Plant Physiol. (1999) 120: 1057-1062
Supply of Fatty Acid Is One Limiting Factor in the Accumulation
of Triacylglycerol in Developing Embryos1
Xiaoming Bao and
John Ohlrogge*
Department of Botany and Plant Pathology, Michigan State
University, East Lansing, Michigan 48824
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ABSTRACT |
The metabolic factors that determine
oil yield in seeds are still not well understood. To begin to examine
the limits on triacylglycerol (TAG) production, developing
Cuphea lanceolata, Ulmus carpinifolia, and Ulmus parvifolia embryos were incubated with factors
whose availability might limit oil accumulation. The addition of
glycerol or sucrose did not significantly influence the rate of TAG
synthesis. However, the rate of 14C-TAG synthesis upon
addition of 2.1 mM 14C-decanoic acid
(10:0) was approximately four times higher than the in vivo rate
of TAG accumulation in C. lanceolata and two times
higher than the in vivo rate in U. carpinifolia and
U. parvifolia. In C. lanceolata embryos,
the highest rate of 14C-TAG synthesis (14.3 nmol
h
1 embryo1) was achieved with the addition
of 3.6 mM decanoic acid. 14C-Decanoic acid was
incorporated equally well in all three acyl positions of TAG. The
results suggest that C. lanceolata, U. carpinifolia, and U. parvifolia embryos have
sufficient acyltransferase activities and glycerol-3-phosphate levels
to support rates of TAG synthesis in excess of those found in vivo.
Consequently, the amount of TAG synthesized in these oilseeds may be in
part determined by the amount of fatty acid produced in plastids.
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INTRODUCTION |
In plants the biosynthesis of storage TAG occurs at high levels
primarily in the seeds, but there is a wide range in the levels of TAG
that accumulate in different plant species. For example, seeds of
species of Zea, Hordeum, or Pisum usually contain
less than 5% to 10% TAG by dry weight, whereas many other species
such as castor accumulate over 50% TAG in seeds. The regulatory or metabolic factors that influence this very wide range of oil
accumulation in seeds are currently unknown (Ohlrogge and Jaworski,
1997
). Considering that over 30 reactions are required to convert
acetyl-CoA to TAG, there could be many steps or genes that control the
yield of end product TAG. To begin to dissect possible limiting
factor(s) in the pathway of TAG biosynthesis, it is useful to
conceptually divide the pathway into two parts: (a) the production of
acyl chains, which occurs in plastids, and (b) the utilization of acyl chains for glycerolipid synthesis in the ER and oilbody. In the second
part, the only unique enzyme for TAG synthesis is DAGAT, which is
responsible for the acylation of 1,2-diacylglycerol at the
sn-3 position (Roughan and Slack, 1982; Stymne and Stobart, 1987). Since DAGAT is located at the branch point that channels diacylglycerol to TAG synthesis, some reports have suggested that DAGAT
may be one rate-limiting enzyme for the accumulation of TAG (Griffiths
et al., 1988; Ichihara et al., 1988; Griffiths and Harwood, 1991; Perry
and Harwood, 1993a, 1993b).
In this study we asked whether the fatty acid supply can influence the
amount of TAG produced in oilseeds. If the supply of fatty acid is a
limiting factor for TAG biosynthesis, then providing exogenous fatty
acid to the developing embryos should increase the rate of TAG
production. Unfortunately, long-chain fatty acids have very low
solubility in aqueous solution, and so addition of long-chain fatty
acids at concentrations sufficient to increase rates of lipid synthesis
is very difficult. However, embryos of Cuphea lanceolata, Ulmus
carpinifolia, and Ulmus parvifolia contain high levels
of decanoic acid in their TAG (80%, 63%, and 71%, respectively),
and, since decanoic acid is easily dissolved in water at millimolar
concentrations, these species provided a convenient model system with
which to test the influence of fatty acid supply on TAG accumulation.
Such in vitro model experiments may provide a useful guide toward the
selection of targets for future metabolic engineering in transgenic
plants.
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MATERIALS AND METHODS |
Plant Materials and Chemicals
Cuphea lanceolata plants were grown in Beal garden on
the Michigan State University campus (East Lansing). C. lanceolata plants typically begin to flower in mid-July and our
experiments were performed in August. Flowers were hand-pollinated and
seeds were harvested at various stages of development. After removal of
the seed coat, the resulting embryos were used immediately for labeling experiments or were stored at 
20°C for later lipid analysis. Embryos were also collected from two species of elm trees (Ulmus carpinifolia and Ulmus parvifolia) growing on the
Michigan State University campus. Elm trees begin flowering in early
May and we collected embryos from the time they were big enough to
dissect until they were mature. The embryos were removed from seed coat and used for labeling experiments immediately or stored at 20°C for
subsequent lipid analysis.
[1-14C]Octanoic acid (55 mCi/mmol),
[1-14C]decanoic acid (55 mCi/mmol), and
[1-14C]oleic acid (55 mCi/mmol) were purchased
from American Radiolabeled Chemicals (St. Louis). Tritridecanoin
(C13:0), L-dipentadecanoyl (C15:0)
-phosphatidylcholine, and lipase (from Rhizopus arrhizus) were obtained from Sigma.
Lipid Analysis
Lipids were extracted from 20 embryos at each developmental stage
according to the method of Bligh and Dyer (1959). Prior to extraction,
tritridecanoin (C13:0) and L-dipentadecanoyl (C15:0) -phosphatidylcholine were added to each sample as internal
standards for GC analysis. TAG was separated from polar lipids by TLC
(20- × 20-cm K6 silica 60-Å plates, Whatman) in hexane:diethyl
ether:acetic acid (70:30:1, v/v). TAG bands were eluted from the silica
gel with chloroform:methanol (1:2, v/v). Fatty acid methyl esters from
TAG were prepared by heating lipids at 90°C for 45 min in 0.3 mL of
toluene and 1 mL of 10% (v/v) boron trichloride/methanol (Sigma). The
resulting fatty acid methyl esters were separated and quantified by GC
analysis.
Feeding Developing Embryos with Exogenous Fatty Acid
Ten pairs of C. lanceolata cotyledons (10 DAF)
were cut in half and incubated at 28°C with gentle shaking in 200 mL
of 0.1 M phosphate (pH 7.2) containing 2.1 mM
[1-14C]decanoic acid in the presence or the absence of
0.125 mM glycerol. The incubation buffer was changed
once after 1 h of incubation. Assays were terminated by removing
the incubation buffer, washing the embryos twice with water, and
initiating lipid extraction. TAG was separated by TLC using a solvent
system composed of hexane:diethyl ether:acetic acid (70:30:1, v/v).
Labeled TAG was quantified using both an imager (Instant Imager,
Packard Instruments) and liquid scintillation counting. In some
experiments, other factors that may influence TAG synthesis, such as
exogenous fatty acid concentration (2.1-80.1
mM), glycerol (with/without 0.125 mM), Suc (0-200 mM), and
pH (6-8), were tested. Only one factor was changed in each treatment.
The position of exogenous fatty acid incorporated in TAG was determined
using TAG lipase from R. arrhizus, which cleaves fatty acids
from the sn-1 and sn-3
positions of TAG, and then the radioactivity remaining in the
sn-2-monoacylglycerol was compared with free fatty acids released from the sn-1 and
sn-3 of TAG by the action of TAG lipase. Purified
TAG was dissolved in 0.5 mL of diethyl ether in 13-mL screw-cap glass
tubes. One milliliter of 0.1 M Tris-HCl (pH 7.8)
buffer containing 5 mM
CaCl2 was added to the tube, then 43,000 units of
lipase was added to the bottom of the tube, bubbled in
N2, and shaken for 10 min at room temperature. Monoacylglycerol and free fatty acid products were extracted and resolved by TLC in hexane:diethyl ether:acetic acid (35:70:1.5, v/v).
The radioactivity in the monoacylglycerol and free fatty acid bands on
the TLC plate were quantified using an imager (Packard Instruments).
In similar experiments, 10 pairs of cotyledons from U. carpinifolia and U. parvifolia embryos at the middle
stage of development were also used for feeding experiments as
described above, except incubation buffer was changed every half hour.
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RESULTS |
Fatty Acid Deposition, Composition, and in Vivo Rate of TAG
Accumulation during Embryo Development
Mature C. lanceolata seeds accumulate TAG, in which
decanoic acid is the predominant fatty acid, reaching a level of 80 mol % (Bafor et al., 1990). Embryos were large enough to isolate at 6 DAF,
and the seeds reached maturity about 20 DAF. As shown in Figure
1, TAG deposition in C. lanceolata embryos was linear from 8 to 12 DAF, and during this
period TAG accumulated at the rate of 2.9 nmol
h1 embryo1. This result
was close to the 2.3 nmol h1
embryo1 measured by Bafor et al. (1990). During
this same period, the relative amount of decanoic acid in TAG increased
from 40 to 75 mol %. The fatty acid composition of TAG in C. lanceolata embryos at the middle stage of TAG accumulation (11 DAF) was similar to the results obtained by Bafor et al. (1990).
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| Figure 1.
TAG accumulation and percentage of decanoic acid
in TAG in developing embryos of C. lanceolata, U. carpinifolia, and U. parvifolia. Twenty embryos
at each stage were analyzed for TAG content. The left scale represents
the TAG content ( ), and the right scale represents the percent of
decanoic acid in TAG ( ).
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In U. carpinifolia and U. parvifolia, it is
difficult to tag flowers on a daily basis because the elm trees are
very tall. Therefore, embryos were collected only when they reached a
stage when lipid analysis was feasible. The first collection was
designated as the zero time point, and later time points were recorded
as days after the first collection. The rate of TAG accumulation of
U. carpinifolia and U. parvifolia embryos was
linear from 7 to 10 d after first collection, and the relative
amount of decanoic acid in TAG increased from 40% to 65% (Fig. 1).
The fatty acid composition of TAG in both elm species at 10 d
after first collection is given in Table
I. At this stage the medium-chain fatty
acids constituted 77% and 85% of the TAG fatty acids in U. carpinifolia and U. parvifolia, respectively.
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Table I.
Fatty acid composition of TAG from 20 U. carpinifolia (2,750 nmol TAG embryo1) and U. parvifolia (3,170 nmol TAG embryo1) embryos at
10 d after first collection
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Rates of TAG Synthesis by Developing Embryos with Addition of
Exogenous Fatty Acid
Bafor et al. (1990) observed that when developing C. lanceolata embryos were incubated with exogenous decanoic acid and
glycerol, the rate of exogenous decanoic acid incorporated into TAG was 33.9 nmol h1 embryo1.
Assuming that decanoic acid could be esterified to all three positions
of glycerol, the rate of 14C-TAG synthesis was at
least 11.3 nmol h1
embryo1, which is four times higher than the in
vivo rate of TAG accumulation (2.9 nmol h1
embryo1). To further examine and extend these
results, we tested whether the addition of exogenous fatty acid alone
could increase the rate of 14C-TAG synthesis in
C. lanceolata developing embryos, and whether these results
could be extended to other species.
Ten pairs of cotyledons of C. lanceolata, U. carpinifolia, and U. parvifolia, harvested when TAG
accumulation was in the linear range, were incubated in buffer
containing 2.1 mM
[1-14C]decanoic acid with or without glycerol.
As shown in Table II, in the absence of
glycerol the average rates of 14C-TAG synthesis
were 12.5, 20.4, and 12.9 nmol h1
embryo1 for C. lanceolata, U. carpinifolia, and U. parvifolia, respectively. Compared
with their respective in vivo rates of TAG accumulation, 2.9 (C. lanceolata), 9.1 (U. carpinifolia), and 7.7 (U. parvifolia) nmol h1
embryo1, the rates of
14C-TAG synthesis were approximately four times
higher for C. lanceolata and two times higher for U. carpinifolia and U. parvifolia embryos upon addition of
exogenous decanoic acid. The addition of 0.125 mM
glycerol to the incubation solution did not strongly influence the
rates of 14C-TAG synthesis (Table II), suggesting
that there is an adequate endogenous supply of glycerol for higher TAG
synthesis in developing C. lanceolata, U. carpinifolia, and U. parvifolia embryos. The level of
14C-decanoic acid incorporated into TAG was used
to calculate the rate of 14C-TAG synthesis in
each species (Table II). This calculation did not include the
contribution derived from the endogenous de novo TAG synthesis from
nonradioactive precursors, so the values given in Table II represent an
underestimation of the total TAG synthesis from both exogenous and
endogenous fatty acids.
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Table II.
Rate of 14C-TAG synthesis upon addition
of 2.1 mM 14C-decanoic acid
Ten pairs of cotyledons at mid-stage of development were incubated with
2.1 mM decanoic acid in 0.2 mL of 0.1 M
phosphate buffer (pH 7.2) at 28°C for 2 h in the absence (G)
or presence (+G) of 0.125 mM glycerol. The incubation
buffer was changed once at 1 h for Cuphea, and every half hour for
Elm. Rates of 14C-TAG synthesis are calculated as nanomoles
of 14C-decanoic acid found in TAG (per hour) divided by
three. Data represent the average of three independent experiments.
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The TAG derived from C. lanceolata embryos incubated
with [1-14C]decanoic acid for 1 h was
treated with TAG lipase from R. arrhizus. The resulting
sn-2-monoacylglycerol and nonesterified fatty
acid (derived from sn-1 and
sn-3 positions of TAG) were separated by TLC.
Radioactivity was detected in both
sn-2-monoacylglycerol and free fatty acid
fractions, indicating that labeled decanoic acid was esterified to the
sn-2 as well as to the sn-1
and sn-3 positions of the glycerol backbone. The
ratio of radioactivity from sn-2-monoacylglycerol
to that of the free fatty acid was 1:2. This suggests that exogenous
decanoic acid was equally distributed among the three positions of TAG.
We recently observed that lauric acid produced in transgenic
Brassica napus can be subject to 
-oxidation (Eccleston
and Ohlrogge, 1998). If this had occurred in the embryos supplemented
with decanoic acid, a loss of lipid-soluble 14C
would have occurred and 14C would have been
detected not only in decanoic acid, but also in the other fatty acids
isolated after the incubations. Neither event was observed and,
furthermore, recoveries of added decanoic acid were at least 75% to
85%. Therefore, -oxidation was not a major fate of the added
decanoic acid.
Factors That Influence the Incorporation of Exogenous Fatty Acid
into TAG
As shown in Table II, the addition of glycerol to the incubation
buffer had no significant effect on the rate of
14C-TAG synthesis. We also examined if other
factors such as exogenous fatty acid concentrations, Suc (0-200
mM), and pH may influence the rate of
14C-TAG synthesis by developing C. lanceolata embryos. Decanoic acid concentrations were varied over
the range from 2.1 to 80 mM. As shown in Figure
2A, the highest rate of
14C-TAG synthesis (14.3 nmol
h1 embryo1) was
obtained with the addition of 3.6 mM decanoic
acid. Concentrations higher than 3.6 mM decanoic
acid apparently had a deleterious effect on embryos and resulted in
lower rates of TAG deposition.
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| Figure 2.
Rate of 14C-TAG synthesis under
different conditions. A, Ten pairs of cotyledons incubated in 0.2 mL of
phosphate buffer (pH 7.2) with 14C-decanoic acid at the
concentrations indicated. B, Ten pairs of cotyledons incubated in 2.1 mM 14C-decanoic acid plus 0.2 mL of phosphate
buffer ranging in pH from 6 to 8.
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Developing C. lanceolata embryos are nonphotosynthetic
tissue, so the ultimate carbon source for TAG synthesis is derived from
Suc. To determine if Suc concentrations may limit TAG formation in
these experiments, different concentrations of Suc (0-200
mM) were added to the basic incubation solution.
Despite minor variations, the rates of 14C-TAG
synthesis in all samples were close to 12 nmol
h1 embryo1, suggesting
that carbon supply in the form of Suc does not limit TAG accumulation
in these short-term experiments.
Developing C. lanceolata embryos were also incubated under a
range of pH from 6.0 to 8.0. As shown in Figure 2B, the optimal pH for
TAG deposition was 7.2, whereas at pH 6.5 or 7.5, the rate of
14C-TAG synthesis decreased to one-half of that
at pH 7.2.
Utilization of Other Exogenous Fatty Acids by C. lanceolata Cotyledons for TAG Synthesis
Each of the 10 pairs of cotyledons of developing C. lanceolata embryos were separately supplied with 2.1 mM [1-14C]octanoic acid,
[1-14C]decanoic acid, or
[1-14C] oleic acid in 200 mL of 0.1 M phosphate (pH 7.2). Triton X-100 (2.0 mM) was used to increase the solubility of the
oleic acid. After 1 h of incubation, 5.8 nmol of octanoic acid,
37.5 nmol of decanoic acid, and a trace amount of oleic acid were
incorporated into TAG in each embryo, respectively. One simple
interpretation of this result is that one or more of the
acyltransferases of C. lanceolata displays a strong
selectivity in favor of decanoic acid (Bafor and Stymne, 1992; Vogel
and Browse, 1996). However, B. napus embryos were also
incubated with oleic acid under the same conditions and the
incorporation of oleic acid into TAG was significantly lower than the
in vivo rate (data not shown). Therefore, for oleic acid, low aqueous
solubility or transport into the tissue may also prevent its rapid
incorporation into TAG by embryos.
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DISCUSSION |
TAG normally accumulates to a high level only in seeds, but a
metabolic understanding of the tissue specificity of oil accumulation is not yet available. One potential explanation is that DAGAT, which
catalyzes the acylation of position 3 of
1,2-diacyl-sn-glycerol, is specifically expressed in seed.
However, there are several observations that argue against this view.
For example, DAGAT activity was found in spinach leaves (Martin and
Wilson, 1983) and was primarily associated with chloroplast envelopes
(Martin and Wilson, 1984). Roughan et al. (1987) reported that
significant amounts of TAG were synthesized when palmitic acid was
applied to the upper surface of expanding spinach leaves. The level of neutral lipids (mainly TAG) increased at least 3-fold during protoplast isolation from Arabidopsis leaves (Browse et al., 1988). Finally, ozone-fumigated spinach leaves produced high proportions of TAG (Sakaki
et al., 1990). These data together suggest that DAGAT not only occurs
in leaves, but also that leaves have the ability to synthesize TAG.
Although expressed in several tissues, higher expression of DAGAT
during seed development might provide one explanation for TAG
accumulation in oilseeds. Ichihara et al. (1988) measured the specific
activity of DAGAT from safflower in vitro and found that it was lower
than expected. They concluded that the DAGAT reaction may be
rate-limiting. When developing safflower and sunflower cotyledons were
incubated with exogenous radiolabeled fatty acid tracers, substantial
amounts of labeled fatty acids were esterified to DAG (Griffiths et
al., 1988). Since DAG is the direct substrate of DAGAT, it was
suggested that DAGAT could be a rate-limiting step. Perry and
Harwood (1993a, 1993b) found that, when developing seeds of B. napus were incubated with [1-14C]acetate
and [2-3H]glycerol, very low accumulation of
the Kennedy pathway intermediates occurred apart from DAG. These
results were also interpreted as indicating that DAGAT is likely to
exert significant flux control over TAG accumulation.
A similar conclusion was drawn by Griffiths and Harwood (1991) from
studies of TAG synthesis in cocoa. However, the accumulation of DAG
might also be explained as a shortage of acyl chain supply rather than
flux control at DAGAT. Because both DAG and acyl-CoA are direct
substrates of DAGAT, lack of one substrate (acyl-CoA) can lead to the
accumulation of the other (DAG) if the DAGAT
Km for acyl-CoA is higher than the
other acyltransferases. Thus, the accumulation of DAG does not
necessarily imply that DAGAT exerts flux control for TAG synthesis.
To begin to examine the limiting step(s) in TAG production for this
study we considered the pathway of TAG biosynthesis in two parts. The
first half can be characterized as fatty acid production inside
plastids; the second half can be considered as the assembly of TAG in
the ER or oilbodies (Cao and Huang, 1986; Settlage et al., 1995). If
the supply of fatty acid is a limiting factor for TAG synthesis, then
the addition of excess exogenous fatty acid should increase the rate of
TAG synthesis. As shown in Figure 1, TAG accumulated at the rate of
2.9, 9.07, and 7.65 nmol h1
embryo1 in vivo for developing embryos of
C. lanceolata, U. carpinifolia, and U. parvifolia, respectively. With addition of exogenous decanoic acid, their rate of 14C-TAG synthesis was 2- to
4-fold higher than the in vivo accumulation rate. This result clearly
indicates that the supply of fatty acid can be one limiting factor for
TAG accumulation.
In agreement with these observations in seeds, the addition of
exogenous phosphatidylcholine liposomes to Chlamydomonas
reinhardtii cultures caused 10-fold increases in TAG accumulation
(Grenier et al., 1991). Thus, it appears that the capacity of these
systems for TAG accumulation is greater than actually used and that
fatty acid supply, rather than the utilization enzymes may limit TAG accumulation in C. lanceolata, U. carpinifolia,
U. parvifolia, and C. reinhardtii. In addition,
we found that Suc and glycerol had no significant influence on the rate
of 14C-TAG synthesis in C. lanceolata.
This implies that both the endogenous carbon source and the glycerol
backbone are in excess and do not limit 14C-TAG
synthesis during these incubations. However, it is important to
emphasize that such short-term incubations may not reflect factors that
control overall long-term accumulation of storage oils. For example,
over the time scale of seed development, many other factors such as the
ability of oilbodies to accommodate increased TAG might become
limiting.
In the present study exogenous decanoic acid was almost equally
distributed among the three positions of TAG from C. lanceolata. This result implied that not only DAGAT, but also
glycerol-3-P acyltransferse and lysophosphatidic acid acyltransferase
could incorporate exogenous decanoic acid at rates several times above their in vivo activity with endogenous substrates. Although our studies
support the concept that increased fatty acid supply can increase TAG
accumulation, they do not rule out that other factors or enzyme
expression levels may have a similar effect. Flux through a metabolic
pathway can often be driven by either stronger source inputs and/or by
stronger sinks pulling on the pathway. The observations of Zou et al.
(1997) that expression of a yeast acyltransferase in B. napus can increase oil yields may represent an example of sink-driven increases in oil accumulation. Furthermore, we recently found that transgenic B. napus seeds that express very high
levels of medium-chain acyl-ACP thioesterase and produce high levels of
lauric acid induce the beta-oxidation pathway to degrade some of the
lauric acid (Eccleston and Ohlrogge, 1998). Because oil yields are not
reduced in these seeds, fatty acid synthesis apparently increased to
provide a constant oil yield.
In oilseeds, fatty acids esterified to TAG can be generally divided
into two groups. One (18:3, 22:1, and 18:1-HO) needs post-plastidial modification, while the other (10:0, 12:0, and 18:1) does not. Our
results clearly show that the supply of fatty acids is one limiting
factor for the rate of 14C-TAG synthesis in
C. lanceolata, U. carpinifolia, and U. parvifolia, which might be generalized representatives of the
second category. However, for the synthesis of TAG containing high
level of post-plastidially modified fatty acids, the involvement of
phospholipids, desaturases, elongases, hydrolases, etc., may be
additional factors that limit TAG accumulation. In addition, the
observation that increased expression of acetyl-CoA carboxylase
resulted in increased oil content of high-erucic rapeseed (Roesler et
al., 1997) suggests that in rapeseed, in which fatty acids are modified
by elongation, increased fatty acid supply can also increase TAG
accumulation.
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FOOTNOTES |
1
This work was supported by the Michigan
Agricultural Experiment Station and by a grant from the Department of
Energy (no. DE-FG02-87ER12729).
*
Corresponding author; e-mail ohlrogge{at}pilot.msu.edu; fax
517-353-1926.
Received January 26, 1999;
accepted April 20, 1999.
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ABBREVIATIONS |
Abbreviations:
DAF, days after flowering.
DAGAT, diacylglycerol
acyltransferase.
TAG, triacylglycerol.
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ACKNOWLEDGMENTS |
We thank Sten Stymne for helpful discussions and for his
original studies on C. lanceolata, which stimulated this
project. Mike Pollard and Jim Todd provided critical reading of the
manuscript. Ellen Crittendon of Beal Gardens provided help in
identifying and accessing C. lanceolata, U. carpinifolia, and U. parvifolia populations on the
campus of Michigan State University.
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Abstract
Introduction