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Identification and Analysis of a Gene from
Calendula officinalis Encoding a Fatty
Acid Conjugase
Xiao Qiu
1
*, Darwin W. Reed, Haiping Hong
1
, Samuel L. MacKenzie, and Patrick S. Covello
Research and Development, Bioriginal Food and Science Corporation, 102 Melville Street, Saskatoon,
Saskatchewan, Canada S7J 0R1 (X.Q., H.H.); and National Research Council of Canada, Plant Biotechnology
Institute, 110 Gymnasium Place, Saskatoon, Saskatchewan, Canada S7N 0W9 (D.W.R., S.L.M., P.S.C.)
Two homologous cDNAs, CoFad2 and CoFac2, were isolated from a Calendula officinalis developing seed by a polymerase
chain reaction-based cloning strategy. Both sequences share similarity to FAD2 desaturases and FAD2-related enzymes. In
C. officinalis plants CoFad2 was expressed in all tissues tested, whereas CoFac2 expression was specific to developing seeds.
Expression of CoFad2 cDNA in yeast (Saccharomyces cerevisiae) indicated it encodes a ⌬12 desaturase that introduces a double
bond at the 12 position of 16:1(9Z) and 18:1(9Z). Expression of CoFac2 in yeast revealed that the encoded enzyme acts as a
fatty acid conjugase converting 18:2(9Z,12Z) to calendic acid 18:3(8E,10E,12Z). The enzyme also has weak activity on the
mono-unsaturates 16:1(9Z) and 18:1(9Z) producing compounds with the properties of 8,10 conjugated dienes.
Hundreds of different fatty acids, many of which
have potential for industrial and pharmaceutical use,
have been identified in nature (Smith, 1970). How-
ever, these fatty acids are mostly produced in the
wild plant species or microorganisms that are not
readily cultivated and cultured commercially. Con-
ventional oilseed crops have high yields and oil con-
tents, but they produce a limited set of fatty acids,
which usually contain less than three double bonds
in their acyl chains.
Generally speaking, in polyunsaturated fatty acids
(PUFA), double bonds tend to be methylene-
interrupted and in the cis configuration. However,
fatty acids containing conjugated double bonds with
various stereochemical configurations do occur in
bacteria, algae, and plants. In the marine algae Boss-
iella orbigniana and Ptilota filicina, a substantial pro-
portion of the PUFA contain conjugated double
bonds (Burgess et al., 1991; Wise et al., 1994). In
plants, various conjugated linolenic acid isomers ac-
cumulate in seeds. Examples includes
␣
-eleostearic
acid [18:3(9Z,11E,13E)] in Momordica charantia (Liu
et al., 1997), punicic acid [18:3(9Z,11E,13Z)] in Pu-
nica granatum and Cayaponia africana, and jarcaric acid
[18:3(8Z,10E,12Z)] in Jacaranda mimosifolia (Chisholm
and Hopkins, 1967b; Hopkins and Chisholm, 1968).
Calendula officinalis is an annual flowering plant that
can accumulate more than 40% of calendic acid
[18:3(8E,10E,12Z)] of the seed lipid fatty acids
(Chisholm and Hopkins, 1967a). Although oils con-
taining conjugated linolenic acids have potential
value as drying oils, only
␣
-eleostearic acid-
containing oil from tung (Aleurites fordii) seeds is
currently of commercial significance.
As compared with conjugated polyunsaturated ac-
ids, conjugated linoleic acids (CLAs) appear less
commonly in nature. A few reports have docu-
mented the occurrence of this fatty acid in the foods
derived from ruminant animals (Fritsche and
Fritsche, 1998) and a number of anaerobic bacteria
such as rumen bacterium Butyrivibrio fibrisolvens (Ke-
pler et al., 1966; Kepler and Tove, 1967). It is believed
that CLAs are originally generated by rumen bacteria
and then absorbed by the animal host (Pariza, 1997).
The diversity of fatty acids in nature is largely due
to various combinations of the numbers and loca-
tions of double and triple bonds and other functional
groups (hydroxyl and epoxy). These are produced by
a family of structurally related enzymes (with three
conservative His-rich motifs), including desaturases
and their diverged forms such as hydroxylases, ep-
oxygenases, acetylenases, and the so-called fatty acid
conjugases (Lee et al., 1998; Shanklin and Cahoon,
1998; Cahoon et al., 1999). For microsomal enzymes
in this category it is believed that they use fatty acids
esterified to complex lipid as the substrate and accept
electrons from an electron transport chain consisting
of NAD(P)H, cytochrome b
5
reductase, and cyto-
chrome b
5
.
Based on the information that microsomal desatu-
rases and related enzymes have similar primary
structure, we undertook a PCR approach to clone
genes that are involved in the biosynthesis of conju-
gated fatty acids in C. officinalis. Two unique cDNAs
(CoFad2 and CoFac2) were identified. Expression of
the two cDNAs in yeast (Saccharomyces cerevisiae) re-
1
Present address: National Research Council of Canada, Plant
Biotechnology Institute, 110 Gymnasium Place, Saskatoon, SK,
Canada S7N 0W9.
* Corresponding author; e-mail xqiu@pbi.nrc.ca; fax 306–975–
4839.
Plant Physiology, February 2001, Vol. 125, pp. 847–855, www.plantphysiol.org © 2001 American Society of Plant Physiologists 847
vealed that CoFAD2 is a ⌬12 desaturase and CoFAC2
is a fatty acid conjugase that could convert the ⌬9
double bond of linoleic acid and, to a lesser extent, of
palmitoleic and oleic acids, into two conjugated dou-
ble bonds at ⌬8 and ⌬10 position. To our knowledge
this is the first example of identification of an enzyme
that can produce CLAs.
RESULTS
C. officinalis is an annual flowering plant that has
recently drawn scientific attention due to health
claims of the essential oil in the flowers and the
industrial potential of calendic acid in the seed oil.
Calendic is the major fatty acid in the seeds, account-
ing for more than 40% of the total fatty acids. We are
interested in the molecular basis for the biosynthesis
of this special fatty acid.
Identification of a cDNA Coding for a Putative Fatty
Acid Conjugase
To identify genes encoding conjugated double
bond-forming enzymes in C. officinalis, a PCR-based
cloning strategy was adopted. Sequencing of PCR
products revealed three types of inserts related to
desaturases. One had high sequence similarity to
-3
desaturases (FAD3). The other two shared amino
acid sequence similarity to various ⌬12 desaturases
(FAD2) and related enzymes, such as an acetylenase
from Crepis alpina (Lee et al., 1998).
To isolate full-length cDNA clones the two types of
Fad2-like inserts were used as probes to screen a
cDNA library from developing seeds, which resulted
in identification of several cDNA clones in each
group. Sequencing identified two unique full-length
of cDNAs, CoFad2 and CoFac2. CoFad2 is 1,411 bp and
codes for 383 amino acids with an M
r
of 44,000.
CoFac2 is 1,301 bp in length and codes for 374 amino
acids with a molecular mass of 43.6 kD. Sequence
comparison revealed 46% amino acid identity be-
tween the two deduced proteins. The identity occurs
all along the polypeptides with the highest among
three conservative His-rich areas (Fig. 1).
Sequence comparisons indicate that CoFAD2
shares 73% to 89% amino acid identity with the ⌬12
desaturases from various plants (Okuley et al., 1994;
Lee et al., 1998; GenBank accession nos. AF188264
and AAC 31698). Whereas CoFAC2 shares approxi-
mately equal sequence identity (50%) both to FAD2
desaturases and to other FAD2-like fatty acid-
modifying enzymes including FAD2 from C. officina-
lis (CoFAD2, this paper), Indian mustard (GenBank
accession Q39287 ), and borage (GenBank accession
no. AAC31698), the ⌬12 acetylenase of C. alpina (Lee
et al., 1998), the bifunctional enzyme (oleate 12-
hydroxylase:12-desaturase) of Lesquerella fendleri
(Broun et al., 1998), the 12,13-epoxygenase of Crepis
palaestina (Lee et al., 1998), and fatty acid conjugases
from C. officinalis (Fritsche et al., 1999), Impatiens
balsamina, and Momordica charantia (Cahoon et al.,
1999).
Phylogenetic analysis indicates that CoFAC2 dis-
tinguishes itself as one of the most deeply branching
within the plant FAD2-like sequences (Fig. 2). Boot-
strap analysis does indicate that this branching pat-
tern is not particularly reliable and it is possible that
CoFAC2 could cluster with other fatty acid conju-
gases, an epoxygenase, and an acetylenase. On the
other hand, CoFAD2 is clearly grouped within a
main branch of FAD2-like enzymes, which includes
the FAD2s per se, as well as the L. fendleri bifunc-
tional enzyme (Broun et al., 1998) and Ricinus com-
munis hydroxylase (van de Loo et al., 1995). These
results suggest the possible functions of CoFAD2 and
CoFAC2 as those of the extraplastidial ⌬12 fatty acid
desaturase and a fatty acid modifier likely to be
involved in calendic acid biosynthesis, respectively.
Northern-Blot Analysis of CoFac2 and CoFad2
Northern-blot analysis indicated that the CoFac2
was exclusively expressed in the developing seeds of
C. officinalis (Fig. 3). It was not expressed in vegeta-
tive tissues such as leaves and in reproductive tissues
such as flower buds. In contrast, CoFad2 was ex-
pressed in all tissues tested such as leaves, flower
buds, and developing seeds, but preferentially in
flower buds and developing seeds. Expression pat-
terns of the two genes were consistent with the pat-
tern of calendic acid accumulation, which occurs
only in seeds. In C. officinalis calendic acid accumu-
lated only in seeds, whereas linoleic acid, the product
of the ⌬12 desaturase (CoFAD2), was present in all
Figure 1. Comparison of CoFad2 and CoFac2 protein sequences of
C.
officinalis
. Vertical bars indicate identical amino acids.
Qiu et al.
848 Plant Physiol. Vol. 125, 2001
three tissues examined, but the flower buds and de-
veloping seeds contain a higher amount of this fatty
acid.
Expression of CoFac2 and CoFad2 in Yeast
To investigate the function of CoFac2 the full-length
cDNA was expressed in the yeast strain AMY-2
␣
in
which the stearoyl-coenzyme A desaturase gene, ole1,
is disrupted. The strain is unable to grow in minimal
media without supplementation of mono-unsaturated
fatty acids and allows for experimental control of the
fatty composition of the yeast. In our experiments the
strain was grown in minimal medium supplemented
with 17:1(10Z), a non-substrate of CoFAC2, which
enabled us to study the substrate specificity of the
enzyme toward various substrates, especially mono-
unsaturates. A number of possible substrates includ-
ing 16:0, 16:1(9Z), 17:1(10Z), 18:0, 18:1(9Z), 18:1(9E),
18:1(11Z), 18:1(11E), 18:1(12Z), 18:1(15Z), 18:2(9Z,
12Z), 18:3(9Z,12Z,15Z), 20:0, 20:2(11Z,14Z), and
22:1(13Z) were tested. As indicated in Figures 4 and
5 and Table I, only 18:2(9Z,12Z) and, to a lesser
extent, 16:1(9Z) and 18:1(9Z) were converted to con-
jugated fatty acids by the enzyme. For cultures sup-
plemented separately with the three substrates, when
gas chromatograms of fatty acid methyl esters
(FAMEs) derived from strains expressing CoFac2
were compared with those for vector controls, extra
peaks were detected as shown in Figure 4. These peaks
were selectively ablated when a Diels-Alder reaction
with 4-methyl-1,2,4-triazoline-3,5-dione (MTAD) was
performed prior to gas chromatography (GC) analy-
sis (data not shown). The sets of m/z peaks indicated
in Figure 5 are highly diagnostic for the original
double bond positions of the conjugated fatty acid
analyte. Mass spectral (MS) analysis of the MTAD
derivatives indicates that the products of 16:1, 18:1,
and 18:2 conversion are 16:2(8, 10) and 18:2(8, 10)
(Fig. 5) and 18:3(8, 10, 12) (data not shown). Assign-
ment of the product of 18:1(9) conversion is also
supported by the agreement of its GC peak retention
Figure 2. Phylogenetic analysis of FAD2-like enzymes. The dendro-
gram represents the result of a neighbor-joining analysis of amino
acid sequence distances with bootstrap values in percent shown at
nodes. The tree was arbitrarily rooted with
Synechococcus
desb
sequence. Sequences used for the analysis were obtained from ac-
cession numbers: AAB61352.1, Sydesb,
-3 desaturase of
Synecho-
coccus
PCC7002; CAB64256.1, CoFac1, (8, 11)-linoleoyl desaturase
from
C. officinalis
; P46313, AtFad2, Arabidopsis ⌬12 desaturase;
CAA76158.1, CaFad2, ⌬12 fatty acid acetylenase from
C. alpina
;
AAF05915.1, IbFac, ⌬12 oleic acid desaturase-like protein from
I.
balsamina
; CAA76157.1, CpFad2, ⌬12 fatty acid desaturase from
Crepis palaestina
; AAC32755.1, LfFdh, bifunctional oleate 12-hy-
droxylase:desaturase from
L. fendleri
; T14269, HaFad2, ⌬12 oleate
desaturase from common sunflower; T09839, RcFah, oleate 12-
hydroxylase from castor bean; CAA76156.1, CpEpo, ⌬12 fatty acid
epoxygenase from
C. palaestina
; AAF05916.1, McFac, ⌬12 oleic
acid desaturase-like protein from
Momordica charantia
; and
AAF08684.1, MaFad2, ⌬12 fatty acid desaturase from
Mortierella
alpina
.
Figure 3. Northern-blot analysis of
CoFad2
and
CoFac2
. A, Autora-
diogram of northern blot hybridized with
CoFad2
and
CoFac2
probes. B, Ethidium bromide gel indicating RNA loading. F, Flower
buds; L, leaves; S, developing seeds (see “Materials and Methods”).
Calendula Fatty Acid Conjugase
Plant Physiol. Vol. 125, 2001 849
time with one of a mixture of standard CLA isomers
(data not shown). The mass spectrum for the analyte
identified as 18:3(8, 10, 12) is consistent with two
compounds for which the Diels-Alder reaction has
occurred at the 8 and 10 positions or the 10 and 12
positions of an 18:3 isomer. This compound has the
same GC retention time as the major FAME derived
from C. officinalis seeds and is in all likelihood 18:
3(8E,10E,12Z). In control experiments with the
AMY2
␣
/pYES2 strain no peaks corresponding to
conjugated fatty acids were detected (Fig. 4 and GC/
MS, data not shown). The peaks in Figure 4 marked
with asterisks were also ablated by reaction with
MTAD. Their retention times and GC/MS analysis of
the FAMES suggests that they are (conjugated) iso-
mers of the major conjugated products. The amounts
of conjugated fatty acids that accumulate in yeast
cultures are shown in Table I. Qualitatively similar
results were obtained in experiments with the un-
supplemented and fatty acid-supplemented INVSc2/
pCoFac2 strain (data not shown). Within the limita-
tions of these in vivo experiments it would appear
that the conjugase has a much higher activity on 18:2
than on the monoenes.
As for the function of CoFad2, the expression of it in
the wild-type yeast strain INVSc2 indicated that the
encoded enzyme is a ⌬12 desaturase that introduces
a double bond at position 12 of 1:1(9Z) and 18:1(9Z)
(data not shown).
DISCUSSION
In this report we described the identification and
characterization of two homologous cDNAs, CoFac2
and CoFad2, from C. officinalis. Both products have
sequence similarity to the FAD2 desaturases and re-
lated enzymes from plants. CoFAD2 has higher
amino acid identity to the FAD2 desaturases (approx-
imately 80%), whereas CoFAC2 has approximately
equal sequence identity (approximately 50%) to both
FAD2 desaturases and FAD2-related enzymes, in-
cluding the ⌬12 acetylenase of C. alpina (Lee et al.,
1998), a bifunctional enzyme (oleate 12-hydroxylase:
12-desaturase) of L. fendleri (Broun et al., 1998), an
epoxygenases from C. palaestina (Lee et al., 1998), fatty
acid conjugases from C. officinalis (Fritsche et al., 1999),
I. balsamina, and M. charantia (Cahoon et al., 1999).
Expression of CoFad2 cDNA in yeast indicated it en-
codes a ⌬12 desaturase, whereas expression of CoFac2
in yeast revealed that the encoded enzyme produced
conjugated linoleic and linolenic acids from 18:1(9Z)
and 18:2(9Z,12Z) substrates, respectively.
The name “conjugase” was previously coined to
refer to enzymes that are responsible for introducing
conjugated double bonds into acyl chains. Two con-
jugases from I. balsamina and M. charantia were found
to be able to convert the ⌬12 double bond of linoleic
acid into two conjugated double bonds at the 11 and
13 positions, resulting in the production of conju-
gated linolenic acid [18:3(9Z,11E,13E)]. Expression
of CoFac2 in yeast showed that this “conjugase” could
convert ⌬9 double bonds of 16:1(9Z), 18:1(9Z), and
18:2(9Z,12Z) into two conjugated double bonds at
the 8 and 10 positions to produce their corresponding
conjugated fatty acids.
Two major routes to the biosynthesis of conjugated
fatty acids have been elucidated. Isomerization of a
common fatty acid into its conjugated counterpart
without the introduction of additional double bonds
was first described during biohydrogenation of lino-
leic acid by anaerobic rumen bacteria (Polan et al.,
1964). In marine algae a recently identified enzyme
can isomerize several PUFA into their corresponding
conjugated polyenoic acids (Wise et al., 1994, 1997).
The process is strictly an isomerization; there is no
oxidized intermediate or net desaturation involved.
In plants the mechanism underlying biosynthesis
of conjugated linolenic acids was studied by radiola-
beling (Crombie and Holloway, 1985; Liu et al., 1997).
Kinetics of the time course of metabolism of the
radiolabeled precursors indicated linoleic acid ester-
ified to phosphatidylcholine is an intermediate pre-
cursor of conjugated linolenic acid, implying that
Figure 4. GC analysis of FAMEs from yeast strain AMY-2
␣
trans-
formed with pCoFac2 (a) or control plasmid pYES2 (b) supplemented
with 17:1(10) and separately with 16:1(9), (1), 18:1(9), (2), or 18:2(9,
12), (3) (see “Materials and Methods”). ⌬ indicates 18:1 and 20:1
impurities in the 16:1 and 18:1 substrates, respectively. An asterisk
indicates peaks corresponding to isomers of the major conjugated
products (see “Results”).
Qiu et al.
850 Plant Physiol. Vol. 125, 2001
there is desaturation involved. Substrate specificity
studies of CoFAD2, along with that of conjugases
from I. balsamina and M. charantia (Cahoon et al.,
1999), favor the hypothesis that conjugated fatty ac-
ids in plants are produced by a process similar to
desaturation, which can result in introduction of one
additional double bond in the existing fatty acid
substrate. Crombie and Holloway (1985) previously
observed that during conversion of linoleic acid to
calendic acid in C. officinalis developing seeds, there
is no loss of labeled hydrogens at C-9, C-10, C-12, and
C-13, but there is a loss of a hydrogen from C-8 and
C-11. Thus, Fritsche et al. (1999) speculated that the
C. officinalis fatty acid conjugase could abstract hy-
drogens at carbon 8 and 11 positions, resulting in two
conjugated double bonds in the 8 and 10. Two genes
have now been cloned from C. officinalis whose prod-
ucts appear to catalyze the production of calendic
acid. However, it is still not clear whether both
cloned enzymes actually act via an “8,11 desatura-
tion” mechanism.
It was unexpected that 18:1 (9) acts as a substrate,
albeit a weak one, for CoFAC2 giving rise to conju-
gated linoleic acid [18:2(8, 10)] in yeast. CLA is a
newly recognized nutraceutical compound that has
recently drawn the attention of the pharmaceutical
and nutraceutical industries because of its various
physiological effects in animals and humans (Hau-
mann, 1996; Ip, 1997; Pariza, 1997). Dietary CLA (two
major isomers: 9Z,11E and 10E,12Z) was shown to
reduce the development of atherosclerosis in rabbits
(Lee et al., 1994) and to inhibit development of vari-
ous cancers in model animals (Pariza et al., 1999).
Feeding CLAs at low concentration (0.5% of diet) to
rodents can enhance immune function (Miller et al.,
1994). In addition, CLAs were recently found to de-
crease fat composition and increase lean body masses
and to improve feed efficiency in chickens and pigs
(Park et al., 1997). With the realization of the benefits
of CLAs, market demand for the product is growing.
There is, unfortunately, no rich natural source for
CLAs. Although some animal foods such as dairy
products and meat derived from ruminants contain
CLAs, the proportion is low. Linoleic acid can be
converted to CLA by chemical methods (Berdeaux et
al., 1998; Chen et al., 1999). However, CLA derived
from the chemical process is a mixture of several
isomers. The two major isomers (9Z,11E and 10E,
12Z) in about equal proportions account for about
80% of the product. The rest are other CLA isomers.
CLA produced by CoFAC2 in yeast is an unusual
isomer with two conjugated double bonds at the 8
and 10 positions. The stereochemistry of the product
remains to be determined. It is likely that it is 8E and
10E, since calendic acid [18:3(8E,10E,12Z)] is also a
product of the enzyme in yeast.
The finding that CoFAC2 can use oleic substrate to
synthesize the CLA has opened up a question regard-
ing the potential uses: does this CLA isomer have any
physiological effects on human and animal as com-
mon CLA does? To answer the question, preparation
of large amounts of the isomer is the first essential step
since feeding experiments and clinical trials would
consume a large amount of the fatty acid. If the effi-
ciency of conversion of 18:1 to CLA could be im-
proved, it may be possible to produce the 8,10 isomer
commercially in genetically modified organisms.
MATERIALS AND METHODS
Plant Materials
Calendula officinalis was grown in a growth chamber at
22°C with a 16-h photoperiod at a photon flux density of
150 to 200
Em
⫺2
s
⫺1
. The developing seeds at 15 to 30 d
after flowering were collected. The embryos were dissected
from seeds and used for RNA isolation.
Construction and Screening of cDNA Library
The total RNA was isolated from developing embryos
according to Qiu and Erickson (1994). The cDNA library
was constructed from the total RNA. The first strand cDNA
was synthesized by superscript II reverse transcriptase
from Gibco-BRL (Gaithersburg, MD). The second strand
cDNA was synthesized by DNA polymerase I from Strat-
agene (La Jolla, CA). After size fractionation, cDNA inserts
larger than 1 kb were ligated into
Uni-Zap XR vector
(Stratagene). The recombinant
DNAs were then packaged
with Gigapack III Gold packaging extract (Stratagene) and
plated on NZ amine-yeast extract plates. The resulting
library represented more than 8 ⫻ 10
6
independent clones.
Screening of the cDNA library was performed according to
standard methods (Sambrook et al., 1989).
Table I.
Conversion of exogenous fatty acids by the yeast strain AMY-2
␣
/pCoFac2
See “Materials and Methods” for culture conditions. Values are the means and SDs (in parentheses)
of three experiments. For control experiments using the AMY-2
␣
/pYES2 strain, no significant peaks were
detected at the retention time of the desaturation product.
Substrate Supplied Substrate Accumulation Product Product Accumulation
% total fatty acids % total fatty acids
16:1(9) 45 (12) 16:2 (8, 10) 0.29 (0.04)
18:1(9) 42 (10) 18:2 (8, 10) 0.09 (0.02)
18:2(9, 12) 18 (3) 18:3 (8
E
,10
E
,12
Z
) 0.56 (0.3)
Calendula Fatty Acid Conjugase
Plant Physiol. Vol. 125, 2001 851
Reverse Transcriptase-PCR
For reverse-transcriptase experiments the single strand
cDNA was synthesized by superscript II reverse transcrip-
tase (Gibco-BRL) from total RNA and was then used as the
template for PCR reaction. Two degenerate primers (the
forward primer: GCXCAC/TGAC/A/GTGC/TGGXCAC/
TC/GA and the reverse primer: CATXGTXG/CA/TG/
AAAXAG/AG/ATGG/ATG) were designed to target the
conserved His-rich domains of desaturases. The PCR ampli-
fication consisted of 35 cycles with 1 min at 94°C, 1.5 min at
55°C, and 2 min at 72°C followed by an extension step at
72°C for 10 min. The amplified products from 400 to 600 bp
Figure 5. GCMS EI spectra of the MTAD derivatives of novel fatty acids in AMY-2
␣
/pCoFac2 cultures supplemented with
16:1(9
Z
), (A) and 18:1(9
Z
), (B). The structures assigned to the derivatives are shown with asterisks indicating the original
position of the double bonds in the fatty acid. The pairs of peaks with
m/z
values 236 and 308 in A and 264 and 308 in B
are diagnostic for the loss of R
1
and R
2
fragments, respectively, for 16:2(8, 10) and 18:2(8, 10) derivatives.
Qiu et al.
852 Plant Physiol. Vol. 125, 2001
were isolated from agarose gel and purified by a kit (Qiaex
II gel purification, Qiagen, Valencia, CA), and subsequently
cloned into the TA cloning vector pCR 2.1 (Invitrogen, Carls-
bad, CA). The cloned inserts were then sequenced by PRISM
DyeDeoxy Terminator Cycle Sequencing System (Perkin
Elmer/Applied Biosystems, Foster City, CA).
Phylogenetic Analysis
For phylogenetic analysis, predicted amino acid se-
quences were aligned using CLUSTALW (version 1.60;
Thompson et al., 1998) with the default parameters, includ-
ing gap open and extension penalties of 10 and 0.05, re-
spectively, for pairwise and multiple alignments. The BLO-
SUM 30 protein weight matrix was used for pairwise
alignments and the BLOSUM series for multiple align-
ments. CLUSTALW was used to determine dendrograms
representing a neighbor-joining analysis of sequence dis-
tances. Bootstrap analysis was performed with 1,000 itera-
tions and visualized with the TreeView program (Page,
1996).
Northern-Blot Analysis
For northern-blot analysis, 7
g of total RNAs isolated
from flower buds, leaves, and developing seeds of C. offi-
cinalis as described above were fractionated in a
formaldehyde-agarose gel. After electrophoresis RNAs
were transferred to Hybond membrane (Amersham Phar-
macia, Uppsala) using 10⫻ SSC transferring solution and
were then fixed to the membrane by UV crosslinking.
Filter-bound RNAs were then hybridized with the radiola-
beled cDNA probes at 68°C for1hinQuickhyb (Strat-
agene). After hybridization the blots were washed once at
room temperature for one-half an hour with a solution of
2⫻ SSC and 1% (w/v) SDS, and once at 65°C for one-half
an hour with a solution of 0.1⫻ SSC and 0.1% (w/v) SDS.
Expression of CoFad2 and CoFac2 in Yeast
(Saccharomyces cerevisiae)
The open reading frames of CoFad2 and CoFac2 were
amplified by PCR using the Precision Plus enzyme (Strat-
agene) and cloned into a TA cloning vector (pCR 2.1,
Invitrogen). Having confirmed that the PCR products were
identical to the original cDNAs by sequencing, the frag-
ments were then released by a BamHI-EcoRI double diges-
tion and inserted into the yeast expression vector pYES2
(Invitrogen) under the control of the inducible promoter
GAL1.
Yeast strains InvSc2 (Invitrogen) and AMY-2
␣
[the ge-
notype: MAT
␣
, CYTb5, ole1(⌬BstEII)::LEU2, trp1-1, can1-
100, ura3-1, ade2-1, HIS3; Mitchell and Martin, 1995] were
transformed with the expression constructs using the lith-
ium acetate method and transformants were selected on
minimal medium plates lacking uracil (Gietz et al., 1992;
Covello and Reed, 1996).
Transformants were first grown in minimal medium
lacking uracil and containing Glc (CM-ura, Ausubel et al.,
1995) at 28°C. After overnight culture the cells were spun
down, washed, and resuspended in distilled water. Mini-
mal medium with 2% (w/v) Gal replacing Glc, and with or
without 0.3 mm substrate fatty acids in the presence of 0.1%
(w/v) Tergitol was inoculated with the yeast transformant
cell suspension and incubated at 20°C for 3 d followed by
15°C for 3 d. For the AMY2
␣
strain media were supple-
mented with 0.3 mm 17:1(10Z) and 0.1% (w/v) Tergitol.
Fatty Acid Analysis
Yeast cultures were pelleted by centrifugation (4,000g,10
min.) and pellets were washed with 10 mL of 1% (w/v)
Tergitol solution and 2 ⫻ 10 mL of water. The yeast pellet
was dried under vacuum at ambient temperature. To the
dried pellet in a glass culture tube was added 1 mL of
methanol and the pellet was dispersed using a high speed
homogenizer. To this mixture was added 2 mL of 0.5 m
sodium methoxide in methanol. The tube was flushed with
nitrogen, sealed, and heated to 50°C for 1 h. The cooled
mixture was extracted with 2 ⫻ 2 mL of hexane. The pooled
hexane was washed with 2 mL of water and was concen-
trated under N
2
for GC or GC/MS analysis.
FAME analysis was carried out using a gas chromato-
graph (6890, Hewlett-Packard, Palo Alto, CA) equipped
with a DB-23 fused silica column (30 m ⫻ 0.25 mm i.d.,
0.25-
m film thickness; J&W Scientific, Fulsom, CA) with a
temperature program of 180°C for 1 min, 4°C/min to
240°C, hold for 15 min.
For conjugated polyene analysis, FAME were derivat-
ized with MTAD (Dobson, 1998). One hundred microliters
of a dilute solution of MTAD (⬍1 mg/mL, slight pink
color) in CHCl
3
at 0°C was added to dry FAME from yeast
cells with agitation for 5 to 10 s. A dilute solution of
1,3-hexadiene (excess) was then added to neutralize reac-
tants (removal of color). The tube was dried under nitrogen
and the residue was re-dissolved in CHCl
3
.
GC/MS analysis was performed in standard EI mode
using a Fisons VG TRIO 2000 mass spectrometer (VG An-
alytical, Manchester, UK) controlled by Masslynx version
2.0 software, coupled to a GC 8000 Series gas chromato-
graph. For FAME analysis, a DB-23 column was used with
the temperature program described above. For MTAD deriv-
ative analysis, a DB-5 column (60 m ⫻ 0.32 mm i.d., 0.25-
m
film thickness, J&W Scientific) that was temperature-
programmed at 50°C for 1 min, increased at 20°C/min to
160°C, then 5°C/min to 350°C and held for 15 min.
In some experiments, C. officinalis oil extracted from
seeds or a mixture of CLAs (Sigma, St. Louis) was used as
the standard.
ACKNOWLEDGMENTS
The authors wish to thank Dr. Ron Wilen for providing
C. officinalis seeds, Dr. Charles Martin for providing yeast
AMY-2
␣
mutant strain, and Stephen Ambrose for GC/MS
analysis.
Received June 6, 2000; returned for revision July 20, 2000;
accepted October 12, 2000.
Calendula Fatty Acid Conjugase
Plant Physiol. Vol. 125, 2001 853
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