From the Plantech Research Institute, 1000 Kamoshida-cho, Aoba-ku, Yokohama 227-0033, Japan
Received for publication, October 21, 2002, and in revised form, November 27, 2002
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Conjugated linolenic acids are present as
major seed oils in several plant species. Punicic acid (or trichosanic
acid) is a conjugated linolenic acid isomer containing
cis- A large number of fatty acid species have been found in plant seed
oils. Typically, plant seeds contain saturated and unsaturated fatty
acids, such as palmitic (16:0), palmitoleic
(16:1 Previous studies have indicated that linoleic acid is the acyl
precursor of It is known that Trichosanthes kirilowii and P. granatum accumulate punicic acid specifically in seeds up to ~40
and ~80% (w/w) of the total seed oil, respectively (11, 12). The
biochemical process resulting in high punicic acid accumulation in the
seeds of these plants, however, has not been clear. In this study, for the first step in understanding CLNA accumulation in plant seed oils,
we isolated cDNAs that encode enzymes associated with the formation
of punicic acid from T. kirilowii and
P. granatum and expressed them in Arabidopsis
plants. We also analyzed the function of these enzymes in yeast
cells. Interestingly the enzymes were demonstrated to possess both
FAD2-related cDNA Isolation--
Total RNA was isolated from
maturing seeds of T. kirilowii and P. granatum by
the methods of Carpenter et al. (13). The first strand
cDNA was synthesized with an oligo-dT primer and Superscript II
reverse transcriptase (Invitrogen) from the total RNA and used for PCR
amplification. To isolate cDNA fragments, degenerate primers were
designed to target conserved amino acid sequences in FAD2-related
enzymes. For T. kirilowii, a set of degenerate primers,
5'-TGYGGNCAYCAYGCNTTYAGYGAYTAYCART-3' (forward primer) and
5'-GGRTGNGTRTGYTGNARNKMNGT-3' (reverse primer) were used to target the
amino acid sequences CGHHAFSDYQ and T(Y/A)LQHTHP, respectively.
For P. granatum, 5'-TGYGGNCAYMRNGCNTTYWSNGAYTAYCAR-3' (forward primer) and 5'-KYNCCNCKNARCCARTYCCAYTC-3' (reverse primer) were used to target the amino acids CGH(H/R)AFSDYQ and
EW(D/N)WLRG(A/N), respectively. PCR amplification was performed with
TaKaRa Ex Taq (Takara Shuzo) with 30 cycles of 30 s at
94 °C, 1 min at 50 °C, and 40 s at 72 °C followed by an
extension step for 10 min at 72 °C. The amplified products (~0.5
kb) were cloned into pGEM-T Easy plasmid vector (Promega) and sequenced
using a PRISM DyeDeoxy Terminator Cycle Sequencing System (Applied
Biosystems). The sequence analysis revealed that two types of cDNA
fragments closely related to FAD2 were isolated in each experiment for
T. kirilowii (TkFac and TkFad2) and P. granatum (PgFac and PgFad2). The second strand cDNA was
synthesized with a Marathon cDNA Amplification kit
(Clontech). Adaptor ligation to the double strand
cDNAs and 5'- and 3'-rapid amplification of cDNA ends was
performed according to the manufacturer's protocol. cDNA fragments
containing 5' and 3' regions were cloned into pGEM-T Easy and
sequenced. Finally, full-length cDNAs of four FAD2-related
cDNAs were isolated by PCR amplification with Pyrobest DNA
polymerase (Takara Shuzo) using a set of primers corresponding to the
sequences in 5'- and 3'-untranslated regions. The PCR amplification
consisted of 25 cycles of 30 s at 94 °C, 1 min at 55 °C, and
2.5 min at 72 °C followed by an extension step for 10 min at
72 °C. The PCR product was incubated with TaKaRa Ex Taq
DNA polymerase (Takara Shuzo) for 10 min at 72 °C. The PCR products
from four independent amplifications for a set of primers were cloned
into pGEM-T Easy and sequenced as described above.
Expression of TkFac and PgFac in Arabidopsis thaliana--
The
coding regions of TkFac and PgFac were amplified by PCR using Pyrobest
DNA polymerase. For amplification of TkFac, the forward primer was
designed with a flanking BamHI site
(5'-taggatccATGGGAGGTTGGTGAAGGAATAG-3'), and the reverse primer
corresponded to the sequence 5-27 bp downstream from the stop codon of
TkFac with a flanking SacI site
(5'-atgagctcGATGATATCATGAAACCAAGAGG-3'). For amplification of PgFac,
the forward primer was designed with flanking
EcoRV/XbaI sites
(5'-tagatatctagaATGGGAGCTGATGGAACAATGTCTC-3'), and the reverse primer
corresponded to the sequence 7-33 bp downstream from the stop codon of
PgFac with a flanking SacI site
(5'-atgagctcGATATTAGGTTCGATTCTAATAAC-3'). The PCR amplification and
cloning into pGEM-T Easy were as described above. pGEM-T Easy/TkFac was
digested with BamHI and SacI and ligated at the
BamHI/SacI site of the pGEM-3Z plasmid vector
(Promega). To generate plant expression vectors with the CaMV 35S
promoter and nos terminator (pKS-TkFac and pKS-PgFac), the cDNA
sequences were released from pGEM-3Z/TkFac and from pGEM-T Easy/PgFac
with XbaHI/SacI digestion and then cloned into
the corresponding sites of a binary vector pLAN421 (14) in which the
GUS gene has been eliminated with
XbaI/SacI digestion. To generate plant expression vectors with a strong seed-specific promoter of the
Brassica napus napin gene (15) (pKN-TkFac and
pKN-PgFac), the XbaI-SacI fragments were cloned
into binary vector pNGKM (16) in which the GUS gene had been eliminated with XbaI/SacI digestion.
A. thaliana ecotype Columbia (Col-0) was transformed with
the conjugase construct by vacuum infiltration methods (17). Five plants per 20-cm2 pot were raised at 21 °C under
fluorescent illumination with a 16-h light/8-h dark cycle.
Agrobacterium strain EHA101 (pEHA) (18) harboring the
conjugase construct was cultured overnight at 30 °C in YEB medium
(0.1% yeast extract, 0.5% beef extract, 0.5% peptone, and 0.5%
sucrose, pH 7.0) supplemented with 50 µg/ml kanamycin, 25 µg/ml
chloramphenicol, 100 µg/ml spectinomycin, and 2.5 µg/ml
tetracycline the day before infiltration. Plants were dipped with
Agrobacterium suspension diluted to
A600 = 0.8 in dipping solution containing 0.02%
Silwet L-77 and 0.044 µM 6-benzylaminopurine for 15 min
under vacuum and then placed in a covered tray. The next day the plants
were uncovered, set upright, and allowed to grow for ~4 weeks in a
growth chamber under the conditions described above. When the siliques
were matured, seeds were harvested and planted for selection of
positive transformants. Seeds were surface-sterilized by immersion for
2 min in 70% ethanol followed by 15 min in 5% bleach solution
containing 1% SDS and then rinsed five times with sterile water.
Transformants were selected for by survival on Murashige-Skoog
medium containing 30 µg/ml kanamycin and 250 µg/ml carbenicillin.
Expression of FAD2-related Enzymes in Saccharomyces
cerevisiae--
The BamHI-SacI fragment of TkFac
cDNA released from pGEM-3Z/TkFac as described above was cloned into
the yeast expression vector pYES2 with the galactose-inducible
GAL1 promoter (Invitrogen) to generate pYES2/TkFac.
pGEM-T Easy/PgFac was digested with EcoRV/SacI and cloned into the plasmid vector pBluescript II (Stratagene), and
then the PgFac cDNA was released with
HindIII/SacI digestion and cloned into pYES2 to
generate pYES2/PgFac. The coding regions of TkFad2 and PgFad2 were
amplified by PCR using Pyrobest DNA polymerase and cloned into pGEM-T
Easy as described above. For amplification of TkFad2, the forward
primer was designed with a flanking SmaI site
(5'-atcccgggATGGAAAAGGGCGTTCAAGAGC-3'), and the reverse primer
corresponded to the sequence 1-24 bp downstream from the stop codon of
TkFad2 with a flanking SacI site
(5'-atgagctcCAATTTTGACTCAAGACAGAC-3'). For amplification of PgFad2, the
forward primer was designed with flanking
HindIII/XbaI sites
(5'-taaagctctagaATGGGAGCCGGTGGAAGAATGACGG-3'), and the reverse primer
corresponded to the sequence 4-29 bp downstream from the stop codon of
PgFad2 with a flanking KpnI site
(5'-atggtaccTTGCGACCAGCAATGTGGTAAATGG-3'). pGEM-T Easy/TkFad2 was
digested with SmaI/SacI, and the released cDNA fragment was cloned into the corresponding site of pBluescript II. The TkFad2 cDNA was then released with
HindIII/SacI digestion and cloned into pYES2. The
PgFad2 cDNA fragment was released from pGEM-T Easy by
HindIII/KpnI digestion and cloned into pYES2. The resulting plasmids as well as pYES2 were introduced into S. cerevisiae D452-2 cells (19) using an S. c.
EasyComp Transformation kit (Invitrogen). Transformed cells were
selected for on yeast synthetic minimal medium plates lacking uracil
(SC-URA) (20).
Individual colonies of the transformed cells were grown in glucose
culture medium (SC-URA/2% glucose) for 1 day at 28 °C with shaking.
Cells were then collected by centrifugation, washed in sterilized
water, and dissolved in galactose culture medium (SC-URA/2% galactose). The cell suspension was diluted to
A600 = 0.2 in galactose culture medium
containing 0.1% (w/v) Tergitol-type Nonidet P-40 (Sigma) and grown at
20 °C for 3 days followed by 15 °C for 3 days with shaking in the
presence or absence of 0.3 mM linoleic, Fatty Acid Analysis--
Yeast cells were harvested by
centrifugation and then washed twice in 1% (w/v) Tergitol solution and
three times in distilled water. The washed cells were aliquoted into
glass tubes and freeze-dried under vacuum. The dried cell pellets were
then incubated with 0.5 M sodium methoxide in methanol at
50 °C for 1 h. After the tubes were cooled to room temperature,
the mixture was extracted with hexane. The pooled extracts were dried
under vacuum and then dissolved in a small volume of hexane, and 1 µl
was used for gas chromatography (GC) or GC-mass spectrometry (MS)
analysis. For fatty acid analysis of Arabidopsis materials,
seeds (2 mg) were ground with a mortar and pestle and then added to 1 ml of 0.5 M sodium methoxide in methanol. Leaf tissues (4 mg) were homogenized in 1 ml of 0.5 M sodium methoxide in
methanol. For T. kirilowii and P. granatum, 2 mg
of seeds without seed coats were used for fatty acid extraction. The
homogenates were transferred to glass tubes and incubated at 50 °C
for 1 h. After the tubes were cooled to room temperature, 1.5 ml
of 0.9% (w/v) sodium chloride was added to the samples, and fatty acid
methyl esters were extracted with 1 ml of hexane. After centrifugation
at 1,000 × g for 5 min, the hexane layer was
transferred to a new tube and dried under vacuum. The dried samples
were dissolved in a small volume of hexane, and 1 µl was used for GC
or GC-MS analysis. Fatty acid methyl esters were analyzed and
quantified using a gas chromatograph (GC18A, Shimadzu) equipped with a
TC-70 fused silica column (60 m × 0.25-mm inner diameter, 0.25-mm
film thickness; GL Science). The oven temperature was programmed to
rise from 150 °C to 240 °C at a rate of 3 °C/min and then hold
for 6 min. GC-MS analysis was performed in standard EI mode
using a JMS-600H MSroute mass spectrometer (JEOL) coupled to a 6890 Series gas chromatograph (Agilent). Samples were separated under
conditions as described above.
Isolation of cDNAs Encoding FAD2-related Enzymes--
Total
fatty acids extracted from maturing seeds of T. kirilowii
and P. granatum were analyzed by GC. Both seeds contained punicic acid at levels of more than ~40% (w/w) (T. kirilowii) and ~70% (w/w) (P. granatum) of total
fatty acids (see Figs. 2D and 4D). We prepared
RNA from these materials and isolated four cDNAs from T. kirilowii (TkFad2 and TkFac) and P. granatum (PgFad2 and PgFac) that encode polypeptides related to FAD2. These polypeptides contain three clusters of histidine residues that are thought to act as
ligands to the catalytic iron atoms that have been proposed to form a
di-iron oxo group (21). Comparison of amino acid sequences of
FAD2-related fatty acid-modifying enzymes (GenBankTM
accession numbers: hydroxylase, T09839 and AAC32755; epoxygenase,
CAA76156; acetylenase, CAA76158; and conjugase, AAF05915, AAF05916,
AAG42259, AAG42260, and AAK26632) with Production of Punicic Acid in A. thaliana--
To examine whether
TkFac and PgFac are associated with the formation of punicic acid, the
full-length cDNAs encoding TkFac and PgFac were expressed in
Arabidopsis plants under control of the constitutive CaMV
35S promoter (pKS-TkFac and pKS-PgFac) or the seed-specific napin
promoter (pKN-TkFac and pKN-PgFac). Transformants (T1) were selected by
drug resistance and by PCR, and then fatty acid methyl esters (FAMEs)
from leaves of T1 plants were analyzed by gas chromatography. Although
there was no difference in the fatty acid composition of vegetative
tissues between transgenic and untransformed plants (data not shown), a
prominent peak of FAME (Fig. 2,
B and C, arrows), which was not
present in seeds from untransformed plant, was found in seeds of
transgenic plants. The peak displayed a gas chromatographic retention
time identical to that of the methyl ester of punicic acid from
T. kirilowii (Fig. 2D) and different from those
of
In all transgenic plants, punicic acid was detected in seeds but not in
vegetative tissues (data not shown). We compared the fatty acid
composition of seed oils in transgenic and untransformed plants (Table
I). Maximal concentration of punicic acid
in seed oils was 10.2% (w/w) in pKN-TkFac transformants and 4.4%
(w/w) in pKN-PgFac transformants (data not shown). The average
concentration of punicic acid was higher in seeds from pKN-TkFac and
pKN-PgFac transformants carrying the napin promoter, 3.5 and 2.3%
(w/w), respectively. In contrast, concentrations in pKS-TkFac and
pKS-PgFac transformants were ~0.4% (w/w). The concentration of the
fatty acid tentatively identified as
20:3 Functional Analyses of FAD2-related Enzymes in S. cerevisiae--
To further characterize the function of the
FAD2-related polypeptides, each full-length cDNA was expressed
under control of the GAL1 promoter in yeast cells.
First, each FAD2-related polypeptide was expressed in yeast cells grown
in culture medium without exogenous fatty acids, and fatty acid
compositions were compared with control yeast cells transformed with
pYES2. In yeast cells expressing TkFad2 and PgFad2, two peaks of FAMEs
were detected that were not present in those of pYES2-transformed
control cells (Fig. 3 and data not
shown). These were determined to be methyl esters of
16:2
In addition to 16:2
Lastly, yeast cells expressing TkFac and PgFac were grown with
exogenous In this study we isolated cDNAs encoding enzymes (TkFac and
PgFac) involved in the formation of conjugated trans- Punicic acid accumulation was examined in transgenic
Arabidopsis plants. All transgenic plants accumulated
punicic acid in seeds (Table I), but not in leaves, even when the
conjugases were driven under control of the CaMV 35S promoter (data not
shown). A similar observation has been reported in
Arabidopsis transformed with Expression of the conjugases under control of either the CaMV S35 or
napin promoter led to accumulation of punicic acid in seeds (Table I).
The concentration of punicic acid in seeds from individual
transformants varied from less than 1% to ~10% (data not shown).
Accompanying punicic acid accumulation, several changes in fatty acid
composition were observed (Table I). Oleic acid (18:1) increased in
concentration (Table I) to a remarkable level in transgenic seeds with
~10% (w/w) punicic acid, reaching a 1.8-fold higher concentration
than in untransformed seeds. In contrast, linoleic (18:2) and
The substrate specificity of the conjugases was revealed by their
expression in yeast cells.
16:2 Fatty acids with conjugated double bonds have been studied in
therapeutic medical applications, and a large number of beneficial effects of dietary supplementation with conjugated linoleic acid (CLA)
have been reported. CLA has been shown to reduce the incidence of
mammalian tumors in mice, inhibit the proliferation of cancer cells in
culture (31-33), and reduce body fat in rodents and humans (34-37).
Recent studies have shown that CLNAs are more cytotoxic to tumor cells
than is CLA (2). In addition, perirenal and epididymal adipose tissues
are reduced in rats fed free fatty acids rich in CLNAs (4). CLNAs have
been suggested to modulate body fat and triacylglycerol metabolism
differently than CLA (4), but the exact mechanism of the
anticarcinogenic and anti-obese action of CLA and CLNA remains unknown.
In addition to increasing our understanding of fatty acid biochemistry,
identification of conjugases may lead to the discovery of methods to
produce CLNAs in common oil crops.
9, trans-
11, cis-
13
double bonds in the C18 carbon chain. Here we report
cDNAs, TkFac and PgFac, isolated from Trichosanthes
kirilowii and Punica granatum, that encode a class of
conjugases associated with the formation of trans-
11,
cis-
13 double bonds. Expression of TkFac and PgFac in
Arabidopsis seeds under transcriptional control of the
seed-specific napin promoter resulted in accumulation of punicic acid
up to ~10% (w/w) of the total seed oils. In contrast, no punicic
acid was found in lipids from leaves even when the conjugases were
driven under control of the cauliflower mosaic virus 35S promoter. In
yeast cells grown without exogenous fatty acids in the culture medium,
TkFac and PgFac expression resulted in punicic acid accumulation
accompanied by 16:2
9cis, 12cis
and 18:2
9cis, 12cis production.
Thus, TkFac and PgFac are defined as bifunctional enzymes having both
conjugase and
12-oleate desaturase activity. Furthermore, we
demonstrate that
16:2
9cis, 12cis and
18:3
9cis, 12cis, 15cis
as well as 18:2
9cis, 12cis are
potential substrates for the conjugases to form trans-
11 and cis-
13 double bonds.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
9cis), stearic (18:0), oleic
(18:1
9cis), linoleic
(18:2
9cis, 12cis), and
-linolenic
(18:3
9cis, 12cis, 15cis)
acids. These are typical fatty acids with all other fatty acids regarded as unusual. Typically, polyunsaturation of fatty acids is
methylene (-CH2-)-interrupted and occurs in
cis-configuration as found in linoleic and linolenic acids.
In contrast, conjugated (non-methylene-interrupted) fatty acids contain
double bonds in cis- or trans-configuration. The
conjugated fatty acids occur as diene, triene, and tetraene in which
the most common conjugated polyenoic acids are octadecatrienoic acids,
termed CLNAs.1 Positional and
geometrical isomers of CLNA, three 8,10,12-trienes and four
9,11,13-trienes, have been reported to occur naturally (1). Five CLNA
isomers occur as major seed oils of several plants:
-eleostearic
(cis-
9, trans-
11, trans-
13),
calendic (trans-
8, trans-
10,
cis-
12), punicic (cis-
9,
trans-
11, cis-
13), jacaric
(cis-
8, trans-
10, cis-
12),
and catalpic (trans-
9, trans-
11,
cis-
13) acids (1). These isomers have closely related structure; for example,
-eleostearic and jacaric acids are
geometrical isomers of punicic and calendic acids, respectively. CLNAs
are major seed oils in plants such as tung (Aleurites
fordii), karela (Momordica charantia), marigold
(Calendula officinalis), and pomegranate (Punica
granatum). Tung oil contains high levels of 9,11,13-triene (
-eleostearic acid) and is used mainly in quick-drying enamels and
varnishes. There also is growing evidence showing that supplementation with CLNA has cytotoxic effects on tumor cells and that uptake of CLNA
has an effect on lipid metabolism (2-4). Conjugated eicosapentaenoic and docosahexaenoic acids with conjugated trienoic structure also exhibit cytotoxic effects on tumor cells (5). It has been further suggested that the biological action of each conjugated fatty acid may
not be equivalent (3).
-eleostearic acid and linoleoyl
phosphatidylcholine is the precursor of
-eleostearoyl
phosphatidylcholine (6). Recently cDNAs encoding enzymes that
catalyze the formation of the conjugated double bonds in CLNA have been
identified. These enzymes were termed conjugases and were shown to be
divergent forms of
12-oleate desaturase (FAD2). Two types of
conjugases associated with the formation of conjugated double bonds in
trans-configuration have been identified: one catalyzes the
conversion of a cis-
12 double bond into the conjugated
trans-
11, trans-
13 double bonds found in
-eleostearic acid (7), and the other modifies a cis-
9 double bond into the trans-
8, trans-
10
double bonds of calendic acid (8, 9). Very recently a class of
conjugase associated with the formation of cis-
11,
trans-
13 double bonds of punicic acid was identified
(10). Although the primary structures of these conjugases are similar,
each enzyme specifically catalyzes the formation of
-eleostearic,
punicic, or calendic acids. Other classes of conjugases associated with
the formation of conjugated double bonds in jacaric and catalpic
acids have not been reported.
12-oleate desaturase and conjugase activities. The finding supports
the idea that conjugases have diverged from
12-oleate desaturase.
The growing number of identified primary structures of fatty
acid-modifying enzymes provides valuable information to understand the
functional divergence of these enzymes.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-linolenic, or
11,14-eicosadienoic acid
(20:2
11cis, 14cis).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
12-oleate desaturases (GenBankTM accession numbers: P46313, T14269, CAA76157,
AAK26633, and AAF78778) revealed that several amino acids at certain positions were strictly conserved in
12-oleate desaturases from a
number of plant species. As these amino acids were conserved in TkFad2
and PgFad2, we supposed that these were
12-oleate desaturases (Fig.
1A). On the other hand, the
phylogenetic analysis indicated that TkFac and PgFac were grouped
within a conjugase branch (Fig. 1B), suggesting that they
were conjugases. TkFad2 and PgFad2 encode 369 and 387 amino acids and
share 63-68 and 68-71% amino acid identity, respectively, with the
12-oleate desaturases. On the other hand, TkFac and PgFac encode 387 and 395 amino acids and share 59-62 and 55-58% amino acid identity
to
12-oleate desaturases, respectively. PgFac shares 42-54%
identity to other conjugases (MomoFadX from M. charantia
(7), ImpFadX from Impatiens balsamina (7), and
CoFac2/CoFADXs from C. officinalis (8, 9)). TkFac shares
sequence identity of 45% to CoFac2/CoFADXs, 55% to ImpFADX, and 55%
to PgFac but 74% to MomoFadX. The higher sequence identity between
TkFac and MomoFadX may reflect that the two species belong to the same
family (Cucurbitaceae).
View larger version (60K):
[in a new window]
Fig. 1.
Sequence analyses of FAD2-related proteins
from T. kirilowii and P. granatum. A, comparison of amino acid
sequences of FAD2-related proteins from T. kirilowii
(TkFad2 and TkFac) and P. granatum
(PgFad2 and PgFac) with FAD2 ( 12-oleate
desaturase) of A. thaliana (AtFad2). Three
clusters of histidine residues are indicated by bars. Amino
acids identical to those in the TkFac sequence are indicated by
shading. Gaps in alignments are indicated by
dashes. B, phylogenetic analysis of FAD2 and
conjugase proteins using the ClustalW program. The distance along the
horizontal axis corresponds to the extent of sequence divergence.
Sequences were obtained from GenBankTM accession numbers:
ImpFadX, I. balsamina conjugase, AAF05915.1;
CoFadX1, CoFadX2, and CoFac2, C. officinalis conjugases, AAG42259, AAG42260, and AAK26632;
MomoFadX, M. charantia conjugase, AAF05916.1;
CpFad2, Crepis palaestina
12-oleate
desaturase, CAA76157.1; CoFad2, C. officinalis
12-oleate desaturase, AAK26633; and AtFad2, A. thaliana
12-oleate desaturase, P46313.
-eleostearic and calendic acids extracted from M. charantia and C. officinalis seeds, respectively (data
not shown). The mass spectrum of this FAME was identical to that of the
methyl punicic acid from T. kirilowii: the molecular ion was
m/z = 292, which was identified as a methyl
ester of an isomer of octadecatrienoic acid (18:3), and other
diagnostic ions were clearly observed (data not shown). From these
results, we concluded that TkFac and PgFac are "conjugases"
associated with the formation of the conjugated trans-
11,
cis-
13 double bonds of punicic acid. In addition to the
peak of methyl punicic acid, an additional small peak of FAME (Fig. 2,
B and C, arrowheads), which was not
found in seeds from untransformed plants, was detected in those from
transgenic plants. The mass spectrum of this FAME was characterized by
an abundant molecular ion at m/z = 320 and other diagnostic ions, which was identified as a methyl ester of an
isomer of eicosatrienoic acid (20:3). This peak displayed a gas
chromatographic retention time different from that of the methyl
20:3
11cis, 14cis, 17cis
present in both untransformed and transformed Arabidopsis
seeds (Fig. 2). We analyzed FAMEs of yeast cells fed with
20:2
11cis, 14cis to test whether
this fatty acid was utilized as a substrate of TkFac and PgFac to
produce conjugated 20:3 isomer. However, we could not find a peak
corresponding to the methyl 20:3 isomer found in Arabidopsis
seeds (data not shown). We supposed that a part of punicic
acid was elongated to form the conjugated 20:3 (20:3
11cis, 13trans, 15cis)
as Arabidopsis seeds possess microsomal fatty acid elongase activity (22, 23).
View larger version (21K):
[in a new window]
Fig. 2.
Gas chromatographic analyses of
FAMEs from transgenic Arabidopsis seeds. FAMEs
from seeds of an untransformed Arabidopsis plant
(A), from those of a T1 plant transformed with TkFac
(B) and PgFac (C), and from T. kirilowii seeds (D) were analyzed by GC. The labeled
peaks corresponding to methyl esters of fatty acids are as follows:
16:0, palmitic acid; 18:0, stearic acid; 18:1, oleic acid; 18:2,
linoleic acid; 18:3, -linolenic acid; 20:0, eicosanoic (arachidic)
acid; 20:1, eicosenoic (gondoic) acid; 20:2, eicosadienoic acid; 20:3,
eicosatrienoic acid; 22:0, docosanoic (behenic) acid; and 22:1,
docosenoic (erucic) acid. Arrows, methyl punicic acid;
arrowheads, a methyl ester of 20:3 isomer, which was
tentatively identified as
20:3
11cis, 13trans, 15cis.
11cis, 13trans, 15cis
was in proportion to that of punicic acid (Table I) and accumulated to
as much as 1% (w/w) of seed oils in the T2 seeds accumulating 10.2%
(w/w) punicic acid (data not shown). Punicic acid accumulation in
Arabidopsis seeds was accompanied by changes in relative
amounts of other fatty acids (Table I). The change was remarkable in seeds of transformants with cDNAs driven by the napin promoter. Relative amounts of linoleic
(18:2
9cis, 12cis) and linolenic
acids
(18:3
9cis, 12cis, 15cis),
which were 30% (w/w) and 19% (w/w) in untransformed seeds, were
significantly lower in T2 seeds, at 23-24 and 12-15% (w/w), respectively. In contrast, the concentration of oleic acid was higher
in seeds of transgenic plants (23-26% (w/w)) than in those of
untransformed plants (15% (w/w)).
Fatty acid composition of Arabidopsis seeds from untransformed plants
and from transgenic plants expressing TkFac and PgFac
9cis, 12cis and
18:2
9cis, 12cis by gas
chromatographic retention time (Fig. 3) and mass spectral analyses
(data not shown). We could not detect the peak corresponding to the
methyl punicic acid in Fad2-expressing cells, although a large amount
of linoleic acid was present (~19% (w/w) of the total fatty acids,
Fig. 3B and data not shown). We concluded that TkFad2 and
PgFad2 were
12-oleate desaturases. Interestingly, yeast cells
expressing PgFac produced
16:2
9cis, 12cis (~1% (w/w))
and 18:2
9cis, 12cis (~0.2%
(w/w)), which were also found in TkFac-expressing cells although in
lower concentrations of ~0.2% (w/w) and ~0.1% (w/w), respectively
(Fig. 3, C and D). Although TkFac and PgFac were demonstrated to be the enzymes associated with punicic acid formation in plants, these enzymes were shown to possess
12-oleate desaturase activity in addition to their conjugase activity.
View larger version (19K):
[in a new window]
Fig. 3.
Gas chromatographic analyses of FAMEs from
yeast cells expressing FAD2-related enzymes that were grown in the
absence of exogenous fatty acids. FAMEs from pYES2-transformed
(A), TkFad2-transformed (B), TkFac-transformed
(C), and PgFac-transformed cells (D) were
analyzed by GC. The labeled peaks corresponding to methyl esters of
fatty acids are as follows: 16:0, palmitic acid; 16:1, palmitoleic
acid; 16:2, hexadecadienoic acid
( 9cis, 12cis); 18:0, stearic
acid; 18:1, oleic acid; and 18:2, linoleic acid. Arrow,
methyl punicic acid; arrowhead, a methyl ester of 16:3
isomer, which was tentatively identified as
16:3
9cis, 11trans, 13cis.
9cis, 12cis
and 18:2
9cis, 12cis, two novel
peaks of FAMEs (Fig. 3D, arrow and
arrowhead) were found in PgFac-expressing cells grown in the
absence of exogenous fatty acids. The peak indicated by the
arrow was determined to be methyl punicic acid by GC-MS analysis (data not shown) and by gas chromatographic retention time
(Fig. 4D). Relative amounts of
punicic acid produced in yeast cells grown with linoleic acid were
greater than in those grown without linoleic acid (Figs. 3 and 4). With
the addition of 0.3 mM linoleic acid to the medium, punicic
acid was produced at concentrations of ~0.1 and ~0.8% (w/w) of the
total fatty acids of TkFac- and PgFac-expressing cells, respectively
(Fig. 4, B and C). In contrast, no or a very
small amount of punicic acid was produced in TkFac- or PgFac-expressing
cells grown without exogenous linoleic acid (Fig. 3, C and
D). Another peak indicated by an arrowhead (Fig. 3) was also found in yeast cells grown with linoleic acid (Fig. 4C, arrowhead). This was characterized by a
molecular ion at m/z = 264 and other
diagnostic ions consistent with that of a methyl ester of
hexadecatrienoic acid (16:3) but showed a different gas chromatographic
retention time from
16:3
9cis, 12cis, 15cis
(data not shown). The relative amount of
16:2
9cis, 12cis produced in
PgFac-transformed cells was smaller in the cells grown with exogenous
linoleic acid than in those grown without linoleic acid probably
because of incorporation of linoleic acid from the medium into the
lipid components of the cells instead of
16:2
9cis, 12cis (Fig. 4).
Accompanying the 16:2
9cis, 12cis
reduction in cells fed linoleic acid, the amount of the 16:3 isomer was
also reduced. This result suggests that the isomer of 16:3 was
16:3
9cis, 11trans, 13cis
specifically produced by PgFac expression. We detected a small amount
of FAMEs of the 16:3 isomer and punicic acid through analysis of a
large amount of FAMEs extracted from TkFac-expressing cells grown
without exogenous fatty acids (data not shown). Therefore, we concluded
that TkFac and PgFac are similar in function to each other.
View larger version (19K):
[in a new window]
Fig. 4.
Gas chromatographic analyses of FAMEs from
yeast cells expressing TkFac and PgFac that were grown in the presence
of exogenous linoleic acid. FAMEs from pYES2-transformed
(A), TkFac-transformed (B), and PgFac-transformed
cells (C) were analyzed by GC. Similarly, FAMEs from
P. granatum seeds were analyzed for reference
(D). The labeled peaks corresponding to methyl esters of
fatty acids are as follows: 16:0, palmitic acid; 16:1, palmitoleic
acid; 16:2, hexadecadienoic acid
( 9cis, 12cis); 18:0, stearic
acid; 18:1, oleic acid; and 18:2, linoleic acid. Arrows,
methyl punicic acid; arrowhead, a methyl ester of 16:3
isomer, which was tentatively identified as
16:3
9cis, 11trans, 13cis.
-linolenic acid, and FAMEs of these cells were compared with those of control cells by gas chromatography. We detected a novel
although very small peak of FAME, which was not found in cells grown
with linoleic acid (data not shown). By mass spectral analysis, the
FAME was identified as a methyl ester of octadecatetraenoic acid
(18:4), which was characterized by a molecular ion at
m/z = 290 and other diagnostic ions (data
not shown). Taken together the conjugases were shown to have the
ability to form conjugated trienoic fatty acids of
C16 and C18 carbon chains and the
conjugated tetraenoic fatty acid of the C18 carbon chain.
No conjugated dienoic fatty acids were detected in cells grown without
exogenous fatty acid in the medium (Fig. 3) or with oleic acid (data
not shown). These results suggest that TkFac and PgFac do not modify
the cis-
9 double bonds of oleic and palmitoleic acids.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
11,
cis-
13 double bonds of punicic acid. Very recently
cDNA encoding PuFADX, an enzyme identical to PgFac, was isolated
from P. granatum (10). We have found a notable feature of
TkFac and PgFac: they have bifunctional enzymatic activity. Yeast cells
transformed with TkFac and PgFac accumulated
16:2
9cis, 12cis and
18:2
9cis, 12cis (Fig. 3). TkFac
and PgFac were thus demonstrated to have both
12-oleate desaturase
and conjugase activities. This observation supports the idea that a
number of fatty acid-modifying enzymes have diverged from fatty acid
desaturases (7, 24, 25). LFAH12 of Lesquerella fendleri has
been shown to be a bifunctional enzyme having both
12-oleate
hydroxylase and
12-oleate desaturase (FAD2) activities (26).
Replacement of seven amino acids in LFAH12 with ones strictly conserved
in FAD2 changed the catalytic preference of the enzyme from hydroxylase
to desaturase (27). It is thus possible that a small number of amino
acids determine the enzymatic specificity of conjugases. Punicic acid
and
-eleostearic acid are closely related isomers in which a double
bond at the
13 position is in cis- or
trans-configuration. Comparison of amino acid sequences of
the conjugases revealed that several amino acids conserved in MomoFADX
and ImpFADX (which form conjugated trans-
11,
trans-
13 double bonds) were replaced in TkFac and PgFac
(which form conjugated trans-
11, cis-
13
double bonds). It is likely that several specific amino acids may be
involved in the divergent functions of these enzymes.
12-oleate hydroxylase (26).
In nature, seed-specific accumulation of unusual fatty acids results
from seed-specific expression of their anabolic enzymes (7-9, 24, 26).
It is not known whether conjugated fatty acids are eliminated from
lipids in vegetative tissues because of their toxicity to cell
function. It has been suggested that hydroxy fatty acids are broken
down at a higher rate than typical fatty acids in vegetative tissues
(26). Ectopic accumulation of very long chain fatty acids in vegetative
tissues was accompanied by abnormalities in cell growth and chloroplast membrane integrity (28).
-linolenic acid (18:3) decreased from 30% (w/w) and 20% (w/w) in
untransformed seeds (Table I) to 19% (w/w) and 8% (w/w) in transgenic
seeds with 10% (w/w) punicic acid, respectively. A similar effect,
suppression of
12-oleate desaturase, was reported in plants
transformed with
12-acyl-modifying enzymes. In
Arabidopsis seeds expressing
12-oleate hydroxylase (26, 29) and
12-linoleate epoxygenase (30) and in somatic soybean embryos expressing conjugases (7), accumulation of modified fatty acids
was accompanied by a marked increase in oleic acid. A recent study
suggested that exogenously expressed
12-acyl-modifying enzymes lead
to an increase in oleic acid by interfering with endogenous
12-oleate desaturase at the translational or post-translational levels (30). In transgenic Arabidopsis seeds, a novel fatty acid occurred that was identified as an isomer of 20:3 by GC-MS analysis. We analyzed the FAMEs of PgFac-expressing yeast cells grown
with 20:2
9cis, 12cis but did not
find the peak corresponding to the 20:3 isomer (data not shown).
Therefore, it is unlikely that the 20:3 isomer resulted from
conjugase-mediated direct conversion of
20:2
9cis, 12cis to conjugated
20:3. Alternatively, as Arabidopsis seeds contain polyunsaturated very long chain fatty acids (Fig. 2), in contrast to
T. kirilowii and P. granatum which accumulate
neither very long chain fatty acids nor
-linolenic acid (Figs.
2D and 4D), it is possible that the 20:3 isomer
resulted from fatty acid elongation of punicic acid by the activity of
microsomal fatty acid elongase (22, 23).
9cis, 12cis occurred in yeast
expressing the conjugases and could be converted into the 16:3 isomer,
which was proposed to be a conjugated fatty acid containing
cis-
9, trans-
11, cis-
13
double bonds (Figs. 3D and 4C). Thus, the
conjugases can utilize fatty acids with C16 carbon
chains as substrates as well as those with C18 carbon chains. In addition, we analyzed the FAMEs in PgFac-expressing yeast
cells grown with
-linolenic acid
(18:3
9cis, 12cis, 15cis)
and found a novel peak representing a methyl ester of 18:4 isomer, which was proposed to be
18:4
9cis, 11trans, 13cis, 15cis.
By GC-MS analysis of FAMEs from transgenic Arabidopsis
seeds, we also found a small amount of methyl ester of 18:4 isomer,
which had a chromatographic retention time identical to that of the 18:4 isomer found in yeast cells (data not shown). In
Arabidopsis seeds,
-linolenic acid composes ~10% (w/w)
of the total seed oils but, in contrast, is absent in seed oils of
T. kirilowii and P. granatum. In addition,
transgenic studies using yeast cells have demonstrated that
-linolenic acid is a possible substrate for conjugases associated
with the formation of both trans-
11, trans-
13 double bonds and trans-
8,
trans-
10 double bonds (7-9). In our study, yeast cells
accumulated large amounts of 16:1
9cis and
18:1
9cis; however, no novel dienoic fatty
acids were found in TkFac- and PgFac-expressing cells (Figs. 3 and 4).
This result indicates that these enzymes exclusively modify a
12
double bond in contrast to CoFac2/CoFADXs, which modifies a
9 double
bond to produce conjugated trans-
8,
trans-
10 double bonds (8, 9).
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Rieko Hakoda for GC analysis and production of transgenic plants. We also thank Yasuyuki Hayashi for providing the yeast D452-2 strain, Hideya Fujimoto for providing T. kirilowii, and others of Plantech Research Institute for help.
![]() |
FOOTNOTES |
---|
* This work was supported by the Research and Development Program for New Bio-industry Initiatives of the Bio-oriented Technology Research Advancement Institution (BRAIN).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/EBI Data Bank with accession number(s) AY178444 (TkFac cDNA), AY178445 (TkFad2 cDNA), AY178446 (PgFac cDNA), and AY178447 (PgFad2 cDNA).
To whom correspondence should be addressed. Tel.: 81-45-963-3520;
Fax: 81-45-962-7492; E-mail: 5505276@cc.m-kagaku.co.jp.
Published, JBC Papers in Press, December 2, 2002, DOI 10.1074/jbc.M210748200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
CLNA, conjugated
linolenic acid;
CaMV, cauliflower mosaic virus;
FAD2, 12-oleate
desaturase;
GC, gas chromatography;
MS, mass spectrometry;
FAME, fatty
acid methyl ester;
CLA, conjugated linoleic acid.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Smith, C. R., Jr. (1970) Prog. Chem. Fats Other Lipids 11, 137-177 |
2. | Igarashi, M., and Miyazawa, T. (2000) Cancer Lett. 148, 173-179[CrossRef][Medline] [Order article via Infotrieve] |
3. | Suzuki, R., Noguchi, R., Ota, T., Abe, M., Miyashita, K., and Kawada, T. (2001) J. Am. Oil Chem. Soc. 36, 477-482 |
4. | Koba, K., Akahoshi, A., Yamasaki, M., Tanaka, K., Yamada, K., Iwata, T., Kamegai, T., Tsutsumi, K., and Sugano, M. (2002) J. Am. Oil Chem. Soc. 37, 343-350 |
5. | Igarashi, M., and Miyazawa, T. (2000) Biochem. Biophys. Res. Commun. 270, 649-656[CrossRef][Medline] [Order article via Infotrieve] |
6. |
Liu, L.,
Hammond, G.,
and Nikolau, B. J.
(1997)
Plant Physiol.
113,
1343-1349 |
7. |
Cahoon, E. B.,
Carlson, T. J.,
Ripp, K. G.,
Schweiger, B. J.,
Cook, G. A.,
Hall, S. E.,
and Kinney, A. J.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
12935-12940 |
8. |
Cahoon, E. B.,
Ripp, K. G.,
Hall, S. E.,
and Kinney, A. J.
(2001)
J. Biol. Chem.
276,
2637-2643 |
9. |
Qiu, X.,
Reed, D. W.,
Hong, H.,
MacKenzie, S. L.,
and Covello, P. S.
(2001)
Plant Physiol.
125,
847-855 |
10. |
Hornung, E.,
Pernstich, C.,
and Feussner, I.
(2002)
Eur. J. Biochem.
269,
4852-4859 |
11. | Joh, Y.-G., Kim, S.-J., and Christie, W. (1995) J. Am. Oil Chem. Soc. 72, 1037-1042 |
12. | Takagi, T., and Itabashi, Y. (1981) Lipids 16, 546-551 |
13. | Carpenter, C. D., and Simon, A. (1998) Methods Mol. Biol. 82, 85-89[Medline] [Order article via Infotrieve] |
14. | Uematsu, C., Murase, M., Ichikawa, H., and Imamura, J. (1991) Plant Cell Rep. 10, 286-290 |
15. |
Josefsson, L.-G.,
Lenman, M.,
Ericson, M. L.,
and Rask, L.
(1987)
J. Biol. Chem.
262,
12196-12201 |
16. | Kohno-Murase, J., Murase, M., Ichikawa, H., and Imamura, J. (1994) Plant Mol. Biol. 26, 1115-1124[Medline] [Order article via Infotrieve] |
17. | Bechtold, N., Ellis, J., and Pelletier, G. (1993) Life Sci. 316, 1194-1199 |
18. | Hood, E. E., Helmer, G. L., Fraley, R. T., and Chilton, M.-D. (1986) J. Bacteriol. 168, 1291-1301[Medline] [Order article via Infotrieve] |
19. | Matsushita, M., and Nikawa, J. (1995) J. Biochem. 117, 447-451[Abstract] |
20. | Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., Struhl, K., Albright, L. M., Coen, D. M., and Varki, A. (1995) Current Protocols in Molecular Biology , John Wiley & Sons, NY |
21. | Shanklin, J., Whittle, E., and Fox, B. G. (1994) Biochemistry 33, 12787-12794[Medline] [Order article via Infotrieve] |
22. |
James, D. W.,
Lim, E.,
Keller, J.,
Plooy, I.,
Ralston, E.,
and Dooner, H. K.
(1995)
Plant Cell
7,
309-319 |
23. | Millar, A. A., and Kunst, L. (1997) Plant J. 12, 121-131[CrossRef][Medline] [Order article via Infotrieve] |
24. | van de Loo, F. J., Broun, P., Turner, S., and Somerville, C. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 6743-6747[Abstract] |
25. |
Lee, M.,
Lenman, M.,
Banas, A.,
Bafor, M.,
Singh, S.,
Schweizer, M.,
Nilsson, R.,
Liljenberg, C.,
Dahlqvist, A.,
Gummeson, P.-O.,
Sjödahl, S.,
Green, A.,
and Stymne, S.
(1998)
Science
280,
915-918 |
26. | Broun, P., Boddupalli, S., and Somerville, C. (1998) Plant J. 13, 201-210[CrossRef][Medline] [Order article via Infotrieve] |
27. |
Broun, P.,
Shanklin, J.,
Whittle, E.,
and Somerville, C.
(1998)
Science
282,
1315-1317 |
28. | Millar, A. A., Wrischer, M., and Kunst, L. (1998) Plant Cell 11, 1889-1902[CrossRef] |
29. |
Broun, P.,
and Somerville, C.
(1997)
Plant Physiol.
113,
933-942 |
30. | Singh, S., Thomaeus, S., Lee, M., Stymne, S., and Green, A. (2001) Planta 212, 872-879[CrossRef][Medline] [Order article via Infotrieve] |
31. | Ip, C., Chin, S. F., Scimeca, J. A., and Pariza, M. W. (1991) Cancer Res. 51, 6118-6124[Abstract] |
32. | Shultz, T. D., Chew, B. P., Seaman, W. R., and Luedecke, L. O. (1992) Cancer Lett. 63, 125-133[CrossRef][Medline] [Order article via Infotrieve] |
33. | Durgam, V. R., and Fernandes, G. (1997) Cancer Lett. 116, 121-130[CrossRef][Medline] [Order article via Infotrieve] |
34. | West, D. B., DeLany, J. P., Camet, O. M., Blohm, F., Truett, A. A., and Scimeca, J. A. (1998) Am. J. Physiol. 275, R667-R672[Medline] [Order article via Infotrieve] |
35. | DeLany, J. P., Blohm, F., Truett, A. A., Scimeca, J. A., and West, D. B. (1999) Am. J. Physiol. 276, R1172-R1179[Medline] [Order article via Infotrieve] |
36. | Zambell, K. L., Keim, N. L., Van Loan, M. D., Gale, B., Benito, P., Kelley, D. S., and Nelson, G. J. (2000) Lipids 35, 777-787[Medline] [Order article via Infotrieve] |
37. |
Blankson, H.,
Stakkestad, J. A.,
Fagertun, H.,
Thom, E.,
Wadstein, J.,
and Gudmundsen, O.
(2000)
J. Nutr.
130,
2943-2948 |