Eur. J. Biochem.

267, 85±96 (2000) q FEBS 2000

Expression in yeast and tobacco of plant cDNAs encoding

acyl CoA:diacylglycerol acyltransferase
Pierrette Bouvier-NaveÂ1, Pierre Benveniste1, Peter Oelkers2, Stephen L. Sturley2 and Hubert Schaller1
1
Institut de Biologie MoleÂculaire des Plantes, Strasbourg, France; 2Institute of Human Nutrition, Columbia University College of Physicians
and Surgeons, New York, USA

During the course of a search for cDNAs encoding plant sterol acyltransferases, an expressed sequence tag clone
presenting substantial identity with yeast and animal acyl CoA:cholesterol acyltransferases was used to screen
cDNA libraries from Arabidopsis and tobacco. This resulted in the isolation of two full-length cDNAs encoding
proteins of 520 and 532 amino acids, respectively. Attempts to complement the yeast double-mutant are1 are2
defective in acyl CoA:cholesterol acyltransferase were unsuccessful, showing that neither gene encodes acyl
CoA:cholesterol acyltransferase. Their deduced amino acid sequences were then shown to have 40 and 38%
identity, respectively, with a murine acyl CoA:diacylglycerol acyltransferase and their expression in are1 are2 or
wild-type yeast resulted in a strong increase in the incorporation of oleyl CoA into triacylglycerols. Incorporation
was 2±3 times higher in microsomes from yeast transformed with these plant cDNAs than in yeast transformed
with the void vector, clearly showing that these cDNAs encode acyl CoA:diacylglycerol acyltransferases.
Moreover, during the preparation of microsomes from the Arabidopsis DGAT-transformed yeast, a floating layer
was observed on top of the 100 000 g supernatant. This fraction was enriched in triacylglycerols and exhibited
strong acyl CoA:diacylglycerol acyltransferase activity, whereas almost no activity was detected in the
corresponding clear fraction from the control yeast. Thanks to the use of this active fraction and
dihexanoylglycerol as a substrate, the de novo synthesis of 1,2-dihexanoyl 3-oleyl glycerol by AtDGAT could
be demonstrated. Transformation of tobacco with AtDGAT was also performed. Analysis of 19 primary
transformants allowed detection, in several individuals, of a marked increase (up to seven times) of
triacylglycerol content which correlated with the AtDGAT mRNA expression. Furthermore, light-microscopy
observations of leaf epidermis cells, stained with a lipid-specific dye, showed the presence of lipid droplets in the
cells of triacylglycerol-overproducer plants, thus illustrating the potential application of acyl CoA:diacylglycerol
acyltransferase-transformed plants.
Keywords: expression in yeast; floating lipid layer; plant diacylglycerol acyltransferase; triacylglycerol synthesis;
transgenic tobacco.

Acyl CoA:diacylglycerol acyltransferase (DGAT; EC 2.3.1.20)
is a membrane-bound enzyme that catalyzes the last and only
committed step in triacylglycerol synthesis, by using a
diacylglycerol and a fatty acyl CoA as its substrates [1,2]. In
animals, triacylglycerol synthesis is involved in numerous
processes such as regulation of plasma triacylglycerol concen-
tration, fat storage in adipocytes and milk production [3]. In
yeast, triacylglycerols and steryl esters coaccumulate under
certain physiological conditions in lipid particles (or lipid
bodies), which constitute a fatty acid and sterol storage pool
Correspondence to P. Bouvier-NaveÂ, Institut de Biologie MoleÂculaire des
Plantes, DeÂpartement de Biologie Cellulaire et MoleÂculaire, Institut de
Botanique, 28 rue Goethe, F-67083 Strasbourg cedex, France.
Fax: + 33 3 8835 8484, E-mail: Pierre.Benveniste@ibmp-ulp.u-strasbg.fr
Abbreviations: ACAT, acyl CoA:cholesterol acyltransferase; ARGP,
ACAT-related gene product; DGAT, diacylglycerol acyltransferase; EREE,
ethylene responsive enhancer element; EST, expressed sequence tag; PGK,
phosphoglycerate kinase; p.f.u., plaque-forming units.
Enzymes: acyl CoA:cholesterol acyl transferase (EC 2.3.1.26); acyl
CoA:diacylglycerol acyltransferase (EC 2.3.1.20).
Note: a web page is available at http://www.ibmp.u-strasbg.fr/
(Received 30 July 1999, revised 1 October 1999, accepted
22 October 1999)
[4,5]. In plants, triacylglycerol synthesis is mainly involved in
the generation of seed oils [6] but triacylglycerols are also
stored in fruits, petals and mature pollen grains [7] where they
might be important as a source of fatty acid for the rapid growth
of the pollen tube [8].
DGAT-catalyzed esterification of diacylglycerol was pro-
posed to be an important step in the control of plant
triacylglycerol synthesis [9,10] and cloning of the DGAT
gene would allow us to ascertain this role. In recent months two
cDNA clones have been reported, the deduced proteins of
which showed identity with known acyl CoA:cholesterol
acyltransferases (ACATs). One called ACAT-related gene
product (ARGP) was isolated from Homo sapiens [11] and
another from Mus musculus [12]. Actually both clones were
shown not to code for an ACAT. The H. sapiens cDNA
encoding ARGP did not complement a yeast are1 are2 double-
mutant defective in the two ACATs (ARE1 and ARE2) present
in wild-type yeast. Moreover, the murine cDNA, when
expressed in H5 insect cells, did not produce any ACAT
activity but was unambiguously shown to encode a DGAT [12].
More recently an Arabidopsis thaliana cDNA presenting
significant identity with murine DGAT was cloned by Hobbs
et al. [13]. This cDNA was expressed in insect cell cultures and
the microsomal preparation of these cells catalyzed the
86 P. Bouvier-Nave et al. (Eur. J. Biochem. 267)
synthesis of [14C] triacylglycerol from [14C]dioleylglycerol and
oleyl-CoA thus demonstrating its function. In addition, these
authors showed that the cDNA AtDGAT is expressed most
strongly in developing embryos and petals [13]. During the
course of a search for an ACAT-like plant cDNA, we happened
to isolate, independently and using a different procedure, the
same AtDGAT, as well as its homolog from Nicotiana tabacum.
Both cDNAs were expressed in yeast where we could
accumulate and thoroughly characterize the product of the
enzymatic reaction. Transgenic tobaccos expressing AtDGAT
under the control of a constitutive promoter were generated in
order to prove the function in planta. A strong increase in the
triacylglycerol content of young growing leaves was shown to
be correlated with the appearance of lipid droplets in the cells.
E X P E R I M E N TA L P R O C E D U R E S
Strains, media and culture conditions
Escherichia coli. XL1blue recA-[recA1, lac-, endA1, gyrA96,
thi, hsdR17, SupE44, relA1 (F 0 proAB, lac1q, lacZ D M15,
Tn10)].
Saccharomyces cerevisiae. Two strains of common genetic
background (can 1±100, his 3±11, 15, leu 2-3, 112, trp 1-1,
ura 3-1) were used: SCY059 (MATa, met14D HpaI-SalI,
are1D NA::HIS3, are2D ::LEU2) and the corresponding wild-
type SCY062 (MATa, ade2±1).
Yeast strains transformed with plasmid pYeDP60, harboring
either no insert or the plant cDNA under study, were always
grown at the same time. For sterol or triacylglycerol analysis,
culture conditions were 3 days at 30 8C on minimal medium
[6.7 g´L21 yeast nitrogen base (Difco), 20 g´L21 galactose]
containing suitable supplements (50 mg´mL21 each), i.e. Ade,
Trp and Met for SCY059 (are1 are2) and His, Trp and Leu for
SCY062 (wild-type). The cultures were then centrifuged and
freeze-dried.
For subcellular fractionation, these transformed strains were
grown for 3 days at 30 8C in minimum medium containing
20 g´L21 glucose and the suitable supplements. The cultures
were then centrifuged under sterile conditions, and cells were
resuspended in a complete medium [10 g´L21 yeast extract
(Difco), 10 g´L21 bactopeptone (Difco), 20 g´L21 galactose]
and grown overnight at 30 8C. This two-step procedure resulted
in a high cell mass, suitable for microsome preparation.
Plasmid for yeast transformation
The plasmid pYeDP60 [14] was used to transform yeast strains.
This plasmid contains an E. coli replication origin, a yeast
2 mm plasmid replication origin, an E. coli ampicillin-
resistance gene and the yeast genes URA3 and ADE2. It
utilizes an expression cassette including a galactose-inducible
hybrid promoter and a phosphoglycerate kinase (PGK)
terminator. Gene expression is driven by the upstream
activating sequence of the yeast GAL10 and CYC4 genes.
Cloning of a cDNA encoding DGAT from A. thaliana
An EST cDNA clone (ABRC DNA Stock Center, Columbia,
OM 43210-1002) showing significant identity with ACATs was
sequenced. This 849 bp EST fragment was used to screen a
cDNA library CD4-15 [15] supplied by the ABRC DNA Stock
Center. In total, 500 000 plaque-forming units (p.f.u.) were
screened with the EST clone labeled with 32P after random
priming leading to 12 cDNA clones of different lengths. Among
them 10 were larger than 1.5 kb. When cut with EcoRI these
q FEBS 2000
clones showed a digestion pattern suggesting that they
possess an identical ORF but differ by the respective length
of the 5 0 -UTR and 3 0 -UTR. Two of them were sequenced, the
first (3D11) contained 1845 bp with an ORF of 1560 bp flanked
by 5 0 -UTR and 3 0 -UTR of, respectively, 90 and 195 bp. The
second (3D71) contained 2062 bp with an identical ORF flanked
by 5 0 -UTR and 3 0 -UTR of, respectively, 173 and 429 bp.
Cloning of a cDNA encoding DGAT from N. tabacum
A cDNA library from a 3-week-old N. tabacum cv. Xanthi line
LAB 1-4 calli derived from leaf protoplasts [16] was screened
with the Arabidopsis EST clone of 849 bp. First, 250 000 p.f.u.
were screened with an ORF from the Arabidopsis cDNA 3D11
leading to two clones (1A111 and 2A113). One of them
(2A113) was sequenced and shown to contain 1354 bp and its
deduced amino acid sequence (317 amino acids) displayed a
strong (. 70%) identity with the Arabidopsis DGAT deduced
protein sequence. This clone, called NtDGAT2, was likely
uncomplete. After digestion with EcoRI and HindIII, a 1-kb
fragment was randomly labeled with [32P]dCTP and used to
screen again the tobacco library (250 000 p.f.u.), seven clones
of different lengths were isolated. NtDGAT2 was again found
among them and was shown to be identical to the cDNA found
previously. Another clone presented a length (2099 bp)
comparable with that of the Arabidopsis DGAT. After
sequencing it was shown to encode a polypeptide of 532
amino acids presenting 68% identity with Arabidopsis DGAT
and 86% identity with NtDGAT2. This cDNA was called
NtDGAT1.
Reformatting and cloning DGAT cDNAs into the yeast
expression vector pYeDP60
Deletion of the 5 0 -UTR and 3 0 -UTR of the DGAT cDNAs was
performed by PCR amplification using specific primers.
Arabidopsis thaliana DGAT. Specific primers were designed to
introduce a BamHI restriction site immediatly upstream of the
initiation codon and a KpnI site immediatly downstream of the
stop codon. Direct primer: 5 0 -atatatggatccATGGCGATTTTG-
GATTCTGC-3 0 . Reverse primer: 5 0 -tatataggtaccTCATGA-
CATCGATCCTTTTCG-3 0 . The DGAT was amplified using
25 thermal cycles (1 min 93 8C, 2 min 56 8C, 3 min 72 8C)
with Pyrococcus furiosus (Pfu) DNA polymerase under the
standard conditions. The PCR product was digested with
BamHI and KpnI and subsequently cloned into Bluescript to
give At DGAT-pSK. The BamHI, KpnI insert in pSK was
extracted and subcloned into BamHI, KpnI of pYeDP60 leading
to AtDGAT±pYeDP60.
A. thaliana FLAG DGAT. In order to evaluate the amount of
Arabidopsis DGAT expressed in yeast, an N-terminal FLAG
epitope (MDYKDDDDK, epitope underlined) was fused to the
DGAT protein. For this purpose a DNA molecule containing at
5 0 -end the appropriated nucleotide sequence was synthesized
by PCR. Direct primer: atatatggatccATGGACTACAAGGAC-
GACGATGACAAGGCGATTTTGGATTCTGCTGG. The reverse
primer is the one used above to amplify the At DGAT. The PCR
product was digested with BamHI and KpnI and subsequently
cloned into pYeDP60 to give AtFLAGDGAT±pYeDP60.
N. tabacum DGAT1. Direct primer: atatatggatccATGGTGAT-
CATGGAATTGCC. Reverse primer tatataggtaccTTAACGTG-
CACTGCTTTTCT. The tobacco DGAT1 was amplified using
q FEBS 2000 Plant cDNAs encoding diacylglycerol acyltransferase (Eur. J. Biochem. 267) 87
25 thermal cycles (1 min at 93 8C, 2 min at 56 8C, 3 min at
72 8C) with Pfu DNA polymerase under the standard
conditions. The PCR product was digested with BamHI and
KpnI and subsequently cloned into pYeDP60 to give
NtDGAT1±pYeDP60.
Caenorhabditis elegans DGAT. cDNA EST yk 453 a2 was
kindly donated by Y. Kohara (Mishima, Japan). After sequen-
cing, this clone perfectly matches a processed gene (Z75526)
arising from the systematic sequencing of the genome of this
nematode. The ORF was amplified by PCR under standard
conditions (see above) using the following primers. Direct
primer: atatatggatccATGCAAATGCGTCAACAAACG. Reverse
primer: tatataggtaccTCAAATACCAACGGTTTGG. The PCR
product was digested with BamHI and KpnI and subcloned into
BamHI, KpnI of pYEDP60 leading to CeDGAT±pYeDP60.
Nucleotide sequence determination
Sequencing of cDNAs AtDGAT, AtFLAGDGAT, NtDGAT1 and
CeDGAT, as well as their ORF derivatives made by PCR, was
performed using an automatic sequencer Perkin-Elmer model
373 with T3, T7 primers, specific oligonucleotide sequence
belonging to the sequenced DNA and a modified Taq
polymerase capable of incorporating fluorescent dNTP. Com-
plete sequencing of both DNA strands was performed. cDNAs
were in pSK or in pYeDP60.
Transformation of yeast
Transformation was performed according to Schiestl and Gietz
[17] with some modifications. A fresh yeast culture (initial
absorbance = 0.2) was grown in complete medium YPG
[10 g´L21 yeast extract (Difco), 10 g´L21 bactopeptone
(Difco), 20 g´L21 glucose] for 5 h. The cells were collected,
washed twice with water and then with 1.5 mL of a 0.1 m
lithium acetate solution in Tris/EDTA buffer (1 mm EDTA,
10 mm Tris/HCl, pH 7.5) and finally resuspended in 200 mL of
the same solution. Salmon sperm was added as a DNA carrier
(100 mg from a 10 mg´mL21 solution in Tris/EDTA) after
sonication (10 s) and boiling (20 min) to the plasmid DNA
(1 mg). Competent yeast cells (50±80 mL) and 50 mL of
40% poly(ethylene glycol), 0.1 m lithium acetate solution in
Tris/EDTA (pH 7.5) were added. The mixture was incubated
for 30 min at 30 8C followed by 15 min at 42 8C. After
centrifugation, the cells were resuspended in YPG (1 mL),
incubated 1 h at 30 8C, collected and then plated (with 100 mL
water) on minimum medium containing suitable supplements
(50 mg´mL21 each).
Plant expression vectors
A cDNA 3D11 encoding the ORF of At DGAT was subcloned
into the BamHI±KpnI sites of a pBD 121 binary vector
derivative engineered as described by Husselstein et al. [18].
In particular, the vector contained between the left and right
T-DNA borders a kanamycin resistance gene and a multiple
cloning site flanked in the 5 0 -region by a duplicated 35S
promoter described by Kay et al. [19] and by a nopaline
synthase terminator at the 3 0 -end. The At DGAT construct
and the corresponding void plasmid were transferred from
E. coli XL1 Blue into Agrobacterium tumefaciens LB4404
[20] by triparental mating with the helper plasmid pRK
2013 [21].
Plant transformation
Tobacco (N. tabacum L. var Xanthi) was transformed via
Agrobacterium tumefaciens according to a modification [22] of
the method reported by Horsch et al. [23]. In brief, leaf pieces
were cocultivated with an overnight A. tumefaciens culture on
Murashige and Skoog medium supplemented with glucose
(30 g´L21), ANA (0.1 mg´L21) and 6-benzylaminopurine
(1 mg´L21) for 2 days and then transferred onto the same
medium supplemented with kanamycin (100 mg´L21) as the
plant-selective agent and cefotaxime (500 mg´L21) to prevent
further bacterial growth. Regenerated shoots were rooted on
Murashige and Skoog medium with a half-reduced concentra-
tion of NH4NO3 and supplemented with sucrose (30 g´L21),
kanamycin (200 mg´L21 ) and cefotaxime (200 mg´L21 ). In
vitro cultures were grown under a 16-h light period at 24 8C
and an 8-h dark period at 20 8C. Primary transformants were
subcultured as T1 vitro-plantlets every 7±8 weeks in order to
generate leaf material in sufficient quantities for analytical
processes. Primary transformants were then transferred to the
greenhouse and grown under standard conditions.
RNA gel blot analysis
Total RNAs from leaf material of 6-week-old in vitro-grown
primary transformants were extracted according to the method
described by Goodall et al. [24]. Northern analysis was carried
out by separating 10 mg RNA samples on formaldehyde gel as
described previously [25] and blotting them onto nylon
filters (Hybond-N+, Amersham). Randomly primed (Strata-
gene) 32 P-labeled probes polymerized from the 3D11 cDNA
were hybridized to the filters as recommended by the
manufacturer. Filters were then washed in 0.2 NaCl/Cit
containing 0.1% SDS at 65 8C before autoradiography. Filters
were further washed in 0.1% SDS at 70 8C for 3 h and then
hybridized to randomly primed 32P-labeled probes polymerized
from a 18S rRNA as an internal standard.
Lipid analysis
Lipids were extracted from freeze-dried yeast cells or tobacco
leaves by immersion in methylene chloride/methanol (2 : 1)
and heating under reflux for 1 h. The extract was submitted to
TLC in methylene chloride into sterol esters (Rf = 0.8±0.7),
triacylglycerols (Rf = 0.5±0.4) and free sterols (Rf = 0.3±0.1)
using cholesteryl palmitate, trioleylglycerol, lanosterol and
cholesterol as references.
Sterol esters were hydrolyzed by heating under reflux for 1 h
with methanolic KOH (6%). The sterols were then extracted
with hexane. Both free sterols and sterols corresponding to
sterol esters were acetylated according to Schmitt and
Benveniste [26]. Sterol acetates were purified by TLC in
methylene chloride, then quantified and identified by GC on a
DB-1 capillary column (according to their relative retention
time to the internal standard cholesterol), as described
previoulsy [22].
Triacylglycerols purified by TLC from the lipid extract of
either yeast or transgenic tobacco leaves were quantified using
the colorimetric assay Perichrom Triglycerides GPO-PAP from
Roche-Diagnostics (Meylan, France), according to McGowan
et al. [27].
GC-MS was performed on a computerized gas-chromato-
graph mass spectrometer (Fison MD500) equipped with an
on-column injector and a capillary column (30 m  0.25 mm
internal diameter) coated with DB5 (J&W Scientific). The
88 P. Bouvier-Nave et al. (Eur. J. Biochem. 267)
different fragments obtained are designated by the ratio m/z and
their relative intensity.
Lipid-specific staining of cells
Leaf epidermis was stained in a satured Sudan IV (dye no.
26106 according to the color index) solution in 70% (v/v)
ethanol. Lipid droplets appear as orange spherical granules on
light-microscopy.
Subcellular fractionation
Yeasts were grown for 3 days in 100 mL glucose minimum
medium followed by 16 h in 200 mL galactose complete
medium (see above). The harvested cells were then disrupted as
described previously [28] except that KCl was omitted from the
washing buffer and BSA (1%) was added to the disruption
medium. The homogenate was centrifuged for 10 min at
12 000 g and the supernatant for 60 min at 100 000 g. The
lipid layer floating at the top of the 100 000 g supernatant from
the plant DGAT-transformed yeasts was carefully withdrawn as
was the corresponding volume (4±6 mL) from the void
plasmid-transformed yeast. The microsomal pellet was resus-
pended (2±5 mg protein´mL21) in 0.1 m Tris/HCl pH 7
containing 20% (v/v) glycerol. In some experiments (Fig. 3A,
C) microsomes and the floating lipid layer were washed by
dilution in Tris/HCl buffer and recentrifugation. Coarse or
washed fractions were kept at 280 8C for months without
significant loss of activity.
Proteins were quantified using the Bio-Rad protein assay
according to Bradford [29]. Western blots of microsomes (6 mg
protein) or the floating lipid layer (5 mL) from AtFLAGDGAT-
transformed yeast were achieved after SDS/PAGE, electro-
transfer to a nylon membrane (Hybond C, Amersham) and
immunoblotting with the anti-FLAG M2 mAb from IBI/Kodak.
Enzymatic assays
ACAT assays were performed according to Billheimer et al.
[30] using either [14C]cholesterol or [14C]oleyl CoA.
DGAT was assayed according to Cases et al. [12] with some
modifications. Diacylglycerol (either dioleylglycerol or dihex-
anoylglycerol, 400 mm) was resuspended in 0.1 m Tris/HCl
buffer pH 7 containing 20% glycerol, by vigorous shaking. The
enzymatic preparation, either microsomes (50 mg protein) or
100 000 g supernatant top fraction (40 mL) was then added. In
the control assays, proteins were omitted. After the addition of
[14C]oleyl CoA (20 mm, 200 000 c.p.m.) the reaction mixture
(100 mL) was incubated for 10 min at 30 8C. Under these
conditions the substrates were shown to be saturating, the
reaction was linear with protein and time and the reaction yield
was kept below 10%. The reaction was stopped by adding a
mixture of methylene chloride (400 mL) and methanol
(100 mL) containing trioleylglycerol (100 mg) and cholesteryl
palmitate (100 mg) as carriers. After further addition of water
(300 mL) and methylene chloride (600 mL), the organic phase
was withdrawn and the water phase was extracted twice more
with methylene chloride. The lipid extract was separated by
TLC with trioleylglycerol as standard and the radioactive
triacylglycerol bands (Rf 0.5±0.4 for incubations with or
without dioleylglycerol, Rf 0.3±0.2 for those with dihexanoyl-
glycerol) were detected with an automatic TLC-linear analyzer
(Berthold), and scrapped off the plate for counting.
A large-scale, high-yield DGAT assay was set up for GC and
GC-MS. The incubation mixture (2 mL) had the same
q FEBS 2000
composition as described above except that unlabeled oleyl
CoA (40 mm) was used and a longer incubation time (2 h) was
applied. After purification, the triacylglycerol formed from
dihexanoylglycerol was quantified by GC using the same
column and temperature program as for steryl acetates. GC-MS
analysis yielded the following spectrum: 552 (M+; 0.1), 534
(M+-H2O; 0.2), 438 (5), 437 (M+-hexanoyloxy; 5), 393
(oleyl + 128; 3), 363 (2), 340 (2), 339 (oleyl + 74; 2), 337
(2), 283 (oleic acid + 1; 5), 271 (M+-oleyloxy; 49), 265 (oleyl;
6), 264 (10), 257 (3), 227 (hexanoyl + 128; 10), 173
(hexanoyl + 74; 21), 99 (hexanoyl; 100). These characteristic
triacylglycerol fragmentation ions, as described by Barber
et al. [31] and Christie [32], correspond unambiguously to
1,2-dihexanoyl 3-oleyl glycerol.
R E S U LT S
Cloning of a cDNA encoding DGAT from A. thaliana
In order to isolate a full-length cDNA clone encoding an
Arabidopsis enzyme able to acylate plant sterols, we started
from an EST clone (GenBank accession number AA042298).
After sequencing, this EST cDNA was shown to contain 849 bp
and the deduced protein sequence had 30 and 25% identity with
H. sapiens [33] and S. cerevisiae [34] ACATs, respectively.
This EST clone was used as a probe to screen an A. thaliana
cDNA library, as described above. After screening
500.000 p.f.u., 12 cDNA clones of different lengths were
isolated. Among them, 10 had a length compatible with those
(< 2 kb) reported for mammalian and baker's yeast ACAT
cDNAs. Two (3D11 and 3D71) were sequenced and contained
1845 and 2162 bp, respectively. They differed only by the
respective length of their 5 0 -UTR and 3 0 -UTR. The 5 0 -UTR and
3 0 -UTR of 3D71 were 173 and 429 bp. Both were shown to
encode a polypeptide of 520 amino acids (58 947 Da)
presenting 25% identity with both H. sapiens and baker's
yeast ACATs and 40% identity with an ARGP from H. sapiens
[11] and a DGAT from Mus musculus [12] (Table 1). It was
identical to that isolated by Hobbs [13].
Cloning of a cDNA encoding DGAT from N. tabacum and
comparison of its deduced protein sequence with those of
other acyltransferases
A cDNA library from a 3-week-old N. tabacum cv. Xanthi line
LAB 1-4 calli derived from leaf protoplasts [16] was screened
using the A. thaliana EST clone. As described above, seven
cDNA clones of different lengths were isolated. Among them
one had a length (2099 bp) comparable with that of the
Arabidopsis DGAT. It was shown to encode a polypeptide of
532 amino acids presenting 68% identity with Arabidopsis
DGAT (Table 1). Strikingly, the first 100 amino acids from the
N-terminal sequence show a low identity (19%) with the
corresponding Arabidopsis sequence. After amino acid 135, the
identity increases abruptly and becomes . 70%. The Kyte
Doolittle hydrophobicity profile is very similar to that of the
Arabidopsis DGAT with a hydrophilic moiety of 120 amino
acids and 10 hydrophobic domains [13]. As in the Arabidopsis
DGAT, and at exactly the same place, four leucines (L230,
L237, L244, L251), are regularly spaced out by six variable
amino acids forming a typical leucine zipper. This cDNA clone
was called Nt DGAT1. Another cDNA clone had a length of
1354 bp. Its deduced amino acid sequence (317 amino acids)
was much shorter than that of NtDGAT1 but had a high identity
(86%) with it. This second cDNA (NtDGAT2) was of course
q FEBS 2000 Plant cDNAs encoding diacylglycerol acyltransferase (Eur. J. Biochem. 267) 89

Table 1. Comparison of the deduced amino acid sequence of A. thaliana DGAT with those of other acyltransferases showing % identity. The names
and accession numbers are those in Fig. 1, except ScACAT1 and 2, which are the Saccharomyces cerevisiae sequences (P25268 and U51790, respectively)
and CeACAT, which is the Caenorhabditis elegans sequence (Z68131).

AtDGAT NtDGAT HsARGP MmDGAT CeDGAT HsACAT1 HsACAT2 ScACAT1 ScACAT2 CeACAT

AtDGAT 100
NtDGAT 68 100
HsAGRP 40 38 100
MmDGAT 39 38 86 100
CeDGAT 36 37 47 47 100
HsACAT1 25 25 26 27 25 100
HsACAT2 24 26 25 26 25 49 100
ScACAT1 26 26 21 23 24 23 25 100
ScACAT2 26 27 23 24 26 28 24 49 100
CeACAT 20 19 24 24 21 34 31 23 25 100

incomplete but its presence in the library showed that there are
at least two genes encoding DGAT in tobacco.
Sequence comparison of the DGATs from Arabidopsis and
tobacco with the two human ACATs [33], murine DGAT [12],
the putative human DGAT called ARGP [11] and an unknown
cDNA from C. elegans (putative DGAT) is shown in Fig. 1.
Remarkably, all these sequences present the same global pattern
and are composed of a strongly hydrophilic moiety of < 100
amino acids (in blue) followed by 10 hydrophobic domains (in
red and boxed) which may correspond, in the case of DGATs,
to nine membrane helix spanning domains as confirmed by the
transmembrane predict program (http://ulrec3.unil.ch/software/
TMPRED-form.html). In addition all these cDNAs have several
invariant domains in the second moiety of the deduced protein.
A remarkably conserved domain (AELLCFGDREFYKDWW)
is situated between amino acids 382 and 400 of the AtDGAT.
This sequence is present in a hydrophilic domain situated
between two supposed transmembrane helices. It is common to
both DGATs and ACATs. Also worthy of interest is the
presence in all DGATs and ACATs of an invariant serine
(Ser254 in AtDGAT) which has been shown to be essential for
ACAT activity [37]. However, the DGAT sequences can easily
be distinguished from the ACAT sequences. The five DGAT
sequences possess a remarkable motif consisting of four
consecutive positively charged amino acids (four Arg in four
of five DGATs, three Arg and one Lys in AtDGAT). This cluster
is absent from all ACATs. Several other motives are also
restricted to the five DGATs, one example is a sequence
IERVLKL starting at Ileu352 of AtDGAT.
Organization of the gene encoding A. thaliana DGAT
In recent months a genomic sequence originated from
chromosome II sequencing appeared in GenBank (AC005917)
and was designated as `putative acyl CoA:cholesterol acyl-
transferase'. According to databases (gene prediction program
grail), this sequence was processed into a polypeptide (441
amino acids) matching partially the protein (520 amino acids)
deduced from the AtDGAT cDNA. Using the GTX and XAG
rule defining introns boundaries for splicing, we reconstituted
from a 5051-bp genomic sequence corresponding to the
polypeptide of 441 amino acids, a cDNA perfectly matching
our AtDGAT cDNA. According to this study the gene encoding
the Arabidopsis DGAT was formed by 16 exons separated by 15
introns (Fig. 2). The sequence (817 bp) situated upstream of
the ATG was submitted to the place signal scan program
(http://www.dna.affrc.go.jp/htdoc/PLACE/WAIS.html) to identify
cis-acting regulatory DNA elements possibly intervening in the
regulation of DGAT expression. Interestingly an ethylene-
responsive element (ATTTCAAA) [36] was shown to be
present 719 bp upstream of the ATG. This important finding
is discussed below.
If one now considers the sequence downstream of the ATG, it
appears that the first exon (130 amino acids) corresponds
exactly to the hydrophilic moiety of the protein. The second
exon fits precisely with the first hydrophobic trans-membrane
segment.
Expression of AtDGAT in a yeast mutant deficient in ACAT
activity
Because this cDNA was first expected to encode an ACAT-like
protein, it was expressed in the yeast double-mutant are1 are2
under control of the GAL10-CYC1 promoter from the vector
pYeDP60. Because in this mutant the two ACAT genes have
been knocked out, sterol ester biosynthesis is absent [33] and
microsomes from this yeast lack ACAT activity [32]. Sterol
ester and free sterol contents were determined for AtDGAT-
transformed are1 are2 and the void plasmid-transformed yeast.
Both were devoid of sterol esters and had similar sterol profile
(data not shown). Microsomes were prepared from both yeasts
and ACAT activity was assayed. It was undetectable under
conditions in which microsomes from the corresponding wild-
type yeast, transformed or not with AtDGAT, displaid
significant activity (160 pmol sterol esters per mg protein per
min). Taken together, these results clearly showed that cDNA
AtDGAT does not encode an ACAT.
Meanwhile, Cases et al. [12] described a mouse cDNA
encoding a DGAT, with which AtDGAT shared 40% identity.
Therefore, the DGAT activities of AtDGAT-transformed are1
are2 and void plasmid-transformed are1 are2 were measured
and compared. Microsomes from both yeasts were incubated
with [14C]oleyl CoA in the absence or presence of dioleyl-
glycerol or dihexanoyl-glycerol (Fig. 3A). The results clearly
show that although microsomes from the void plasmid-
transformed yeast display a significant DGAT activity due to
the yeast enzyme, microsomes from the AtDGAT-transformed
yeast show a higher (more than double) activity, corresponding
to the expression of both the yeast and the plant genes. These
results further show that yeast and plant DGAT activities are
measurable without added diacylglycerols, presumably because
diacylglycerols are present in the enzymatic preparations or
produced during the course of the incubation. The addition of
dioleylglycerol or dihexanoylglycerol significantly increases
90 P. Bouvier-Nave et al. (Eur. J. Biochem. 267) q FEBS 2000

Fig. 1. Sequence alignment of various putative or demonstrated diacylglycerol or acyl CoA cholesterol acyltransferases. DGAT, acyl
CoA:diacylglycerol acyltransferase; ACAT, acyl CoA:cholesterol acyltransferase; ARGP, ACAT-related gene product. Alignments were performed using
the pileup program of the GCG package run with default parameters and the seqvu program. AtDGAT is the A. thaliana sequence (GenBank accession
number AF051849). NtDGAT1 is the N. tabacum sequence (GenBank accession number AF129003). MmDGAT is the M. musculus sequence
(GenBank accession number AF078752). HsARGP is the H. sapiens sequence (GenBank accession number AF059202). CeDGAT is the C. elegans
sequence (GenBank accession number Z75526). HsACAT1 is the H. sapiens sequence (GenBank accession number L21934). HsACAT2 is the
H. sapiens sequence (GenBank accession number AF059203). Hydrophilic domains are in blue. Ten hydrophobic segments (red) are boxed. Signs are
placed under important amino acids (e.g. the Ser254 of the Arabidopsis cDNA) or highly conserved motives such as the invariant
FGDREFYRDWWN segment. Clusters of arginines present in DGATs are underlined. Leucines involved in a putative leucine zipper are circled.

both yeast and plant activities. Expression of AtDGAT in the
corresponding wild-type yeast gave identical results (data not
shown). Thus expression of cDNA AtDGAT in yeast produces a
2±3 times increase of DGAT activity in the microsomal
fraction.
Expression of AtDGAT, AtFLAGDGAT and NtDGAT1 in
wild-type yeast
The wild-type yeast was transformed with AtDGAT, a FLAG
epitope-tagged version of AtDGAT called AtFLAGDGAT, a
N. tabacum homolog of AtDGAT, called NtDGAT1, or the void
plasmid. The triacylglycerol content of these strains was
determined. The three DGAT-transformed yeasts had signifi-
cantly higher triacylglycerol contents than the void plasmid-
transformed yeast (25±80 versus 9 ^ 3 nmol triacylglycerol
per mg dry weight).
DGAT activity was measured in microsomes from yeast
transformed with either AtDGAT, AtFLAGDGAT or NtDGAT1
in the presence of [14C]oleyl CoA and dioleyl-glycerol as
substrates and shown to be significantly higher than that of the
void plasmid-transformed yeast (20±24 versus 6 nmol triacyl-
glycerol formed per mg protein per h). Taken together, these
results obtained with the wild-type yeast confirm those obtained
q FEBS 2000 Plant cDNAs encoding diacylglycerol acyltransferase (Eur. J. Biochem. 267) 91

Fig. 2. Organization of the gene encoding the diacylglycerol acyltransferase from A. thaliana. The part of the gene corresponding to the coding region
(3020 bp) contains 16 exons and 15 introns. Exons are shown by continuous heavy lines, introns are shown by continuous thin lines. Parts of the gene
upstream and downstream of the coding region are shown by broken lines. The numbers under the exons and above the introns are in bp. The motif
ATTTCAAA at 2719 bp is a putative EREE. The arrow indicates the translation start point determined by Hobbs et al. [13]. S,E,A: SacI, EcoRI, AseI.
Letters b and e mark the 5 0 -end and the 3 0 -end of the longest cDNA (3D71).

for AtDGAT with the mutant are1 are2, indicate that the FLAG
epitope-tagged version of AtDGAT produces a functional
protein and clearly show that NtDGAT1 encodes a DGAT.
The microsomal fractions from AtDGAT-transformed and
AtFLAGDGAT-transformed yeast cells, were submitted to
SDS/PAGE followed by a Western blot analysis using
commercial anti-FLAG serum (Fig. 3B). The blot revealed a
tagged protein with a molecular mass (54 kDa) close to that
(59 kDa) calculated for AtDGAT according to its sequence.
added diacylglycerols, probably for the same reasons. However,
the addition of dioleylglycerol or dihexanoylglycerol signifi-
cantly increases the activity of the plant enzyme expressed in
yeast (Fig. 3C). Dihexanoylglycerol was again a good substrate
and, because of its low molecular mass, the resulting
triacylglycerol (Rf = 0.2) could be easily separated from the

Expression of AtDGAT in the floating lipid layer

from the transformed yeast: identification of the
enzymatic product
In the course of the preparation of microsomes from plant
DGAT-transformed yeasts, we consistently observed the
formation of a cloudy floating layer on top of the 100 000 g
supernatants from the AtDGAT-tranformed, AtFLAGDGAT-
transformed or, to a lesser extent, NtDGAT1-transformed
yeasts, whereas the supernatant from the void plasmid-
transformed yeast remained clear. These floating layers and
the corresponding region of the 100 000 g supernatant from
the void plasmid-transformed yeast were taken from the
centrifugation tubes for analysis.
Extraction of lipids and purification of triacylglycerols by
TLC revealed that the floating layer from the AtDGAT-
transformed yeast had a much higher triacylglycerol mass
(assessed by I2 visualization) than the 100 000 g supernatant
top fraction from the void plasmid-transformed yeast (data not
shown). Furthermore, the floating lipid layers from yeast
transformed with AtDGAT, AtFLAGDGAT or NtDGAT1 were
shown to display much higher DGAT activities than the
100 000 g supernatant top from the void plasmid-transformed
yeast (5, 4 and 1 versus 0.1 nmol´mL21´h21). The presence of
plant DGAT in the floating lipid layer from AtFLAGDGAT-
transformed yeast was confirmed by SDS/PAGE followed by
Western blot analysis (Fig. 3B).
The DGAT activities of the 100 000 g supernatant top
fractions from yeast transformed with AtDGAT or the void
plasmid were studied further. The activities were measured with
[14C]oleyl CoA in the absence or presence of dioleylglycerol or Fig. 3. DGAT enzymatic activities in subfractions of yeast expressing
dihexanoylglycerol (Fig. 3C). The 100 000 g supernatant top AtDGAT. Yeasts were transformed with B the void pYeDP60 plasmid, B
from the control yeast is almost devoid of activity, whereas the AtDGAT in pYeDP60, or AtFLAGDGAT in pYeDP60. (A) DGAT activities
floating lipid layer from the AtDGAT-transformed yeast (nmol triacylglycerol per mg protein per h) in washed microsomes from
contains significant activity, 200±600 times higher than that void plasmid-transformed or AtDGAT-transformed are1 are2 mutant. (B)
of the control. Hence the floating lipid layer obtained from Expression of the FLAG-tagged DGAT. Microsomes (6 mg protein) from
AtDGAT-transformed yeast exhibits a DGAT activity almost wild-type yeast transformed with (1) AtFLAGDGAT or (2) AtDGAT and
exclusively due to the plant gene. Because of the presence of 100 000 g supernatant top fractions (5 mL) from wild-type yeast trans-
BSA in the 100 000 g supernatant, specific activities could not formed with (3) AtFLAGDGAT or (4) AtDGAT were resolved by SDS/PAGE.
be determined for this fraction. Its total DGAT activity Proteins were submitted to immunoblotting with an anti-FLAG serum.
represented one half to one sixth, according to the experiment, (C) DGAT activities (nmol triacylglycerol´mL21´h21) in twice-washed
that of the corresponding microsomal fraction. 100 000 g supernatant top fractions from void plasmid-transformed or
As observed with the microsomal preparations (Fig. 3A) the At DGAT-transformed wild-type yeast. DOG, dioleylglycerol; DHG,
DGAT activity of the floating lipid layer is measurable without dihexanoylglycerol.
92 P. Bouvier-Nave et al. (Eur. J. Biochem. 267)
usual long-chain fatty acid triacylglycerols (Rf = 0.4) using
TLC. This latter TLC band corresponds to unlabeled pre-
existing triacylglycerols together with labeled triacylglycerols
formed during the incubation from endogenous diacylglycerols.
In order to identify unambiguously the product of the reaction
catalyzed by the A. thaliana DGAT, we scaled up (20 times)
the usual incubation mixture with dihexanoylglycerol and
the floating lipid layer from AtDGAT-transformed yeast,
lengthened the incubation time (to 2 h) and could thus purify
20 mg of the short-chain fatty acid triacylglycerol. GC-MS
analysis of this pure product clearly confirmed its structure as
1,2-dihexanoyl 3-oleyl glycerol (see MS in Experimental
procedures). It must be noted that the same high-scale, long-
time incubation with the 100 000 g supernatant top fraction
from the control yeast yielded 0.5 mg of this low molecular
mass triacylglycerol. Under these conditions, the floating lipid
layer from the AtDGAT-transformed yeast produced 40 times
Fig. 5. Light micrographs ( 850) of petiole or limb epidermis cells after staining in a solution of Sudan IV. (A) Wild-type petiole epidermis; (B) wild-
type limb epidermis; (C) petiole epidermis of triacylglycerol-overproducer line 5; (D) line 5 limb epidermis. triacylglycerol dosage gave the following values
for the samples shown in this figure: wild-type leaf material, 3.1 nmol triacylglycerol per mg dry weight; line 5 leaf material, 11.5 nmol triacylglycerol per
mg dry weight.
q FEBS 2000
Fig. 4. Expression of At DGAT in N. tabacum.
Lanes 1±19, primary transformants; lane 20,
wild-type tobacco. (A) Triacylglycerol content.
Triacylglycerols were extracted from young
leaves, purified by TLC and quantified using a
colorimetric assay. The column in lane 20
corresponds to the mean of three different
wild-type plants (21 ^ 5 nmol triacylglycerol
per mg dry weight). (B) Northern analysis: total
leaf RNA from primary transformants (around
10 mg) was hybridized with a probe derived
from the Arabidopsis DGAT. The arrow denotes
the DGAT 1.5 kb transcript. (C) 18S rRNA
hybridization of the same blot is shown to
illustrate the relative amount of RNA loaded
and transferred.
more 1,2-dihexanoyl 3-oleyl glycerol, which definitely proves
the function of the protein encoded by AtDGAT.
Expression of AtDGAT in N. tabacum
AtDGAT cDNA encoding the ORF of the gene was introduced
into N. tabacum under the control of a strong constitutive
promoter (tandem CaMV 35S). Nineteen primary transformants
grown in vitro on kanamycin-supplemented Murashige and
Skoog medium were analyzed and compared with wild-type
tobacco. The upper leaves from 6-week-old plants were
separated into two batches: one for triacylglycerol dosage and
the other for RNA gel blots.
The results shown in Fig. 4 clearly show that, in all cases, the
expression of AtDGAT in tobacco is associated with a
significant increase in the triacylglycerol content. In the best
case, this increase was up to seven times the mean value
q FEBS 2000 Plant cDNAs encoding diacylglycerol acyltransferase (Eur. J. Biochem. 267) 93
measured for wild-type leaf material. The specificity of the
expression was attested to by the fact that control tobacco did
not exhibit any AtDGAT expression, showing that under our
experimental conditions the AtDGAT probe did not hybridize
with the endogenous NtDGAT RNA and allowed quantification
of transgene expression only.
The primary transformants were then transferred to the
greenhouse and grown under standard conditions. Leaf material
was collected at the flowering stage (3.5 months old) and
submitted to triacylglycerol dosage. The results were in full
agreement with those obtained with the corresponding in vitro
plantlets, i.e. an accumulation of triacylglycerols up to five
times the control value (data not shown). To address the
question of the intracellular location of the overproduced
triacylglycerols, we stained petiole and limb epidermis peels
from the wild-type and transgenic lines in an ethanolic solution
of Sudan IV, a lipid-specific dye. Light-microscopy obser-
vations showed the presence of lipid droplets that appeared as
orange spheres in the cytoplasm of stained cells from transgenic
triacylglycerol-overproducer plants (Fig. 5C,D); control lines,
as well as low-triacylglycerol-containing transgenic plants,
showed very few (or none) of these lipid droplets (Fig. 5A,B).
Taken together these data demonstrate that, as expected,
AtDGAT is involved in triacylglycerol synthesis in planta.
DISCUSSION
As shown here cDNA clones encoding DGAT from Arabidopsis
(AtDGAT ) and tobacco (NtDGAT ) have been characterized.
This was carried out using their functional expression in yeast
and in tobacco. At the time when this was performed, a similar
study on the functional expression of AtDGAT in insect cells
was published [13]. However, our work is original in the
following aspects.
Protein sequences deduced from the cDNA clones
As shown in Table 1 the protein sequences deduced from the
cDNAs or genes considered here can be divided into two
families. The first is composed of proteins having < 35±75%
identity with the At DGAT. In this group we find the identified
DGATs from Arabidopsis, tobacco and mouse, the human clone
called ARGP, which was shown not to be an ACAT and
suggested to be a DGAT [11], and a cDNA clone from
C. elegans, which can be considered as a putative DGAT
because of its relatively high identity (36%) with that of the
Arabidopsis DGAT. The second group is composed of proteins
having < 25% identity with AtDGAT. In that group we find the
two human ACATs (HsACAT1 and HsACAT2) and the two
yeast ACATS (ScACAT1 and ScACAT2). As shown in the
sequence alignments (Fig. 1), divergences between DGATs and
ACATs are concentrated in the first moiety starting from the
N-terminal part of the protein, while the second moiety of the
protein is remarkably conserved among the seven sequences.
These strong identities show clearly that ACATs and DGATs
are closely related evolutionarily. This is not surprising because
both enzymes catalyze the acylation by a fatty acyl CoA ester
of strongly hydrophobic alcohols associated with endoplasmic
reticulum: diacylglycerol for DGAT and cholesterol for ACAT.
The other acyl CoA acyltransferases known to date, glycero-
phosphate acyltransferase, lysophosphatidate acyltransferase
[38] and vindoline acetyltransferase [39], did not show any
significant sequence identity suggesting that they are unrelated.
Expression of the Arabidopsis and tobacco DGATs in yeast
Whereas expression of the Arabidopsis cDNA cloned here, in
the double mutant (are1 are2) defective in sterol acyltrans-
ferases, did not lead to either complementation of the mutation
or detection of any ACAT activity at the microsomal level, a
strong increase in the triacylglycerol content of the yeast and in
the DGAT activity of its microsomes were observed. Because
no yeast mutant defective in triacylglycerol synthesis has been
reported to date [5] we used either are1 are2 or a wild-type
yeast. Fortunately under the conditions used, the in vitro
incorporation of [14C]oleyl CoA into triacylglycerols was
significantly higher in microsomes from yeast transformed
with AtDGAT-pYeDP60 than with the void vector (Fig. 3A).
Under these conditions the increment of DGAT activity due to
the expression of the plant cDNA culminated at . 40 nmol
triacylglycerol´h21´mg protein21, a value 102 times higher than
that (300 pmol triacylglycerol´h21´mg protein21) reported in
the insect expression system [13]. As significant endogenous
DGAT activity was observed in control yeast, we tried to devise
conditions to decrease this yeast activity. Such a goal was
achieved using Triton X-100 in the incubation medium. Indeed
the detergent almost completely inhibited the endogenous
DGAT activity, but also decreased the plant DGAT activity in
the AtDGAT-transformed yeast by a factor of 2 or more
according to the experiments (results not shown).
The best results were obtained after the discovery that the
floating lipid layer appearing during the course of the
microsome preparation from yeast transformed with AtDGAT
exhibited strong DGAT activity, whereas the corresponding
zone of the 100 000 g supernatant from the yeast transformed
with the void vector was almost devoid of activity (Fig. 3C).
Such selective expression of AtDGAT in the floating lipid
layer from the transformed yeast allowed us to demonstrate
the net synthesis by AtDGAT of a specific triacylglycerol
(1,2-dihexanoyl 3-oleyl glycerol), which could be separated
thoroughly from endogenous triacylglycerols by TLC and
clearly identified by GC-MS. Finally, the use of a N-terminal
FLAG epitope in the cDNA AtDGAT allowed us to show that
the enzymatic activity measured in transformed yeast can be
ascribed to the presence of a protein with a molecular mass
(54 kDa) compatible with that (59 kDa) deduced from the
proteic sequence. As expected, this protein was shown to be
present both in the microsomal fraction and in the floating lipid
layer (Fig. 3B). Therefore, the yeast system appears to be
appropriate for the expression of AtDGAT. It also allowed
identification of a new DGAT from tobacco. The charac-
terization of a DGAT from C. elegans is in progress in the same
expression system.
As mentioned above, DGAT is a membrane-bound enzyme.
In plants it is thought to be located in the endoplasmic
reticulum [40±42] but its activity has also been suggested in oil
body fractions from Brassica napus [43]. This dual localization
of DGAT was also suggested in the case of yeast [44] and
described in an oleaginous fungus [45]. As in animals, the lipid
bodies (or oil bodies) of plants and yeast are believed to arise
from specific microdomains of the endoplasmic reticulum
membrane that contain the full set of triacylglycerol bio-
synthesis enzymes [7,42,46]. Hence, some of these enzymes
might remain attached to the membrane of the lipid (oil) bodies
after their budding [7]. Indeed such a dual localization has been
described in yeast for some enzymes involved in triacylglycerol
synthesis, glycerol 3-phosphate acyltransferase and 1-acyl-
glycerol 3-phosphate acyltransferase [5], as well as enzymes of
sterol biosynthesis, sterol methyl transferase ERG6 [47] and
94 P. Bouvier-Nave et al. (Eur. J. Biochem. 267)
squalene epoxidase ERG1 [48]. The localization of the plant
DGAT in At DGAT-transformed yeast is therefore not surprising
even thought yeast DGAT is localized mainly in microsomes in
our conditions of culture.
Expression of AtDGAT in tobacco
Although the gene AtDGAT was efficiently expressed in yeast
to give an enzymatically active protein, this result did not give
any information about the activity of this protein inside the
plant. For a complete characterization it was necessary to show
that the gene could be expressed in plants. To this purpose
transgenic tobaccos expressing AtDGAT cDNA and over-
producing triacylglycerols have been characterized. Although
DGAT enzymatic assays were not performed here, it seems
reasonable to assume that triacylglycerol overproduction
corresponds to increased DGAT activity. Relevant to these
considerations, an EMS-mutant of Arabidopsis showing a
correlation between lower DGAT activity and poor triacyl-
glycerol formation has been reported previously [49].
The accumulation of triacylglycerols in leaf material from
transgenic DGAT plants was shown to be associated with the
presence of lipid droplets in the cytoplasm of their cells
(Fig. 5). Although this does not prove directly that the droplets
contain exclusively the overproduced triacylglycerols and
correspond to the well-defined plant lipid bodies [7], it is
most likely that they are budding from the endoplasmic
reticulum where triacylglycerols are synthetized. Synthesis
and storage of triacylglycerols, for example in seeds, have been
widely documented as a process in which these compounds
accumulate in the endoplasmic reticulum membrane and bud
off as droplets surrounded by a phospholipid monolayer [7]. We
previously observed lipid droplets similar to those shown in
Fig. 5, in the cytoplasm of cells from sterol-overproducer plant
lines. Sterols, which are biosynthetized in the endoplasmic
reticulum as triacylglycerols are, were shown to accumulate, in
these plant lines, as fatty acid steryl esters [16,50,51].
Triacylglycerol accumulation has already been described in
the case of a plant (Arabidopsis) expressing a yeast gene
(encoding lysophosphatidic acid acyltransferase) [52]. Never-
theless, the present work constitutes the first report to show an
accumulation of triacylglycerols in a transgenic plant expres-
sing a plant gene (DGAT ). This result suggests that DGAT
activity would be limiting, at least in tobacco leaves, which
normally produce small amounts of triacylglycerols. That the
same stimulation of triacylglycerol synthesis that occurs in
tissues such as tobacco flowers and seeds, but also in fruits and
tubers in other species, remains to be shown.
These considerations raise the interesting problem of the
regulation of the AtDGAT gene. The DGAT gene corresponding
to the cDNA described here was sequenced recently on
BAC F27F23 (GenBank accession number AC003058) and
BAC F3 P11 (GenBank accession number AC005917) and was
shown to be positioned on chromosome II. The region upstream
of the translation starting point possesses interesting features.
As shown recently [13] the transcription starting point is
situated at 2225 bp, leading to a leader sequence much longer
than the average in higher plant genes. A similar situation has
been observed in the gene encoding D7-sterol-C5-desaturase in
Arabidopsis [18]. As shown previously [53] sequences
contained within the 5 0 -transcribed, UTR may play a key role
in regulation of hydroxymethyl glutaryl CoA reductase
expression in Arabidopsis, therefore similar studies should be
conducted in the DGAT gene. A characteristic ethylene
responsive enhancer element (EREE) ATTTCAAA has been
q FEBS 2000
found in the promoter of genes activated by ethylene in
carnation flower petals [36] and in ripening tomato fruit [54].
The same EREE motif has been found in the DGAT gene at
2719 bp upstream to ATG. As triacylglycerols have been
shown to accumulate in flower petals and DGAT was shown to
be strongly expressed in Brassica napus flower petals [13], it
may be suggested that the EREE motif would play an important
role in the regulation of triacylglycerol synthesis.
Finally, it appears from the literature that triacylglycerol and
steryl ester biosynthesis in animals and plants are closely
interwoven. Doubtless the DGAT cDNAs described here
constitute potent tools for studying these interactions. However,
plant genes encoding the enzyme catalyzing the acylation of
plant sterols have still to be characterized. Whilst a revision of
this manuscript after it's review was in progress, another paper
describing A. thaliana DGAT in the mutant line AS11 was
published [55].
ACKNOWLEDGMENTS
We are grateful to Dr Y. Kohara (National Institute of Genetics,
Mishima, Japan) who kindly gave the EST cDNA yk453a2 from
C. elegans and to Dr D. Pompon (Center National de la Recherche
Scientifique, Gif sur Yvette) for allowing us to use the plasmid pYeDP60.
We warmly acknowledge the skillful assistance of M. Schmitz for the
transformation and in vitro culture of tobacco, A. Hoeft for the GC-MS,
B. Bastian and L. Thiriet for typing the manuscript. We also wish to
thank T. Husselstein, I. Benveniste and R. Bronner for their advice
on yeast transformation, Western blotting and microscopic observation,
respectively.
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