From DuPont Nutrition and Health, Experimental Station, Wilmington, Delaware 19880-0402
Received for publication, October 9, 2000, and in revised form, November 1, 2000
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ABSTRACT |
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Divergent forms of the plant
The common polyunsaturated fatty acids of plant seed oils contain
cis-double bonds that are separated by a methylene group. The primary examples of such fatty acids are linoleic acid
(18:2 We have recently demonstrated that the conjugated
trans- Calendic acid, the primary fatty acid of Calendula
officinalis seeds (8-10), is a conjugated trienoic fatty acid,
like In this report, we have undertaken a genomics-based approach to
characterize the biosynthetic origin of the conjugated double bonds of
calendic acid. By sequencing of random cDNAs derived from
developing C. officinalis seeds, we have identified
cDNAs for two closely related variant forms of FAD2. Expression of
either cDNA in somatic soybean embryos results in the accumulation
of calendic acid. These findings thus demonstrate that FAD2-type enzymes can catalyze not only the modification of the
cDNA Library Construction--
Total RNA was isolated from
developing seeds of C. officinalis variety Dwarf Gem
(Burpee) plants using the method described by Jones et al.
(15). Poly(A)+ RNA was enriched from the total RNA and used
for cDNA library construction as described previously (4). The
resulting library consisted of cDNA inserts cloned directionally
(5' to 3') in the EcoRI and XhoI sites of
pBluescript II SK(+) and was maintained in Escherichia coli
DH10B cells (Life Technologies, Inc.). Bacterial cells harboring the
libraries in plasmid form were stored as glycerol stocks at Generation of ESTs and Identification of Divergent FAD2
cDNAs--
Plasmids for EST analysis were prepared from randomly
picked colonies from the C. officinalis cDNA library
using the QIAGEN REAL Prep 96 system according to the manufacturer's
protocol. Nucleotide sequence was obtained from the 5'-ends of
cDNAs in pBluescript II SK(+) using the M13 reverse priming site
and dye terminator cycle sequencing with an ABI 377 DNA fluorescence
sequencer. Partial nucleotide sequences were obtained for 3036 random
cDNAs from the C. officinalis developing seed library
using this methodology. Putative identities were assigned to these
cDNAs by comparison of their partial sequences with translated
sequences in the public data bases using the NCBI BLASTX program
(16).
From this analysis of the C. officinalis developing seed
library, full-length cDNAs for two closely related divergent forms of FAD2 were identified. The polypeptides encoded by these cDNAs were designated CoFADX-1 and CoFADX-2. Nucleotide sequences were determined from both strands of the CoFADX-1 and CoFADX-2 cDNAs in
pBluescript II SK(+) by dye terminator sequencing using the instrumentation described above.
Expression of CoFADX-1 and CoFADX-2 cDNAs in Somatic Soybean
Embryos--
The vector pKS67 was used for expression of cDNAs for
CoFADX-1 and CoFADX-2 in soybean (Glycine max) somatic
embryos. This vector contains a unique NotI site for cloning
of transgenes that is flanked by the seed-specific promoter of the gene
for the
The coding sequences for CoFADX-1 and CoFADX-2 were amplified by PCR
using Pfu polymerase (Stratagene) to generate flanking NotI sites for subcloning into the pKS67 expression vector.
Full-length cDNAs for CoFADX-1 and CoFADX-2 were used as templates
for PCRs. For amplification of CoFADX-1, the following oligonucleotide
primer combination was used:
5'-ttgcggccgcTACACCTAGCTACGTACCATG-3' (sense) and
5'-ttgcggccgTCACGGTACTGATGATGGCAC-3' (antisense). The CoFADX-2 cDNA
was amplified using the following primer combination:
5'-agcggccgcTATACCATGGGCAAG-3' (sense) and 5'-tgcggccgcTATGTTAAACTTC-3'
(antisense). Note that the sequences shown in lowercase letters contain
an added NotI site along with additional bases to facilitate
restriction enzyme digestion. The resulting PCR products were subcloned
into the intermediate vector pCR-Script AMP SK(+) (Stratagene)
according to the manufacturer's protocol. The amplified coding
sequence for CoFADX-1 or CoFADX-2 was then released with
NotI digestion and subcloned into the corresponding site of
the soybean expression vector pKS67.
Gene fusions of the CoFADX-1 and CoFADX-2 cDNAs with the
Fatty Acid Analysis of Transgenic Soybean Embryos--
Fatty
acid methyl esters were prepared from transgenic soybean embryos by
transesterification in 1% (w/v) sodium methoxide in methanol. Single
soybean embryos were homogenized with a glass stirring rod in 0.5 ml of
the sodium methoxide solution and incubated at room temperature for 20 min. At the end of this period, 0.5 ml of 1 M sodium
chloride was added, and fatty acid methyl esters were extracted with
0.5 ml of heptane. Fatty acid methyl esters were separated and
quantified using a Hewlett-Packard 5890 gas chromatograph fitted with
an Omegawax column (30 m × 0.32 mm (inner diameter); Supelco
Inc.). The oven temperature was programmed from 220 °C (2-min hold)
to 240 °C at a rate of 20 °C/min, and carrier gas was supplied by
a Whatman hydrogen generator. Fatty acid methyl esters were also
analyzed by GC-MS using a Hewlett-Packard 6890 gas chromatograph
interfaced with a Hewlett-Packard 5973 mass selective detector. Samples
were separated with an INNOWax column (30 m × 0.25 mm (inner
diameter); Hewlett-Packard Co.) or with an HP-5 column (30 m × 0.25 mm (inner diameter); Hewlett-Packard Co.). The oven temperature
was programmed from 185 °C (3.5-min hold) to 215 °C (5-min hold)
at a rate of 2 °C/min and then to 230 °C at a rate of
5 °C/min. The structures of fatty acid methyl esters with conjugated
double bonds were also characterized by GC-MS following Diels-Alder
derivatization by reaction with 4-methyl-1,2,4-triazoline-3,5-dione (MTAD) (Aldrich) (21). For these studies, fatty acid methyl esters from
transgenic soybean embryos were reacted for 10 s on ice with 0.5 ml of 5 mM MTAD in dichloromethane. The reaction was
stopped by the addition of 50 µl of 2,4-hexadiene (Aldrich). The
derivatized samples were then dried under nitrogen and resuspended in
heptane for GC-MS analysis. MTAD adducts were resolved using either a
DB-1 Ht column (15 m × 0.25 mm (inner diameter); J & W
Scientific) or an HP-5 column (30 m × 0.25 mm (inner diameter)). The oven temperature was programmed from 185 °C (3-min hold) to 275 °C at a rate of 2.5 °C/min. For identification of calendic acid, the mass spectra of Diels-Alder derivatives prepared from transgenic soybean embryos were compared with those of calendic acid
adducts generated from fatty acid methyl esters of C. officinalis seeds.
Expression of CoFADX-1 and CoFADX-2 in Saccharomyces
cerevisiae--
The activities of CoFADX-1 and CoFADX-2 were
characterized by expression of the corresponding full-length cDNAs
in S. cerevisiae behind the GAL1 promoter in the
vector pESC-URA (Stratagene). The coding sequences of CoFADX-1 and
CoFADX-2 were removed as BamHI/XhoI fragments
from pBluescript II SK(+) and cloned into the corresponding sites of
pESC-URA. The resulting plasmids were introduced into S. cerevisiae INVSc1 cells (Invitrogen) by lithium acetate-mediated
transformation (22). Transformed cells were selected for their ability
to grow on medium lacking uracil. Individual colonies of transformed
cells were then grown for 2 days at 30 °C in medium lacking uracil
(0.17% (w/v) yeast nitrogen base without amino acids (Difco), 0.5%
(w/v) ammonium sulfate, and 0.18% (w/v) SC-URA (Bio 101, Inc.))
supplemented with glycerol and glucose to final concentrations of 5%
(v/v) and 0.5% (w/v), respectively. Cells were then washed twice in
the growth medium described above with galactose at a final
concentration of 2% (w/v) as the carbon source. The washed cells were
then diluted to A600 Northern Blot Analysis--
Total RNA was extracted from leaves
and developing seeds of C. officinalis using Trizol (Life
Technologies, Inc.) according to the manufacturer's protocol. Total
RNA (20 µg) from each tissue and RNA standards were electrophoresed
on a 1% (w/v) agarose gel containing formaldehyde. Following
electrophoresis, RNA was transferred from the gel to Bright Star-Plus
nylon membrane (Ambion Inc.) using NorthernMax transfer buffer (Ambion
Inc.). The RNA was fixed to the membrane by UV cross-linking. The
membrane was rinsed with 2× SSC and then hybridized with
32P-labeled probes for 18 h at 42 °C in NorthernMax
hybridization buffer (Ambion Inc.). Probes were prepared from cDNAs
for CoFADX-1 or CoFADX-2 and labeled using random hexamer priming (17).
Following incubation with probes, blots were washed for 15 min with 2×
SSC and 0.1% SDS at room temperature, then washed for 15 min at room temperature with 0.2× SSC and 0.1% SDS, and finally washed for 15 min
at 42 °C with 0.2× SSC and 0.1% SDS. Radioactivity on filters was
detected by phosphorimaging. Message sizes were estimated based on
mobility relative to a 0.24-9.5 kilobase RNA ladder (Life Technologies, Inc.).
Given the high degree of identity between the open reading frames of
the CoFADX-1 and CoFADX-2 cDNAs, probes were prepared primarily
from the 3'-untranslated regions of these cDNAs to more specifically distinguish between the expression patterns of the corresponding genes. The probes used for Northern analysis were generated by PCR amplification using Pfu polymerase, and
cDNAs for CoFADX-1 and CoFADX-2 were used as templates. PCR
products were purified by agarose gel electrophoresis prior to use in
labeling reactions. For amplification of the CoFADX-1-specific probe
(292 base pairs), the following oligonucleotides were used:
5'-GATTTGAAGTTTCAAATAATC-3' (sense) and 5'-GATAACGCCTTTATTATACTG-3'
(antisense). For amplification of the CoFADX-2-specific probe (149 base
pairs), the following oligonucleotides were used:
5'-AAAATAAGACTTGAAGTTTAAC-3' (sense) and 5'-GGATAACTCCTTTATTATAC-3' (antisense).
Identification of Divergent FAD2 cDNAs in C. officinalis
Seeds--
An EST approach was used to determine the biosynthetic
origin of calendic acid in C. officinalis seeds. DNA
sequences were obtained from the 5'-ends of >3000 randomly selected
cDNAs from a C. officinalis developing seed library.
From this pool of ESTs, 12 cDNAs that encode FAD2-related
polypeptides were identified by BLAST homology. Based on sequence
comparisons, five of these cDNAs corresponded to polypeptides that
were more closely related to the "typical" plant FAD2 that is
associated with the cis-
Using Northern blot analysis, expression of genes for CoFADX-1 and
CoFADX-2 was detected in developing seeds, but was not detected in
leaves of C. officinalis (Fig.
3). This expression profile is consistent
with the seed-specific occurrence of calendic acid in C. officinalis (8).2
Functional Characterization of Divergent C. officinalis FAD2
Enzymes in Transgenic Plants--
FAD2-related polypeptides are
microsomal enzymes that are typically recalcitrant to in
vitro assay in solubilized membrane extracts (6). As an
alternative method of functional characterization, CoFADX-1 and
CoFADX-2 were expressed in somatic soybean embryos to examine their
effect on fatty acid content. Like seeds, somatic soybean embryos are
rich in triacylglycerols, and the fatty acid composition of transgenic
somatic embryos is completely predictive of the fatty acid composition
of seeds obtained from regenerated plants (25). In these experiments,
expression of cDNAs for CoFADX-1 and CoFADX-2 was placed under the
control of the strong seed-specific promoter of the gene for the
Two additional fatty acids (corresponding to peaks
a and b in Fig. 4B) were detected in
low amounts in the transgenic soybean embryos. The methyl ester of
peak a in Fig. 4B displayed a mass spectrum
identical to that of the methyl ester of calendic acid (data not
shown). However, its gas chromatographic retention time on polar phases
was slightly longer than that of methyl calendic acid. Based on these
properties, this fatty acid was tentatively identified as the
trans-
In somatic soybean embryos expressing CoFADX-2, calendic acid
accumulated to as high as 15-22% (w/w) of the total fatty acids (Table I). Surprisingly, this level of
calendic acid accumulation had little effect, if any, on the oleic acid
(18:1 Substrate Specificities of CoFADX-1 and CoFADX-2 in S. cerevisiae--
CoFADX-1 and CoFADX-2 were expressed in S. cerevisiae to examine the substrate specificities of these
enzymes. For these experiments, cDNAs encoding CoFADX-1 and
CoFADX-2 were introduced behind the GAL1 promoter in the
expression vector pESC-URA. In galactose-induced cells transformed with
cDNAs for either polypeptide, calendic acid accumulation was
observed only when exogenous linoleic acid was included in the growth
medium (Fig. 6A). These
results thus confirm that linoleic acid is the precursor of calendic
acid via the
Calendic acid accumulation was also dependent on the growth temperature
of the cultures, as little or no calendic acid was detected in induced
cells maintained at 30 °C. However, the accumulation of this fatty
acid was enhanced by reduced growth temperatures (e.g.
16 °C). This temperature dependence of calendic acid accumulation is
similar to what has been previously observed with linoleic acid
production in S. cerevisiae expressing
Arabidopsis FAD2 (26). Calendic acid accounted for as much
as 4.5% (w/w) of the total fatty acids of induced S. cerevisiae cells maintained at 16 °C (Table
II). Of note, the amount of calendic acid
detected in cells expressing CoFADX-1 was at least comparable to that
found in cells expressing CoFADX-2 (data not shown).
More detailed characterization of substrate specificity was conducted
using S. cerevisiae cells expressing CoFADX-1. No conjugated dienoic fatty acids were detected in cells grown without exogenous fatty acid or with added oleic acid (data not shown). These results suggest that CoFADX-1 has no or relatively low activity with oleic acid
or with the palmitoleic acid (16:1
The inclusion of
To examine the relative activity of CoFADX-1 for linoleic and
We have identified cDNAs for two highly expressed FAD2-related
polypeptides (CoFADX-1 and CoFADX-2) from C. officinalis
seed, a tissue that is enriched in calendic acid
(18:3 The involvement of FAD2-related enzymes in the modification of the
It should be noted that a cDNA for a FAD2-related enzyme
(CoDES) from C. officinalis was recently identified
by Fritsche et al. (28) and was reported to be a calendic
acid-producing desaturase. Note that CoDES is identified as
CoFad2 in Fig. 2. However, no gas chromatographic or mass
spectral evidence was provided to support this functional
identification of CoDES. Interestingly, CoDES shares only 50% amino
acid sequence identity with CoFADX-1 and CoFADX-2, but is instead most
related to a Crepis FAD2 acetylenase (76% identity) (24).
This observation may explain the lack of convincing evidence that
calendic acid is produced when this cDNA is expressed in S. cerevisiae (28).2 In addition, we were unable
to detect any copies of cDNAs for CoDES in the ~3000 random
cDNAs that were sequenced from C. officinalis seeds. In
contrast, cDNAs for CoFADX-1 and CoFADX-2 accounted for ~0.23%
of the C. officinalis seed ESTs. Of note, we have identified expressed genes encoding FAD2-related polypeptides that share 80-90%
amino acid sequence identity with CoDES in a variety of other
Asteraceae species, including those that do not accumulate conjugated fatty acids in their seed oils.2 Thus, it
seems unlikely that CoDES encodes a seed fatty acid conjugase.
Among the unexpected results from the transgenic production of calendic
acid in somatic soybean embryos was the lack of an accompanying high
oleic acid phenotype. This finding is in contrast to that
previously observed with unusual fatty acid synthesis resulting from
the transgenic expression of divergent FAD2 enzymes, including the
castor and Lesquerella hydroxylases and the
Momordica Another unexpected result from the transgenic somatic soybean embryos
was the accumulation of small amounts of an 18:4 isomer that we
tentatively identified as
18:4 Amounts of calendic acid in somatic soybean embryos expressing CoFADX-1
and CoFADX-2 were as high as 4% (w/w) and 22% (w/w), respectively, of
the total fatty acids of these tissues. Differences in levels of
calendic acid accumulation in these lines may be due to factors
associated with the transgenic expression of CoFADX-1 and CoFADX-2 or
to a lower specific activity of CoFADX-1 in the transgenic soybean
embryos. Regardless, amounts of calendic acid resulting from the
expression of either CoFADX-1 or CoFADX-2 are likely sufficient to
increase the oxidation rate of triacylglycerols in the transgenic
soybean embryos. In this regard, seed oils such as tung oil that are
enriched in polyunsaturated fatty acids with conjugated double bonds
are used as drying agents in coating materials (e.g. paints,
varnishes, and inks) because of their high rates of oxidation (2, 3).
Therefore, the transgenic expression of CoFADX-1 or CoFADX-2 may
ultimately be useful for the production of improved drying oils in
existing oilseed crops such as soybean.
12-oleic-acid desaturase (FAD2) have previously
been shown to catalyze the formation of acetylenic bonds, epoxy groups,
and conjugated
11,
13-double bonds by
modification of an existing
12-double bond in
C18 fatty acids. Here, we report a class of FAD2-related enzymes that modifies a
9-double bond to produce the
conjugated
trans-
8,trans-
10-double
bonds found in calendic acid
(18:3
8trans,10trans,12cis),
the major component of the seed oil of Calendula
officinalis. Using an expressed sequence tag approach, cDNAs
for two closely related FAD2-like enzymes, designated CoFADX-1 and
CoFADX-2, were identified from a C. officinalis developing
seed cDNA library. The deduced amino acid sequences of these
polypeptides share 40-50% identity with those of other FAD2 and
FAD2-related enzymes. Expression of either CoFADX-1 or CoFADX-2 in
somatic soybean embryos resulted in the production of calendic acid. In
embryos expressing CoFADX-2, calendic acid accumulated to as high as
22% (w/w) of the total fatty acids. In addition, expression of
CoFADX-1 and CoFADX-2 in Saccharomyces cerevisiae was
accompanied by calendic acid accumulation when induced cells were
supplied exogenous linoleic acid
(18:2
9cis,12cis). These
results are thus consistent with a route of calendic acid synthesis
involving modification of the
9-double bond of linoleic
acid. Regiospecificity for
9-double bonds is
unprecedented among FAD2-related enzymes and further expands the
functional diversity found in this family of enzymes.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
9cis,12cis) and
-linolenic acid
(18:3
9cis,12cis,15cis).1
In contrast, the seed oils of a number of plant species contain polyunsaturated fatty acids with conjugated (or
non-methylene-interrupted) double bonds (1). Examples of these unusual
fatty acids include
-eleostearic acid
(18:3
9cis,11trans,13trans),
-parinaric acid
(18:4
9cis,11trans,13trans,15cis),
punicic acid
(18:3
9cis,11trans,13cis),
and calendic acid
(18:3
8trans,10trans,12cis)
(1). Seed oils that contain fatty acids with conjugated double bonds
display high rates of oxidation compared with oils that contain
unsaturated fatty acids with methylene-interrupted double bonds (2).
Because of this property, seed oils such as tung oil that are enriched
in fatty acids with conjugated double bonds are used commercially as
drying agents in paints and varnishes (3).
11- and
trans-
13-double bonds of
-eleostearic and
-parinaric acids in seeds of Momordica charantia and
Impatiens balsamina, respectively, are synthesized by
divergent forms of the
12-oleic-acid desaturase (FAD2;
oleate desaturase, EC 1.3.1.35), which we have termed
"conjugases" (4). These enzymes catalyze the conversion of an
existing cis-
12-double bond into conjugated
trans-
11- and
trans-
13-double bonds (4, 5). This activity
contrasts with that of the typical FAD2 desaturase of plants, which
introduces a cis-
12-double bond into oleic
acid. In M. charantia seeds,
-eleostearic acid is formed
by modification of the cis-
12-double bond of
linoleic acid by a FAD2 conjugase (4). Similarly, the synthesis of
-parinaric acid in I. balsamina seeds arises from the
conjugase-catalyzed modification of the
cis-
12-double bond of
-linolenic acid (4).
These reactions use fatty acids bound to phosphatidylcholine as
substrates (5), as has been shown for other FAD2-type enzymes (6). In
addition, based on the mechanism proposed for conjugated double bond
synthesis in red algae (7), the production of
-eleostearic and
-parinaric acids probably involves removal of a hydrogen atom from
the C-11 and C-14 methylene groups that flank the
cis-
12-double bond of linoleic and
-linolenic acids.
-eleostearic acid, but contains conjugated
trans-
8-,
trans-
10-, and
cis-
12-double bonds. This fatty acid composes
50-60% (w/w) of the seed oil of C. officinalis, but is
absent from leaves of this plant (8).2 In common with
-eleostearic acid synthesis, linoleic acid has been shown to be the
biosynthetic precursor of calendic acid (11, 12). Unlike
-eleostearic acid synthesis, however, the conjugated trans-
8- and
trans-
10-double bonds of calendic acid arise
from modification of the cis-
9-double bond of
linoleic acid (11, 12). Based on our previous studies (4) and the
proposed mechanism of conjugases (7), it seemed likely that the
conjugated trans-
8- and
trans-
10-double bonds of calendic acid are
formed by a fatty acid desaturase-like enzyme. The involvement of a
FAD2-related enzyme in the modification of a
cis-
9-double bond, however, has not been
previously demonstrated.
12-position, but also the
9-position of
fatty acid substrates. In addition, we show that calendic acid
accumulation in somatic soybean embryos is not accompanied by large
increases in oleic acid content, which is in contrast to the phenotype
generally observed with the expression of other divergent FAD2 enzymes
in transgenic plants (4, 13, 14).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
80 °C
until used for expressed sequence tag
(EST)3 analysis.
'-subunit of
-conglycinin (17) and phaseolin termination
sequence (18). Bacterial selection with this vector is conferred by a
hygromycin B phosphotransferase gene (19) under the control of the T7
RNA polymerase promoter, and plant selection is conferred by a second hygromycin B phosphotransferase gene under the control of the cauliflower mosaic virus 35 S promoter.
-conglycinin promoter and phaseolin termination sequences in vector pKS67 were introduced into soybean embryos of cultivar A2872 or Jack
using the particle bombardment method of transformation (4, 20).
Selection and propagation of the transgenic somatic soybean embryos
have been described previously (4, 20). Expression of CoFADX-1 or
CoFADX-2 was confirmed by PCR amplification using sequence-specific
primers and first-strand cDNA prepared from total RNA isolated from
the transgenic somatic soybean embryos.
0.2 in the galactose
growth medium that also contained Tergitol type NP-40 (Sigma) at a
concentration of 0.2% (w/v). Aliquots of these cells were grown in a
volume of 3 ml without exogenous fatty acids or with the addition of
oleic acid (18:1
9cis), linoleic acid
(18:2
9cis,12cis), or
-linolenic acid
(18:3
9cis,12cis,15cis)
at a final concentration of 0.7 mM. Experiments were also
conducted with both linoleic and
-linolenic acids added to the
medium, each at a concentration of 0.35 mM.
Galactose-induced cultures were maintained at 16 °C with shaking
(350 rpm). Cells were harvested by centrifugation when cultures reached
densities of A600
3-4. Cell pellets from
the 3-ml cultures were washed with water and dried under vacuum. The
pellets were then resuspended in 0.4 ml of 1% (w/v) sodium methoxide
in methanol and incubated at room temperature for 20 min. Fatty acid
methyl esters resulting from this direct transesterification of cell
pellets were extracted and analyzed by GC and GC-MS as described above
for analysis of somatic soybean embryos. Fatty acid methyl esters were
also reacted with MTAD, and the resulting Diels-Alder adducts were
analyzed by GC-MS as described above.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
12-desaturation of
oleic acid. The remaining seven cDNAs were found to encode two
closely related polypeptides that were designated CoFADX-1 and
CoFADX-2. Of these cDNAs, six encoded CoFADX-1, and one encoded
CoFADX-2. The longest full-length cDNAs corresponding to CoFADX-1
and CoFADX-2 contained 1457 and 1295 base pairs, respectively. The
amino acid sequences of CoFADX-1 and CoFADX-2 deduced from full-length
cDNAs share 94% identity (Fig. 1).
These polypeptides, however, share <51% identity with all reported
FAD2 and FAD2-like enzymes, including hydroxylases (14, 23),
epoxygenases (24), acetylenases (24), and
12-specific
conjugases (4) (Figs. 1 and 2).
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Fig. 1.
Comparison of amino acid sequences of
divergent forms of FAD2 from C. officinalis (CoFADX-1
and CoFADX-2) with a FAD2 desaturase from Arabidopsis
thaliana (AtDes) and FAD2
12-conjugases from M. charantia and I. balsamina
(McConj and IbConj,
respectively). Colons indicate amino acids that are
identical to those in the CoFADX-1 sequence. Gaps in the alignments are
maintained with dashes.
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Fig. 2.
Dendrogram of FAD2 and FAD2-related
polypeptides derived from alignment of amino acid sequences using the
ClustalX program. The distance along the horizontal
axis corresponds to the degree of sequence divergence. The
GenBankTM/EBI accession numbers of the amino acid sequences
represented in the phylogenetic tree are as follows: Ricinus
communis hydroxylase (RcOH), T09839;
Arachis hypogaea FAD2 desaturase (AhDES),
AAB84262; Glycine max FAD2 desaturase (GmDES),
P48630; C. officinalis FAD2 (CoFad2), CAB64256;
Crepis palaestina epoxygenase (CpEPOX), CAA76156;
Crepis alpina acetylenase (CaACET), CAA76158;
CoFADX-1, AF310155; CoFADX-2, AF310156; I. balsamina
12-conjugase (IbCONJ), AAF05915; M. charantia
12-conjugase (McConj),
AAF05916; A. thaliana FAD2 desaturase (AtDES), P46313;
Lesquerella fendleri hydroxylase (LfOH),
AAC32755; C. palaestina FAD2 desaturase (CpDES),
CAA76157; Petroselinum crispum FAD2 desaturase
(PcDES), T15042; Solanum commersonii FAD2
desaturase (ScDES), T10480; and Helianthus annuus
FAD2 desaturase (HaDES), T14269. CoFad2
corresponds to the CoDES sequence described by Fritsche et
al. (28).
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Fig. 3.
Northern blot analysis of the expression of
genes for CoFADX-1 and CoFADX-2. Radiolabeled probes
derived from cDNAs for CoFADX-1 and CoFADX-2 were hybridized to 20 µg of total RNA isolated from leaves (L) and developing
seeds (S) of C. officinalis as shown in
A. The ethidium bromide-stained gels corresponding to the
blots in A are shown in B. kb,
kilobase.
'-subunit of
-conglycinin (17). Soybean embryos transformed with
expression constructs for either CoFADX-1 or CoFADX-2 were found to
accumulate several fatty acids that were not detected in untransformed
embryos (Fig. 4). The methyl ester of the
most abundant of these fatty acids displayed a gas chromatographic
retention time identical to that of the calendic acid methyl ester in
extracts from C. officinalis seeds (Fig. 4). In addition,
the mass spectrum of this fatty acid methyl ester was identical to that
of methyl calendic acid and was characterized by an abundant molecular
ion at m/z = 292 (data not shown). To further
characterize the identity of this novel fatty acid in soybean embryos
transformed with CoFADX-1 or CoFADX-2, fatty acid methyl esters from
the transgenic embryos were reacted with MTAD and then analyzed by
GC-MS. This reagent readily forms Diels-Alder adducts with conjugated
trans,trans-double bonds (21). The product formed
from fatty acid methyl esters of the transgenic soybean embryos
displayed a mass spectral fragmentation pattern identical to that of
the Diels-Alder adduct of calendic acid methyl ester prepared from
C. officinalis seeds (Fig. 5).
As shown by the mass spectra in Fig. 5, the primary adduct detected
resulted from derivatization of the trans-
8-
and trans-
10-double bonds of the calendic
acid methyl ester, which is consistent with the properties of
Diels-Alder reactions (21). These data from transgenic soybean embryos
thus demonstrate that CoFADX-1 and CoFADX-2 are associated with the
formation of the conjugated trans-
8- and
trans-
10-double bonds of calendic acid.
View larger version (28K):
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Fig. 4.
Shown in A and B are the
results from gas chromatographic analyses of fatty acid methyl esters
prepared from an untransformed somatic soybean embryo and a transgenic
embryo expressing CoFADX-2, respectively. Shown in C is a
mixture of fatty acid methyl esters from seeds that accumulate
different isomers of C18 conjugated trienoic fatty acids.
This mixture includes fatty acid methyl esters from seeds of
Punica granatum, M. charantia, and C. officinalis. P. granatum seeds accumulate punicic acid
(18:3 9cis,11trans,13cis);
M. charantia seeds accumulate
-eleostearic acid
(18:3
9cis,11trans,13trans);
and C. officinalis seeds accumulate calendic acid
(18:3
8trans,10trans,12cis).
Peaks a and b in B were tentatively
identified as methyl esters of
18:3
8trans,10trans,12trans
and
18:4
8trans,10trans,12cis,15cis,
respectively. The labeled peaks correspond to methyl esters of the
following fatty acids: 16:0, palmitic acid; 18:0,
stearic acid; 18:1, oleic acid; 18:2, linoleic
acid; 18:3,
-linolenic acid; 20:0, eicosanoic
acid; and 22:0, docosanoic acid. R.T., retention
time.
View larger version (29K):
[in a new window]
Fig. 5.
Mass spectral analysis of Diels-Alder adducts
of the methyl ester of calendic acid from C. officinalis
seeds (A) and from transgenic somatic soybean
embryos expressing CoFADX-2 (B). Adducts were
prepared by reaction of fatty acid methyl esters with MTAD, which
preferentially reacts with the conjugated
trans- 8- and
trans-
10-double bonds of methyl calendic acid
as shown in A. A similar mass spectrum was obtained from
analysis of MTAD derivatives prepared from transgenic soybean embryos
expressing CoFADX-1 (data not shown).
8,trans-
10,trans-
12-isomer
of calendic acid. The mass spectrum of the fatty acid methyl ester
corresponding to peak b in Fig. 4B was
characterized by a prominent molecular ion at m/z = 290, which is consistent with that of a methyl 18:4 isomer. Based on
substrate feeding studies with S. cerevisiae cells
expressing CoFADX-1 or CoFADX-2 (described below), this fatty acid was
tentatively identified as
18:4
8trans,10trans,12cis,15cis,
resulting from the activity of these enzymes with
-linolenic acid.
These tentatively identified
18:3
8trans,10trans,12trans
and
18:4
8trans,10trans,12cis,15cis
isomers accounted for <0.5% (w/w) and <0.9% (w/w), respectively, of
the total fatty acids of the transgenic somatic soybean embryos. The
small amounts of these fatty acids in the transgenic plant tissues
limited more detailed characterization of their structures.
9cis) content of the transgenic embryos
relative to untransformed controls (Fig. 4 and Table I). This result is
in marked contrast to previous reports in which the production of
unusual fatty acids from FAD2-like enzymes in transgenic seeds was
accompanied by large increases in the relative amounts of oleic acid
(4, 13, 14). In addition to calendic acid production, the most notable effect on fatty acid composition of soybean embryos expressing CoFADX-2
was a decrease in linoleic acid content compared with untransformed
embryos (Table I). This alteration is consistent with linoleic acid
serving as the precursor of calendic acid as described below. Small
decreases in palmitic acid content were also observed in transgenic
embryos expressing either CoFADX-1 or CoFADX-2 (Table I).
Fatty acid composition of somatic soybean embryos from an untransformed
line and transgenic lines expressing cDNAs for CoFADX-1 and
CoFADX-2
9-double bond-modifying activity of
CoFADX-1 and CoFADX-2.
View larger version (20K):
[in a new window]
Fig. 6.
Gas chromatographic analyses of fatty acid
methyl esters from S. cerevisiae cells expressing
CoFADX-1 in medium containing linoleic or
-linolenic acid. The gas chromatograms shown
in A and C were derived from S. cerevisiae cells containing only the expression vector pESC-URA
and grown in medium supplemented with linoleic or
-linolenic acid,
respectively. The gas chromatograms shown in B and
D were derived from cells expressing the CoFADX-1 cDNA
in vector pESC-URA and grown in medium containing linoleic or
-linolenic acid, respectively. Shown in E are fatty acid
methyl esters prepared from C. officinalis seeds. The peak
labeled 18:4 in D was tentatively identified as
the methyl ester of
18:4
8trans,10trans,12cis,15cis.
Peaks labeled with numbers represent methyl esters of the
following fatty acids: peak 1, palmitic acid (16:0);
peak 2, palmitoleic acid (16:1
9cis);
peak 3, stearic acid (18:0); peak 4, oleic acid
(18:1
9cis); peak 5, linoleic acid
(18:2
9cis,12cis); and
peak 6,
-linolenic acid
(18:3
9cis,12cis,15cis).
Fatty acid composition of S. cerevisiae cells expressing the cDNA
for CoFADX-1 behind the GAL1 promoter in medium supplemented with
polyunsaturated fatty acids
-linolenic acid (+18:3), or both linoleic and
-linolenic acids (+18:2/18:3). The concentration of linolenic and
-linolenic acids in the medium was 0.7 mM in the +18:2
and +18:3 treatments. In the +18:2/18:3 experiment, the concentration
of each fatty acid in the medium was 0.35 mM. For each
experiment, the values shown are the means ± S.D. of the fatty
acid compositions of three independent cultures. Of note, no calendic
acid or 18:4 was detected in control cultures containing the pESC-URA
vector without cDNA insert.
9cis) found in
high levels in cells not provided with exogenous fatty acids.
-linolenic acid in the medium resulted in the
production of a novel fatty acid by cells expressing CoFADX-1 (Fig.
6D). The methyl ester of this fatty acid displayed a
retention time identical to that of peak b in
Fig. 4B in gas chromatograms of somatic soybean embryos
expressing the divergent C. officinalis FAD2 enzymes. In
addition, the mass spectrum of this fatty acid methyl ester contained
an abundant molecular ion at m/z = 290 (data not
shown), which is indicative of an 18:4 isomer. Furthermore, reaction of
this novel fatty acid methyl ester with MTAD resulted in the formation
of a Diels-Alder adduct (as determined by GC-MS) with a molecular ion
at m/z = 403 (data not shown), which is consistent with
an 18:4 isomer that contains conjugated double bonds. Given these
results and our demonstration that CoFADX-1 and CoFADX-2 convert the
cis-
9-double bond of linoleic acid into
trans-
8- and
trans-
10-double bonds, the 18:4 isomer formed
from
-linolenic acid
(18:3
9cis,12cis,15cis)
is probably
18:4
8trans,10trans,12cis,15cis.
This fatty acid accounted for ~1.6% (w/w) of the total fatty acids
of yeast cells expressing CoFADX-1 in the presence of exogenous
-linolenic acid (Table II). The 18:4 isomer was also detected under
similar growth conditions in S. cerevisiae cells expressing CoFADX-2 (data not shown).
-linolenic acids, cells expressing this enzyme were grown with both
fatty acids included in the medium. Although the fatty acids were
provided in equal concentrations,
-linolenic acid was incorporated by cells to amounts nearly twice that of linoleic acid (46%
versus 28% of the total fatty acids) (Table II). Despite
this difference, the accumulation of calendic acid (1.8% of the total
fatty acids), via activity of CoFADX-l with linoleic acid, was 2-fold
greater than the accumulation of 18:4 (0.9% of the total fatty acids), via activity of CoFADX-1 with
-linolenic acid (Table II). Similar results were obtained when this experiment was repeated with cells expressing CoFADX-2 (data not shown). These results thus suggest that
CoFADX-1 and CoFADX-2 are more active with linoleic acid than with
-linolenic acid. This observation is consistent with the higher
amounts of calendic acid versus 18:4 that accumulated in the
transgenic soybean embryos (Fig. 4B) and in yeast cells expressing CoFADX-1 in the presence of either linoleic acid or
-linolenic acid (Table II). Given the recalcitrant nature of FAD2-type enzymes during purification and in vitro assay
(6), determination of more detailed kinetic parameters such as
Km and Vmax for CoFADX-1 and
CoFADX-2 with linoleic and
-linolenic acids was not attempted.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
8trans,10trans,12cis),
an unusual conjugated trienoic fatty acid. Expression of cDNAs for
CoFADX-1 and CoFADX-2 in soybean somatic embryos or in S. cerevisiae cells was accompanied by the accumulation of calendic acid. Production of this fatty acid in S. cerevisiae cells
was dependent on the inclusion of linoleic acid in the medium. These results demonstrate that CoFADX-l and CoFADX-2 are FAD2 conjugases whose expression results in the conversion of the
cis-
9-double bond of linoleic acid into
trans-
8- and
trans-
10-double bonds. This activity thus
gives rise to the three conjugated double bonds
(trans-
8, trans-
10,
and cis-
12) found in calendic acid.
9-position of fatty acid substrates was unexpected given
that all previously described FAD2-type enzymes modify the
12-position of acyl chains. For example, FAD2 desaturase
enzymes introduce double bonds 12 carbons from the carboxyl end
of substrates that contain a
9-double bond (26, 27). A
similar regiospecificity is also displayed by FAD2 hydroxylases from
castor (23) and Lesquerella (14). In addition, FAD2-related
enzymes modify the cis-
12-double bond of
linoleic acid in the synthesis of epoxy groups (24), acetylenic bonds
(24), and conjugated
11,
13-double bonds
(4). Modification of the cis-
9-double bond of
linoleic and
-linolenic acids by CoFADX-1 and CoFADX-2 is thus an
unprecedented activity among FAD2-related enzymes. Based on the
mechanism proposed for conjugated fatty acid synthesis in red algae
(7), these enzymes probably function by removal of hydrogen atoms from
the
8- and
11-carbons of linoleic and
-linolenic acids (28). Given their unique catalytic properties, the
primary structures of CoFADX-1 and CoFADX-2 may provide useful
comparative information for understanding the structural basis of
regiospecificity in FAD2-type enzymes. In this regard, the amino acid
sequences of these enzymes contain several insertions and deletions
relative to other FAD2-type enzymes (Fig. 1). It is interesting to
speculate that these structural features may be associated with the
variant regiospecificities of CoFADX-1 and CoFADX-2.
12-conjugase (4, 13, 14). In
transgenic seeds and somatic soybean embryos that express these
enzymes, the accumulation of unusual fatty acids is typically
accompanied by 2-4-fold increases in the relative content of oleic
acid (4, 13, 14). In contrast to calendic acid, unusual fatty acids
such as ricinoleic acid (12-OH-18:1
9cis) and
-eleostearic acid
(18:3
9cis,11trans,13trans)
result from chemical modifications of the
12-position of
the C18 fatty acid chain. It is thus possible that unusual
fatty acids with modifications of the
12-position
directly or indirectly inhibit oleic acid desaturation on
phosphatidylcholine in transgenic seeds or embryos. This inhibition apparently does not occur or is more limited with the transgenic production of fatty acids that have similar modifications of the
9-position.
8trans,10trans,12cis,15cis.
Based on expression studies with S. cerevisiae, this fatty
acid results from the modification of the
9-double bond
of
-linolenic acid by CoFADX-1 or CoFADX-2 activity. To our
knowledge, the occurrence of 18:4 in Calendula seeds has not
been previously reported. The lack of detectable 18:4 production is
almost certainly due to the fact that
-linolenic acid typically composes <1% (w/w) of the total fatty acids of Calendula
seeds (Fig. 6E). Therefore, in contrast to somatic soybean
embryos, substrate pools of
-linolenic acid in Calendula
seeds are likely insufficient for 18:4 synthesis.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Maureen Dolan, Will Krespan, and others of DuPont Genomics for sequencing of cDNA libraries. We also thank Christine Hainey for assistance with cDNA library preparation; Bruce Schweiger, George Cook, and Christine Howells for transforming somatic soybean embryos; Kevin Stecca for providing vector pKS67; and Dr. Brian McGonigle and Rebecca Cahoon for critical reading of the manuscript.
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FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF310155 (CoFADX-1 cDNA) and AF310156 (CoFADX-2 cDNA).
To whom correspondence should be addressed: DuPont Experimental
Station, E402/4212, Wilmington, DE 19880-0402. Tel.: 302-695-4348; Fax:
302-695-8480; E-mail: Edgar.B.Cahoon@usa.dupont.com.
Published, JBC Papers in Press, November 6, 2000, DOI 10.1074/jbc.M009188200
1
The fatty acid nomenclature used in this work is
as follows. X:Y indicates that the fatty acid
contains X numbers of carbon atoms and Y numbers
of double bonds. z indicates that a double bond is located
at the zth carbon atom relative to the carboxyl end of the
fatty acid.
2 E. B. Cahoon, K. G. Ripp, S. E. Hall, and A. J. Kinney, unpublished results.
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ABBREVIATIONS |
---|
The abbreviations used are: EST, expressed sequence tag; PCR, polymerase chain reaction; GC-MS, gas chromatography-mass spectrometry; MTAD, 4-methyl-1,2,4-triazoline-3,5-dione.
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