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Plant Physiol. (1998) 117: 979-987
(+)-Abscisic Acid Metabolism, 3-Ketoacyl-Coenzyme A Synthase Gene
Expression, and Very-Long-Chain Monounsaturated Fatty Acid Biosynthesis
in
Brassica napus Embryos1
Qungang Qi,
Patricia A. Rose,
Garth D. Abrams,
David C. Taylor,
Suzanne R. Abrams, and
Adrian J. Cutler*
Plant Biotechnology Institute, National Research Council of Canada,
110 Gymnasium Place, Saskatoon, Saskatchewan, Canada S7N 0W9
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ABSTRACT |
Microspore-derived embryos of
Brassica napus cv Reston were used to examine the
effects of exogenous (+)-abscisic acid (ABA) and related compounds on
the accumulation of very-long-chain monounsaturated fatty acids
(VLCMFAs), VLCMFA elongase complex activity, and induction of the
3-ketoacyl-coenzyme A synthase (KCS) gene encoding the condensing
enzyme of the VLCMFA elongation system. Of the concentrations tested,
(+)-ABA at 10 µM showed the strongest effect. Maximum activity of the elongase complex, observed 6 h after 10 µM (+)-ABA treatment, was 60% higher than that of the
untreated embryos at 24 h. The transcript of the KCS gene was
induced by 10 µM (+)-ABA within 1 h and further
increased up to 6 h. The VLCMFAs eicosenoic acid (20:1) and
erucoic acid (22:1) increased by 1.5- to 2-fold in embryos treated with
(+)-ABA for 72 h. Also, (+)-8 -methylene ABA, which is metabolized
more slowly than ABA, had a stronger ABA-like effect on the KCS gene
transcription, elongase complex activity (28% higher), and level of
VLCMFAs (25-30% higher) than ABA. After 24 h approximately 60%
of the added (+)-[3H]ABA (10 µM) was
metabolized, yielding labeled phaseic and dihydrophaseic acid. This
study demonstrates that (+)-ABA promotes VLCMFA biosynthesis via
increased expression of the KCS gene and that reducing ABA catabolism
would increase VLCMFAs in microspore-derived embryos.
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INTRODUCTION |
The phytohormone (+)-ABA (Fig. 1,
structure 1) plays regulatory roles in many physiological processes,
such as control of stomatal aperture, seed dormancy, seed development
and germination, synthesis of seed storage protein and lipid, and
stress tolerance (Zeevaart and Creelman, 1988 ; Davies and Jones, 1991;
Hetherington and Quatrano, 1991; Thomas, 1993). ABA regulates the
expression of many genes, including those encoding oil-body proteins in
microspore-derived and zygotic embryos of Brassica napus
(Vance and Huang, 1988; Wilen et al., 1990; Holbrook et al., 1991,
1992; Taylor and Weber, 1994), and (±)-ABA promotes VLCMFA
accumulation in zygotic embryos of B. napus (Finkelstein and
Somerville, 1989). Recently, Zou et al. (1995) demonstrated that
exogenously applied (+)-ABA and its metabolite 8-OH ABA (Fig. 1,
structure 2) induced the transcripts of oleosin and
 15 desaturase genes, as well as the
accumulation of VLCMFAs in microspore-derived embryos of B. napus cv Reston, whereas ( )-PA (Fig. 1, structure 3) had little
effect. These results suggested that ABA and/or 8-OH ABA may regulate
the accumulation of VLCMFAs in developing oilseed embryos.
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| Figure 1.
Chemical structures of (+)-ABA (1), 8-OH ABA (2),
()-PA (3), ()-DPA (4), and (+)-8-methylene ABA (5).
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The biosynthesis of VLCMFAs occurs by successive condensations of
malonyl-CoA with 18:1-CoA to give eicosenoyl (20:1) and erucoyl (22:1)
moieties. The synthesis is catalyzed by a microsomal fatty acid
"elongase complex" composed of four enzymes, beginning with the
condensing enzyme (von Wettstein-Knowles, 1982; Fehling and Mukherjee,
1991). The Arabidopsis Fatty Acid Elongation1
(FAE1) gene was shown to share homology with three
condensing enzymes: chalcone synthase, stilbene synthase, and
 -ketoacyl carrier protein synthase III. Based on this homology and
the functional studies of this gene (Millar and Kunst, 1997), it was
proposed that the gene identified by the FAE1 mutation
encodes KCS, the seed-specific condensing enzyme, which catalyzes the
first reaction of the microsomal fatty acid elongation system involved
in the biosynthesis of VLCMFAs (James et al., 1995). Studies in
Arabidopsis (Millar and Kunst, 1997) and in other Brassicaceae
spp. (Lassner et al., 1996) have shown that the condensing enzyme is
limiting for the accumulation of VLCMFAs. It was also demonstrated that
in the elongase complex, it is the specificity of the condensing enzyme
that determines which VLCMFAs accumulate (Lassner et al., 1996; Millar
and Kunst, 1997). In this study we expect to use the FAE1
gene as a probe to detect the transcript levels of KCS genes in
microspore-derived embryos of B. napus.
Since completing much of the experimental work described here, the
B. napus KCS homolog of the Arabidopsis KCS gene (identified by the lesion at the FAE1 locus) has been reported (Clemens
and Kunst, 1997). The Arabidopsis and B. napus KCS genes are
85% homologous at the DNA level and 84 to 86% similar at the protein
level, as assessed by the DNAStar suite of programs. This high homology explains the very strong hybridization signal observed in our studies,
in which B. napus KCS transcript levels were monitored by
probing with the Arabidopsis KCS (FAE1) homolog.
The major pathway of metabolism of natural (+)-(S)-ABA (Fig.
1, structure 1) involves oxidation at the 8-methyl group to produce
8-OH ABA (Fig. 1, structure 2), which cyclizes to form ()-PA (Fig.
1, structure 3). ()-PA may then be reduced to DPA (Fig. 1, structure
4) (Gillard and Walton, 1976; Loveys and Milborrow, 1984; Balsevich et
al., 1994; Sorce et al., 1996). PA in either the natural or racemic
form has been found to possess ABA-like activity in a few bioassays
(Dashek et al., 1979; Robertson et al., 1994; Hill et al., 1995), but
in most cases PA and DPA have minimal biological activity (Dashek et
al., 1979; Ho, 1983; Balsevich et al., 1994; Robertson et al., 1994;
Hill et al., 1995; Zou et al., 1995; Sorce et al., 1996).
ABA 8 hydroxylase (the enzyme that converts ABA to PA via 8-OH ABA)
has been shown to be induced by ABA in several experimental systems
(e.g. Uknes and Ho, 1984; Cutler et al., 1997), providing a homeostatic
mechanism to reduce high ABA levels. The content of ABA in seeds
changes markedly during development (Hetherington and Quatrano, 1991),
but the role of ABA 8 hydroxylase as a controlling factor is unknown.
Increasing ABA degradation is potentially important for modulating
endogenous concentrations (Zeevaart and Creelman, 1988; Kende and
Zeevaart, 1997). If changes in the rate of ABA turnover are a
major determinant of ABA concentration in B. napus embryos,
then genetically reducing ABA degradation has the potential to enhance
ABA-like effects on VLCMFA production and lipid accumulation.
(+)-8-Methylene ABA (Fig. 1, structure 5), a new ABA analog, is
metabolized more slowly than ABA in corn cells, resulting in enhanced
biological activity relative to ABA (Abrams et al., 1997). In fact,
8-methylene ABA has been shown to be more effective than (+)-ABA in
several biological assays because it provides the equivalent of an
extended pulse of ABA (Abrams et al., 1997). The consequences of
reduced ABA degradation can therefore be inferred by comparing the
hormonal activity of 8-methylene ABA with that of ABA itself. The
present study was undertaken to determine if it will be feasible in
future transgenic experiments to elevate VLCMFA production by blocking
ABA catabolism. To this end we have extended previous studies (Zou et
al., 1995) by measuring ABA metabolism and examining the effects of ABA
and related substances on the expression of the B. napus KCS
condensing enzyme and the resulting VLCMFA production in
microspore-derived embryos of B. napus cv Reston.
8-Methylene ABA has been used to provide an indication of how VLCMFA
biosynthesis is affected by reduced ABA catabolism.
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MATERIALS AND METHODS |
Chemical Reagents
(+)-ABA (Fig. 1) was obtained by preparative HPLC resolution of
(±)-methyl abscisate followed by hydrolysis of the resolved esters, as
described previously (Dunstan et al., 1992). ()-PA, the naturally
occurring enantiomer (Fig. 1), was obtained from the medium of
suspension cultures of corn (Zea mays L. cv Black Mexican
Sweet) that had been supplied with (+)-ABA, according to the procedure
of Balsevich et al. (1994). DPA (Fig. 1) was prepared from the isolated
PA as described by Zeevaart and Milborrow (1976).
(+)-[3H]ABA was synthesized according to a
reported procedure (Balsevich et al., 1994). (+)-8-Methylene ABA was
synthesized as described previously by Abrams et al. (1997).
[1-14C]Oleic acid (58 mCi/mmol) was purchased
from Amersham and converted to
[1-14C]oleoyl-CoA by an enzymatic method
described previously (Taylor et al., 1990). Neutral lipid standards
were obtained from NuChek Prep., Inc. (Elysian, MN), and polar lipids
were purchased from Sigma. HPLC-grade solvents (Omni-Solv, BDH
Chemicals, Poole, UK) were used throughout these studies. All other
chemicals were of reagent grade.
Plant Material, Microspore Culture, and Hormone Treatments
Brassica napus L. cv Reston, a high-erucic acid variety
accumulating both C20 and C22 fatty acids in developing seeds, was obtained from the University of Manitoba (Winnipeg, Canada). Plants were grown in controlled-environment growth chambers as described by
Zou et al. (1995). Microspores were isolated and cultured according to
the methods described previously (Taylor et al., 1990, 1992; Holbrook
et al., 1992; Zou et al., 1995). At 16 to 19 d in culture, microspore-derived embryo preparations enriched in early- to
mid-cotyledonary stages were obtained by filtration through a sterile,
0.2-µm filter and replated at a density of 0.25 to 0.3 g fresh
weight in 10 mL of medium in 100- × 10-mm Petri plates. After a 24-h
equilibration period, embryo cultures were supplemented with either 10 µM (+)-ABA, (+)-8-methylene ABA, ()-PA, or ()-DPA in
0.1% (v/v) ethanol (hormone treatment) or 0.1% ethanol only as a
control treatment, and maintained in the dark at 25°C on a rotary
shaker at 50 rpm. After 0, 1, 2, 4, 6, 12, 24, 48, or 72 h of
treatment, individual plates of embryos for each treatment (three to
six Petri plates) were harvested by suction filtration and rinsed
thoroughly with distilled water, and the fresh weight was recorded. The
medium was stored at 70°C for subsequent analysis. A portion of the harvested samples was removed for dry-weight determination after desiccation at 100°C for 48 h. The remainder was used to prepare homogenates as described below for determination of total lipid content, fatty acid composition, total protein, and VLCMFA elongase complex activity studies. Total homogenate protein was estimated by the
method of Bradford (1976) using BSA as a standard.
Because there were not enough microspore-derived embryos supplied at
one time for all experiments in the study, different batches of early-
to mid-cotyledonary embryos were used in individual experiments.
All experiments were performed at least twice. Data shown are from
representative experiments.
Metabolism Studies Using (+)-[3H]ABA
(+)-[3H]ABA at the initial concentration
of 10 µM was supplied to the equilibrated B. napus embryo cultures. The mass of embryos in the medium at the
time of hormone addition was about 300 mg fresh weight per plate in 10 mL of medium. Each treatment was replicated four to six times. In
time-course experiments microspore-derived embryos were harvested by
vacuum filtration at the following time intervals after the addition of
10 µM (+)-[3H]ABA: 0, 2, 6, 24, 48, or 72 h; they were then rinsed thoroughly with distilled water
and the fresh weight recorded. Aliquots of harvested embryos intended
for metabolite analysis were immediately frozen in liquid
N2 and stored at 70°C. The medium at each
time point was also stored at 70°C for further analysis.
Frozen embryos (300 mg fresh weight) were ground with a mortar and
pestle under liquid N2 for extraction and
analysis of [3H]ABA and its metabolites. The
ground sample was extracted overnight with 30 mL of solution containing
95% isopropanol and 5% glacial acetic acid, filtered, and further
extracted twice with 15 mL of the same solution for 10 min each time.
The bulked filtrate was concentrated by a rotary evaporator. The
residue was dissolved in 2 mL of 1 N NaOH solution and then
washed three times with 3 mL of methylene chloride to remove lipophilic
materials. The aqueous fraction was brought to approximately pH 3.5 with 1 N HCl and partitioned three times into EtOAc; the
EtOAc extracts were then combined and evaporated under
N2. The dried residue was then dissolved in 100 µL of methanol for analysis. An aliquot (25 µL) of sample was
applied to a Silica Gel GF254 TLC plate for
separation of ABA and its metabolites. The TLC plate was developed with
toluene:EtOAc:acetic acid (25:15:2, v/v). Sample radioactivity was
located by autoradiography and identified by co-chromatography with
known standards. The bands corresponding to ABA, PA, and DPA standards
were excised and their radioactivity measured by liquid-scintillation
counting.
Aliquots of the medium (10 mL) were acidified to pH 3.5 with 1 N HCl and extracted three times with 20 mL of EtOAc. The
combined EtOAc fractions were washed once with 30 mL of saturated NaCl, and then anhydrous Na2SO4
was added to remove water. The filtrate was evaporated to dryness. The
residue was dissolved in 100 µL of methanol and analyzed via
TLC/autoradiography as described above. In an initial experiment, it
was determined that (+)-[3H]ABA was stable in
both embryo-free and spent media.
Identification of ABA and Metabolites by MS
B. napus microspore-derived embryos from cultures
supplemented with 100 µM (+)-ABA were harvested and
extracts prepared as described above. Samples were treated with
diazomethane and analyzed by GC-MS as described previously (Sorce et
al., 1996), with the exception that samples were analyzed by ammonia
chemical ionization and detection of DPA was facilitated through
further derivatization by co-injection with
N,O-bis-(trimethylsilyl)-acetamide. GC retention times and MS data were consistent with those of standard samples of
ABA, PA, and DPA.
Extraction of Lipids from Microspore-Derived Embryos and Analysis
of Fatty Acyl Composition
Embryo homogenates were prepared as described previously (Holbrook
et al., 1992; Zou et al., 1995) and adjusted to give a fresh weight
equivalent of 100 mg/mL homogenate. The filtered homogenates (cell-free
extract) were used directly for isolation and analysis of total lipid
and fatty acid composition, and for in vitro assays of VLCMFA elongase
complex activity.
Total acyl lipids were extracted immediately from fresh
microspore-derived embryo homogenates prepared as described above (0.5 mL, equivalent to 50 mg fresh weight), according to the method described previously (Zou et al., 1995). A portion of the TLE was
transmethylated directly for assay of total acyl composition. An
internal standard of 17:0 free fatty acid was added to the TLE to
permit quantitative fatty acid analysis. The acyl composition of the
fatty acid methyl esters was determined on a gas chromatograph (model
5880, Hewlett-Packard) fitted with a DB-23 column (30 m × 0.25 mm; film thickness 0.25 µm; J&W Scientific, Folsom, CA). The GC
conditions were as described by Holbrook et al. (1992). The remaining
portion of the TLE was further separated into its polar and neutral
lipid components by TLC on Silica Gel H as described previously (Taylor
et al., 1992), and individual lipid classes were transmethylated and
analyzed for acyl composition by GC.
Assay for VLCMFA Elongase Complex Activity
The elongation assay method was adapted from that originally
described by Agrawal and Stumpf (1985). This assay measures the cumulative activity of all four enzymes of the elongase complex, including KCS, the condensing enzyme. A portion of embryo homogenate (equivalent to 0.2-0.25 mg of protein) was used as a source of protein
in elongase assays. The reaction mixture components and conditions and
14C-fatty acid extraction and methylation were as
described by Zou et al. (1995). The 14C-fatty
acid methyl esters were separated and quantified by radio HPLC as
described by Holbrook et al. (1992). The VLCMFA elongase activity was
calculated on the basis of the known specific activity (10 nCi/nmol) of
the [14C]oleoyl-CoA substrate and expressed in
milligrams of protein.
Northern Analysis
A plasmid construct, pNAPIN-FAE1, carrying a cDNA clone
encoding the FAE1 gene (encoding KCS or the condensing
enzyme of the elongase complex) from Arabidopsis was generously
provided by Dr. Ljerka Kunst (Department of Botany, University of
British Columbia, Vancouver, Canada). cDNA inserts were excised by
treatment with SacI and XbaI restriction enzymes,
the DNA fragments were purified with the Sephaglas Band Prep Kit
(Pharmacia), labeled with [32P]dCTP using the
High Prime Kit (Boehringer Mannheim) as described by the manufacturer,
and used as a probe in northern analyses. Total RNA was prepared using
Trizol Reagent (GIBCO-BRL). Total RNAs (about 6-7 µg) were subjected
to electrophoresis according to the method described by Pelle and
Murphy (1993) and blotted onto Hybond-N+ nylon
membranes (Amersham). Ethidium bromide-stained RNA gel was used as a
control for loading and transfer. The blot was hybridized to the
randomly primed FAE1 probe using QuikHyb hybridization solution (Stratagene) at 68°C, and washed at high stringency (0.1× SSC and 0.1% SDS at 60°C).
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RESULTS AND DISCUSSION |
(+)-ABA Metabolism
The metabolism of (+)-ABA in many plants or cell-culture systems
has been well documented, and the major oxidized metabolites have been
identified as PA (Uknes and Ho, 1984; Dunstan et al., 1992; Balsevich
et al., 1994) or DPA (Lehmann et al., 1983; Kubik et al., 1992; Aneja
et al., 1996; Sorce et al., 1996). However, little is known about ABA
metabolism in the microspore-derived embryo system. A previous study
has demonstrated that upon treatment with exogenously applied ABA, the
level of ABA significantly increased in microspore-derived embryos of
B. napus within 8 h (Zou et al., 1995), showing that
ABA is readily taken up by the embryos. To investigate the relationship
between ABA metabolism, the regulation of KCS gene expression, and
accumulation of gene products during VLCMFA synthesis, the major ABA
metabolites were identified and a time-course study using
(+)-[3H]ABA was performed.
Initial GC-MS analysis of microspore-derived embryos treated with 100 µM (+)-ABA for 24 h confirmed PA and DPA as the
major products of metabolism. Microspore-derived embryos were then
treated with 10 µM (+)-[3H]ABA
and both embryo and culture medium samples were extracted and analyzed
via TLC/autoradiography over the course of 72 h. Radiolabeled PA
and DPA were observed as the two major metabolites.
[3H]ABA content in the embryos was shown to
increase rapidly, reaching a maximum concentration 6 h after
treatment (Fig. 2A), with the medium
showing a concurrent decrease in [3H]ABA
content during the same period (Fig. 2B). After 24 h of treatment,
approximately 85% of the [3H]ABA was depleted
from the medium. [3H]PA and
[3H]DPA were detected in embryos and media as
early as 2 h after the hormone treatment and accumulated with
time, with [3H]PA levels decreasing as
[3H]DPA levels increased in the embryos,
consistent with the expected metabolic pathway in which PA is reduced
to DPA. In the medium, [3H]PA levels continued
to increase over time, but at a slower rate than levels of
[3H]DPA, suggesting that the metabolites were
being released from embryos to the medium. These results indicate that
microspore-derived embryos have an active system to catabolize (+)-ABA
to DPA via PA and therefore suggest that reduced catabolism could
greatly increase the time during which a hormonally effective ABA
concentration would be maintained in the embryos.
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| Figure 2.
Time course of metabolism of
(+)-[3H]ABA (10 µM) by microspore-derived
B. napus embryos. A, Metabolite profiles in embryos. B,
Metabolite profiles in the medium. Values shown are means ± SE (n = 4).  , [3H]ABA;
 , [3H]PA; and  , [3H]DPA. FW, Fresh
weight.
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The first metabolite in the pathway from ABA to DPA is 8-OH ABA, which
is in equilibrium with PA. Exogenously supplied 8-OH ABA has been
shown to have high activity in the induction of oleosin and
15 desaturase genes in microspore-derived
embryos of B. napus (Zou et al., 1995) and in the induction
of group 3 Late Embryogenesis Abundant mRNA in wheat
seedling roots (Walker-Simmons et al., 1997). Due to the transient
nature of 8-OH ABA (typically converted to PA during extraction
processes), the levels present in the embryos at the time of sampling
could not be determined. Since 8-OH ABA shows high biological
activity, the possibility of its being the active plant hormone could
not be discounted. The assays described in this paper cannot separate
the effects of exogenously applied (+)-ABA from those of the initially
formed metabolite, 8-OH ABA. This will be the topic of future
research; the present discussion will focus on the relationship between
ABA turnover and VLCFMA production, including the effects of KCS gene
expression and of elongase complex enzyme activity.
The conversion of ABA to PA and then DPA in plants is catalyzed by two
enzymes assumed to be sequential, ABA 8 hydroxylase and PA reductase
(Gillard and Walton, 1976). Babiano (1995) demonstrated that PA
accumulation in germinating chickpea seeds treated with ABA is
correlated with an increase in ABA 8 hydroxylase activity. Similarly,
Uknes and Ho (1984) and Cutler et al. (1997) showed by in vivo studies
that ABA 8 hydroxylase is induced by ABA. Further studies using in
vitro assays have shown that induction of the hydroxylase by ABA occurs
in corn cells (Krochko et al., 1997).
Induction of KCS Gene Transcripts by (+)-ABA and 8-Methylene
ABA
To assess the effects of (+)-ABA and related compounds on VLCMFA
synthesis at the gene level, northern analyses were performed to
measure the transcript levels of the gene encoding the B. napus KCS using the Arabidopsis FAE1 (KCS) gene as a
probe. All concentrations applied, ranging from 1 to 100 µM (+)-ABA, significantly induced the KCS gene transcript
compared with the control treatments (Fig. 3). (+)-ABA treatment (10 µM) gave the highest level of transcript.
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| Figure 3.
Response of KCS gene transcription to various
concentrations of (+)-ABA supplied to microspore-derived B. napus embryos in culture for 24 h. The Arabidopsis
FAE1 gene was used as a probe. The RNA gel-blot analysis
of total RNA (7 µg) was described in ``Materials and Methods''. The
bottom panel shows ethidium bromide-stained RNA gel as a control for
loading.
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Time-course studies showed that in as little as 1 h after 10 µM (+)-ABA treatment, the transcript of the
KCS-condensing enzyme gene was strongly induced and rapidly increased
with time of treatment up to 6 h (Fig.
4). This strong induction signal remained
throughout the sampling period despite the decrease in embryo ABA
content at later time points, as shown in Figure 2. Similarly, in the presence of 10 µM (+)-8-methylene ABA, strong induction
of the KCS message was evident within 2 h and remained at a higher
level than in the control throughout the 72-h sampling period (Fig. 5). In the absence of hormone, the
transcript of the KCS gene remained relatively low during the 72-h
sampling period. In contrast to (+)-ABA and 8-methylene ABA, PA and
DPA had only a slight or no effect on the KCS gene transcript relative
to the control (Fig. 6). Similarly, PA
had no effect on induction of oleosin and 15
desaturase transcripts in microspore-derived embryos (Zou et al.,
1995).
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| Figure 4.
Time course of the effect of 10 µM
(+)-ABA treatment (compared with control treatment) on the KCS
condensing enzyme gene transcript in microspore-derived B. napus embryos. The Arabidopsis FAE1 gene was used
as a probe. Six micrograms of total RNA was loaded onto each lane in
this experiment. The top half of the control and ABA panels shows
results of northern-blot analysis. The bottom half of these panels
shows the corresponding ethidium bromide-stained RNA gel as a control
for loading.
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| Figure 5.
The accumulation of the KCS gene transcript after
microspore-derived B. napus embryos were treated with 10 µM (+)-8-methylene ABA. The Arabidopsis
FAE1 gene was used as a probe. Seven micrograms of total
RNA from each sample was loaded. The bottom panel shows ethidium
bromide-stained RNA gel as a control for loading. c, 0.1% ethanol
treatment as controls; m, 10 µM (+)-8-methylene ABA.
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| Figure 6.
Comparison of induction of the KCS gene transcript
level by (+)-ABA, ()-PA, ()-DPA, and (+)-8-methylene ABA after
24 h. The Arabidopsis FAE1 gene was used as a
probe. Seven micrograms of total RNA from each treatment was loaded.
The bottom panel shows the ethidium bromide-stained RNA gel as a
control for loading.
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Fatty Acyl Composition and VLCMFA Elongase Complex Activity
Previous work (Holbrook et al., 1992; Zou et al., 1995) has shown
that exogenously supplied (±)- or (+)-ABA altered fatty acid
composition and dramatically increased the content of VLCMFAs (20:1 and
22:1) in early- to mid-cotyledonary microspore-derived B. napus embryos in culture. Similar results were obtained in zygotic
embryos of B. napus (Finkelstein and Somerville, 1989). In
those studies, however, no attempt was made to study the concentration dependence of the ABA effect on VLCMFA accumulation. After a 48-h treatment, all concentrations of (+)-ABA tested in this study caused an
increase in the amounts of total fatty acids detected (data not shown),
but the strongest and most significant effect was observed on the
levels of 20:1 and 22:1, which were generally 40 to 70% higher on a
milligrams-of-protein basis and 4 to 8% higher on a mole-percentage
basis, respectively, than those of the control (Table
I). Of the (+)-ABA concentrations tested, 10 µM gave maximum VLCMFA accumulation. This is
consistent with the data for induction of the KCS transcript shown in
Figure 3. The specific activity of the elongase increased between 1 and 10 µM (+)-ABA and appeared to saturate thereafter (Fig.
7).
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Table I.
Effect of various concentrations of (+)-ABA on
VLCMFA accumulation
Microspore-derived B. napus embryos were incubated with
various concentrations of (+)-ABA in culture. After 48 h of
treatment, embryos were harvested, total lipids isolated, and fatty
acid composition determined as described in ``Materials and Methods''. The data shown are means ± SE of four
replicates.
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| Figure 7.
Effect of various concentrations of (+)-ABA on
VLCMFA elongase complex activity in microspore-derived B. napus embryos. Embryos were subjected to various concentrations of
(+)-ABA in culture for 24 h and homogenized to assay for
[14C]20:1 and [14C]22:1 biosynthesis from
[14C]oleate (18:1)-CoA in vitro as described in
``Materials and Methods''. , 20:1 + 22:1; , 22:1; and ,
20:1. Values shown are means ± SE
(n = 4).
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A time-course study was carried out to examine further the effect of
ABA and its metabolites on the elongase complex activity and
accumulation of VLCMFAs. Microspore-derived embryos were incubated with
the optimal concentration (10 µM) of (+)-ABA and
harvested at various times. Within 6 h, an increase in the
accumulation of 20:1 and 22:1 could be detected, and embryos showed a
strong, nearly linear accumulation of VLCMFAs for 72 h on a
milligrams-of-protein basis, relative to the control embryos (Fig.
8). The stimulation of VLCMFA
accumulation by 10 µM (+)-8-methylene ABA was
approximately 20 to 30% higher than that produced by the
(+)-ABA treatment after 24 h. This is in agreement with the higher
KCS gene transcript level induced by (+)-8-methylene ABA compared with
that induced by ABA (Fig. 6).
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| Figure 8.
Time course of effect of (+)-ABA and 8-methylene
ABA on the accumulation of VLCMFAs in microspore-derived B. napus embryos. The early-cotyledonary microspore-derived embryos
were incubated with 10 µM (+)-ABA () and 8-methylene
ABA (), or only 0.1% ethanol (control, ). At various times,
embryos were harvested and VLCMFA content was measured as described in
``Materials and Methods''. Values shown are averages ± SE (n = 4).
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The same trend of increase in VLCMFA accumulation caused by ABA and
8-methylene ABA was also observed on a dry-weight basis (data not
shown). The findings are consistent with the data for the induction of
VLCMFA elongase complex activity presented in Figure
9. After as little as 2 h in the
presence of (+)-ABA, the activity of VLCMFA elongase complex (expressed
as activity per milligram of protein) was significantly stimulated and
this activity increased further up to 6 h. After 6 h, the
activity declined, but remained about 50% higher than that of
corresponding controls (Fig. 9). In this experiment the maximum
specific activity of the elongase at 6 h after 10 µM
(+)-ABA treatment was 138 pmol min 1
mg1 protein, 60% higher than that of the
control (87 pmol min1
mg1 protein) at 24 h. After 48 h of
treatment, the specific activities of the elongase complex in 10 µM (+)-ABA- and 8-methylene ABA-treated embryos were 47 and 75%, respectively, higher than those of the control. A decrease in
the elongase activity in the ABA-treated samples after 6 h of
treatment (especially if control activities are subtracted) may be
related to ABA catabolism, since ABA levels in microspore-derived
embryos also decreased after 6 h (Fig. 2), although (as we noted
above) levels in KCS transcripts remained high from 6 to 72 h
(Fig. 4).
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| Figure 9.
Time course of the effect of 10 µM
(+)-ABA () and 8-methylene ABA () on VLCMFA elongase complex
activity in microspore-derived B. napus embryos. Each data
point represents the mean ± SE of four replicates.
, Control.
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|
The idea that declining embryo ABA content is related to the decrease
in elongase activity after 6 h is consistent with the fact that
addition of fresh (+)-ABA (10 µM) into the treatment medium at 24 h restored the maximum activity of the elongase
enzyme (data not shown). Also, treatment with the more slowly
metabolized (+)-8-methylene ABA resulted in a more prolonged induction
of elongase activity (Fig. 9). The ABA metabolites that accumulate in
the embryo make no significant contribution, since it has been shown
that PA had only a slight effect on the elongase activity (Zou et al.,
1995). In the present study, DPA had essentially no effect (data not
shown). When the elongase data were expressed in terms of total
activity (picomoles per minute), a similar decrease was also observed
in (+)-ABA- and (+)-8-methylene ABA-treated embryos after 6 h of
treatment (data not shown). However, it should be noted that an
increase (10-14%) in embryo protein content over the course of the
experiment may also contribute to the apparent decrease in elongase
activity, when expressed per milligram of protein, at later time
points in ABA- and (+)-8-methylene ABA-treated samples.
Accumulation of TAG and Polar Lipids
To test the effects of (+)-ABA and (+)-8-methylene ABA on storage
lipids and the incorporation of VLCMFAs into TAGs in microspore-derived B. napus embryos, the content of TAG and polar lipids and
the distribution pattern of VLCMFAs in these lipids were determined in
(+)-ABA- and 8-methylene ABA-treated embryos versus the control embryos. After a 48-h treatment with 10 µM (+)-ABA,
8-methylene ABA, and DPA, total fatty acids in the TAG fraction in
embryos were about 50, 70, and 8%, respectively, higher than those of the control treatment on a milligrams-of-protein basis (Table II). A similar trend was obtained on a
dry-weight basis (data not shown). However, there was no significant
difference in total fatty acid content in the polar lipid pool between
the control and hormone treatments. VLCMFA content in the TLE of
embryos treated with (+)-ABA, (+)-8-methylene ABA, or DPA were about
55, 85, and 10%, respectively, higher than that of the control
embryos. As expected, 80 to 85% of the VLCMFAs accumulated during
hormone treatments were found in the TAG fraction (Table II). The data presented here demonstrate that 8-methylene ABA produces a stronger ABA-like effect on the accumulation of TAG and VLCMFAs.
DPA had little or no effect on the induction of TAG (8% increase) and VLCMFA (10% increase) accumulation.
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|
Table II.
Accumulation of VLCMFAs in TLE from
microspore-derived embryos incubated with 10 µM (+)-ABA,
DPA, or 8-methylene ABA for 48 h
|
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| |
CONCLUSIONS |
The results presented here are the first, to our knowledge, to
integrate the effects of ABA and its metabolites on the processes involved in VLCMFA accumulation at the transcript, gene-product (enzyme
activity), and enzyme-product (VLCMFAs) levels. We have shown that the
effect of (+)-ABA on VLCMFA biosynthesis and storage lipid accumulation
is maximal at 10 µM, and therefore, compared lipid
biosynthesis with ABA degradation at this ABA concentration. The
observation that 8-methylene ABA produces stronger effects than ABA in
the experiments reported here implies that catabolic removal of ABA
restricts VLCMFA production.
Overall, the experiments described here allow us to propose the working
hypothesis that ABA catabolism limits VLCMFA production during
embryogenesis, especially when exogenous ABA is greater than 10 µM. To test this hypothesis, future experiments will
explore how ABA catabolism, as measured by ABA 8 hydroxylase activity, affects and is affected by changes in embryo ABA content.
| |
FOOTNOTES |
1
This is National Research Council of Canada
publication no. 40727.
*
Corresponding author; e-mail acutler{at}pbi.nrc.ca; fax
1-306-975-4839.
Received December 15, 1997;
accepted April 14, 1998.
| |
ABBREVIATIONS |
Abbreviations:
DPA, dihydrophaseic acid.
EtOAc, ethyl acetate.
KCS, 3-ketoacyl-CoA synthase.
8-OH ABA, 8-hydroxy ABA.
PA, phaseic
acid.
TAG, triacylglycerol.
TLE, total lipid extract.
VLCMFA, very-long-chain monounsaturated fatty acids.
X:Y, a fatty acyl group
containing X carbon atoms and Y cis double bonds.
| |
ACKNOWLEDGMENTS |
The authors thank Dr. Ljerka Kunst (University of British
Columbia) for kindly supplying plasmid pNAPIN-FAE1, Dr. John J. Balsevich (Plant Biotechnology Institute) for supplying tritiated (+)-ABA, and Dr. Alison Ferrie (Transgenic Plant Center, Saskatoon) for
supplying microspore-derived B. napus embryos. We also
gratefully acknowledge Lawrence Hogge and Doug Olson (Mass
Spectrometry Laboratory, Plant Biotechnology Institute) for GC-MS
analyses.
| |
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Top
Abstract
Introduction