Table of Contents
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
- BIOASSEMBLY OF TAGs CONTAINING VLCFAs IN BRASSICA NAPUS L.: THE MICROSPORE-DERIVED EMBRYO MODEL SYSTEM
- ENZYME TARGETS FOR MANIPULATION OF VLCFA LEVELS IN B. NAPUS
- Lyso-Phosphatidic Acid Acyltransferase (LPAT)
- CONCLUDING REMARKS
- Table 1
- Table 2
- Table 3
- Table 4
- Table 5
- Table 6
- Table 7
- Table 8
- Fig. 1
- Fig. 2
- Fig. 3
- Fig. 4
- Fig. 5
- 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.
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.
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
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).
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.
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
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* 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
zModified from Van Dyne et al. (1990).
|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|
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.
zProportions (wt %) data for other fatty acids not shown.
||Wt % fatty acid|
|Developmental stage ||Embryo |
|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|
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.
zIn vivo rate estimated from measurements of TAG and total protein
in developing mid-late cotyledonary stage embryos during the rapid phase of TAG
||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%)|
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
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).
||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|
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.
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.
||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|
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
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
zData for other fatty acids not shown. Modify from Kunst et al.
||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|
yn = 5
Table 8. Elongase activities in cell-free extracts of developing seed from wild type (WT) and mutant (AC56) A. thalianaz.
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.
|Reaction tested seed line ||Activity (nmol 14C |
(% 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|
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