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Examines the accumulation of novel Delta[sup 6]-unsaturated fatty acids in transgenic tobacco lines expressing the borage Delta[sup 6]-fatty acid desaturase. Presence of the gamma-linolenic acid and octadecatetraenoic acid in a range of seed and non-seed tissues; Distribution of the fatty acids among the various lipid classes of the tobacco leaf.

A cDNA isolated from the prymnesiophyte micro-alga Isochrysis galbana, designated IgASE1, encodes a fatty acid elongating component that is specific for linoleic acid (C18:2n-6) and alpha-linolenic acid (C18:3n-3). Constitutive expression of IgASE1 in Arabidopsis resulted in the accumulation of eicosadienoic acid (EDA; C20:2n-6) and eicosatrienoic acid (ETrA; C20:3n-3) in all tissues examined, with no visible effects on plant morphology. Positional analysis of the various lipid classes indicated that these novel fatty acids were largely excluded from the sn-2 position of chloroplast galactolipids and seed triacylglycerol, whereas they were enriched in the same position in phosphatidylcholine. EDA and ETrA are precursors of arachidonic acid (C20:4n-6), eicosapentaenoic acid (C20:5n-3), and docosahexaenoic acid (C22:6n-3) synthesized via the so-called omega6 Delta8 desaturase and omega3 Delta8 desaturase biosynthetic pathways, respectively. The synthesis of significant quantities of EDA and ETrA in a higher plant is therefore a key step in the production of very long chain polyunsaturated fatty acid in oil-seed species. The results are further discussed in terms of prokaryotic and eukaryotic pathways of lipid synthesis in plants.

We report the production of two very long chain polyunsaturated fatty acids, arachidonic acid (AA) and eicosapentaenoic acid (EPA), in substantial quantities in a higher plant. This was achieved using genes encoding enzymes participating in the omega3/6 Delta8 -desaturation biosynthetic pathways for the formation of C20 polyunsaturated fatty acids. Arabidopsis thaliana was transformed sequentially with genes encoding a Delta9 -specific elongating activity from Isochrysis galbana, a Delta8 -desaturase from Euglena gracilis and a Delta5 -desaturase from Mortierella alpina. Instrumental in the successful reconstitution of these C20 polyunsaturated fatty acid biosynthetic pathways was the I. galbana C18-Delta9 -elongating activity, which may bypass rate-limiting steps present in the conventional Delta6 -desaturase/elongase pathways. The accumulation of EPA and AA in transgenic plants is a breakthrough in the search for alternative sustainable sources of fish oils.

IgASE1, a C18-Delta9-polyunsaturated fatty acid-specific fatty acid elongase component from Isochrysis galbana, contains a variant histidine box (his-box) with glutamine replacing the first histidine of the conserved histidine-rich motif present in all other known equivalent proteins. The importance of glutamine and other variant amino acid residues in the his-box of IgASE1 was determined by site-directed mutagenesis. Results showed that all the variation in amino acid sequence between this motif in IgASE1 and the consensus sequences of other elongase components was required for optimum enzyme activity. The substrate specificity was shown to be unaffected by these changes suggesting that components of the his-box are not directly responsible for substrate specificity.

We have isolated a cDNA encoding the Delta(8) sphingolipid desaturase from the plant Aquilegia vulgaris L. via a PCR-based strategy using primers designed to target the conserved histidine box regions of microsomal desaturases. The function of the cDNA was confirmed by expression in the yeast, Saccharomyces cerevisiae. Analysis of the long-chain sphingoid bases as their dinitrophenyl derivatives by reverse-phase HPLC demonstrated the accumulation of cis - and trans -desaturated sphingoid bases which were not present in the wild-type yeast cells. The Delta(8) desaturated products co-eluted with known Delta(8)-desaturated phytosphingenine and the molecular mass of these products was confirmed by liquid chromatography-MS. The Delta(8) long-chain base desaturase was also able to desaturate dihydrosphingosine substrates. This is the first report of the functional characterization of an A. vulgaris gene product.

Isochrysis galbana, a marine prymnesiophyte microalga, is rich in long chain polyunsaturated fatty acids such as docosahexaenoic acid (C22:6n-3, Delta(4,7,10,13,16,19)). We used a polymerase chain reaction-based strategy to isolate a cDNA, designated IgASE1, encoding a polyunsaturated fatty acid-elongating activity from I. galbana. The coding region of 263 amino acids predicts a protein of 30 kDa that shares only limited homology to animal and fungal proteins with elongating activity. Functional analysis of IgASE1, by expression in Saccharomyces cerevisiae, was used to determine its activity and substrate specificity. Transformed yeast cells specifically elongated the C18-Delta(9) polyunsaturated fatty acids, linoleic acid (C18:2n-6, Delta(9,12)) and alpha-linolenic acid (C18:3n-3, Delta(9,12,15)), to eicosadienoic acid (C20:2n-6, Delta(11,14)) and eicosatrienoic acid (C20:3n-3, Delta(11,14,17)), respectively. To our knowledge this is the first time such an elongating activity has been functionally characterised. The results also suggest that a major route for eicosapentaenoic acid (C20:5n-3, Delta(5,8,11,14,17)) and docosahexaenoic acid syntheses in I. galbana may involve a Delta(8) desaturation pathway.

Aquilegia vulgaris seed oil contains high levels of the rare fatty acid columbinic acid (18:3 Delta(5,9,12)), which is unusual in having the double bond at the Delta(5) carbon in the trans configuration. Columbinic acid was found to be a seed-specific fatty acid not only present in the storage oil but also in membrane lipids. Several putative gene fragments have been isolated from plant RNA with sequences similar to previously characterized 'front-end' desaturases. Functional characterization of the Aquilegia cDNA is underway.

Genes encoding two distinct fatty acid delta9-desaturases were isolated from strains of the oleaginous fungus Mortierella alpina. Two genomic sequences, delta9-1 and delta9-2, each containing a single intron, were cloned from strain CBS 528.72 while one cDNA clone, LM9, was isolated from strain CBS 210.32. The delta9-1 gene encoded a protein of 445 aa which shared 99% identity with the LM9 gene product. These proteins also showed 40-60% identity to the delta9-desaturases (Ole1p) of other fungi and contained the three conserved histidine boxes, C-terminal cytochrome b5 fusion and transmembrane domains characteristic of endoplasmic reticulum membrane-bound delta9-desaturases. LM9 and delta9-1 are therefore considered to represent the same gene (ole1). The ole1 gene was transcriptionally active in all M. alpina strains tested and its function was confirmed by complementation of the Saccharomyces cerevisiae ole1 mutation. Fatty acid analysis of yeast transformants expressing the CBS 210.32 ole1 gene showed an elevated level of oleic acid (18:1) compared to palmitoleic acid (16:1), the major fatty acid component of wild-type S. cerevisiae. This indicated that the M. alpina delta9-desaturase had a substrate preference for stearic acid (18:0) rather than palmitic acid (16:0). Genomic clone delta9-2 (ole2) also encoded a protein of 445 aa which had 86% identity to the delta9-1 and LM9 proteins and whose ORF also complemented the yeast ole1 mutation. The transcript from this gene could only be detected in one of the six M. alpina strains tested, suggesting that its expression may be strain-specific or induced under certain physiological conditions.

The past few years have witnessed a major upsurge in research towards the goal of modifying the lipid composition of plants. Genes encoding a range of different fatty acid desaturase activities have been cloned, and the evolutionary relationships between and within different classes of enzymes have tentatively been established. The effects of expressing some of these desaturases in heterologous hosts have also been studied, often producing unexpected results which contribute further to our understanding of plant lipid modification. It is to be hoped that, in the near future, the goal of producing unusual and valuable fatty acids in transgenic oilseeds will be achieved on a commercial scale.

We have identified a cDNA from the nematode worm Caenorhabditis elegans that encodes a fatty acid delta5 desaturase. Saccharomyces cerevisiae expressing the full-length cDNA was able to convert di-homo-gamma-linolenic acid to arachidonic acid, thus confirming delta5 desaturation. The 1341 bp delta5 desaturase sequence contained an N-terminal cytochrome b5 domain and was located within a kilobase of the C. elegans delta6 desaturase on chromosome IV. With an amino acid identity of 45% it is possible that one of these genes arose from the other by gene duplication. This is the first example of a delta5 desaturase gene isolated from an animal.

Lipid metabolism was studied in 2-d-old liquid cultures of Mucor circinelloides grown at 25 degrees C. Under these conditions, oil accumulated to 0.5 g l-1 with a gamma-linolenic acid content (gamma 18:3) of 60 mg l-1. The major labelled lipids in cultures incubated with [14C]acetate were triacylglycerol (TAG), phosphatidylcholine (PC) and phosphatidylethanolamine (PE). The proportion of label declined in the phospholipids and increased in TAG with time. [14C]18:1 and [14C]18:2 rapidly appeared in PC and PE and later accumulated in [14C]gamma 18:3. TAG-synthesizing capacity was greatest in the microsomal membrane fraction, which accumulated high levels of phosphatidic acid in the presence of glycerol 3-phosphate and acyl-CoA substrates at pH 7.0. Further metabolism of phosphatidic acid to diacylglycerol and TAG was achieved by increasing the pH to 8.0. Lysophosphatidic acid: acyl-CoA acyltransferase (LPAAT) activity was particularly high and may have accounted for the rapid accumulation of phosphatidic acid in the membranes. The glycerol-3-phosphate: acyl-CoA acyltransferase (GPAAT) and LPAAT were non-specific for a range of saturated and unsaturated species of acyl-CoA although the GPAAT showed a marked selectivity for palmitoyl-CoA and the LPAAT for oleoyl- and linoleoyl-CoA. gamma-Linolenic acid was detected at all three positions of sn-TAG and was particularly enriched at the sn-3 position. The preparation of active in vitro systems (microsomal membranes) capable of the complete biosynthetic pathway for TAG assembly may be valuable in understanding the assembly of oils in future transgenic applications.

Arachidonic acid (C20:4 Delta5,8,11,14) is a polyunsaturated fatty acid synthesized by the Delta5-fatty acid desaturation of di-homo-gamma-linolenic acid (C20:3 Delta8,11,14). In mammals, it is known to be a precursor of the prostaglandins and the leukotrienes but it is also accumulated by the filamentous fungus Mortierella alpina. We have isolated a cDNA encoding the Delta5-fatty acid desaturase from M. alpina via a polymerase chain reaction-based strategy using primers designed to the conserved histidine box regions of microsomal desaturases, and confirmed its function by expression in the yeast Saccharomyces cerevisiae. Analysis of the lipids from the transformed yeast demonstrated the accumulation of arachidonic acid. The M. alpina Delta5-desaturase is the first example of a cloned Delta5-desaturase, and differs from other fungal desaturases previously characterized by the presence of an N-terminal domain related to cytochrome b5.

The biosynthesis of C18 polyunsaturated fatty acids has been studied in the fungus Mucor circinelloides. Microsomal membrane preparations contained delta9, delta12 and delta6 desaturase activities. The delta9 desaturase exhibited characteristics similar to those of the animal and yeast delta9 desaturases in being membrane bound and utilising stearoyl-CoA as substrate. Cytochrome b5 (a soluble form lacking the 20-amino-acid hydrophobic C-terminus) stimulated desaturation and was identified as a major cytochrome component of the membranes. A high ferricyanide reductase activity (indicative of NADH:cytochrome b5 reductase activity) coupled to inhibition by cyanide further supported the similarity with the mammalian and yeast enzymes. Time-course studies with radiolabelled oleoyl-CoA showed that the oleate [18:1(9)] was transferred to position sn-2 of phosphatidylcholine (PtdCho) and was desaturated to linoleoyl-PtdCho. Removal of the excess oleoyl-CoA from the membranes prior to addition of reductant confirmed that oleoyl-PtdCho is a substrate for the delta12 desaturase. The entry of oleate at this position of the phospholipid was facilitated by the activity of lyso-PtdCho:acyl-CoA acyltransferase (LPCAT), which readily transferred oleate from oleoyl-CoA to lyso-PtdCho. Desaturation of oleate at the sn-1 position of PtdCho was also demonstrated after the entry of oleate in to the phospholipid by the enzymes of the Kennedy pathway. Thus oleate at sn-1 and sn-2 positions served as substrate for the delta12 desaturase and is consistent with observations in oil seed tissues. LPCAT activity was substantially higher than that observed with lysophosphatidylethanolamine:acyl-CoA acyltransferase (LPEAT) indicating that oleate is less effectively channelled to phosphatidylethanolamine for linoleate synthesis. No desaturation on phosphatidylinositol could be demonstrated. Delta6 desaturase utilised linoleate at the sn-2 position of exogenously supplied PtdCho presented to the membranes in the presence of reductant. Thus, the entry of substrates into PtdCho via LPCAT and the synthesis of linoleate [18:2(9,12)] and gamma-linolenate [18:3(6,9,12)] on this phospholipid is similar to that reported for oil seed membranes.

gamma-Linolenic acid (GLA; C18:3 delta(6,9,12)) is a component of the seed oils of evening primrose (Oenothera spp.), borage (Borago officinalis L.), and some other plants. It is widely used as a dietary supplement and for treatment of various medical conditions. GLA is synthesized by a delta6-fatty acid desaturase using linoleic acid (C18:2 delta(9,12)) as a substrate. To enable the production of GLA in conventional oilseeds, we have isolated a cDNA encoding the delta6-fatty acid desaturase from developing seeds of borage and confirmed its function by expression in transgenic tobacco plants. Analysis of leaf lipids from a transformed plant demonstrated the accumulation of GLA and octadecatetraenoic acid (C18:4 delta(6,9,12,15)) to levels of 13.2% and 9.6% of the total fatty acids, respectively. The borage delta6-fatty acid desaturase differs from other desaturase enzymes, characterized from higher plants previously, by the presence of an N-terminal domain related to cytochrome b5.

Background: Polyunsaturated fatty acids (n-6) are reported to selectively kill malignant cells. Most investigations, however, did not compare neoplastic with non-neoplastic cells from the same tissue type. Here we evaluate the effects of n-6 fatty acids on a non-neoplastic epithelium cell line (CAPE) and a spontaneous carcinoma cell line (CPA) derived from the canine prostate., Methods: Cell lines were cultured in DME in the presence of fatty acids and their effects on cell proliferation monitored by coulter counting. Lipids were extracted and quantitized by gas chromatography., Results: Cell proliferation was reduced more in CAPE. A neoplastic strain (CPA-GLA) tolerant to prolonged culture in 18:3n-6 was isolated. CPA grown in an 18:2n-6 or 18:3n-6 supplemented media accumulated 20:3n-6 and contained little 20:4n-6., Conclusions: Polyenoic n-6 fatty acids are not specifically inhibitory to neoplastic cells which exhibited a marked alteration in the metabolism of 20:4n-6.

Oil-bodies, from the immature cotyledons of sunflower (Helianthus annuus L.), were difficult to purify to homogeneity using conventional techniques. The major protein contaminants were albumin and globulin storage proteins. A protocol has been developed, therefore, based upon the stringent washing of the oil-body fraction in 9 M urea, which effectively removed almost all the contaminating protein as judged by SDS/PAGE. The urea-washed oil-bodies were enriched in two major proteins of M(r) 19000 and 20000. These proteins were oleosins as demonstrated by their amino acid compositions and the sequence analysis of peptides produced by CNBr cleavage. Far-UV CD spectra of the oleosins in trifluoroethanol, trifluoroethanol/water mixtures and as mixed micelles in SDS, were typical of alpha-helical proteins with alpha-helical contents of some 55%. The phospholipid content of the urea-washed preparations was less than 0.1% of that required to form a half-unit membrane surrounding the oil-body. The oil-body surface therefore appears to be an unusual and novel structure, covered largely by an oleosin protein coat or pellicle rather than a conventional fluid membrane, half-unit or otherwise.

Two distinct cDNAs encoding oleosins (oil body proteins) have been identified by degenerate PCR as transcripts present in the developing seeds of sunflower (Helianthus annuus L. cv. Dwarf Sunbred). One (pSOM) of these is closely related to a reported sunflower oleosin, whilst the other (pSO5) has not been previously described. Different expression patterns were observed for the two cDNAs, pSO5 being expressed earlier than pSOM in seed maturation and oil deposition. The results support the contention that oleosin proteins are synthesised either during or closely after the formation of the oil body. Translation in vitro of synthetic oleosin transcripts was enhanced by the addition of microsomes but suppressed by the addition of purified signal recognition particle (SRP) complex. Deletion of 62 amino acid residues at the C-terminus of the oleosin did not alter the in vitro targeting of the protein to the microsomal membrane. Taken together these data support the idea that oleosins are targeted to the ER membrane as part of oil body biogenesis.

Polymerase chain reaction (PCR) was used to amplify transcripts encoding cytochrome b5 from cDNA synthesised from RNA isolated from developing seeds of tobacco (Nicotiana tabacum L.). The sequence of the amplified products indicated that the clones encoded a second form of tobacco cytochrome b5, different from that previously characterised (Smith et al. 1994, Plant Mol Biol 25:527-537). Rapid amplification of cDNA ends (RACE)-PCR was used to amplify the 5' and 3' ends of the transcript. Northern blotting and RNAse protection assays of RNA samples isolated from different tobacco tissues indicated that this second cytochrome b5 form was expressed only in developing seeds. Therefore, it seems likely that this message is the product of a tobacco cytochrome b5 gene specifically expressed in seeds.

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.

A full-length clone encoding cytochrome b5 has been isolated from a tobacco leaf cDNA library in lambda gt11 by PCR using degenerate primers. This cDNA encodes a protein of 139 residues which exhibits a high degree of homology to other cytochrome b5s, the message for which is expressed predominantly in developing seeds and in pigmented flower tissue. In the developing tobacco seed the mRNA is abundant at very early stages (< 10 days after flowering). Southern analysis indicated that more than one gene encodes cytochrome b5 in the tobacco genome. In vitro transcription and translation studies of the cDNA indicated that the protein inserts into the ER membrane by a non-SRP-mediated pathway and that the C-terminus of the protein is required for targeting and insertion.

Embryos of Cuphea lanceolata have more than 80 mol% of decanoic acid ('capric acid') in their triacylglycerols, while this fatty acid is virtually absent in phosphatidylcholine (PtdCho). Seed development was complete 25-27 days after pollination, with rapid triacylglycerol deposition occurring between 9 and 24 days. PtdCho amounts increased until day 15 after pollination. Analysis of embryo lipids showed that the diacylglycerol (DAG) pool consisted of mainly long-chain molecular species, with a very small amount of mixed medium-chain/long-chain glycerols. Almost 100% of the fatty acid at position sn-2 in triacylglycerols (TAG) was decanoic acid. When equimolar mixtures of [14C]decanoic and [14C]oleic acid were fed to whole detached embryos, over half of the radioactivity in the DAG resided in [14C]oleate, whereas [14C]decanoic acid accounted for 93% of the label in the TAG. Microsomal preparations from developing embryos at the mid-stage of TAG accumulation catalysed the acylation of [14C]glycerol 3-phosphate with either decanoyl-CoA or oleoyl-CoA, resulting in the formation of phosphatidic acid (PtdOH), DAG and TAG. Very little [14C]glycerol entered PtdCho. In combined incubations, with an equimolar supply of [14C]oleoyl-CoA and [14C]decanoyl-CoA in the presence of glycerol 3-phosphate, the synthesized PtdCho species consisted to 95% of didecanoic and dioleic species. The didecanoyl-glycerols were very selectively utilized over the dioleoylglycerols in the production of TAG. Substantial amounts of [14C]oleate, but not [14C]decanoate, entered PtdCho. The microsomal preparations of developing embryos were used to assess the acyl specificities of the acyl-CoA:sn-glycerol-3-phosphate acyltransferase (GPAT, EC 2.3.1.15) and the acyl-CoA:sn-1-acyl-glycerol-3-phosphate acyltransferase (LPAAT, EC 2.3.1.51) in Cuphea lanceolata embryos. The efficiency of acyl-CoA utilization by the GPAT was in the order decanoyl = dodecanoyl greater than linoleoyl greater than myristoyl = oleoyl greater than palmitoyl. Decanoyl-CoA was the only acyl donor to be utilized to any extent by the LPAAT when sn-decanoylglycerol 3-phosphate was the acyl acceptor. sn-1-Acylglycerol 3-phosphates with acyl groups shorter than 16 carbon atoms did not serve as acyl acceptors for long-chain (greater than or equal to 16 carbon atoms) acyl-CoA species. On the basis of the results obtained, we propose a schematic model for triacylglycerol assembly and PtdCho synthesis in a tissue specialized in the synthesis of high amounts of medium-chain fatty acids.

The synthesis of triacylglycerols was investigated in microsomes (microsomal fractions) prepared from the developing cotyledons of sunflower (Helianthus annuus). Particular emphasis was placed on the mechanisms involved in controlling the C18- unsaturated-fatty-acid content of the oils. We have demonstrated that the microsomes were capable of: the transfer of oleate from acyl-CoA to position 2 of sn-phosphatidylcholine for its subsequent desaturation and the return of the polyunsaturated products to the acyl-CoA pool by further acyl exchange; the acylation of sn-glycerol 3-phosphate with acyl-CoA to yield phosphatidic acid, which was further utilized in diacyl- and tri-acylglycerol synthesis; and (3) the equilibrium of a diacylglycerol pool with phosphatidylcholine. The acyl exchange between acyl-CoA and position 2 of sn-phosphatidylcholine coupled to the equilibration of diacylglycerol and phosphatidylcholine brings about the continuous enrichment of the glycerol backbone with C18 polyunsaturated fatty acids for triacylglycerol production. Similar reactions were found to operate in another oilseed plant, safflower (Carthamus tinctorius L.). On the other hand, the microsomes of avocado (Persea americana) mesocarp, which synthesize triacylglycerol via the Kennedy [(1961) Fed. Proc. Fed. Am. Soc. Exp. Biol. 20, 934-940] pathway, were deficient in acyl exchange and the diacylglycerol in equilibrium phosphatidylcholine interconversion. The results provide a working model that helps to explain the relationship between C18- unsaturated-fatty-acid synthesis and triacylglycerol production in oilseeds.

Laevulinic acid (Lev) was used to control the rate of protocholorophyllide (PChl) regeneration in the leaves of dark-grown seedlings of barley (Hordeum vulgare) after a brief light treatment. In the leaves given Lev, at concentrations that severely block the resynthesis of protochlorophyllide, there was a massive overproduction of delta-aminolaevulinic acid (AmLev) that was well in excess of that required for the regeneration of PChl observed in the control leaves. Lev, at low concentrations, slightly delayed regeneration and held up, rather than inhibited, the utilization of the AmLev, which accumulated in the tissues. The overproduction and uncontrolled formation of AmLev also occurred in dark-grown leaves treated with a high concentration of Lev and given a light treatment of just sufficient energy to photoreduce only small quantities of the endogenous PChl. Experiments in which a high level of free PChl was induced by incubating the leaves in AmLev indicated that the active species of PChl was that associated with, and bound to, the PChl reductase protein. The results strongly demonstrate a close relationship between the PChl-protein complex and the ability of the leaves to synthesize AmLev.

Microsomal membrane preparations from the developing cotyledons of safflower (Carthamus tinctorius L.) seed catalyse the formation of triacylglycerol fromsn-glycerol 3-phosphate and linoleoyl-CoA. Conditions of incubation were achieved in which the rate of triacylglycerol synthesis approached activities which were compatible with oil accumulation observed in vivo. Reaction mixtures which contained the microsomes took on a white soup-like appearance as triacylglycerol synthesis proceeded and sufficient oil was produced to form a white fat-pad at the surface after centrifugation. The development of the oil bodies in the microsomal membranes was studied by electron microscopy and showed that lipid droplets were formed in or on the membrane surface and were then released as apparently naked entities into the surrounding medium. The ontogeny of the oil droplet in vitro is discussed in terms of oil-body formation in vivo.

Amaranthus caudatus L. seedlings produce less amaranthin in the presence of gibberellic acid A3 (GA3). This phenomenon has been tested as a bioassay for gibberellic acid and has been compared to the bioassay based on lettuce hypocotyl extension. The inhibition of pigment synthesis is much more sensitive to GA3 at concentrations from 1 to 0.01 μg/ml. Of the gibberellins tested GA1, GA3, GA4, and GA7 are the most active in the described bioassay.

Microsomes isolated from the developing cotyledons of the seeds of the safflower varieties, very-high-linoleate, Gila and high-oleate, were capable of exchanging the acyl groups in acyl-CoA with the fatty acids in position 2 of phosphatidylcholine. The specificity of the 'acyl-exchange' towards the acyl moiety in acyl-CoA was selective in the order: oleate greater than linoleate greater than linolenate. Stearoyl-CoA was completely selected against when presented in a mixed substrate with unsaturated 18-carbon acyl-CoAs. Microsomes, of the very-high-linoleate safflower variety, rapidly desaturated in situ-labelled [14C]oleoylphosphatidylcholine in the presence of NADH. Little oleate desaturation, however, was observed in the microsomes of the high-oleate variety. Microsomes of the Gila and high-oleate varieties of safflower rapidly synthesised phosphatidic acid by the acylation of glycerol 3-phosphate with acyl-CoA. The phosphatidic acid was metabolised to diacylglycerol, which was further acylated to triacylglycerol. A strong selectivity for linoleoyl-CoA was found for the acylation of glycerol 3-phosphate in both the Gila and high-oleate microsomes. On the basis of these results, we propose that the pattern of 18-carbon unsaturated fatty acids in the triacylglycerols of all 'oil'-producing seeds is a direct reflection of the fatty acids in the acyl-CoA pool. This, in turn, is governed by: A, the rate and specificity of the acyl exchange between acyl-CoA and phosphatidylcholine; B, the rate of oleate (and linoleate) desaturation in phosphatidylcholine; and C, the rate and specificity of the glycerophosphate acyltransferase.

Microsomal preparations from the developing cotyledons of safflower (Carthamus tinctorius) catalyse the acylation of sn-glycerol 3-phosphate in the presence of acyl-CoA. Under these conditions the radioactive glycerol in sn-glycerol 3-phosphate accumulates in phosphatidic acid, phosphatidylcholine, diacyl- and tri-acylglycerol. The incorporation of glycerol into phosphatidylcholine is via diacylglycerol and probably involves a cholinephosphotransferase. The results show that the glycerol moiety and the acyl components in phosphatidylcholine exchange with the diacylglycerol during the biosynthesis of diacylglycerol from phosphatidic acid. The continuous reversible transfer of diacylglycerol with phosphatidylcholine, which operates during active triacylglycerol synthesis, will control in part the polyunsaturated-fatty-acid quality of the final seed oil.

The utilisation of [(14)C]glycerol 3-phosphate and [(14)C]linoleoyl-CoA in the synthesis of triacylglycerol has been studied in the microsomal preparations of developing cotyledons of safflower seed. The results confirm that the glycerol backbone, which flows towards triacylglycerol from phosphatidic acid through the Kennedy pathway, can enter phosphatidylcholine from diacylglycerol. The equilibration between diacylglycerol and phosphatidylcholine offers a mechanism for the return of oleate to phosphatidylcholine for desaturation to linoleate. We have established that the oleate entering position 1 of sn-phosphatidylcholine from diacylglycerol is desaturated in situ to linoleate. The results indicate that the diacylglycerol phosphatidylcholine interconvertion coupled to the acyl exchange between acyl-CoA and position 2 of sn-phosphatidylcholine brings about the continuous enrichment of the glycerol backbone with C18-polyunsaturated fatty acids and hence these enzymes are of major importance in regulating the acyl quality of the accumulating triacylglycerols. Microsomal preparations from avocado mesocarp, however, did not have detectable acyl exchange between acyl-CoA and phosphatidylcholine or diacylglycerol phosphatidylcholine interconversion despite the high activity of the enzymes of the Kennedy pathway. A scheme is presented which incorporates many of the observations on triacylglycerol synthesis and provides a working model for the regulation of acyl quality in linoleate-rich vegetable oils.

Microsomal preparations from the developing cotyledons of safflower (Carthamus tinctorius) catalysed the acylation of sn-glycerol 3-phosphate in the presence of acyl-CoA. The resulting phosphatidate was further utilized in the synthesis of diacyl- and tri-acylglycerol by the reactions of the so-called 'Kennedy pathway' [Kennedy (1961) Fed. Proc. Fed. Am. Soc. Exp. Biol. 20, 934-940]. Diacylglycerol equilibrated with the phosphatidylcholine pool when glycerol backbone, with the associated acyl groups, flowed from phosphatidate to triacylglycerol. The formation of diacylglycerol from phosphatidate through the action of a phosphatidate phosphohydrolase (phosphatidase) was substantially inhibited by EDTA and, under these conditions, phosphatidate accumulated in the microsomal membranes. The inhibition of the phosphatidase by EDTA was alleviated by Mg2+. The presence of Mg2+ in all incubation mixtures stimulated quite considerably the synthesis of triacylglycerol in vitro. Microsomal preparations incubated with acyl-CoA, sn-glycerol 3-phosphate and EDTA synthesized sufficient phosphatidate for the reliable analysis of its intramolecular fatty acid distribution. In the presence of mixed acyl-CoA substrates the sn-glycerol 3-phosphate was acylated exclusively in position 1 with the saturated fatty acids, palmitate and stearate. The polyunsaturated fatty acid linoleate was, however, utilized largely in the acylation of position 2 of sn-glycerol 3-phosphate. The affinity of the enzymes involved in the acylation of positions 1 and 2 of sn-glycerol 3-phosphate for specific species of acyl-CoA therefore governs the non-random distribution of the different acyl groups in the seed triacylglycerols. The acylation of sn-glycerol 3-phosphate in position 1 with saturated acyl components also accounts for the presence of these groups in position 1 of sn-phosphatidylcholine through the equilibration of diacylglycerol with the phosphatidylcholine pool, which occurs when phosphatidate is utilized in the synthesis of triacylglycerol. These results add further credence to our previous proposals for the regulation of the acyl quality of the triacylglycerols that accumulate in developing oil seeds [Stymne & Stobart (1984) Biochem. J. 220, 481-488; Stobart & Stymne (1985) Planta 163, 119-125].

Microsomal preparations from developing linseed (Linum usitatissimum L.) cotyledons catalyzed i) acyl exchange between acyl-CoA and position 2 of sn-phosphatidylcholine, ii) acylation of sn-glycerol 3-phosphate to yield phosphatidic acid, and iii) the utilisation of phosphatidic acid in the production of diacylglycerol and triacylglycerol. Selectivity studies for C18 acyl species of acyl-CoA indicated a bias for the channelling of oleate to phosphatidylcholine for, presumably, its desaturation, and the utilisation of the polyunsaturated fatty-acid products in the acyl-CoA pool for phosphatidic acid and subsequent triacylglycerol synthesis. The microsomal preparations were capable of returning glycerol backbone with associated acyl components to phosphatidylcholine from diacylglycerol where it may be further enriched with polyunsaturated C18 acids by desaturation. The acyl quality in linolenate-rich oilseeds appears to be under similar control to that found in linoleate-rich species.

Microsomal membrane preparations from rat lung catalyse the incorporation of radioactive linolenic acid from [14C]linolenoyl-CoA into position 2 of sn-phosphatidylcholine. The incorporation was stimulated by bovine serum albumin and free CoA. Free fatty acids in the incubation mixtures were not utilised in the incorporation into complex lipids. Fatty acids were transferred to the acyl-CoA pool during the incorporation of linolenic acid into phosphatidylcholine. An increase in lysophosphatidylcholine occurred in incubations containing both bovine serum albumin and free CoA and in the absence of acyl-CoA. The results were consistent with an acyl-CoA: lysophosphatidylcholine acyltransferase operating in both a forwards and backwards direction and thus catalysing the acyl exchange between acyl-CoA and position 2 of sn-phosphatidylcholine. In incubations with mixed species of acyl-CoAs, palmitic acid was the major fatty acid substrate transferred to phosphatidylcholine in acyl exchange, whereas this acid was completely selected against in the acylation of added lysophosphatidylcholine. The selectivity for palmitoyl-CoA was particularly enhanced when the mixed acyl-CoA substrate was presented to the microsomes in molar concentrations equivalent to the molar ratios of the fatty acids in position 2 of sn-phosphatidylcholine. During acyl exchange, the predominant fatty acid transferred to phosphatidylcholine from acyl-CoA was palmitic acid, whereas arachidonic acid was particularly selected for in the reverse reaction from phosphatidylcholine to acyl-CoA. A hypothesis is presented to explain the differential selectivity for acyl species between the forward and backward reactions of the acyltransferase that is based upon different affinities of the enzyme for substrates at high and low concentrations of acyl donor. Acyl exchange between acyl-CoA and phosphatidylcholine offers, therefore, a possible mechanism for the acyl-remodelling of phosphatidylcholine for the production of lung surfactant.

Developing cotyledons of safflower (Carthamus tinctorius L.) and sunflower (Helianthus annuus L.) readily utilised exogenously supplied (14)C-labelled fatty-acid substrates for the synthesis of triacylglycerols. The other major radioactive lipids were phosphatidylcholine and diacylglycerol. In safflower cotyledons, [(14)C]oleate was rapidly transferred to position 2 of sn-phosphatidylcholine and concomitant with this was the appearance of radioactive linoleate. The linoleate was further utilised in the synthesis of diacyl- and triacyl-glycerol via the reactions of the so-called Kennedy pathway. Supplying [(14)C]linoleate, however, resulted in a more rapid labelling of the diacylglycerols than from [(14)C]oleate. In contrast, sunflower cotyledons readily utilised both labelled acyl substrates for rapid diacylglycerol formation as well as incorporation into position 2 of sn-phosphatidylcholine. In both species, however, [(14)C]palmitate largely entered sn-phosphatidylcholine at position 1 during triacylglycerol synthesis. The results support our previous in-vitro observations with isolated microsomal membrane preparations that (i) the entry of oleate into position 2 of sn-phosphatidylcholine, via acyl exchange, for desaturation to linoleate is of major importance in regulating the level of polyunsaturated fatty acids available for triacylglycerol formation and (ii) Palmitate is largely excluded from position 2 of sn-phosphatidylcholine and enters this phospholipid at position 1 probably via the equilibration with diacylglycerol. Specie differences appear to exist between safflower and sunflower in relation to the relative importance of acyl exchange and the interconversion of diacylglycerol with phosphatidylcholine as mechanisms for the entry of oleate into the phospholipid for desaturation.

Acyl exchange between acyl-CoA and position 2 of sn-phosphatidylcholine occurs in the microsomal preparations of developing safflower cotyledons. Evidence is presented to show that the acyl exchange is catalysed by the combined back and forward reactions of an acyl-CoA:lysophosphatidylcholine acyltransferase (EC 2.3.1.23). The back reaction of the enzyme was demonstrated by the stimulation of the acyl exchange with free CoA and by the observation that the added CoA was acylated with acyl groups from position 2 of sn-phosphatidylcholine. Re-acylation of the, endogenously produced, lysophosphatidylcholine with added acyl-CoA occurred with the same specificity as that observed with added palmitoyl lysophosphatidylcholine. A similar acyl exchange, catalysed by an acyl-CoA:lysophosphatidylcholine acyltransferase, occurred in microsomal preparations of rat liver. The enzyme from safflower had a high specificity for oleate and linoleate, whereas arachidonate was the preferred acyl group in the rat liver microsomal preparations. The rate of the back reaction was 3-5% and 0.2-0.4% of the forward reaction in the microsomal preparations of safflower and rat liver respectively. Previous observations, that the acyl exchange in safflower microsomal preparations was stimulated by bovine serum albumin and sn-glycerol 3-phosphate, can now be explained by the lowered acyl-CoA concentrations in the incubation mixture with albumin and in the increase in free CoA in the presence of sn-glycerol 3-phosphate (by rapid acylation of sn-glycerol 3-phosphate with acyl groups from acyl-CoA to yield phosphatidic acid). Bovine serum albumin and sn-glycerol 3-phosphate, therefore, shift the equilibrium in acyl-CoA:lysophosphatidylcholine acyltransferase-catalysed reactions towards the rate-limiting step in the acyl exchange process, namely the removal of acyl groups from phosphatidylcholine. The possible role of the acyl exchange in the transfer of acyl groups between complex lipids is discussed.

Exogenous gibberellic acid, A3 (GA3) inhibits phytochrome mediated betacyanin synthesis in seedlings of Amaranthus caudatus. The growth retardants, β-chloroethyl-trimethylammonium chloride (CCC), 'isopropyl-4'-(triethylammonium chloride)-5'-methylphenyl piperidine carboxylate (AMO 1618) and tributyl-2,4,-dichlorobenzylphosphonium chloride (phosphon D) enhance pigment synthesis. Retardant stimulation of pigment synthesis is overcome by GA3 application. Besides lowering endogenous GA levels the retardants inhibit protein synthesis by as much as 25%. Retardant inhibition of protein synthesis is not overcome by GA3. The results suggest that amaranthin synthesis in Amaranthus caudatus can be directly controlled by endogenous GA. GA3 has no effect on kinin induced dark pigment synthesis. Kinins, however, do not overcome GA3 inhibition of pigment synthesis in the light.

Microsomal membrane preparations from the maturing cotyledons of common borage (Borago officinalis) exhibit delta 12- and delta 6-desaturase activities, which resulted in the synthesis of linoleate and gamma-linolenate respectively. The desaturase enzymes utilized the complex lipid substrate phosphatidylcholine. The activity of these enzymes was sufficiently high to allow the monitoring of the mass changes in the endogenous oleate, linoleate and gamma-linolenate in the microsomal phosphatidylcholine in the presence of NADH (i.e. under desaturating conditions). The results illustrate that the delta 12-desaturase uses the oleate substrate at both the sn-1 and -2 positions of sn-phosphatidylcholine, whereas the delta 6-desaturase is almost totally restricted to the linoleate at position 2 of the complex lipid. Estimate of the acyl-substrate pool size at position 2 of sn-phosphatidylcholine for both desaturases indicated that some 50% of the oleate and linoleate was available to the enzymes. The microsomes (microsomal fractions) had a somewhat impaired Kennedy [(1961) Fed. Proc. Fed. Am. Soc. Exp. Biol. 20, 934-940] pathway for the formation of triacylglycerols when compared with other oil-rich plant species that have been studied [Stymne & Stobart (1987) The Biochemistry of Plants: a Comprehensive Treatise (Stumpf, P.K., ed.), vol. 10, chapter 8, pp. 175-214, Academic Press, New York]. In the presence of sn-glycerol 3-phosphate and acyl-CoA, large quantities of phosphatidic acid accumulated in the membranes. Acyl-selectivity studies on the glycerol-acylating enzymes showed that gamma-linolenate could be acylated to both the sn-1 and sn-2 positions of sn-glycerol 3-phosphate. However, stereochemical analysis of the acyl components of the sn-triacylglycerol obtained from mature seeds indicated that, whereas no gamma-linolenate was present at the sn-1 position, it accounted for over 50% of the fatty acids at position sn-3. The results indicate that the diacylglycerol acyltransferase (EC 2.3.1.20) may show a strong selectivity for gamma-linolenoyl-CoA and hence result in the efficient removal of this fatty acid from the acyl-CoA pool in vivo, leaving negligible substrate for utilization by the sn-glycerol 3-phosphate acyltransferase (EC 2.3.1.15).

The developing seeds of Borago officinalis (common borage) accumulate a triacylglycerol oil that is relatively rich in the uncommon fatty acid gamma-linolenate (octadec-6,9,12-trienoic acid). Incubation of developing, whole, cotyledons with [14C]oleate and [14C]linoleate showed that the gamma-linolenate was synthesized by the sequential desaturation of oleate----linoleate----gamma-linolenate. Microsomal membrane preparations from the developing cotyledons contained an active delta 6-desaturase enzyme that catalysed the conversion of linoleate into gamma-linolenate. Experiments were designed to manipulate the [14C]linoleate content of the microsomal phosphatidylcholine. The [14C]linoleoyl phosphatidylcholine labelled in situ was converted into gamma-linolenoyl phosphatidylcholine in the presence of NADH. The substrate for the delta 6-desaturase in borage was, therefore, the linoleate in the complex microsomal lipid phosphatidylcholine, rather than, as in animals, the acyl-CoA. This was further confirmed in experiments that compared the specific radioactivity of the gamma-linolenate, in acyl-CoA and phosphatidylcholine, that was synthesized when [14C]linoleoyl-CoA was incubated with microsomal membranes, NADH and non-radioactive gamma-linolenoyl-CoA. The delta 6-desaturase was positionally specific and only utilized the linoleate in position 2 of sn-phosphatidylcholine. Analysis of the positional distribution of fatty acids in the endogenous microsomal sn-phosphatidylcholine showed that, whereas position 1 contained substantial linoleate, only small amounts of gamma-linolenate were present. The results shed further light on the synthesis of C18 polyunsaturated fatty acids in plants and in particular its relationship to the regulation of the acyl quality of the triacylglycerols in oilseeds.

The photoreduction of protochlorophyllide (Pchl) in dark-grown leaves of barley (Hordeum vulgare) brings about the synthesis of delta-aminolaevulinic acid (AmLev). Manipulation of the Pchl level in the leaves by incubation in AmLev indicated that the production of AmLev was intimately related to the state of the Pchl reductase ternary complex. Free Pchl reductase that is unassociated with substrate/product appeared at first to be essential for the photoinduction of AmLev synthesis. Experiments on the photoreduction of Pchl in dark-grown leaves exposed to low-energy red-light, however, showed that photoreduction and AmLev synthesis would occur when the Pchl reductase, together with substrate, was maintained at relatively high endogenous concentration. Under such conditions the availability of free reductase protein would be negligible. An alternative scheme is presented, therefore, that can explain many, if not all, of the observations on AMLev synthesis and its close relationship to Pchl reduction, and which is based on a common supply of NADPH for the reduction of glutamate to AmLev and the synthesis of chorophyll(-ide).

2.4-dichlorophenoxyacetic acid (2.4-D) and (2-chloroethyl)-trimethylammonium chloride (CCC) inhibit chlorophyll synthesis and protochlorophyllide 652 regeneration in 6-8 day old barley leaves whilst having little effect on the rates of protochlorophyll 632 synthesis from exogenous δ-aminelevulinic acid (ALA) and ALA-dehydratase activity. Longer pretreatments with 2.4-D and CCC show it is only after 50 to 60 hr that the rates of P632 production from exogenous ALA and ALA-dehydratase activity are affected. Similar response times were obtained for chloramphenicol (CAP). The results indicate that 2.4-D and CCC may act by directly inhibiting specific plastid-protein synthesis similar to CAP. Hence it seems that it is only those proteins (enzymes) having a rapid turnover that are affected first i.e. the enzymes necessary for ALA synthesis in the plastid.

Both gibberellic acid and abscisic acid inhibit the light induced synthesis of amaranthin in Amaranthus tricolor seedlings. The auxin, indolyl-3-acetic acid has no effect. The protein/RNA inhibitors, cycloheximide and 8-azaguanine, also reduced the levels of amaranthin produced.

Dormancy of intact sycamore (Acer pseudoplatanus) seeds was broken by chilling (5°C) for several weeks in moist conditions. Treatment of unchilled seeds with kinetin induced some germination, but gibberellin was ineffective. This stimulation by kinetin was not suppressed by the added presence of abscisic acid during incubation.The chilling requirement of intact seeds was eliminated by removal of the testa, and the naked embryos developed with no morphological abnormalities. During early growth of isolated embryos in the light, two distinct developmental processes were recognised. One involved initial elongation of the radicle accompanied by geotropic curvature and was stimulated by kinetin but not by gibberellin, while the other involved unrolling of the cotyledons, which was accelerated by gibberellin but much less by kinetin. Abscisic acid strongly suppressed both developmental processes when applied alone, inhibited cotyledon expansion in the presence of gibberellin, but failed to overcome the promotory effects of kinetin on radicle growth. Experiments with CCC indicated that under natural conditions the unrolling of the cotyledons is dependent upon endogenous gibberellin. Radicle growth of isolated embryos was unimpaired by incubation in the dark, but cotyledon expansion of water incubated embryos was poor, and although it was accelerated by gibberellin, the responses in all treatments were slower than in the corresponding light grown samples.It is suggested that endogenous cytokinins are primary factors in the initiation of radicle growth, while gibberellins are important in cotyledon expansion. Abscisic acid appears to have an inhibitory role in both processes, and the interactions of these regulators in the control of germination and development are discussed.