Identification and Analysis of a Gene from Calendula officinalis Encoding a Fatty Acid Conjugase
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Identification and Analysis of a Gene from
Calendula officinalis Encoding a Fatty
Acid Conjugase
Xiao Qiu1*, Darwin W. Reed, Haiping Hong1, 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 taining conjugated linolenic acids have potential
have potential for industrial and pharmaceutical use, value as drying oils, only ␣-eleostearic acid-
have been identified in nature (Smith, 1970). How- containing oil from tung (Aleurites fordii) seeds is
ever, these fatty acids are mostly produced in the currently of commercial significance.
wild plant species or microorganisms that are not As compared with conjugated polyunsaturated ac-
readily cultivated and cultured commercially. Con- ids, conjugated linoleic acids (CLAs) appear less
ventional oilseed crops have high yields and oil con- commonly in nature. A few reports have docu-
tents, but they produce a limited set of fatty acids, mented the occurrence of this fatty acid in the foods
which usually contain less than three double bonds derived from ruminant animals (Fritsche and
in their acyl chains. Fritsche, 1998) and a number of anaerobic bacteria
Generally speaking, in polyunsaturated fatty acids such as rumen bacterium Butyrivibrio fibrisolvens (Ke-
(PUFA), double bonds tend to be methylene- pler et al., 1966; Kepler and Tove, 1967). It is believed
interrupted and in the cis configuration. However, that CLAs are originally generated by rumen bacteria
fatty acids containing conjugated double bonds with and then absorbed by the animal host (Pariza, 1997).
various stereochemical configurations do occur in The diversity of fatty acids in nature is largely due
bacteria, algae, and plants. In the marine algae Boss- to various combinations of the numbers and loca-
iella orbigniana and Ptilota filicina, a substantial pro- tions of double and triple bonds and other functional
portion of the PUFA contain conjugated double groups (hydroxyl and epoxy). These are produced by
bonds (Burgess et al., 1991; Wise et al., 1994). In a family of structurally related enzymes (with three
plants, various conjugated linolenic acid isomers ac- conservative His-rich motifs), including desaturases
cumulate in seeds. Examples includes ␣-eleostearic and their diverged forms such as hydroxylases, ep-
acid [18:3(9Z, 11E, 13E)] in Momordica charantia (Liu oxygenases, acetylenases, and the so-called fatty acid
et al., 1997), punicic acid [18:3(9Z, 11E, 13Z)] in Pu- conjugases (Lee et al., 1998; Shanklin and Cahoon,
nica granatum and Cayaponia africana, and jarcaric acid 1998; Cahoon et al., 1999). For microsomal enzymes
[18:3(8Z, 10E, 12Z)] in Jacaranda mimosifolia (Chisholm in this category it is believed that they use fatty acids
and Hopkins, 1967b; Hopkins and Chisholm, 1968). esterified to complex lipid as the substrate and accept
Calendula officinalis is an annual flowering plant that electrons from an electron transport chain consisting
can accumulate more than 40% of calendic acid of NAD(P)H, cytochrome b5 reductase, and cyto-
[18:3(8E, 10E, 12Z)] of the seed lipid fatty acids chrome b5.
(Chisholm and Hopkins, 1967a). Although oils con- Based on the information that microsomal desatu-
rases and related enzymes have similar primary
1
Present address: National Research Council of Canada, Plant
structure, we undertook a PCR approach to clone
Biotechnology Institute, 110 Gymnasium Place, Saskatoon, SK, genes that are involved in the biosynthesis of conju-
Canada S7N 0W9. gated fatty acids in C. officinalis. Two unique cDNAs
* Corresponding author; e-mail xqiu@pbi.nrc.ca; fax 306 –975– (CoFad2 and CoFac2) were identified. Expression of
4839. the two cDNAs in yeast (Saccharomyces cerevisiae) re-
Plant Physiology, February 2001, Vol. 125, pp. 847–855, www.plantphysiol.org © 2001 American Society of Plant Physiologists 847
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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 Figure 1. Comparison of CoFad2 and CoFac2 protein sequences of C.
Acid Conjugase officinalis. Vertical bars indicate identical amino acids.
To identify genes encoding conjugated double
bond-forming enzymes in C. officinalis, a PCR-based
cloning strategy was adopted. Sequencing of PCR from C. officinalis (Fritsche et al., 1999), Impatiens
products revealed three types of inserts related to balsamina, and Momordica charantia (Cahoon et al.,
desaturases. One had high sequence similarity to -3 1999).
desaturases (FAD3). The other two shared amino Phylogenetic analysis indicates that CoFAC2 dis-
acid sequence similarity to various ⌬12 desaturases tinguishes itself as one of the most deeply branching
(FAD2) and related enzymes, such as an acetylenase within the plant FAD2-like sequences (Fig. 2). Boot-
from Crepis alpina (Lee et al., 1998). strap analysis does indicate that this branching pat-
To isolate full-length cDNA clones the two types of tern is not particularly reliable and it is possible that
Fad2-like inserts were used as probes to screen a CoFAC2 could cluster with other fatty acid conju-
cDNA library from developing seeds, which resulted gases, an epoxygenase, and an acetylenase. On the
in identification of several cDNA clones in each other hand, CoFAD2 is clearly grouped within a
group. Sequencing identified two unique full-length main branch of FAD2-like enzymes, which includes
of cDNAs, CoFad2 and CoFac2. CoFad2 is 1,411 bp and the FAD2s per se, as well as the L. fendleri bifunc-
codes for 383 amino acids with an Mr of 44,000. tional enzyme (Broun et al., 1998) and Ricinus com-
CoFac2 is 1,301 bp in length and codes for 374 amino munis hydroxylase (van de Loo et al., 1995). These
acids with a molecular mass of 43.6 kD. Sequence results suggest the possible functions of CoFAD2 and
comparison revealed 46% amino acid identity be- CoFAC2 as those of the extraplastidial ⌬12 fatty acid
tween the two deduced proteins. The identity occurs desaturase and a fatty acid modifier likely to be
all along the polypeptides with the highest among involved in calendic acid biosynthesis, respectively.
three conservative His-rich areas (Fig. 1).
Sequence comparisons indicate that CoFAD2 Northern-Blot Analysis of CoFac2 and CoFad2
shares 73% to 89% amino acid identity with the ⌬12
desaturases from various plants (Okuley et al., 1994; Northern-blot analysis indicated that the CoFac2
Lee et al., 1998; GenBank accession nos. AF188264 was exclusively expressed in the developing seeds of
and AAC 31698). Whereas CoFAC2 shares approxi- C. officinalis (Fig. 3). It was not expressed in vegeta-
mately equal sequence identity (50%) both to FAD2 tive tissues such as leaves and in reproductive tissues
desaturases and to other FAD2-like fatty acid- such as flower buds. In contrast, CoFad2 was ex-
modifying enzymes including FAD2 from C. officina- pressed in all tissues tested such as leaves, flower
lis (CoFAD2, this paper), Indian mustard (GenBank buds, and developing seeds, but preferentially in
accession Q39287 ), and borage (GenBank accession flower buds and developing seeds. Expression pat-
no. AAC31698), the ⌬12 acetylenase of C. alpina (Lee terns of the two genes were consistent with the pat-
et al., 1998), the bifunctional enzyme (oleate 12- tern of calendic acid accumulation, which occurs
hydroxylase:12-desaturase) of Lesquerella fendleri only in seeds. In C. officinalis calendic acid accumu-
(Broun et al., 1998), the 12,13-epoxygenase of Crepis lated only in seeds, whereas linoleic acid, the product
palaestina (Lee et al., 1998), and fatty acid conjugases of the ⌬12 desaturase (CoFAD2), was present in all
848 Plant Physiol. Vol. 125, 2001
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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
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”).
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-
Figure 2. Phylogenetic analysis of FAD2-like enzymes. The dendro- plemented separately with the three substrates, when
gram represents the result of a neighbor-joining analysis of amino
gas chromatograms of fatty acid methyl esters
acid sequence distances with bootstrap values in percent shown at
nodes. The tree was arbitrarily rooted with Synechococcus desb
(FAMEs) derived from strains expressing CoFac2
sequence. Sequences used for the analysis were obtained from ac- were compared with those for vector controls, extra
cession numbers: AAB61352.1, Sydesb, -3 desaturase of Synecho- peaks were detected as shown in Figure 4. These peaks
coccus PCC7002; CAB64256.1, CoFac1, (8, 11)-linoleoyl desaturase were selectively ablated when a Diels-Alder reaction
from C. officinalis; P46313, AtFad2, Arabidopsis ⌬12 desaturase; with 4-methyl-1,2,4-triazoline-3,5-dione (MTAD) was
CAA76158.1, CaFad2, ⌬12 fatty acid acetylenase from C. alpina; performed prior to gas chromatography (GC) analy-
AAF05915.1, IbFac, ⌬12 oleic acid desaturase-like protein from I. sis (data not shown). The sets of m/z peaks indicated
balsamina; CAA76157.1, CpFad2, ⌬12 fatty acid desaturase from in Figure 5 are highly diagnostic for the original
Crepis palaestina; AAC32755.1, LfFdh, bifunctional oleate 12-hy- double bond positions of the conjugated fatty acid
droxylase:desaturase from L. fendleri; T14269, HaFad2, ⌬12 oleate
analyte. Mass spectral (MS) analysis of the MTAD
desaturase from common sunflower; T09839, RcFah, oleate 12-
hydroxylase from castor bean; CAA76156.1, CpEpo, ⌬12 fatty acid
derivatives indicates that the products of 16:1, 18:1,
epoxygenase from C. palaestina; AAF05916.1, McFac, ⌬12 oleic and 18:2 conversion are 16:2(8, 10) and 18:2(8, 10)
acid desaturase-like protein from Momordica charantia; and (Fig. 5) and 18:3(8, 10, 12) (data not shown). Assign-
AAF08684.1, MaFad2, ⌬12 fatty acid desaturase from Mortierella ment of the product of 18:1(9) conversion is also
alpina. supported by the agreement of its GC peak retention
Plant Physiol. Vol. 125, 2001 849
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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
Figure 4. GC analysis of FAMEs from yeast strain AMY-2␣ trans- conjugated linoleic and linolenic acids from 18:1(9Z)
formed with pCoFac2 (a) or control plasmid pYES2 (b) supplemented and 18:2(9Z, 12Z) substrates, respectively.
with 17:1(10) and separately with 16:1(9), (1), 18:1(9), (2), or 18:2(9, The name “conjugase” was previously coined to
12), (3) (see “Materials and Methods”). ⌬ indicates 18:1 and 20:1
refer to enzymes that are responsible for introducing
impurities in the 16:1 and 18:1 substrates, respectively. An asterisk
indicates peaks corresponding to isomers of the major conjugated
conjugated double bonds into acyl chains. Two con-
products (see “Results”). 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-
time with one of a mixture of standard CLA isomers gated linolenic acid [18:3(9Z, 11E, 13E)]. Expression
(data not shown). The mass spectrum for the analyte of CoFac2 in yeast showed that this “conjugase” could
identified as 18:3(8, 10, 12) is consistent with two convert ⌬9 double bonds of 16:1(9Z), 18:1(9Z), and
compounds for which the Diels-Alder reaction has 18:2(9Z, 12Z) into two conjugated double bonds at
occurred at the 8 and 10 positions or the 10 and 12 the 8 and 10 positions to produce their corresponding
positions of an 18:3 isomer. This compound has the conjugated fatty acids.
same GC retention time as the major FAME derived Two major routes to the biosynthesis of conjugated
from C. officinalis seeds and is in all likelihood 18: fatty acids have been elucidated. Isomerization of a
3(8E, 10E, 12Z). In control experiments with the common fatty acid into its conjugated counterpart
AMY2␣/pYES2 strain no peaks corresponding to without the introduction of additional double bonds
conjugated fatty acids were detected (Fig. 4 and GC/ was first described during biohydrogenation of lino-
MS, data not shown). The peaks in Figure 4 marked leic acid by anaerobic rumen bacteria (Polan et al.,
with asterisks were also ablated by reaction with 1964). In marine algae a recently identified enzyme
MTAD. Their retention times and GC/MS analysis of can isomerize several PUFA into their corresponding
the FAMES suggests that they are (conjugated) iso- conjugated polyenoic acids (Wise et al., 1994, 1997).
mers of the major conjugated products. The amounts The process is strictly an isomerization; there is no
of conjugated fatty acids that accumulate in yeast oxidized intermediate or net desaturation involved.
cultures are shown in Table I. Qualitatively similar In plants the mechanism underlying biosynthesis
results were obtained in experiments with the un- of conjugated linolenic acids was studied by radiola-
supplemented and fatty acid-supplemented INVSc2/ beling (Crombie and Holloway, 1985; Liu et al., 1997).
pCoFac2 strain (data not shown). Within the limita- Kinetics of the time course of metabolism of the
tions of these in vivo experiments it would appear radiolabeled precursors indicated linoleic acid ester-
that the conjugase has a much higher activity on 18:2 ified to phosphatidylcholine is an intermediate pre-
than on the monoenes. cursor of conjugated linolenic acid, implying that
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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 (8E, 10E, 12Z) 0.56 (0.3)
there is desaturation involved. Substrate specificity 12Z) in about equal proportions account for about
studies of CoFAD2, along with that of conjugases 80% of the product. The rest are other CLA isomers.
from I. balsamina and M. charantia (Cahoon et al., CLA produced by CoFAC2 in yeast is an unusual
1999), favor the hypothesis that conjugated fatty ac- isomer with two conjugated double bonds at the 8
ids in plants are produced by a process similar to and 10 positions. The stereochemistry of the product
desaturation, which can result in introduction of one remains to be determined. It is likely that it is 8E and
additional double bond in the existing fatty acid 10E, since calendic acid [18:3(8E, 10E, 12Z)] is also a
substrate. Crombie and Holloway (1985) previously product of the enzyme in yeast.
observed that during conversion of linoleic acid to The finding that CoFAC2 can use oleic substrate to
calendic acid in C. officinalis developing seeds, there synthesize the CLA has opened up a question regard-
is no loss of labeled hydrogens at C-9, C-10, C-12, and ing the potential uses: does this CLA isomer have any
C-13, but there is a loss of a hydrogen from C-8 and physiological effects on human and animal as com-
C-11. Thus, Fritsche et al. (1999) speculated that the mon CLA does? To answer the question, preparation
C. officinalis fatty acid conjugase could abstract hy- of large amounts of the isomer is the first essential step
drogens at carbon 8 and 11 positions, resulting in two since feeding experiments and clinical trials would
conjugated double bonds in the 8 and 10. Two genes consume a large amount of the fatty acid. If the effi-
have now been cloned from C. officinalis whose prod- ciency of conversion of 18:1 to CLA could be im-
ucts appear to catalyze the production of calendic proved, it may be possible to produce the 8,10 isomer
acid. However, it is still not clear whether both commercially in genetically modified organisms.
cloned enzymes actually act via an “8,11 desatura-
tion” mechanism.
It was unexpected that 18:1 (9) acts as a substrate, MATERIALS AND METHODS
albeit a weak one, for CoFAC2 giving rise to conju-
gated linoleic acid [18:2(8, 10)] in yeast. CLA is a Plant Materials
newly recognized nutraceutical compound that has Calendula officinalis was grown in a growth chamber at
recently drawn the attention of the pharmaceutical 22°C with a 16-h photoperiod at a photon flux density of
and nutraceutical industries because of its various 150 to 200 E m⫺2 s⫺1. The developing seeds at 15 to 30 d
physiological effects in animals and humans (Hau- after flowering were collected. The embryos were dissected
mann, 1996; Ip, 1997; Pariza, 1997). Dietary CLA (two from seeds and used for RNA isolation.
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). Construction and Screening of cDNA Library
Feeding CLAs at low concentration (0.5% of diet) to
rodents can enhance immune function (Miller et al., The total RNA was isolated from developing embryos
1994). In addition, CLAs were recently found to de- according to Qiu and Erickson (1994). The cDNA library
crease fat composition and increase lean body masses was constructed from the total RNA. The first strand cDNA
and to improve feed efficiency in chickens and pigs was synthesized by superscript II reverse transcriptase
(Park et al., 1997). With the realization of the benefits from Gibco-BRL (Gaithersburg, MD). The second strand
of CLAs, market demand for the product is growing. cDNA was synthesized by DNA polymerase I from Strat-
There is, unfortunately, no rich natural source for agene (La Jolla, CA). After size fractionation, cDNA inserts
CLAs. Although some animal foods such as dairy larger than 1 kb were ligated into Uni-Zap XR vector
products and meat derived from ruminants contain (Stratagene). The recombinant DNAs were then packaged
CLAs, the proportion is low. Linoleic acid can be with Gigapack III Gold packaging extract (Stratagene) and
converted to CLA by chemical methods (Berdeaux et plated on NZ amine-yeast extract plates. The resulting
al., 1998; Chen et al., 1999). However, CLA derived library represented more than 8 ⫻ 106 independent clones.
from the chemical process is a mixture of several Screening of the cDNA library was performed according to
isomers. The two major isomers (9Z, 11E and 10E, standard methods (Sambrook et al., 1989).
Plant Physiol. Vol. 125, 2001 851
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Figure 5. GCMS EI spectra of the MTAD derivatives of novel fatty acids in AMY-2␣/pCoFac2 cultures supplemented with
16:1(9Z), (A) and 18:1(9Z), (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 R1 and R2 fragments, respectively, for 16:2(8, 10) and 18:2(8, 10) derivatives.
Reverse Transcriptase-PCR TC/GA and the reverse primer: CATXGTXG/CA/TG/
For reverse-transcriptase experiments the single strand AAAXAG/AG/ATGG/ATG) were designed to target the
cDNA was synthesized by superscript II reverse transcrip- conserved His-rich domains of desaturases. The PCR ampli-
tase (Gibco-BRL) from total RNA and was then used as the fication consisted of 35 cycles with 1 min at 94°C, 1.5 min at
template for PCR reaction. Two degenerate primers (the 55°C, and 2 min at 72°C followed by an extension step at
forward primer: GCXCAC/TGAC/A/GTGC/TGGXCAC/ 72°C for 10 min. The amplified products from 400 to 600 bp
852 Plant Physiol. Vol. 125, 2001
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were isolated from agarose gel and purified by a kit (Qiaex 1995) at 28°C. After overnight culture the cells were spun
II gel purification, Qiagen, Valencia, CA), and subsequently down, washed, and resuspended in distilled water. Mini-
cloned into the TA cloning vector pCR 2.1 (Invitrogen, Carls- mal medium with 2% (w/v) Gal replacing Glc, and with or
bad, CA). The cloned inserts were then sequenced by PRISM without 0.3 mm substrate fatty acids in the presence of 0.1%
DyeDeoxy Terminator Cycle Sequencing System (Perkin (w/v) Tergitol was inoculated with the yeast transformant
Elmer/Applied Biosystems, Foster City, CA). 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.
Phylogenetic Analysis
For phylogenetic analysis, predicted amino acid se- Fatty Acid Analysis
quences were aligned using CLUSTALW (version 1.60;
Thompson et al., 1998) with the default parameters, includ- Yeast cultures were pelleted by centrifugation (4,000g, 10
ing gap open and extension penalties of 10 and 0.05, re- min.) and pellets were washed with 10 mL of 1% (w/v)
spectively, for pairwise and multiple alignments. The BLO- Tergitol solution and 2 ⫻ 10 mL of water. The yeast pellet
SUM 30 protein weight matrix was used for pairwise was dried under vacuum at ambient temperature. To the
alignments and the BLOSUM series for multiple align- dried pellet in a glass culture tube was added 1 mL of
ments. CLUSTALW was used to determine dendrograms methanol and the pellet was dispersed using a high speed
representing a neighbor-joining analysis of sequence dis- homogenizer. To this mixture was added 2 mL of 0.5 m
tances. Bootstrap analysis was performed with 1,000 itera- sodium methoxide in methanol. The tube was flushed with
tions and visualized with the TreeView program (Page, nitrogen, sealed, and heated to 50°C for 1 h. The cooled
1996). mixture was extracted with 2 ⫻ 2 mL of hexane. The pooled
hexane was washed with 2 mL of water and was concen-
trated under N2 for GC or GC/MS analysis.
Northern-Blot Analysis FAME analysis was carried out using a gas chromato-
graph (6890, Hewlett-Packard, Palo Alto, CA) equipped
For northern-blot analysis, 7 g of total RNAs isolated
with a DB-23 fused silica column (30 m ⫻ 0.25 mm i.d.,
from flower buds, leaves, and developing seeds of C. offi-
0.25-m film thickness; J&W Scientific, Fulsom, CA) with a
cinalis as described above were fractionated in a
temperature program of 180°C for 1 min, 4°C/min to
formaldehyde-agarose gel. After electrophoresis RNAs
240°C, hold for 15 min.
were transferred to Hybond membrane (Amersham Phar-
For conjugated polyene analysis, FAME were derivat-
macia, Uppsala) using 10⫻ SSC transferring solution and
ized with MTAD (Dobson, 1998). One hundred microliters
were then fixed to the membrane by UV crosslinking.
of a dilute solution of MTAD (⬍1 mg/mL, slight pink
Filter-bound RNAs were then hybridized with the radiola-
color) in CHCl3 at 0°C was added to dry FAME from yeast
beled cDNA probes at 68°C for 1 h in Quickhyb (Strat-
cells with agitation for 5 to 10 s. A dilute solution of
agene). After hybridization the blots were washed once at
1,3-hexadiene (excess) was then added to neutralize reac-
room temperature for one-half an hour with a solution of
tants (removal of color). The tube was dried under nitrogen
2⫻ SSC and 1% (w/v) SDS, and once at 65°C for one-half
and the residue was re-dissolved in CHCl3.
an hour with a solution of 0.1⫻ SSC and 0.1% (w/v) SDS.
GC/MS analysis was performed in standard EI mode
using a Fisons VG TRIO 2000 mass spectrometer (VG An-
Expression of CoFad2 and CoFac2 in Yeast alytical, Manchester, UK) controlled by Masslynx version
(Saccharomyces cerevisiae) 2.0 software, coupled to a GC 8000 Series gas chromato-
graph. For FAME analysis, a DB-23 column was used with
The open reading frames of CoFad2 and CoFac2 were the temperature program described above. For MTAD deriv-
amplified by PCR using the Precision Plus enzyme (Strat- ative analysis, a DB-5 column (60 m ⫻ 0.32 mm i.d., 0.25-m
agene) and cloned into a TA cloning vector (pCR 2.1, film thickness, J&W Scientific) that was temperature-
Invitrogen). Having confirmed that the PCR products were programmed at 50°C for 1 min, increased at 20°C/min to
identical to the original cDNAs by sequencing, the frag- 160°C, then 5°C/min to 350°C and held for 15 min.
ments were then released by a BamHI-EcoRI double diges- In some experiments, C. officinalis oil extracted from
tion and inserted into the yeast expression vector pYES2 seeds or a mixture of CLAs (Sigma, St. Louis) was used as
(Invitrogen) under the control of the inducible promoter the standard.
GAL1.
Yeast strains InvSc2 (Invitrogen) and AMY-2␣ [the ge-
notype: MAT␣, CYTb5, ole1(⌬BstEII)::LEU2, trp1-1, can1- ACKNOWLEDGMENTS
100, ura3-1, ade2-1, HIS3; Mitchell and Martin, 1995] were
The authors wish to thank Dr. Ron Wilen for providing
transformed with the expression constructs using the lith-
C. officinalis seeds, Dr. Charles Martin for providing yeast
ium acetate method and transformants were selected on
AMY-2␣ mutant strain, and Stephen Ambrose for GC/MS
minimal medium plates lacking uracil (Gietz et al., 1992;
analysis.
Covello and Reed, 1996).
Transformants were first grown in minimal medium Received June 6, 2000; returned for revision July 20, 2000;
lacking uracil and containing Glc (CM-ura, Ausubel et al., accepted October 12, 2000.
Plant Physiol. Vol. 125, 2001 853
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Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.Qiu et al.
LITERATURE CITED Kepler CR, Hirons KP, McNeill JJ, Tove SB (1966) Inter-
mediates and products of biohydrogenation of linoleic
Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman
acid by Butyrivibrio fibrsolvens. J Biol Chem 241(6):
JG, Smith JA, Struhl K, Albright LM, Coen DM, Varki
1350–1354
A (1995) Current Protocols in Molecular Biology. John
Kepler CR, Tove SB (1967) Biohydrogenation of unsatur-
Wiley & Sons, New York
ated fatty acids, III purification and properties of a li-
Berdeaux O, Voinot L, Angioni E, Juaneda P, Sebedio JL
noleate ⌬12-cis, ⌬11 trans-isomerase from Butyrivibrio
(1998) A simple method of preparation of methyl trans-
fibrsolvens. J Biol Chem 212(24): 5686–5692
10, cis-12 and cis-9, trans-11-octadecadienoates from
Lee KN, Kritchevsky D, Pariza MW (1994) Conjugated
methyl linoleate. JAOCS 75(12): 1749–1755
linoleic acids and atherosclerosis in rabbits. Atheroscle-
Broun P, Boddupalli S, Somerville C (1998) A bifunctional
rosis 108: 19–25
oleate 12-hydroxylase: desaturase from Lesquerella
Lee M, Lenman M, Banas A, Bafor M, Singh S, Schweizer
fendleri. Plant J 13(2): 201–210
M, Nilsson R, Liljenberg C, Dahlqvist A, Gummeson P,
Burgess JR, de la Rosa RI, Jacobs RS, Butler A (1991) A
Sjoedahl S, Green A, Stymne S (1998) Identification of
new eicosapentaenoic acid formed from arachidonic acid
non-heme diiron proteins that catalyze triple bond and
in the coralline red algae Bossiella orbigniana. Lipids 26:
162–165 epoxy group formation. Science 280(5365): 915–918
Cahoon EB, Carlson TJ, Ripp KG, Schweiger BJ, Cook Liu L, Hammond EG, Nikolau BJ (1997) In vivo studies of
GA, Hall SE, Kinney AJ (1999) Biosynthetic origin of the biosynthesis of ␣-eleostearic acid in the seed of Mo-
conjugated double bonds: production of fatty acid com- mordica charantia L. Plant Physiol 113: 1343–1349
ponents of high-value drying oils in transgenic soybean Miller CC, Park Y, Pariza MW, Cook ME (1994) Feeding
embryos. Proc Natl Acad Sci USA 96: 12935–12940 conjugated linoleic acid to animals partially overcomes-
Chen CA, Lu W, Sih CJ (1999) Synthesis of 9Z, 11E- catabolic responses due to endotoxin injection. Biochem
octadecadienoic and 10E, 12Z-octadecadienoic acids, the Biophys Res Commun 198: 1107–1112
major components of conjugated linoleic acid. Lipids Mitchell AG, Martin CE (1995) A novel cytochrome b5-like
34(8): 879–884 domain is linked to the carboxyl terminus of the Saccha-
Chisholm MJ, Hopkins CY (1967a) Calendic acid in seed romyces cerevisiae ⌬-9 fatty acid. J Biol Chem 270:
oils of the genus Calendula. Can J Biochem 45: 251–255 29766–29772
Chisholm MJ, Hopkins CY (1967b) Conjugated fatty acids Okuley J, Lightner J, Feldman KA, Yadav NS, Lark E,
in some Cucurbitaceae seed oils. Can J Biochem 45: Browse J (1994) Arabidopsis FAD2 gene encodes the en-
1081–1086 zyme that is essential for polyunsaturated lipid synthe-
Covello PS, Reed DW (1996) Functional expression of the sis. Plant Cell 6: 147–158
extraplastidial Arabidopsis thaliana oleate desaturase gene Page RDM (1996) TREEVIEW: an application to display
(FAD2) in Saccharomyces cerevisiae. Plant Physiol 111: phylogenetic trees on personal computers. Computer
223–226 Appl Biosci 12: 357–358
Crombie L, Holloway JH (1985) The biosynthesis of calen- Park Y, Albright KJ, Liu W, Storkson JM, Cook ME,
dic acid, octadeca-(8E, 10E, 12E)-trienoic acid, by devel- Pariza MW (1997) Effect of conjugated linoleic acid on
oping marigold seeds: origin of (E,E,Z) and (Z,E,Z) con- body composition in mice. Lipids 32: 853–858
jugated triene acids in higher plants. J Chem Soc Perkin Pariza MW (1997) Conjugated linoleic acid, a newly recog-
Trans 1: 2425–2434 nized nutrient. Chem Industry 16: 464–466
Dobson G (1998) Identification of conjugated fatty acids by Pariza MW, Park Y, Cook ME (1999) Conjugated linoleic
gas chromatography-mass spectrometry of 4-methyl- acid and the control of cancer and obesity. Toxicol Sci 52:
1,2,4-triazoline-3,5-dione adducts. JAOCS 75(2): 137–142 107–110
Fritsche K, Hornung E, Peitzsch N, Renz A, Feussner I Polan CE, McNeill JJ, Tove SB (1964) Biohydrogenation of
(1999) Isolation and characterization of a calendic acid unsaturated fatty acids by rumen bacteria. J Biol Chem
producing (8, 11)-linoleoyl desaturase. FEBS Lett 462: 88(4): 1056–1064
249–253 Qiu X, Erickson L (1994) A simple and effective method for
Fritsche S, Fritsche J (1998) Occurrence of conjugated li- isolating RNA from alfalfa pollen. Plant Mol Biol Re-
noleic acid isomers in beef. JAOCS 75: 1449–1451 porter 12: 209–214
Gietz D, St. Jean A, Woods RA, Schiestl RH (1992) Im- Sambrook J, Fritsch EF, Maniatis T (1989): Molecular
proved method for high efficiency transformation of in- Cloning: A Laboratory Manual. Cold Spring Harbor Lab-
tact yeast cells. Nucleic Acids Res 20: 1425 oratory Press, Cold Spring Harbor, NY
Haumann BF (1996) Conjugated linoleic acid offers re- Shanklin J, Cahoon EB (1998) Desaturation and related
search promise. Inform 7: 152–159 modifications of fatty acids. Annu Rev Plant Physiol
Hopkins CY, Chisholm MJ (1968) A survey of the conju- Plant Mol Biol 49: 611–641
gated fatty acids of seed oils. JAOCS 45: 176–182 Smith CR (1970) Occurrence of unusual fatty acids in
Ip C (1997) Review of the effects of trans fatty acids, oleic plants. In RT Holman, ed, Progress in the Chemistry of
acid, n-3 polyunsaturated fatty acids, and conjugated Fats and Other Lipids. Vol XI, part 1. Pergamon Press,
linoleic acid on mammary carcinogenesis in animals. Oxford, pp 137–177
Am J Clin Nutr 66: S1523–S1529 Thompson JD, Higgins DG, Gibson TJ (1998) CLUSTAL
854 Plant Physiol. Vol. 125, 2001
Downloaded on May 3, 2021. - Published by https://plantphysiol.org
Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.Calendula Fatty Acid Conjugase
W: improving the sensitivity of progressive multiple se- Wise ML, Hamberg M, Gerwick WH (1994) Biosynthesis
quence alignment through sequence weighting, position- of conjugated triene-containing fatty acids by a novel
specific gap penalties and weight matrix choice. Nucleic isomerase from the red marine alga Ptilota filicina. Bio-
Acids Res 22: 4673–4680 chemistry 33: 15223–15232
van de Loo FJ, Broun P, Turner S, Somerville C (1995) An Wise ML, Hamberg M, Gerwick WH (1997) Characteriza-
oleate 12-hydroxylase from Ricinus communis L. is a fatty tion of the substrate binding site of polyenoic fatty acids
acyl desaturase homolog. Proc Natl Acad Sci USA 92(15): isomerase, a novel enzyme from the marine alga Ptilota
6743–6747 filicina. Biochemistry 36: 2985–2992
Plant Physiol. Vol. 125, 2001 855
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