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Taylor, D.C., L. Kunst, and S.L. MacKenzie. 1993. Bioassembly of storage lipids in oilseed crops; Target: Trierucin. p. 181-191. In: J. Janick and J.E. Simon (eds.), New crops. Wiley, New York.

Bioassembly of Storage Lipids in Oilseed Crops; Target: Trierucin*

David C. Taylor, Ljerka Kunst, and Samuel L. MacKenzie

  1. BIOASSEMBLY OF TAGs CONTAINING VLCFAs IN BRASSICA NAPUS L.: THE MICROSPORE-DERIVED EMBRYO MODEL SYSTEM
  2. ENZYME TARGETS FOR MANIPULATION OF VLCFA LEVELS IN B. NAPUS
    1. Lyso-Phosphatidic Acid Acyltransferase (LPAT)
    2. Elongase
  3. CONCLUDING REMARKS
  4. REFERENCES
  5. Table 1
  6. Table 2
  7. Table 3
  8. Table 4
  9. Table 5
  10. Table 6
  11. Table 7
  12. Table 8
  13. Fig. 1
  14. Fig. 2
  15. Fig. 3
  16. Fig. 4
  17. Fig. 5
  18. Fig. 6
The long-range goal of the Plant Biotechnology Institute's Seed Oil Modification Project is the development, through recombinant DNA technology, of new plant cultivars capable of producing specifically-designed seed oils not attainable by conventional breeding methods. As a model, we intend to genetically modify Brassica napus L. to produce seed oils rich in erucic acid (22:1, cis-13-docosenoic acid) and other very long chain fatty acids (VLCFAs). VLCFAs are valued as industrial feedstocks for the production of surfactants, plasticizers, and surface coatings, while trierucin is an excellent high temperature lubricant (Princen and Rothfus 1984) and can also be used as a novel treatment for adrenoleukodystrophy (Van Dyne et al. 1990). While the primary use of high erucic oils is currently for the production of erucamide, employed as a slip and anti-block agent in the manufacture of plastic films, the expanded utilization of such seed oils for industrial applications has been forecast to increase with the advent of the USDA-sponsored High Erucic Acid Oil Project (USDA Co-operative State Research Service 1990; Van Dyne et al. 1990). Indeed, more than 200 patented applications have been catalogued for the C22 oleochemicals erucic acid, behenic acid (22:0, docosanoic acid) and their derivatives, and such compounds have been cited as strategic industrial feedstocks for the 21st century (Sonntag 1991). Some of these potential applications are listed in Table 1.

The scientific rationale for our model study to manipulate the bioassembly of triacylglycerols (TAGs) containing VLCFAs is as follows: (1) VLCFAs, such as erucic acid, are confined almost exclusively to the neutral lipid (chiefly triacylglycerol) fraction in developing oilseeds and are not components of membrane (phospho- or glyco-) lipids (Roughan and Slack 1982; Griffiths et al. 1988); (2) 22:1 is an excellent seed storage lipid marker for studying biochemical mechanisms specific to TAG assembly (Taylor et al. 1991); (3) Molecular-genetic modification of seed oils to enhance levels of VLCFAs will be TAG-specific, and should avoid any potentially-lethal interference with membrane lipid metabolism.

This report describes the combined biochemical and molecular-genetic approaches we have utilized to identify two target genes from oilseeds which, when isolated and transgenically (over) expressed in B. napus, might lead to seed oils high in strategic VLCFAs (e.g. trierucin). The first challenge lies in the fact that the lyso-phosphatidic acid acyltransferase or LPAT in Brassica species cannot insert erucic acid at the middle (sn-2) position on the glycerol backbone. However, other plant species do possess this function and therefore constitute targets for gene retrieval and transgenic expression to give B. napus this capability. It may also be necessary to increase the capacity for VLCFA biosynthesis to provide adequate levels of these fatty acids for incorporation into storage oils. Thus, our second target is the "elongase" system, which creates VLCFAs by sequentially elongating C18 fatty acyl precursors.

BIOASSEMBLY OF TAGs CONTAINING VLCFAs IN BRASSICA NAPUS L.: THE MICROSPORE-DERIVED EMBRYO MODEL SYSTEM

The TAGs found in B. napus L. and other oilseeds of the Brassicaceae have an acyl composition typical of that shown in Fig. 1. VLCFAs such as eicosenoic (20:1) and erucic (22:1) acids are esterified to the sn-1 and sn-3 positions, but not the sn-2 position (Brockerhoff 1971; Norton and Harris 1983). Rather, the latter position is usually esterified with C18 fatty acids such as oleic (18:1) acid. While it is generally accepted that in higher plants, C16 and C18 fatty acyl moieties are incorporated into TAGs via the G-3-P pathway according to Kennedy (Barron and Stumpf 1962; Stymne and Stobart 1987) (Fig. 2), until recently, the mechanism involved in the biosynthesis of TAGs containing VLCFAs was not fully understood, despite a number of studies which attempted to elucidate the pathway in developing oilseeds (Pollard and Stumpf 1980a,b; Mukherjee 1986; Sun et al. 1988; Battey and Ohlrogge 1989; Bernerth and Frentzen 1990; Fehling and Mukherjee 1990). Primarily, the difficulty was due to the fact that, in typical metabolism studies, radiolabeled erucic acid or erucoyl-CoA were very poorly metabolized by developing zygotic embryo preparations in vitro.

In contrast to zygotic embryos, microspore-derived (MD) embryos are haploid and derived, as the name implies, from immature male microspores. Via techniques of tissue culture, the microspores can be "reprogrammed" to undergo embryogenesis in a manner similar to developing zygotic embryos in a fertilized seed (Fan et al. 1988; Pechan and Keller 1988). In particular, MD embryos developed in the high erucic acid cultivar Reston have been shown to accumulate VLCFAs such as 22:1 in TAGs in a manner similar to developing zygotic embryos of the same cultivar (Table 2) (Taylor et al. 1990b; Pomeroy et al. 1991; Taylor et al. 1991; Weber et al. 1992). More importantly, recent studies performed in this laboratory have demonstrated that, in comparison to its zygotic counterpart, the Reston MD embryo system actively metabolizes and incorporates radiolabeled erucoyl moieties into TAGs in vitro (Table 3). We have shown that the MD embryo system possesses all of the enzymes necessary for TAG bioassembly (Taylor et al. 1990b, 1991, 1992a).

We have exploited this model system to demonstrate for the first time, that TAGs containing erucoyl moieties at both the sn-1 and sn-3 positions in B. napus are bioassembled via the Kennedy Pathway, with G-3-P as the initial acceptor and erucoyl-CoA as the acyl donor (Fig. 3) (Taylor et al. 1992a). Furthermore, this mechanism is intimately linked, possibly via metabolite channeling, to de novo VLCFA biosynthesis from oleoyl-CoA via two-carbon extensions in the presence of malonyl-CoA and reducing equivalents. As the 14C very long chain acyl-CoAs are synthesized in vitro, they are incorporated onto the glycerol backbone of G-3-P, into the Kennedy pathway intermediates LPA, PA, DAG and PC, and accumulate in TAGs (Fig 3). In the absence of exogenous G-3-P, there is a dramatic decrease in the incorporation of newly-synthesized VLCFAs into all glycerolipids but especially TAGs, and a concomitant build-up of newly-synthesized very long chain acyl-CoAs (Fig. 3, 2 -G-3-P), a finding which strongly supports the key role of G-3-P as the initial acyl acceptor. The Kennedy pathway as the mechanism for the bioassembly of TAGs containing VLCFAs has also been confirmed in zygotic embryos of B. napus cv. Reston (Taylor et al. 1992a) and is supported by studies in other cruciferous oilseeds (Fehling et al. 1990). Stereospecific analyses of TAGs biosynthesized in vitro by the Reston MD embryo system following de novo VLCFA biosynthesis from 14C oleoyl-CoA or 14C 20:1-CoA, show that radiolabeled erucoyl moieties are incorporated into the sn-1 and sn-3 positions, but not the sn-2 position (Table 4). This pattern is similar to that found in endogenous TAGs of both MD and zygotic Reston embryos (Taylor et al. 1991). Furthermore, this finding confirms that B. napus lacks the enzymic capacity for placing erucoyl moieties into the sn-2 position, and indicates that the theoretical breeding limit for erucic acid content in B. napus is 2/3 or 66 mole %. Recent breeding efforts at the University of Manitoba have yielded a high erucic acid variety, Hero (Scarth et al. 1991), with an upper limit of about 56% 22:1 (Dr. Rachel Scarth pers. commun.).

ENZYME TARGETS FOR MANIPULATION OF VLCFA LEVELS IN B. NAPUS

Lyso-Phosphatidic Acid Acyltransferase (LPAT)

High erucic acid B. napus does not have erucoyl moieties at the sn-2 position in its seed oil (Brockerhoff 1971), and does not contain trierucin (Fig. 4A). However, within nature there exist examples of species which do have significant proportions of erucic acid at the sn-2 position (Table 5). Nasturtium (Tropaeolum majus) seed oil contains about 75% erucic acid of which about one third is esterified to the sn-2 position (Mattson and Volpenhein 1961). Furthermore, trierucin is the major TAG species in nasturtium, as confirmed in our laboratory by direct probe mass spectrometry of the TAG fraction isolated from mature seed (Fig. 4B). This is encouraging as it indicates that there is no stereochemical constraint preventing the biosynthesis of this triacylglycerol by developing plant embryos. Perhaps the best example of apparent sn-2 erucoyl specificity is meadowfoam (Limnanthes douglasii) which inserts about two thirds of its erucic acid into this position (Phillips et al. 1971) (Table 5).

Studies in this laboratory (Taylor et al. 1990a) (Table 6) and others (Cao et al. 1990; Löhden et al. 1990) have demonstrated that the accumulation of erucic acid at the sn-2 position of TAGs in meadowfoam species is due to the high erucoyl-CoA specificity of the lyso-phosphatidic acid acyltransferase (LPAT). Based on relative specific activity, the 22:1-CoA:LPAT activity in homogenates or microsomal fractions from meadowfoam was 50 to 100-fold greater than the corresponding activity in B. napus. However, the 18:1-CoA:LPAT activities of meadowfoam and rapeseed were essentially identical (Table 6), suggesting that an LPAT highly specific for erucoyl moieties is present in meadowfoam. It is perhaps surprising that the in vitro erucoyl specificity of the nasturtium LPAT was not much higher than that observed in B. napus and about an order of magnitude lower than that present in meadowfoam (Taylor et al. 1990a).

We are currently taking two approaches to isolate and characterize the gene encoding the erucoyl-specific LPAT from meadowfoam. The first is a biochemical approach involving the isolation and purification of LPATs from various developing oilseeds followed by the use of either an antibody or oligonucleotide probe (designed after microsequencing) to screen a cDNA library from developing Limnanthes douglasii seed. The first step in purifying the enzyme was to find a subcellular fraction enriched in the protein of interest. The erucoyl-CoA specific LPAT from meadowfoam, while probably extra-plastidic, was found to be enriched in a 10,000xg pellet fraction, which contained 95% of the total activity originally measured in a homogenate. While the specific activity of this fraction was somewhat lower than that found in a 100,000xg fraction, the overall recovery of total activity in the 10,000xg pellet made it the obvious choice as an enriched fraction for beginning enzyme purification. Thus far, the general protocol shown in Fig. 5 has been used to purify LPATs from developing embryos of meadowfoam and safflower and has yielded enzyme preparations which are purified nearly 1000-fold compared to the crude homogenates. The key factor in the protocol has been the "selective solubilization" of the 10,000xg pellet activity into a 10,000xg supernatant fraction using a combination of detergent treatment and physical dispersion methods. This step alone has yielded an apparent purification, based on specific activity, of >300-fold and has enabled further purification by gel filtration and ion exchange chromotographies. We are now approaching homogeneity with our LPAT preparations.

The second, more recent approach, is a molecular one, in which we hope to functionally complement an LPAT-deficient mutant of E. coli (Coleman 1990) with an oilseed gene encoding LPAT. This might be accomplished by direct transformation of competent cells of the mutant microbial host with a plasmid cDNA library, or by infection of the microbial mutant with specialized plant cDNA expression libraries (e.g. lambda YES Arabidopsis library, Elledge et al. 1991).

Once we acquire the meadowfoam LPAT gene, our goal is then to transform B. napus to allow the production of trierucin (Fig. 6). Two recent pieces of biochemical evidence from our laboratory are encouraging in this regard: (1) The meadowfoam LPAT will recognize 22:1-lyso-phosphatidic acid (22:1-LPA) and can insert erucic acid into the sn-2 position (Table 6). As we have shown, 22:1-LPA is an intermediate in the bioassembly of TAGs containing VLCFAs in B. napus (Fig. 3). (2) An in vitro experiment designed to simulate transformation of B. napus with the meadowfoam LPAT was conducted, in which homogenates or microsomal fractions from B. napus MD embryos were supplied with 14C erucoyl-CoA and the non-indigenous 1,2-dierucin. Under these conditions, 14C-labeled trierucin was produced by the B. napus system, proving that the 1,2-diacylglycerol acyltransferase (DGAT) was capable of utilizing diacylglycerol with 22:1 at both the sn-1 and sn-2 positions (Taylor et al. 1992b). Furthermore, the DGAT in MD embryos of Brassica napus exhibits a greater specificity for erucoyl-CoA over oleoyl-CoA at concentrations above 5 µM in vitro (Weselake et al. 1991).

Elongase

If the supply of erucic acid for TAG bioassembly is limiting, the targeted transformation of B. napus with meadowfoam LPAT as depicted in Fig. 6, may primarily result in a redistribution of existing erucoyl moieties, rather than an accumulation of sufficient trierucin. Thus, in order to be able to manipulate VLCFA levels in transgenic B. napus, our second target is the gene encoding the elongase responsible for VLCFA biosynthesis from oleoyl-CoA.

The approach to obtaining this gene is a molecular-genetic one. It involves the isolation of mutants in a small crucifer Arabidopsis thaliana deficient in VLCFA biosynthesis, characterization of the mutants to determine whether the elongase gene is marked by a mutation, and if so, cloning this gene using the technique of chromosome walking (Kunst and Underhill 1990). This technique takes advantage of the fact that A. thaliana has the smallest known higher plant genome (ca. 100,000 Kb) and possesses very little repetitive DNA (Meyerowitz 1989).

An EMS-mutagenized population of A. thaliana seed has been screened by GC and six mutant lines have been isolated with stably-inherited changes in the VLCFA content of seed lipids. Of these, four mutants contained less than 1% 20:1 (wild-type seed contains 18% 20:1), a reduced level of 20:0 and no detectable 22:1 (Kunst and Underhill 1990). Genetic analyses have shown that all the described changes in fatty acid composition of these four mutants are caused by a mutation at the same nuclear locus, FAE1 (Lemieux et al. 1990). In addition, reciprocal crosses between one of these VLCFA mutants, designated AC56 (Kunst et al. 1992), and wild type have shown that F1 progeny have VLCFA levels which are intermediate between the wild-type and mutant (Table 7). This incomplete dominance suggests that the amount of available gene product, i.e. "elongase," limits elongation, i.e. VLCFA biosynthesis. In vitro biochemical characterization of AC56, has revealed that, relative to wild-type, seeds of the mutant are deficient in the capacity to biosynthesize 14C labeled 20:1 from 14C 18:1-CoA, 14C 22:1 from 14C 20:1-CoA, and 14C 20:0 from 14C 18:0-CoA, in the presence of malonyl-CoA and reducing equivalents (Table 8) (Kunst et al. 1992).

Since the FAE1 gene product is involved in the synthesis of all the VLCFAs in A. thaliana, we have chosen the FAE1 gene as the target for isolation by chromosome walking. In preparing to chromosome walk to the FAE1 locus, its postion has been mapped to chromosome 4 in the region encompassed by the cer2 and ap2 morphological markers (Kunst and Underhill 1990). The next step, high resolution mapping relative to appropriate RFLP markers, is currently underway. The RFLP closest to the elongase gene will serve as the starting site for the chromosome walk.

Once the elongase gene from Arabidopsis has been isolated, we intend to use it as a probe to isolate the corresponding gene in B. napus. Overexpression of the elongase function responsible for VLCFA biosynthesis may be required to produce sufficient trierucin in a transgenic B. napus housing the meadowfoam LPAT.

CONCLUDING REMARKS

Using the Reston MD embryo model system, we have established a biochemical baseline for the mechanism by which TAGs containing VLCFAs are made in high erucic acid B. napus. Modification of rapeseed through biotechnology would offer the opportunity to produce ultra-high erucic acid seed oils which can provide renewable, biodegradable industrial feedstocks and allow agricultural diversification for the Canadian farmer. A number of strategies combining techniques in biochemistry, genetics, and molecular biology are being exploited to achieve this goal in a program focused on the isolation and transgenic expression of genes encoding two target enzymes: meadowfoam LPAT and B. napus elongase. Clearly, the B. napus germplasm of choice for transformation with these target genes will be cultivars already optimized for the maximum erucic acid content (approaching 66 mol %) through plant breeding efforts.

REFERENCES

* National Research Council of Canada 33534. The authors gratefully acknowledge the contributions of Drs. M.K. Pomeroy, N. Weber and R.J. Weselake and the technical assistance of D. Barton, M. Giblin, L. Hogge, J. Magus, D. Olson, and D. Reed.
Table 1. Industrial uses of trierucin, erucic acid, and derivativesz.

Trierucin Pharmaceuticals, lubricants, waxes, heat transfer fluids, dielectric fluids
Erucic acid Erucamide: slip agent, plasticizers
Amines: surfactants, antistats, flotation agents, corrosion inhibitors
Behenic acid Antifriction coatings, mold release agents, flow improvers, mixing and processing aids
Erucyl alcohol Surfactants, slip and coating agents
Behenyl alcohol Surfactants, slip and coating agents
Wax esters Lubricants, cosmetics
Brassylic Acid Nylons, perfumes, plasticizers, polyesters, synthetic lubricants, paints and coatings
Pelargonic Acid Plasticizers, plastics, coatings, flavors, perfumes, cosmetics
zModified from Van Dyne et al. (1990).

Table 2. Proportions of very long chain fatty acids in total lipids from microspore-derived (MD) and zygotic (Z) embryos of B. napus cv Reston at different stages of developmentz.

Wt % fatty acid
Developmental stage Embryo
source		 20:0 20:1 22:0 22:1
Microspores 1.4 0.9 1.0 ---y
Heart MD 1.9 --- 2.0 ---
Z --- --- --- ---
Torpedo MD 1.8 0.7 1.1 ---
Z 1.3 1.0 1.2 1.2
Early cotyledonary MD 1.7 2.6 0.3 1.0
Z 1.3 1.8 0.2 0.6
Mid- cotyledonary MD 0.9 6.9 0.3 5.0
Z 1.5 4.0 0.6 2.3
Late cotyledonary MD 0.8 8.2 0.4 11.1
Z 1.0 11.9 0.2 13.0
Very late cotyledonary MD 0.8 11.8 0.3 21.8
Z 0.7 12.2 0.3 26.4
Seed 0.9 11.2 0.4 32.7
zProportions (wt %) data for other fatty acids not shown.
y---not detected. Modified from Pomeroy et al. (1991).

Table 3. Comparison of in vivo and in vitro rates of TAG biosynthesis in developing zygotic and MD embryos of B. napus cv. Reston.

Rate of triacylglycerol biosynthesis (pmol•min-1•mg protein-1)
in vivoz in vitroy
Embryo system homogenate microsomes
Zygotic 75 6 (8%)x 12 (16%)
Microspore-derived 142 166 (117%) 333 (234%)
zIn vivo rate estimated from measurements of TAG and total protein in developing mid-late cotyledonary stage embryos during the rapid phase of TAG accumulation.
yIn vitro rate measured in homogenates or microsomal preparations from mid-late cotyledonary stage embryos using the reaction system:
G-3-P + 14C 22:1-CoA, and assuming one erucoyl moiety incorporated per TAG (18:1/18:1/14C 22:1) synthesized.
xValues in parentheses express the in vitro rate as a percentage of the estimated in vivo rate. Modified from Taylor et al. (1991).

Table 4. Stereospecific distribution of radioactive acyl moieties in TAGs formed by homogenates of B. napus cv. Reston MD embryosz.

Distribution of radioactivity (%)y
Position on the glycerol backbone
Reaction tested 14C acyl species sn-1 sn-2 sn-3 Total
14C 18:1-CoA + Malonyl-CoA 18:1 31 65 4 100
20:1 17 10 73 100
22:1 12 --- 88 100
14C 20:1-CoA + Malonyl-CoA 20:1 8 9 83 100
22:1 13 --- 87 100
zHomogenates prepared from developing MD embryos of B. napus cv. Reston were incubated with G-3-P and either 14C 18:1-CoA or 14C 20:1-CoA in the presence of malonyl-CoA and reducing equivalents. The radiolabeled TAG fraction was isolated and stereospecific analyses performed as described by Taylor et al. (1992a).
yData is expressed as % distribution of each 14C fatty acyl moiety over all three sn- positions on the glycerol backbone. Modified from Taylor et al. (1992a).

Table 5. Content of erucic acid and its sn-2 distribution in various oilseeds.

Species Common name Total mol % 22:1 in TAGs Mol % 22:1 at sn-2 position Reference
B. napus H.E.A. Rapeseed 40-57 Tr-2 Brockerhoff (1971); Norton and Harris (1983); Taylor et al. (1991)
C. abyss. Abyssinian kale 51 3 Mattson and Volpenhein (1961)
T. majus Nasturtium 70-75 34 Mattson and Volpenhein (1961)
L. dougl. Meadowfoam 14-17 67 Phillips et al. (1971)

Table 6. Comparison of relative LPAT activities in vitro in preparations from developing seed of meadowfoam, nasturtium and rapeseed (B. napus)z.

Relative 22:1-CoA:LPAT activityz Relative 18:1-CoA:LPAT activityx
Seed Homogenate Microsomal fraction Homogenate
Meadowfoam 100 (45)y 100 100
Nasturtium 12 7 ndw
Rapeseed 2 (0)y 1 95
zUnless otherwise indicated, 22:1-CoA:LPAT reactions were assayed in the presence of 15 µM 14C 22:1-CoA + 45 µM 18:1-LPA; 100% activity for homogenate preparation = 30 pmol/min/mg protein; 100% activity for microsomal fraction = 103 pmol/min/mg protein.
yNumbers in brackets indicate activity assayed in the presence of 15 µM 14C 22:1-CoA + 45 µM 22:1-LPA, relative to activity measured in the 15 µM 14C 22:1-CoA + 45 µM 18:1-LPA reaction system.
x18:1-CoA:LPAT activity was assayed in the presence of 15 µM 14C 18:1-CoA + 45 µM 18:1-LPA; 100% activity = 185 pmol/min/mg protein.
wNot determined. Modified from Taylor et al. (1990a).

Table 7. Very long chain fatty acid composition of total lipids of wild type (WT), mutant (AC56), and F1 (WT x AC56) seeds of A. thaliana grown at 22°Cz.

Content (mol %±SD)y
Fatty acid Wild type F1 AC56
20:0 1.8±0.2 1.1±0.2 0.5±0.2
20:1 18.2±1.2 9.7±1.2 0.8±0.1
22:1 1.6±0.2 0.7±0.1 0.0±0.0
zData for other fatty acids not shown. Modify from Kunst et al. (1992).
yn = 5

Table 8. Elongase activities in cell-free extracts of developing seed from wild type (WT) and mutant (AC56) A. thalianaz.

Reaction tested seed line Activity (nmol 14C
acyl product
h-1mg protein-1)		 Relative activity
(% of WT)
14C 18:1-CoA elongation
WT 12.9 100
AC56 0.2 1.6
14C 20:1-CoA elongation
WT 2.0 100
AC56 0 0
14C 18:0-CoA elongation
WT 6.8 100
AC56 0.5 7.4
zAliquots of filtered seed homogenate (50 to 200 ug protein) were incubated at 30°C for 1 hour. Values are the means of 2 to 3 replicates in 2 to 4 independent experiments. 14C 20:1, 14C 22:1 and 14C 20:0 were the products of 14C 18:1-CoA, 14C 20:1-CoA and 14C 18:0-CoA elongation reactions, respectively, conducted in the presence of malonyl-CoA and reductant.


Fig. 1. Structure of a triacylglycerol typical of that found in the Brassicaceae (e.g. B. napus), indicating the stereo-chemically-distinct sn-1, sn-2 and sn-3 positions. Erucic acid (22:1) can be esterified at both the sn-1 and -3 positions, but is virtually excluded from the sn-2 position.

Fig. 2. Scheme for triacylglycerol bioassembly (Kennedy) pathway in developing oilseeds. 18:1-CoA, oleoyl-coenzyme A; 18:2, linoleic acid; G-3-P, glycerol-3-phosphate; G-3-P AT, glycerol-3-phosphate acyltrans-ferase; LPA, lyso-phosphatidic acid; LPAT, lyso-phosphatidic acid acyltransferase; PA, phosphatidic acid; PA phosphatase, phosphatidic acid phosphatase; PC, phosphatidylcholine; DAG, diacylglycerol; DGAT, diacyl-glycerol acyltransferase; TAG, triacylglycerol; CPT, sn-1,2-diacylglycerol cholinephosphotransferase. After desaturation on the PC backbone, polyunsaturated C18 fatty acids can enter the acyl-CoA pool via the enzyme acyl-CoA:lyso-phosphatidylcholine acyltransferase (not shown). (Modified from Stymne and Stobart 1987).

Fig. 3. Incorporation of newly-synthesized very long chain acyl-CoAs into glycerolipids via the Kennedy pathway in B. napus cv Reston MD embryos. A 15,000xg particulate fraction prepared from mid-cotlyedonary embryos was incubated with 40 µM 14C 18:1-CoA and 1 mM malonyl-CoA in the presence of 200 µM G-3-P, 0.5 mM NADH and 0.5 mM NADPH. 2 hour incubations were also conducted under identical conditions except that G-3-P was ommitted from the reaction mixtures (2 -G3P). At each time point, 14C-labeled acyl-CoAs and 14C glycerolipid species containing newly-synthesized VLCFA moieties were isolated and analyzed as described by Taylor et al. (1992a). 14C 18:1-CoA incorporation data not shown. Modified from Taylor et al. (1992a).


Fig. 4. Direct-probe electron impact mass spectrometry of triacylglycerol (TAG) fractions isolated from developing seed of (A) high erucic acid B. napus, cv Reston at 6 weeks post-anthesis; (B) Tropaeolum majus (nasturtium), cv dwarf cherry rose at 4 weeks post-anthesis. Mass spectrometry was performed as described by Taylor et al. (1991). Only the molecular ion (M+) region is shown. Acyl group assignments were confirmed by MS/MS daughter ion analyses (not shown). The major TAG species are designated as EOE, EiOE etc. for TAGs containing E=erucoyl, Ei=eicosenoyl, and O=oleoyl moieties. Each molecular ion cluster represents TAGs containing combinations of VLCFAs with 18:1, 18:2 or 18:3 on the glycerol backbone. B. napus (A) contains molecular ions for monoeicosenoyl (M+=913), monoerucoyl, (M+=941), monoeicosenoyl, monoerucoyl (M+=969), dierucoyl (M+=997) and dierucoyl, monoeicosenoyl (M+=1025) TAG species. Trierucin (M+=1053) is not detected. In contrast, trierucin (EEE, M+=1053), is the major TAG species detected in T. majus (B). All major TAG species display characteristic [M-18]+ fragmentation.

Fig. 5. General protocol for the purification of lyso-phosphatidic acid acyltransferase (LPAT) from developing oilseeds.

Fig. 6. Scheme for transformation of B. napus (breeding limit 66 mol % erucic acid) with a gene for meadowfoam LPAT expressing a high degree of erucoyl specificity, to produce a new ultra-high erucic acid cultivar capable of making trierucin. From National Research Council of Canada Plant Biotechnology Institute Annual Report, 1989-1990.
Last update September 9, 1997 aw