA sequence in the parental species and in the hybrids. In T. perfoliatum there were approximately 10(5) copies per haploid genome, and the range in the hybrids was 1-37% of the value in T. perfoliatum. When the nuclear DNA content of the regenerated shoots was analysed we found partial as well as symmetric hybrids. Even though the rooting and establishment of hybrid shoots in the greenhouse were difficult, resulting in the death of many plants, 19 plants were cultured to full maturity. Seeds obtained from 15 plants were analysed to determine whether they contained nervonic acid, and 5 of the hybrids were found to contain significantly greater amounts of nervonic acid than B. napus.

Regio- and stereoselectivity of cytochrome P-450 and peroxygenase-dependent formation of cis-12,13-epoxy-9(Z)-octadecenoic acid (vernolic acid) in Euphorbia lagascae.

Two oxygenases associated with microsomes prepared from Euphorbia lagascae developing seeds were found to convert linoleic acid into cis-12,13-epoxy-9(Z)-octadecenoic acid (vernolate): a cytochrome P-450 and a peroxygenase. The cytochrome P-450 dependent epoxidation is characterized by a remarkable regio- and enantioselectivity, i.e. only the 12(S),13(R)-enantiomer is formed in the endosperm. In germinating seeds, peroxygenase was active but no cytochrome P-450 epoxidase could be detected. Moreover, because of the very high enantioselectivity of the fatty acid epoxide hydrolase, which is also found in these tissues and which preferentially hydrates the 12(R),13(S)-epoxide enantiomer, 12(S),13(R)-epoxy-9(Z)-octadecenoic acid is the only isomer which can accumulate in E. lagascae.

Biosynthesis of vernoleate (cis-12-epoxyoctadeca-cis-9-enoate) in microsomal preparations from developing endosperm of Euphorbia lagascae.

Epoxidated fatty acids are the major constituents of the triacylglycerols in a few plant species. We have investigated the biosynthesis of vernolic acid (cis-12-epoxyoctadeca-cis-9-enoic acid) in the seed oil of Euphorbia lagascae. Microsomes were isolated from developing endosperm. The membrane lipids were labeled in situ with [14C]oleate or [14C]linoleate, which mainly were recovered in phosphatidylcholine (PC), and the metabolization of the radioactive fatty acids was followed in incubations with or without NADPH. In the presence of NADPH, [14C]vernoleate was formed. After short incubations, most of the vernolic acid was found in PC, but with increasing incubation times, the free acid dominated. The synthesis of vernoleate was inhibited by carbon monoxide, but not by cyanide. The presence of anticytochrome b5 antibodies inhibited both the desaturation of [14C]oleate to [14C]linoleate and the epoxidation of [14C]linoleate to [14C]vernoleate. Free linoleic acid did not serve as substrate for epoxidation. The results indicate that, in the endosperm of E. lagascae, vernoleate is synthesized on PC from linoleate, and that the epoxidation is catalyzed by a cytochrome P450 and involves cytochrome b5.

Evidence for cytochrome b5 as an electron donor in ricinoleic acid biosynthesis in microsomal preparations from developing castor bean (Ricinus communis L.).

The major b-type cytochrome in microsomal membrane preparations from developing endosperm of castor bean (Ricinus communis) was cytochrome b5. Cytochrome P-450 was also present. The microsomal membranes had delta 12-hydroxylase activity and catalysed the NAD(P)H-dependent hydroxylation of oleate to yield ricinoleic acid. CO had no effect on the hydroxylase activity. Rabbit polyclonal antibodies were raised against the hydrophilic cytochrome b5 fragment purified from cauliflower (Brassica oleracea) floret microsomes. The anti-(cytochrome b5) IgG inhibited delta 12-hydroxylase, delta 12-desaturase and cytochrome c reductase activity in the microsomes. The results indicate that electrons from NAD(P)H were transferred to the site of hydroxylation via cytochrome b5 and that cytochrome P-450 was not involved.

Biosynthesis of linolenate in developing embryos and cell-free preparations of high-linolenate linseed (Linum usitatissimum) and low-linolenate mutants.

Biosynthesis of alpha-linolenate was investigated in developing embryos of the high-linolenic (45%) linseed cv. Glenelg, two mutant lines (M1589 and M1722) having reduced linolenic acid content (30%), and a very low linolenic (2%) genotype (Zero) obtained by recombination of the M1589 and M1722 mutations. Glenelg embryos showed an exponential rate of linolenate synthesis that paralleled their exponential pattern of triacylglycerol accumulation. The Zero line, although showing a pattern of triacylglycerol accumulation similar to that of Glenelg, accumulated linolenate at only a very low and constant rate throughout embryo development. An NADH- and O2-dependent decrease in oleate and increase in linolenate content of phosphatidylcholine was observed in dilute homogenates prepared from Glenelg embryos at 21 days after flowering, indicating active oleoyl- and linoleoyl-phosphatidylcholine desaturases in these preparations. While oleate decreased similarly in both sn positions of phosphatidylcholine, the increase in linolenate was confined mostly to the sn-2 position. Homogenates prepared from the mutant lines showed decreases in oleoyl-phosphatidylcholine similar to those of the wild-type Glenelg, whereas the increase in linolenoyl-phosphatidylcholine was substantially lower in M1589 and M1722 and barely detected in Zero. In vivo labeling experiments with detached embryos at 17 days after flowering, as well as analysis of endogenous linolenate content in various lipids, indicated that only delta 15-phospholipid desaturases, and not delta 15-galactolipid desaturases, were affected by the mutations. Embryos from M1722 had amounts of both radioactive and endogenous linolenate at position sn-1 of phosphatidylcholine that were close to those of the wild-type embryos, whereas M1589 had only 30 and 50% of these levels, respectively. The regulation of linolenic acid content in oilseeds is discussed on the basis of the results obtained.