Plant Molecular Biology 52: 31–41, 2003.

© 2003 Kluwer Academic Publishers. Printed in the Netherlands.

31

Expression pattern of diacylglycerol acyltransferase-1, an enzyme

involved in triacylglycerol biosynthesis, in Arabidopsis thaliana

Chaofu Lu Lu1 , Shen Bayon de Noyer, Douglas H. Hobbs, Jinling Kang∗ , Yancheng Wen2 ,
Dieter Krachtus and Matthew J. Hills∗
John Innes Centre, Norwich Research Park, Colney Lane, Norwich NR4 7UH, UK (∗ author for correspondence; e-
mail matthew.hills@bbsrc.ac.uk); 1 Present address: Institute of Biological Chemistry, Washington State University,
WA 99164, USA; 2 Permanent address: Henan Academy of Agricultural Sciences, Zhenzhou, Henan Province,
China

Received 29 May 2002; accepted in revised form 8 October 2002

Key words: Arabidopsis, diacylglycerol acyltransferase, expression pattern, β-glucuronidase (GUS), triacylglyc-
erol synthesis

Abstract
Triacylglycerol (TAG) is the major carbon storage reserve in oilseeds such as Arabidopsis. Acyl-
CoA:diacylglycerol acyltransferase (DGAT) catalyses the final step of the TAG synthesis pathway. Although TAG
is mainly accumulated during seed development, and DGAT has presumably the highest activity in developing
seeds, we show here that TAG synthesis is also actively taking place during germination and seedling development
in Arabidopsis. The expression pattern of the DGAT1 gene was studied in transgenic plants containing the reporter
gene β-glucuronidase (GUS) fused with DNA sequences flanking the DGAT1 coding region. GUS activity was
not only detected in developing seeds and pollen, which normally accumulate storage TAG, but also in germinat-
ing seeds and seedlings. Western blots showed that DGAT1 protein is present in several tissues, though is most
abundant in developing seeds. In seedlings, DGAT1 is expressed in shoot and root apical regions, correlating with
rapid cell division and growth. The expression of GUS in seedlings was consistent with the results of RNA gel blot
analyses, precursor feeding and DGAT assay. In addition, DGAT1 gene expression is up-regulated by glucose and
associated with glucose-induced changes in seedling development.

Introduction has also been identified in plants and animals (Cases

Triacylglycerol (TAG) is the most efficient en-
ergy storage compound found in many eukaryotic
organisms (Ohlrogge and Browse, 1995; Farese
et al., 2000). TAG is derived from diacylglycerol
(DAG) through acylation of the sn-3 position in the
glycerol backbone. This step is mainly catalysed
by acyl-CoA:diacylglycerol acyltransferase (DGAT)
(Ohlrogge and Browse, 1995). In addition, there
is recent evidence that phospholipid:DAG acyltrans-
ferase also can catalyse this final acylation step
(Dahlqvist et al., 2000). In Arabidopsis, the DGAT1
(or At2g19450) gene has been cloned (Hobbs et al.,
1999; Routaboul et al., 1999; Zou et al., 1999). The
DGAT2 gene, which is sequence-unrelated to DGAT1,
et al., 2001; Lardizabal et al., 2001).
In animals, TAG synthesis occurs in several cell
types including adipocytes and hepatocytes, and may
also play a role in lipoprotein assembly and the regu-
lation of plasma TAG concentration and the cellular
DAG levels (Farese et al., 2000; Nishizuka, 1992).
In plants, TAG mainly accumulates in seeds, pollen
and fruits of a number species (Murphy et al., 1993;
Murphy, 2001) and serves as an important carbon and
energy source during seed and pollen germination.
However, several lines of evidence suggest that TAG
synthesis and DGAT activity are not restricted to the
embryo and anthers. DGAT activity has been found
in spinach leaves (Martin and Wilson, 1983) and ger-
32
minating soybean cotyledons (Wilson and Kwanyuen,
1986) and TAG synthesis has been measured in leaves
exposed to ozone treatment (Sakaki et al., 1990). TAG
synthesis has also been found in senescing leaves
of Arabidopsis, which appears to be an intermedi-
ate step in the conversion of thylakoid fatty acids to
phloem-mobile sucrose (Kaup et al., 2002). In addi-
tion, DGAT1 transcripts have been detected in many
tissues in Arabidopsis including developing siliques,
flowers, germinating seeds and young seedlings (Zou
et al., 1999). By using RNA gel blot analyses, our
previous studies also detected low levels of DGAT1
expression in leaves and stems besides its strong ex-
pression in embryo and flowers in Brassica napus
(Hobbs et al., 1999). These observations suggested
that TAG may also play roles other than its ‘classical’
role as a storage reserve in plants.
DGAT1 has been shown to play a major role
in TAG synthesis in Arabidopsis. The mutants de-
ficient in DGAT1 activity only accumulate 55–75%
of the wild-type seed oil level (Katavic et al., 1999;
Routaboul et al., 1999). More interestingly, we found
that these mutants also displayed increased sensitivity
to sugars and various stress conditions during germi-
nation and seedling development (Lu and Hills, 2002).
To better understand the roles of DGAT and TAG
synthesis in plants, especially during post-embryonic
stages, we investigated the expression pattern of the
DGAT1 gene in Arabidopsis using transgenic plants
containing the DGAT1 gene promoter-GUS chimeric
gene. We show here that DGAT1 exhibits a tissue-
specific and development-associated expression pat-
tern.
Materials and methods
DGAT-GUS gene fusions
A reporter gene construct (pDGAT:GUS) was made by
combining the gene for the histochemical marker β-
glucuronidase (GUS) with nucleotide sequences flank-
ing the coding region of DGAT. A DNA fragment
representing ca. 2.1 kb of 5 -flanking sequence was
isolated from the genomic DNA (Col) by polymerase
chain reaction (PCR) amplification. This DNA frag-
ment, which contains 1845 bp of the 5 -flanking region
and 225 bp of the untranslated leader sequence, was
initially cloned into the pBluescript KS+ phagemid
vector (Stratagene) and then fused to the GUS re-
porter gene in the plasmid OPgus (Keddie et al.,
1994) cassette to yield construct pDGAT:GUS:NOS.
The downstream sequence directly after the DGAT
coding region was cloned as a 1207 bp fragment
by PCR into pBluescript KS+ and then fused into
pDGAT:GUS:NOS in place of NOS terminator to
yield the plasmid pDGAT:GUS. The DGAT promoter-
GUS region of pDGAT:GUS was cloned into the
binary vector pBIN19 (Bevan, 1984), and then
were transferred to Agrobacterium tumefaciens strain
GV3101 by electroporation.
Plant material and growth conditions
Arabidopsis thaliana ecotype Col was used for trans-
formation by the Agrobacterium-mediated transfor-
mation method (Clough and Bent, 1998). Transfor-
mants (T1 generation) were selected in half-strength
Murashige and Skoog (MS) medium (Murashige and
Skoog, 1962) supplemented with 50 mg/l kanamycin
before transfer to soil in the greenhouse. The number
of T-DNA loci within each line was determined by
segregation for kanamycin resistance in the progeny
(T2 ). The tag1 mutants, tag1-2 and tag1-1, were
kindly provided by Drs Lepiniec (INRA) and Kunst
(UBC, Vancouver), respectively. For germination and
seedling development tests, wild-type (Col-0 and Ws-
0) and mutant seeds were surface-sterilized with 30%
household bleach for 7 min and rinsed with sterile
water at least 3 times, and plated on the above media
without kanamycin. Plates were kept at 4 ◦ C for 4 days
and then transferred to 23 ◦ C for germination under
a 16 h light/8 h dark cycle. For western blotting, the
tag1-1 and tag1-2 plants together with their respective
parent ecotypes Col and Ws were grown in a growth
chamber under a 16 h light 20 ◦ C/8 h dark 15 ◦ C cycle.
GUS activity assays
Histochemical analyses were performed with leaves,
flowers and siliques of the primary (T1 ) transformants
carrying the pDGAT:GUS construct, and with germi-
nating seeds and whole seedlings of the (T2 ) progeny.
GUS activity was detected histochemically by sub-
merging plant materials in GUS histochemical buffer
(100 mM sodium phosphate, 0.5 mM K3 Fe(CN)6 ,
0.5 mM K4 Fe(CN)6 , 10 mM Na2 -EDTA, 0.1% Tri-
ton X-100, and 1 mg/ml 5-bromo-4-chloro-3- indoyl
glucuronide (X-gluc)) and incubating at 37 ◦ C un-
til colour developed (2–12 h). Samples were cleared
with 70% v/v ethanol and were mounted on micro-
scope slides and observed either under a dissecting

microscope or under a compound microscope (Carl
Zeiss).
Precursor feeding experiment
Seedlings were grown on half-strength MS medium
with 1% w/v sucrose under the above conditions in
the culture room. Ten 2-day old seedlings were har-
vested and incubated in 50 µl 30mM MES-NaOH
pH 5.0 with 37 kBq [14 C]-sodium acetate. After in-
cubation overnight at room temperature, 1 ml chloro-
form/methanol (2:1 v/v) was added. The tubes were
incubated for 20 min before 250 µl 0.88% KCl was
added. After vortexing for 5 s, the phases were sepa-
rated by centrifugation, 10 s pulse (Microfuge). The
organic phase was rescued and evaporated to dry-
ness under a nitrogen atmosphere. The lipidic residue
was dissolved in 50 µl chloroform and resolved by
thin-layer chromatography (TLC) with hexane/diethyl
ether/acetic acid (80:20:2, v/v). An autoradiograph
was produced overnight and bands corresponding to
known standards scraped and counted in 3 ml scintil-
lation fluid.
Antibody production and purification
DNA encoding the first 130 N-terminal amino acids
of DGAT1 was amplified by PCR and cloned into the
expression vector pET15b from Novagen (Madison,
WI). The peptide, including an N-terminal histidine
tag, was expressed in Escherichia coli strain BL21
by induction with isopropyl thio-β-D-galactosidase in
accordance with manufacturer’s instructions. The pep-
tide was purified in a nickel column and eluted with an
imidazole buffer as previously described (Brown et al.,
1998). The histidine tag was cleaved from the peptide
by using thrombin from Novagen with conditions for
efficient cleavage as described by the manufacturer.
The peptide solution was then concentrated using an
Amicon YM3 cut-off filter to produce a final solution
of 1.2 mg/ml. Antibodies were raised in two rabbits
by standard protocols by Sigma-Genosys (Cambridge,
UK). The antibodies were purified by affinity chro-
matography in a column to which the 130 amino acid
DGAT1 peptide had been covalently bound with an
AminoLink Plus Immobilization Kit (Pierce, USA)
according to the manufacturer’s instructions. The anti-
bodies (3 ml) were diluted 1:10 by addition of 10 mM
Tris pH 7.5 and passed through the column. The anti-
bodies were eluted with 100 mM glycine pH 2.5 and
the antibody containing fractions pooled as previously
described (Pongdontri and Hills, 2001).
33
Homogenization of plant material
The homogenization medium contained 400 mM su-
crose, 100 mM Hepes-NaOH pH 7.5, 10 mM KCl,
1 mM MgCl2 , 5 mM EDTA, 2 mM DTT, and a
protease inhibitor cocktail tablet from Roche (Lewes,
UK). Plant material was homogenized using a glass
mortar and pestle. The homogenate was spun at 20 000
× g for 15 min and the supernatant fraction was
again spun at 356 000 × g for 15 min to obtain the
microsomal membrane fraction. This fraction was re-
suspended in 50 µl of homogenizing medium and
the protein concentration was determined by a dye-
binding assay (Bradford, 1976).
SDS-PAGE and western blot analysis
Proteins were separated by SDS-PAGE on a Laemmli
gel system with a Mini-Protean duel slab cell (Bio-
Rad, Hemel Hempstead, UK). A 50 µg protein sample
was loaded as previously described (Brown et al.,
1998). Pre-stained SDS-PAGE standards (BioRad,
UK) were used as molecular mass markers. After
electrophoresis, proteins were transferred from the
gel onto nitrocellulose membrane (Hybond-C, 45 µm;
Amersham, UK) by means of a Mini-Transblot ap-
paratus according to the manufacturer’s instructions
(BioRad, UK). The membrane was blocked with 2.5%
w/v dry milk powder in TBS (10 mM Tris pH 7.4,
150 mM NaCl) and incubated with the purified pri-
mary antibody at 1:300 dilution and then washed as
previously described (Pongdontri and Hills, 2001).
The blot was then incubated in blocking buffer con-
taining secondary antibody from Sigma (Poole, UK) at
1:2000 dilution. The blot was washed again and pres-
ence of the alkaline phosphatase conjugated secondary
antibody was visualized with Western Blue stabilized
substrate (Promega).
DGAT activity
DGAT activity was determined by measuring the in-
corporation of label from [14 C]oleoyl-CoA into TAG
(Bouvier-Nave et al., 2000). Assay mixtures (100 µl)
contained 100 mM Tris-HCl pH 7.4, 20% v/v glyc-
erol, 200 µM 1,2-dioleoylglycerol and 22.8 µM
[14 C]oleoyl-CoA (2.11 GBq/mmol) and reactions
were started by the addition of 10 µg microsomal
membrane protein. The assays were incubated in glass
tubes for 5 min at 30 ◦ C with shaking at 130 r.p.m.
The 1,2-dioleoylglycerol was prepared as a 400 µM
34

Figure 1. Expression of the DGAT:GUS chimeric gene in Arabidopsis transgenic plants. A. Schematic representation of the DGAT1
(At2g19450) gene on chromosome 2 of A. thaliana with respect to the flanking annotated genes At2g19440 and At2g19460 within the BAC
clone F27F23, and the construct of the DGAT1 promoter-GUS fusion gene. B. GUS expression in developing seeds (insert showing staining in
embryo). C. GUS expression in flower (insert showing GUS staining in pollen).

stock in 100 mM Tris-HCl pH 7.4, 20% v/v glyc-
erol by vigorous shaking. The reaction was stopped
by the addition of 1.875 ml methanol/chloroform (2:1)
and 400 µl water and brief shaking of the tube. Next
613 µl each of water and chloroform were added and
the mixture was vortexed, spun at 2000 × g for 2 min.
The lower phase was transferred to another tube, the
solvent removed under a stream of N2 and the lipid
applied to a silica gel TLC plate and the lipids resolved
with a solvent containing hexane/diethyl ether/acetic
acid (70:30:1). The radioactive TAG bands, identified
with a standard, were scraped and 14 C determined by
scintillation counting.
Results
Isolation and sequence analysis of the DGAT1
promoter
The gene, At2g19450, that encodes DGAT1 in Ara-
bidopsis lies on the BAC clone F27F23 on chro-
mosome 2 (Hobbs et al., 1999). Sequencing and
annotation of the clone (GenBank accession number
AC003058) revealed that two open reading frames
(At2g19440 and At2g19460) are about 6.5 kb up-
stream and 1.7 kb downstream of the coding region
of At2g19450, respectively. A ca. 2.1 kb upstream
fragment corresponding to the XbaI restriction site to
the ATG start codon of the At2g19450 gene was iso-
lated by PCR from Arabidopsis (ecotype Col) genomic
DNA and completely sequenced to ensure no error
was introduced. This fragment was fused to the cod-
ing sequence for β-glucuronidase (GUS). To include
possible regulatory elements in the 3 sequences of the
gene, a 1.2 kb downstream sequence of the At2g19450
coding region including the fragment from the gene
stop codon to the SalI restriction site was also fused
to the 3 of the GUS gene (Figure 1A). The result-
ing construct was transformed into Arabidopsis plants,
and the primary transformants were analysed for GUS
activity with the histochemical substrate 5-bromo-
4-chloro-3-indolyl β-glucuronide (Xgluc) (Jefferson
et al., 1987). GUS expression was determined in 10 to
 35

Figure 2. Nucleotide sequence of the 5 -upstream region of the DGAT1 gene. Putative TATA box and promoter elements described in the text
are labeled and underlined. The symbol (−) denotes the reverse orientation. The arrow indicates the transcription start site (Hobbs et al., 1999).
The beginning of the DGAT1 gene open reading frame is shown in lower-case letters.

20 independent transformants. Transformants showed
intense staining in maturing embryos (>7 days after
flowering) and pollen, as shown in Figure 1B and C.
GUS activity was not detected during early embryo-
genesis, i.e. before pattern formation was completed
(Goldberg et al., 1994), or in desiccating seeds (data
not shown). This expression pattern is consistent with
the fact that TAG synthesis is actively taking place
during the embryo expansion phase (Mansfield and
Briarty, 1991), and that pollen also synthesizes TAG
during development (Piffanelli et al., 1997). In our
previous report, northern blot analyses showed that
DGAT1 was also highly expressed in embryo and flo-
ral tissues in Brassica napus (Hobbs et al., 1999).
Therefore, the DNA sequence flanking the At2g19450
gene in the reporter construct contains regulatory ele-
ments necessary for appropriate expression of DGAT1
during the phases of seed and pollen development.
The sequence of the 2.1 kb upstream region con-
tains a number of putative promoter elements (Fig-
ure 2). Several of these elements are in common
with those found in the promoter of the napA gene,
which encodes the seed-specific storage protein napin
in Brassica napus (Ericson et al., 1991). For example,
the napin motif TACACAT (Ericson et al., 1991), is
present in the 5 -flanking region of the DGAT1 gene at
position −1484. The octomer (CA)n element CCAA-
CACC (Ellerstrom et al., 1996) required for embryo-
and endosperm-specific transcription of the napA gene
is present at −289. The E box of the napA gene (Stal-
berg et al., 1996) is present in the DGAT1 promoter
region 12 times, 6 in the forward and 6 in the re-
verse orientation (not shown). Another element for
seed-specific napA expression, the RY repeat CAT-
GCA (Ezcurra et al., 1999), is found at −1799 and
−67 but in the reverse orientation. These findings
suggest that the DGAT1 gene shares some regulatory
elements with the napA gene for expression in seeds
as both are responsible for synthesizing seed storage
reserves. The hexamer motif AAATGA that has been
shown to be a cis-acting element directing expression
in tobacco pollen (Weterings et al., 1995) is found
36

Figure 3. GUS histochemical staining of transgenic Arabidopsis plants during germination and seedling development. A. Germinating seed, 0
day after imbibition (dpi). B. 1 dpi seedling showing GUS staining in cotyledon and hypocotyl. C. 5 dpi seedling showing GUS staining in the
shoot apex and root tips. D. 15 dpi seedling showing GUS staining in young leaves. E. 15 dpi seedling showing GUS staining in root tips. F.
Primary root tip. G. Emerging lateral root showing GUS staining in the tip.

at the position of −402. Interestingly, the ethylene-
responsive element (ERE) ATTTCAAA (Montgomery
et al., 1993; Itzhaki et al., 1994) in present three times
in the 5 -flanking region of the DGAT1 gene, two in
the forward (−1721 and −720) and one in the re-
verse (−1051) orientation. This suggests that DGAT1
expression may be regulated by ethylene during seed
filling and maturation.
Post-embryonic DGAT1 expression in transgenic
Arabidopsis plants
To obtain information about the DGAT1 gene pro-
moter activity during post-embryonic phases, GUS
assays were performed with progeny from six in-
dependently derived transgenic lines, all of which
showed strong staining of embryos and pollen. A sig-
nificant level of GUS activity was already detectable
in seeds after imbibition on germination medium at
4 ◦ C for 4 days (Figure 3A). High GUS activity was
detected in cotyledons and hypocotyls in germinating
seedlings (Figure 3B). GUS staining was equally in-
tense in both cotyledons and hypocotyls even when
they were incubated in the staining solution for a
short time (1–2 h). However, after cotyledon expan-
sion GUS activity began to decline in cotyledons but
remained in hypocotyls until the first true leaves ap-
peared. GUS activity was, in turn, observed in shoot
apices and root tips (Figure 3C). In seedlings, GUS
activity was detected in young leaves (Figure 3D) and
root tips (Figure 3E). The initiating lateral roots devel-
oping from the pericycle also showed GUS staining
shortly after emerging from the central root (Fig-
ure 3G). GUS activity was not detected in mature
leaves (Figure 3D). However, GUS activity remained
in roots 15 days after germination, where primary and
lateral root tips were intensely stained (Figure 3F).
Therefore, DGAT1 expression was regulated in a
tissue- and time- dependent manner. It was possible
that the staining in germinating seeds and seedlings
was caused by GUS protein that had been expressed
during seed development. However, it was previously
reported that DGAT1 mRNA was present at high lev-
els in many different tissues of Arabidopsis, including
germinating seeds, young seedlings, roots and leaves
(Zou et al., 1999). Similar results were obtained in this
study, except that we observed a low level of DGAT1
mRNA in leaves compared to that in other tissues (Fig-
ure 4). These results are consistent with the DGAT1
promoter-GUS expression pattern. This study shows
that although the location of DGAT1 promoter expres-
sion changes during seedling development the amount
of DGAT1 mRNA in the whole seedling remains fairly
constant.

Figure 4. Northern blot analysis of DGAT1 expression in Arabidop-
sis seedlings. Total RNA was extracted from whole seedlings at 1, 3,
5, 7, 10 and 15 dpi, and leaves and roots of 10 dpi seedlings. Total
RNA (10 µg) was loaded for northern blot and hybridized to the
DGAT cDNA probe. Ethidium bromide staining is shown for equal
loading.
Figure 5. Western blot analysis of DGAT1 in a range of tissues from
Arabidopsis. Each lane contained 50 µg of microsomal membrane
protein. A. Developing seed tissue from the Arabidopsis wild types
Columbia (Col) and Wassilewskija (Ws) and mutants tag1-1 and
tag1-2. B. Col: DS, developing seed; I, inflorescence; S, stem; L,
leaf; 7ds, 7-day old seedling. C. Developing seeds of ecotype Col.
dpa, days after anthesis.
In this study, GUS activity was not detected in
other tissues such as stems and senescing leaves, in
which TAG is also synthesized (Kaup et al., 2002).
One possibility is that the regulatory elements speci-
fying DGAT1 gene expression in these tissues are not
included in the DNA sequences of this study. We have
previously shown that the At2g19450 gene contains
15 introns (Hobbs et al., 1999). We cannot exclude
the possible roles of the introns in regulation of the
DGAT1 gene expression.
Expression of DGAT1 protein in Arabidopsis
Antibodies raised against the soluble 130 amino acid
N-terminal domain of DGAT1 were tested against mi-
crosomal membrane extracts from developing seeds
of the DGAT1 mutants tag1-1 (Zou et al., 1999)
37
and tag1-2 (Routaboul et al., 1999) and against their
parental ecotypes Col and Ws by western blotting
(Figure 5A). The western blot showed a band at
51 kDa that was found in developing seeds from the
mutants and wild types (Figure 5A) and all other tis-
sues examined (data not shown) but this band did not
correspond to DGAT1 as predicted previously by ex-
pression of the DGAT1 cDNA in insect cell cultures
(Hobbs et al., 1999). A band at 54 kDa was present in
the wild types but was absent in the deletional mutant
tag1-2 indicating that the band at 54 kDa is DGAT1.
The difference in molecular mass estimates between
this and our previously published result may be due to
the use of different batches of pre-stained marker pro-
teins. It was recently reported that DGAT1 appeared
mainly as a 29 kDa band on western blots with little
of the full-length protein being detected (Kaup et al.,
2002). This was attributed to instability of the protein.
We did not observe the band at 29 kDa but this may
be due to the fact that the antibodies used in that study
were raised against a C-terminal peptide. A band at
ca. 56 kDa was visible in the lane corresponding to
membranes from the tag1-1 mutant (Figure 5A). This
result confirms that the tag1-1 contains an insertion
and is expressed as a protein.
A western blot of microsomal membranes from
a range of tissues show that DGAT1 protein is ex-
pressed in developing seed, inflorescence, and 7-day
seedlings, and at a lower level in the stem, but is not
detected in the leaf (Figure 5B). During embryo devel-
opment, DGAT1 protein is already present at 5 days
after anthesis, increases to a peak at 7–9 days, and
then gradually declines, though is still detectable at 23
days when the seeds are turning brown and desiccating
(Figure 5C). It has previously been reported that oil
is synthesized at maximum rates from days 7–13 after
anthesis (Focks and Benning, 1998), thus DGAT1 pro-
tein is present during the period of oil synthesis. The
persistence of DGAT1 protein after the period of oil
synthesis has stopped is in contrast to other enzymes
that are involved in oil synthesis which are usually
rapidly degraded during the seed desiccation phase
(Pongdontri and Hills, 2001).
TAG synthesis in Arabidopsis seedlings
Strong expression of the DGAT1 gene in germinating
seedlings is unexpected since at this stage of devel-
opment TAG is being catabolized (Eastmond et al.,
2000). In order to determine whether TAG synthesis
occurs in seedlings, seedlings were fed 2 days after
38
Table 1. DGAT activity and TAG synthesis in Arabidopsis seedlings.

A. TAG synthesis in Arabidopsis seedlings B. DGAT activity in seedling membranes

(nmol per mg protein per minute)
2-day seedling 2nd leaf Exp. 1 Exp. 2

Col WT 6.7%∗ 1.3% 201 ± 1 116 ± 18

tag1-1 2.3%∗ 1.2% 2± 3 7± 4
Ws WT 5.2%∗ 1.2% 260 ± 29 138 ± 11
tag1-2 2.8%∗ 1.2% 4± 1 8± 2

A. Seedlings at 2 days after germination and 2nd leaves from 15-day plants were incubated
with [14 C]acetate. Lipids extracted from the plant material were resolved by thin layer
chromatography and the amount of radioactivity incorporated into TAG was calculated as
a proportion of that incorporated into total lipids. Data represent the mean value from 3
independent experiments with 3 replications. ∗ Significant at P < 0.01.
B. Microsomal membranes isolated from 2-day seedlings were assayed for DGAT activ-
ity measuring incorporation of [14 C]oleoyl-CoA into TAG as described in Materials and
methods. Results from two independent experiments are presented.

germination with [14 C]acetate and the incorporation
of label into TAG was determined. Since the incor-
poration of label into TAG in whole tissue would not
prove the presence of DGAT1 activity, the tag1 mu-
tants were used as controls. Table 1A shows that in
both Col and Ws wild-type seedlings, about 5–7% of
[14 C]acetate was incorporated into TAG as a propor-
tion of label incorporated into total lipids. However, in
the tag1 mutants, incorporation of label into TAG was
significantly reduced with only 2–3% of [14C]acetate
being incorporated. These results suggest that about
50% of the capacity for TAG synthesis in the seedlings
is catalysed by DGAT1, as has been shown in devel-
oping seeds (Katavic et al., 1995; Routaboul et al.,
1999). This suggests that the remaining TAG synthesis
activity in the mutants may be conferred by DGAT2
(Lardizabal et al., 2001), or by other enzymes such as
phospholipid:diacylglycerol acyltransferase (PDAT),
which can also catalyse TAG synthesis by an acyl-
CoA-independent reaction (Dahlqvist et al., 2000).
Leaves from 15-day old plants accumulated about
1% of the [14 C]acetate in TAG as a proportion of
the total label incorporated into lipid. The tag1-1
and tag1-2 mutants were not affected in the synthe-
sis of TAG in the leaf, which was expected since
DGAT1 is expressed at only a low level in leaves
(Figure 4). This TAG is most likely synthesized by
DGAT2 or by a non-DGAT-catalysed route (Dahlqvist
et al., 2000; Lardizabal et al., 2001). We also directly
measured DGAT activity in microsomal membranes
isolated from 2-day old seedlings of tag1-1 and tag1-
2 and their respective parent ecotypes. DGAT activity
in the mutants was less than 10% of that of the re-
spective wild types (Table 1B). This suggests that the
DGAT1 activity predominates over DGAT2 in devel-
oping seedlings and that the TAG synthesis observed
in the acetate feeding experiments was likely to be
formed by the action of PDAT or transacylases. How-
ever, the possibility that the assay conditions used here
discriminated against DGAT2 activity should not be
excluded.
DGAT1 expression is up-regulated by glucose
After germination, TAG is catabolized and converted
to sugar to support seedling growth. The tissue-
specific DGAT1 expression pattern suggested that it
might be regulated in response to metabolism and de-
velopment of the seedlings. The expression level of
DGAT1 in 2-day seedlings grown on medium sup-
plemented with 2% glucose was about 4-fold higher
than that of the seedlings grown on sugar-free medium
(Figure 6A). Seedling growth was arrested on medium
containing 6% glucose since cotyledons failed to turn
green and showed little expansion, similar to the ob-
servation in previous studies (Jang and Sheen, 1994;
Gibson, 2000; Laby et al., 2000). The DGAT1 expres-
sion level in these seedlings was about 10-fold higher
than that of seedlings grown on sugar-free medium
(Figure 6A). GUS expression was similarly greatly
increased in transgenic seedlings grown on medium
containing 6% glucose (Figure 6B). Previous studies
have shown that ethylene antagonizes sugar signals
in sugar-induced stress effects. Seedlings treated with
1-aminocyclopropane-1- carboxylic acid (ACC), the
immediate precursor for ethylene biosynthesis, could
overcome the developmental arrest (Zhou et al., 1998).
We similarly found that Arabidopsis seedlings devel-

Figure 6. DGAT1 expression is up-regulated by glucose. A. Northern blot of RNA from seedlings 3 days after germination and grown on 0%
glucose (lane 1), 2% glucose (lane 2), 6% glucose and 50 µM ACC (lane 3), and 6% glucose media. Total RNA (10 µg) was loaded for each
sample. Ethidium bromide staining is shown for equal loading. B. GUS staining of seedlings 2 days after germination on media containing the
1/2 MS salt (left), 1/2 MS + 6% glucose (middle) and 1/2 MS + 6% glucose + 50 µM ACC (right).
oped green cotyledons and true leaves on the media
containing 6% glucose when 50 µM ACC was added.
At the same time, the DGAT1 promoter driven GUS
expression level was restored to normal, as was the
DGAT1 mRNA transcript (Figure 6).
Discussion
The primary role of DGAT1 is to catalyse the final
step of storage TAG synthesis. It has been shown
that the DGAT1-deficient Arabidopsis mutants accu-
mulate less TAG in their seeds (Katavic et al., 1995;
Routaboul et al., 1999). The fact that the promoter
of the DGAT1 gene shares several common regulatory
elements with the promoter of the seed-specific napA
gene (Figure 2) also suggests that DGAT1 mainly
functions in developing seeds. However, unlike the
napin genes, DGAT1 gene expression is not restricted
to seeds. Sequence analysis showed that the two RY
repeat motifs (CATGCA) found in the DGAT1 gene
promoter are far apart and are present in a reverse
orientation (Figure 2). Although the function of the
CATGCA motif in the DGAT1 gene promoter remains
speculative, the RY repeat has been well studied and it
is present in many seed-specific storage protein genes
(Baumlein et al., 1992; Dickinson et al., 1988; Ezcurra
et al., 1999; Reidt et al., 2000; Riggs et al., 1989).
Mutations in the RY element abolished the seed spe-
cific enhancing activity of the upstream regulatory
sequences of the β-conglycinin α-subunit, the 7S soy-
bean seed storage protein, and caused expression of
the chimeric promoter in leaves (Fujiwara and Beachy,
1994).
This study reveals an interesting expression pattern
of the DGAT1 gene in Arabidopsis during germination
and seedling growth besides its expression in develop-
ing seeds and pollen. The promoter-GUS reporter gene
39
expression was confirmed by northern and western
blots, precursor feeding and DGAT1 assays. Before
true leaves appeared, the DGAT1:GUS expression was
found to be equally strong in cotyledons and hypocotyl
(Figure 3B). After germination, seed oil serves as the
major carbon and energy source for seedling devel-
opment before photoautotrophism is successfully es-
tablished. The DGAT1 activity during post-embryonic
stages may have a role to sequester fatty acids released
from the storage TAG and to coordinate metabolism
with seedling development as it responds to changes
in its environment. For instance, alteration of the car-
bon to nitrogen ratio cause large changes in the rate
of storage TAG catabolism in developing Arabidopsis
seedlings (Martin et al., 2002). It is possible that un-
der conditions where fatty acid oxidation is inhibited,
fatty acids released from TAG by the action of lipase
would need to be recycled back to TAG and DGAT1
could catalyse that reaction. In older seedlings, GUS
activity was detected mainly in shoot apex and root
tips and DGAT1 protein was present at relatively high
levels in microsomal membranes isolated from whole
7-day seedlings (Figure 5B). This expression pattern
suggests that DGAT1 activity correlates with rapid cell
division and growth. Membrane lipid synthesis is im-
portant in these regions, and DGAT1 may play a role
in balancing the demand for DAG and acyl-CoAs with
their supply. The amount of DGAT1 protein in 7-day
old seedlings was similar to that in developing seeds
(Figure 5B) while TAG degradation is completed by
day 4 (Lu and Hills, 2002). This suggests that ei-
ther the DGAT1 activity is strongly regulated in the
seedlings or that TAG synthesized in the young plant
is very rapidly turned over. Previous work has shown
that DGAT1 expression in Brassica cell cultures is
up-regulated by increasing the sucrose concentration
(Nykiforuk et al., 2002). Transcription of the DGAT1
40
gene was significantly increased in seedlings grown in
the presence of exogenous glucose and expression in-
creased 10-fold during seedling growth arrest induced
by 6% glucose. However, when the seedling growth
was resumed by adding ACC, the ethylene synthesis
precursor, the DGAT1 gene expression was restored
to normal level (Figure 6). It has been suggested that
a high sugar concentration induces the young Ara-
bidopsis seedling to revert to the ‘storage mode’ of
the embryo whilst its development is stalled (Rook
et al., 2001). Given the important role of DGAT1 in
storage oil synthesis in developing seeds, the ethyl-
ene reversible, glucose induced increase in DGAT1
expression fits with this hypothesis.
Acknowledgements
We thank Drs Loic Lepiniec and Ljerka Kunst for sup-
plying the tag1 mutants. This work was supported by
the Biotechnology and Biological Sciences Research
Council through the Competitive Strategic Grant to the
John Innes Centre and through a research grant un-
der the ‘Genome Analysis of Agriculturally Important
Traits’ initiative.
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