Tài liệu Báo cáo khoa học: Purification and characterization of glutamate N-acetyltransferase involved in citrulline accumulation in wild watermelon doc

Purification and characterization of glutamate
N-acetyltransferase involved in citrulline accumulation
in wild watermelon
Kentaro Takahara, Kinya Akashi and Akiho Yokota
Graduate School of Biological Sciences, Nara Institute of Science and Technology, Japan
Drought in the presence of strong light is a major
environmental stress that reduces plant productivity
[1]. To adapt to this adverse condition, numerous bio-
chemical and physiological tolerance mechanisms are
expressed in plant cells [2]. One such response involves
accumulation of small organic metabolites, such as
mannitol, proline and glycine betaine, which are collec-
tively referred to as compatible solutes [3]. Compatible
solutes are thought to play important roles in drought
tolerance in plants, acting as mediators of osmotic
adjustment, stabilizers of subcellular structures, and
scavengers of active oxygen radicals [4]. The mecha-
nisms of proline, mannitol, and glycine betaine accu-
mulation are highly regulated through activation of
biosynthesis and ⁄ or suppression of catabolism [3–5].
Wild watermelon plants, which inhabit the Kalahari
Desert, Botswana, exhibit high drought ⁄ strong-light
stress tolerance [6]. They are able to maintain their
photosynthetic apparatus during prolonged periods of
drought in strong light, suggesting the presence of
Keywords
citrulline; drought/strong-light stress;
glutamate N-acetyltransferase;
thermostability; wild watermelon
Correspondence
A. Yokota, Nara Institute of Science and

Technology, Graduate School of Biological
Sciences, 8916-5 Takayama, Ikoma,
Nara 630-0101, Japan
Fax: +81 743 72 5569
Tel: +81 743 72 5560
E-mail:
(Received 12 July 2005, revised 18 August
2005, accepted 23 August 2005)
doi:10.1111/j.1742-4658.2005.04933.x
Citrulline is an efficient hydroxyl radical scavenger that can accumulate at
concentrations of up to 30 mm in the leaves of wild watermelon during
drought in the presence of strong light; however, the mechanism of this
accumulation remains unclear. In this study, we characterized wild water-
melon glutamate N-acetyltransferase (CLGAT) that catalyses the trans-
acetylation reaction between acetylornithine and glutamate to form
acetylglutamate and ornithine, thereby functioning in the first and fifth
steps in citrulline biosynthesis. CLGAT enzyme purified 7000-fold from
leaves was composed of two subunits with different N-terminal amino acid
sequences. Analysis of the corresponding cDNA revealed that these two
subunits have molecular masses of 21.3 and 23.5 kDa and are derived from
a single precursor polypeptide, suggesting that the CLGAT precursor is
cleaved autocatalytically at the conserved ATML motif, as in other glutam-
ate N-acetyltransferases of microorganisms. A green fluorescence protein
assay revealed that the first 26-amino acid sequence at the N-terminus of
the precursor functions as a chloroplast transit peptide. The CLGAT
exhibited thermostability up to 70 °C, suggesting an increase in enzyme
activity under high leaf temperature conditions during drought ⁄ strong-light
stresses. Moreover, CLGAT was not inhibited by citrulline or arginine at
physiologically relevant high concentrations. These findings suggest that
CLGAT can effectively participate in the biosynthesis of citrulline in wild

watermelon leaves during drought ⁄ strong-light stress.
Abbreviations
AOD, acetylornithine deacetylase; CLGAT, Citrullus lanatus glutamate N-acetyltransferase; DRIP-1, drought-induced polypeptide 1;
DTT, dithiothreitol; GAT, glutamate N-acetyltransfease; GFP, green fluorescence protein.
FEBS Journal 272 (2005) 5353–5364 ª 2005 FEBS 5353
mechanisms that allow them to tolerate oxidative stress
arising from excess light energy absorbed by the leaves.
Drought ⁄ strong-light stresses result in an accumulation
of a novel compatible solute, citrulline, in the leaves
[6]. The concentration of citrulline in the stressed
leaves reaches up to 30 mm, compared to only 0.6 mm
in unstressed leaves [6]. Among known compatible sol-
utes, citrulline is one of the most efficient scavengers
for hydroxyl radicals [7]. These findings suggest that
citrulline functions as a hydroxyl radical scavenger in
the presence of strong light.
In addition to citruline, concentration of arginine
increases from 0.3 mm in unstressed conditions to
7mm under drought ⁄ strong-light stress in the leaves of
wild watermelon [6]. Although arginine is the final
product of the arginine biosynthetic pathway, wild
watermelon plants accumulate larger quantities of
citrulline, an intermediate in this pathway, during
drought ⁄ strong-light stress. Arginine is a key meta-
bolite in regulation of this pathway in plants [8]. How-
ever, the mechanism of accumulation for these
metabolites in wild watermelon remains unclear.
Regulation of citrulline and arginine synthesis has
been studied extensively in prokaryotes and Saccharo-
myces cerevisiae [9,10]. The pathway starts with acety-

lation of glutamate into N-acetylglutamate, which is
then converted into N-acetylornithine by three con-
secutive enzymatic steps, namely, phosphorylation,
reduction, and transamination (Fig. 1). In the fifth
step, N-acetylornithine is converted into ornithine,
which is used for synthesis of citrulline and arginine in
the urea cycle. Two different enzymes are known to be
required for catalysis of this fifth step; one is acetylorni-
thine deacetylase (AOD, EC 3.5.1.16), which catalyses
deacetylation of N-acetylornithine yielding ornithine
and acetate [11]. This linear pathway is regulated
by arginine-induced feedback-inhibition of N-acetyl-
glutamate synthase, the first-step enzyme in the
pathway [12]. AOD is found in Enterobacteriaceae such
as Escherichia coli [9,13]. The second enzyme is
glutamate N-acetyltransferase (GAT, EC 2.3.1.35),
which catalyses transfer of the acetyl group from
N-acetylornithine into glutamate yielding ornithine
and N-acetylglutamate. Glutamate N-acetyltransferase
therefore recycles the acetyl moiety of N-acetyl-
ornithine, regenerating N-acetylglutamate in citrulline
and arginine biosynthesis. This enzyme is functional in
all other microorganisms characterized so far, such as
Bacillus subtilis and S. cerevisiae [14,15]. In this acetyl-
recycling pathway, both the first- and the second-step
enzymes, N-acetylglutamate synthase and N-acetylglut-
amate kinase, respectively, are inhibited by arginine.
Glutamate N-acetyltransferase is also weakly inhibited
by arginine [16,17]. As a result, the concentration of cit-
rulline and arginine is kept low through these feedback

inhibitions in the microorganisms examined so far.
How wild watermelon is able to accumulate high levels
of citrulline is therefore an intriguing question.
In plants, knowledge on the pathway of citrulline
and arginine biosynthesis is still fragmentary [8,18].
The genome project revealed that both GAT and
AOD-homologous genes exist in Arabidopsis thaliana
[19]. Our previous study showed that a novel protein,
drought-induced polypeptide 1 (DRIP-1), which shares
sequence homology with bacterial AOD, is strongly
induced by drought ⁄ strong-light stress in wild water-
melon [6]. However, the catalytic property of DRIP-1
remains to be determined, and it is not known whether
DRIP-1 contributes to massive accumulation of citrul-
line in wild watermelon.
As a first step to understand the mechanism of citrul-
line and arginine accumulation in wild watermelon, we
focused on the fifth step of citrulline biosynthesis, at
which point DRIP-1 was expected to function as
AOD. However, GAT activity, not AOD activity, was
detected in wild watermelon leaves in which DRIP-1
Fig. 1. The pathway of citrulline and arginine biosynthesis. AGS,
N-acetylglutamate synthase; AGK, N-acetylglutamate kinase; AGPR,
N-acetylglutamate 5-phosphate reductase; AOAT, N-acetylornithine
transaminase; GAT, glutamate N-acetyltransferase; AOD, N-acetyl-
ornithine deacetylase; OCT, ornithine carbamoyltransferase; ASS,
argininosuccinate synthase and ASL, argininosuccinate lyase.
Wild watermelon glutamate N-acetyltransferase K. Takahara et al.
5354 FEBS Journal 272 (2005) 5353–5364 ª 2005 FEBS
had been strongly induced. This paper reports the

purification and characterization of GAT from wild
watermelon leaves, and discusses its function during
drought ⁄ strong-light stress on the basis of its two
unique enzymatic properties; thermotolerance and
insensitivity to inhibition by downstream products, cit-
rulline and arginine.
Results
The enzyme involved in catalysis of the fifth step
of citrulline biosynthesis in wild watermelon
leaves
During citrulline biosynthesis, N-acetylornithine is con-
verted into ornithine by AOD and ⁄ or GAT (Fig. 1).
To examine contribution of these two enzymes to cit-
rulline synthesis in wild watermelon leaves, we assayed
their activities in extracts of wild watermelon leaves
during progression of drought ⁄ strong-light stress. At
each time point investigated, AOD activity was below
the detection limit (< 0.02 nmolÆmin
)1
Æmg protein
)1
;
Fig. 2A). Although bacterial AODs are activated by a
divalent metal ion such as Co
2+
or Zn
2+
[20], no
AOD activity was detected in extracts from wild
watermelon leaves even if these metal ions were inclu-

ded in the reaction mixture (data not shown). In a pos-
itive control experiment, we could detect similar AOD
activity in the extract of E. coli to that reported in the
literature [11], demonstrating that the assay procedures
were valid for detecting AOD activity. The undetect-
able AOD activity in extracts from wild watermelon
leaves is in contrast to the strong expression of DRIP-
1 during stress (Fig. 2).
On the contrary, GAT activity was detected in
leaves of wild watermelon (Fig. 2A). The specific activ-
ity of GAT in unstressed leaves was approximately
3.2 nmolÆmin
)1
mg protein
)1
, and this did not change
significantly during stress. This constant GAT activity
did not correlate with the induction of DRIP-1 protein
during drought ⁄ strong-light stress (Fig. 2B).
Purification of GAT from wild watermelon leaves
To characterize the GAT in detail, it was purified from
wild watermelon leaves (Table 1). The purification
procedure was developed by taking advantage of the
thermal stability of the GAT activity in crude extracts.
After centrifugation of the total leaf extract to remove
cell debris, the supernatant was heated at 70 °C for
10 min and centrifuged. Negligible loss of enzymatic
activity and 24-fold purification were achieved. Heat
treatment was followed by six chromatography steps
comprising hydrophobic interaction, anion and cation

exchange, gel filtration, and hydroxyapatite chromato-
graphies, resulting in more than a 7000-fold purifica-
tion of GAT. When analysed by SDS ⁄ PAGE, two
polypeptides of  27 kDa were detected in the sample
(Fig. 3A). Protein sequencing analysis revealed that the
N-terminal amino acid sequences of the small (a) and
large (b) polypeptide were XATNEAANYLPEAP and
XMLGVVTTDAVVACDVWRKMVQISVDRSFNQI
TVD, respectively; X represents unidentified amino
A
B
Fig. 2. Enzymatic activities of AOD and GAT and accumulation of
DRIP-1 protein during the progressing drought ⁄ strong-light stress in
wild watermelon leaves. (A) Changes in AOD (j) and GAT (h)
activity. Data points represent means from three independent
experiments and vertical bars are SD. (B) Immunoblot analysis of
DRIP-1 in the total soluble proteins (20 lg per lane) isolated from
plants before (0 day) and after 1, 2, 3 and 5 days of drought ⁄
strong-light treatment.
Table 1. Purification of GAT from wild watermelon leaves.
Step
Protein
(mg)
Total
activity
(U)
Specific
activity
(UÆmg
)1

)
Purification
(fold)
Crude extract 5500 37 0.00067 1
Heat treatment 280 46 0.017 24
Butyl sepharose 15 35 0.23 340
Mono Q (pH 8.0) 0.94 11 1.2 1700
Sephadex 200 0.24 4.6 1.9 2800
Mono Q (pH 7.0) 0.13 3.4 2.6 3900
Mono S 0.07 3.0 4.3 6400
Hydroxyapatite < 0.02 0.94 > 4.7 > 7000
K. Takahara et al. Wild watermelon glutamate N-acetyltransferase
FEBS Journal 272 (2005) 5353–5364 ª 2005 FEBS 5355
acids. The N-terminal amino acid sequence of the a
peptide was identical to a portion of the amino acid
sequence predicted from a watermelon EST clone
(accession number AI563351).
Cloning of watermelon GAT cDNA
To isolate a full-length cDNA clone of the GAT, gene-
specific primers were designed from the sequence of
watermelon EST AI563351 and used for 5¢- and
3¢-RACE. The cloned cDNA encoded a protein
composed of 460 amino acids. The amino acid
sequence had homology to At2g37500 from A. thaliana
(70% identity), B. subtilis GAT (38%) and S. cerevisiae
GAT (26%). Two regions of the deduced sequence
(residues 27–42 and 239–273) were identical to the
N-terminal amino acid sequences of the a and b pep-
tides determined by Edman sequencing, respectively
(Fig. 4); the enzyme was designated CLGAT (Citrullus

lanatus glutamate acetyltransferase). The first 26-amino
acid sequence at the N-terminus of CLGAT was pre-
dicted to function as a chloroplast transit peptide using
the chlorop program [21].
Genomic Southern blot analysis indicated that there
are two copies of the GAT gene in wild watermelon
(data not shown). To confirm that the cDNA cloned
above was that of the GAT purified in this study, we
screened a cDNA library (5 · 10
6
primary plaques) pre-
pared from the mixture of stressed and unstressed
leaves using the EST clone mentioned above as a probe.
Sequences of all nine clones isolated from the library
were identical with the EST and the RACE-derived
clone described above, indicating that only one type of
GAT mRNA is transcribed in wild watermelon leaves.
Citrullus lanatus glutamate acetyltransferase
(CLGAT) possessed the conserved AT(M ⁄ L)L motif
for the GAT family, where the precursor polypeptide
is self-cleaved between alanine and threonine residues
(Fig. 4, asterisk [22,23]);. In fact, the N-terminal resi-
due of the b peptide from purified CLGAT matched
the second residue of this conserved motif. These
results strongly suggest that the precursor peptide of
CLGAT in wild watermelon is also self-cleaved at the
ATML sequence in a manner similar to those in other
GATs reported so far. The molecular masses of the a
and b peptides calculated from the cDNA were 21.3
and 23.5 kDa, respectively – lower than those deter-

mined by SDS ⁄ PAGE (Fig. 3A). However, MALDI-
MS analysis of CLGAT detected two major peaks at
m ⁄ z 21.3 and 23.5 kDa, which corresponded to the
masses deduced from the cDNA (data not shown).
The molecular mass of native GAT was determined by
gel filtration chromatography as  90 kDa (Fig. 3B),
suggesting that CLGAT is a heterotetramer composed
of two each of the a and b subunits.
Analysis of the N-terminal transit peptide of
CLGAT
Analysis of the CLGAT cDNA predicted a chloroplast
transit peptide upstream from the N-terminus of
CLGAT. To examine whether the precursor of
CLGAT is imported into the chloroplasts, we prepared
a plasmid in which the first 26 codons of the CLGAT
A
B
Fig. 3. Molecular mass of GAT purified from leaves of wild water-
melon. (A) SDS ⁄ PAGE of GAT. The peak fraction obtained after
hydroxylapatite chromatography was analysed by PAGE (12% w ⁄ v,
polyaclylamide) and detected by silver staining. (B) Molecular mass
of the native form of wild watermelon GAT was determined from
the plot between the e lution volumes of the enzyme and marker
proteins in Superdex 200 gel chromatography. Thy, thyroglobulin;
Fer, ferritin; Ald, aldolase; Ova, ovalbumin.
Wild watermelon glutamate N-acetyltransferase K. Takahara et al.
5356 FEBS Journal 272 (2005) 5353–5364 ª 2005 FEBS
cDNA were fused in-frame to the coding sequence of
green fluorescent protein (GFP). This fusion protein
was transiently expressed in tobacco leaves and the

pattern of GFP fluorescence was analysed by confocal
microscopy. Chlorophyll autofluorescence was used
as the chloroplast marker. When the putative GAT
transit sequence–GFP fusion protein was expressed in
the leaves, green fluorescence was superimposed on the
chlorophyll autofluorescence giving yellow tint, but
this was undetectable in the cytoplasm (Fig. 5A). In
contrast, when nonfusion GFP was introduced into
the cells, the fluorescence was detected both in the cyto-
sol and nucleus but was excluded from chloroplasts
(Fig. 5B). These observations strongly suggest that the
Fig. 4. Comparison of the amino acid sequence of CLGAT (C; accession no. AB212224) with those from At2g37500 from A. thaliana (A),
B. subtilis GAT (B) and S. cerevisiae GAT(S) (accession nos. AAC98066, NP389002, and NP012464, respectively). Consensus identical (black)
and similar (grey) amino acids are shaded. The sequences of the two N-terminal sequences of the purified CLGAT subunits determined by
Edman sequencing are indicated by lines above the alignment. The conserved motif involved in self-catalysed cleavage is marked by aster-
isks. Filled and open arrows indicate the predicted cleavage sites of the watermelon GAT precursor polypeptide that occur as a result of
stromal processing peptidase and self-catalysed cleavage, respectively.
K. Takahara et al. Wild watermelon glutamate N-acetyltransferase
FEBS Journal 272 (2005) 5353–5364 ª 2005 FEBS 5357
precursor of CLGAT can be targeted to the chloro-
plasts, by the N-terminal transit sequence.
Kinetic analysis of GAT purified from wild
watermelon
The enzyme activity of CLGAT was maximal at pH 7.0
(Fig. 6A). The K
m
for the forward reaction at pH 7.0
was 3.4 mm for N-acetylornithine and 17.8 mm for
glutamate (Fig. 6B,C). To examine the regulation of
CLGAT by downstream products, activity was meas-

ured at a physiologically relevant concentration of
citrulline or arginine. Citrullus lanatus glutamate acetyl-
transferase activities in the presence of 30 mm citrulline
or 7 mm arginine were 100 and 98% of the original
activities, respectively, showing that CLGAT is not
inhibited by these downstream products. In this study,
the effect of ornithine was not determined because the
concentration of ornithine in wild watermelon leaves is
very low and constant before or after drought ⁄ strong
light stress, whereas concentrations of citrulline and
arginine in the leaves increased greatly during the
stress [6].
Surprisingly, CLGAT activity was maximum at
70 °C (Fig. 6D). To determine its thermostability, the
enzyme was incubated for 30 min at various tempera-
tures between 30 and 90 °C and residual activity was
determined (Fig. 6E). Citrullus lanatus glutamate acetyl-
transferase retained 98% of the original activity after
incubation at 70 °C, and still showed about 15% of the
original activity after incubation at 80 °C.
The thermotolerance of CLGAT observed above
prompted us to examine the leaf temperature under
drought conditions in the presence of strong light at
an atmospheric temperature of 35 °C. Under
unstressed conditions, the rate of leaf transpiration
was about 450 mmol H
2
OÆm
)2
Æs

)1
, and the leaf tem-
perature was about 30 °C (Fig. 7). In contrast,
drought ⁄ strong-light stress for five days decreased the
rate of transpiration to  15 mmol H
2
OÆm
)2
Æs
)1
, and
raised the leaf temperature to  44 °C.
Discussion
Citrulline is the most efficient hydroxyl radical scaven-
ger among all known compatible solutes, and its role
in oxidative stress resistance in wild watermelon has
been suggested [7]. In microorganisms examined so far,
the concentration of citrulline is kept low as a result of
rigid regulations such as feedback inhibition and tran-
scriptional regulation [8,9,24]. In contrast, wild water-
melon is unique for the massive accumulation of
citrulline in the leaves in response to drought ⁄ strong-
light stress. However, knowledge of the mechanism of
this accumulation is limited. It was previously sugges-
ted that the stress-induced AOD homologue, DRIP-1,
is involved in citrulline accumulation [25], but its func-
tion remains unclear. Unexpectedly, it is revealed in
this study that AOD activity was not detected in
stressed leaves where DRIP-1 was expressed at a high
level. Instead, GAT activity was detected in leaves,

suggesting that the fifth step of the citrulline pathway
A
B
Fig. 5. CLGAT is a chloroplast protein the
import of which depends on its N-terminal
transit peptide. Fluorescence microscopy
pictures of tobacco leaf cell transiently
expressing a CLGAT-GFP fusion (A) or GFP
alone as a control (B).
Wild watermelon glutamate N-acetyltransferase K. Takahara et al.
5358 FEBS Journal 272 (2005) 5353–5364 ª 2005 FEBS
is mainly catalysed by GAT in wild watermelon.
CLGAT was subsequently purified and identified from
wild watermelon. This is the first report dealing with
GAT purified from plant sources.
Under drought ⁄ strong-light conditions, plants close
their stomata to avoid loss of water, thereby increasing
leaf temperature [26]. In this study, the temperature of
wild watermelon leaves increased from 30 to 44 °C
under experimental stress conditions (Fig. 7). Under
natural desert conditions, leaf temperature of stressed
wild watermelon plants rises up to 60 °C [27]. In
this study, the optimum temperature of CLGAT was
revealed to be 70 °C, which was comparable to that
reported from the thermophilic microorganism B. stearo-
thermophilus (Table 2). Moreover, CLGAT retained
about 15% of its original activity after incubation at
80 °C for 30 min, whereas GAT from B. stearothermo-
philus completely lost its activity after incubation at
75 °C for 30 min [28]. These results suggest that CLGAT

has adapted to the thermogenic condition of leaf tissue
under drought in the presence of strong light.
In stressed leaves of watermelon, citrulline and
arginine accumulate massively to about 30 and 7 mm,
respectively [6], raising the question as to what extent
biosynthetic enzymes are inhibited by the downstream
products. The present results revealed that CLGAT
was not inhibited by either citrulline or arginine. This
is in contrast to GATs from other organisms: GAT
from Thermus aquaticus ZO5 is strongly inhibited
(K
i
¼ 1.75 mm [29]); and that from S. cerevisiae is
moderately inhibited by arginine (10% inhibition with
5mm arginine [30]). Therefore, CLGAT can function
without a loss of activity in the presence of high con-
centrations of citrulline and arginine. We did not
examine whether CLGAT is inhibited by ornithine,
another downstream product in the pathway.
Although several microbial GATs have been shown to
be inhibited by ornithine [28], the K
i
for ornithine in
Fig. 6. Kinetic analysis of CLGAT. (A) The
pH dependency of CLGAT activity. Enzyme
activity was determined between pH 4.2
and 7.8 in citrate ⁄ sodium phosphate buffer
(j) and Tris ⁄ HCl buffer (n). Activity is
expressed as a percentage of the maximal
activity. (B and C) Saturation kinetics of

CLGAT for N-acetylornithine (B) and glutam-
ate (C). The concentration of N-acetyl-
ornithine was varied from 0.5 to 15 m
M
with glutamate fixed at 10 mM in (B), and in
(C) that of glutamate was changed between
0.5 and 30 m
M with N-acetylornithine fixed
at 10 m
M. Double reciprocal plot of CLGAT
activity against the concentration of sub-
strates are shown in the insets. The fitted
linear regression lines and parameters are
also presented. The K
m
was estimated
based on these parameters. (D) Tempera-
ture dependency of CLGAT activity. Enzyme
activity was measured by incubating the
reaction mixtures at 20–90 °C. Activity is
expressed as percentage of the maximal
activity. (E) Thermostability of CLGAT. The
enzyme was incubated for 30 min at the
indicated temperatures and residual enzyme
activity was measured at 30 °C. Values
represent the mean ± SE from three
independent measurements.
K. Takahara et al. Wild watermelon glutamate N-acetyltransferase
FEBS Journal 272 (2005) 5353–5364 ª 2005 FEBS 5359
these GATs is between 1 and 3 mm, much higher than

the physiological concentration of ornithine (approxi-
mately 0.1 mm) in the leaves of wild watermelon under
drought and strong light stress [6].
The kinetics parameters obtained in this study
enabled us to discuss the role of CLGAT in citrulline
accumulation. Although CLGAT activity was
unchanged during drought stress in the presence of
strong light, an elevated leaf temperature from 30 to
44 °C would enhance the CLGAT reaction by about
two times that under unstressed conditions as shown
in Fig. 6D. Moreover, an increased concentration of
glutamate, a substrate for CLGAT, from 2.5 to
7.5 mm under drought ⁄ strong-light conditions [6]
would further elevate the CLGAT reaction by about
2.6-fold. As CLGAT catalyses not only the fifth step,
but also the first step of citrulline biosynthesis, these
estimations suggest that the influx of glutamate carbon
skeletons into the urea cycle is increased about fivefold
during drought ⁄ strong-light stress. Thus, CLGAT can
effectively participate in the citrulline biosynthesis
under drought conditions through its unique proper-
ties, namely, high thermostability and insensitivity to
inhibitions induced by the downstream products such
as citrulline and arginine.
A GFP-localization assay (Fig. 5) suggested that
CLGAT is a chloroplastic enzyme. In S. cerevisiae,
ornithine biosynthetic enzymes including GAT are
localized in mitochondria [15,30,31], and ornithine is
exported to the cytosol to be converted into citrulline
and arginine [31,32]. In plants, information on the

localization of citrulline and arginine biosynthetic
enzymes is scarce, but it has been reported that ornith-
ine carbamoyltransferase, an enzyme required for the
sixth step in the citrulline and arginine pathway, is
localized in chloroplasts in Canavalia lineate [33], rais-
ing the possibility that citrulline biosynthesis in plants
is catalysed in chloroplasts. In fact, cDNAs for all
six citrulline biosynthetic enzymes in Arabidopsis were
predicted as having chloroplast-targeting sequences
using the various programs [18].
A
B
Fig. 7. (A) Change in the stomatal conductance (h) and leaf tem-
perature (d) of wild watermelon plants during drought. Data
points represent the means from three independent experiments
and vertical bars are the SD. (B) Visualization of the thermal distri-
bution in wild watermelon plants. Images 1 and 2 represent
photographs of wild watermelon plants watered daily and subjec-
ted to drought for 5 days, respectively, and images 3 and 4 show
their thermal distributions analysed using an infrared thermal
camera, respectively.
Table 2. Comparison of the kinetic parameters for GAT from wild watermelon, S. cerevisiae and B. stearothermophilus. ND, not deter-
mined.
Wild watermelon S. cerevisiae
a
B. stearothermophilus
b
K
m glutamate
(mM) 17.8 7.2 19.2

K
m N-acetylornithine
(mM) 3.4 1.0 2.3
Temperature for maximum activity (°C) 70 ND 75
The pH for maximum activity 7.0 7.5 8.0
Activity in the presence of 5 m
M arginine
c
100 < 90 ND
a
[15, 30].
b
[28].
c
Expressed as a percentage of the maximum activity.
Wild watermelon glutamate N-acetyltransferase K. Takahara et al.
5360 FEBS Journal 272 (2005) 5353–5364 ª 2005 FEBS
The CLGAT enzyme was composed of two
subunits, a and b, derived from a single precursor
polypeptide (Fig. 3). The N-terminal amino acid
sequence of the b subunit from purified CLGAT coin-
cided with the cleavage site of the conserved motif,
ATML, in the sequence predicted from its cDNA.
This suggests that the CLGAT precursor is self-
cleaved as proposed for other GATs generating two
subunits that assemble as an a2b2 heterotetramer
[22,23,28]. Although such self-cleavage has been sug-
gested in several proteins other than GAT in plants
[34], CLGAT is the first example of the self-cleavage
for a chloroplastic protein.

The function of DRIP-1 remains to be determined.
In this study, no AOD activity was detected in wild
watermelon leaves in which abundant DRIP-1 accumu-
lation was seen (Fig. 2). Moreover, recombinant
DRIP-1 expressed in E. coli had no detectable AOD
activity (data not shown). One possible explanation for
this discrepancy is that DRIP-1 requires a cofactor or
activator for its catalysis, although we tested Co
2+
and Zn
2+
in the present enzymatic assays. The second
possibility is that DRIP-1 possesses a different func-
tion unrelated to AOD.
In this study, we demonstrated that CLGAT,
which catalyses the first and fifth steps of the citrul-
line biosynthetic pathway, can contribute effectively
to citrulline biosynthesis under drought conditions
owing to its high thermostability and insensitivity to
the inhibition induced by citrulline and arginine.
However, many different enzymes are involved in the
metabolism of citrulline (Fig. 1), and their kinetic
properties and ⁄ or expression pattern remain to be
examined. Comprehensive analysis of the citrulline
biosynthesis pathway is required to fully understand
the mechanism of citrulline accumulation in wild
watermelon.
Experimental procedures
Materials
N-Acetylornithine was purchased from Sigma (St. Louis,

MO, USA). Other chemicals and reagents were purchased
from Nakalai (Kyoto, Japan).
Plant material
Wild watermelon (C. lanatus L. sp. no. 101117-1) was
grown in a growth chamber (16 ⁄ 8 h light ⁄ dark regime at
temperatures of 35 ⁄ 25 °C, 50 ⁄ 60% humidity and 800 lmol
photonsÆm
)2
Æs
)1
) in 500-mL paper pots. Soil for horti-
culture was purchased from PROTOLEAF (Tokyo, Japan).
Plants were watered daily at 9 a.m. (1 h after the start of
the light period). Two-week-old plants with fully expanded
fourth leaves were used in the experiments. Drought treat-
ment was started by stopping watering. For GAT purifica-
tion, the plants were grown in a greenhouse at a
temperature between 25 and 35 °C for 2 months from June
to August in 2002.
Analysis of leaf transpiration and temperature
Transpiration of attached fourth leaves was measured at a
light intensity of 800 lmol photonsÆ m
)2
Æs
)1
at 35 °C using a
porometer (type AP4; AT delta-T device, Cambridge, UK).
Leaf temperature was measured using a thermometer
(model AP-320, 0.25K-J1M1, Anritu meter, Tokyo), and
thermal images were obtained using an infrared camera

(TVS-8500, Nippon Avionics Ltd, Tokyo, Japan). Data
were collected around 15:00 (7 h after the start of the light
period).
Enzyme assays
Acetylornithine deacetylase and GAT activity were
measured by quantifying the production of ornithine
from N-acetylornithine using the colorimetric ninhydrin
procedure as described previously [11,15]. The AOD
assay mixture (200 lL) contained 100 mm potassium
phosphate buffer pH 7.0, 6 mm N-acetylornithine,
0.5 mm metal salt (CoCl
2
or ZnCl
2
) and the enzyme.
The GAT assay mixture (200 lL) contained 100 mm
potassium phosphate buffer (pH 7.0), 6 mm N-acetylorni-
thine, 6 mm glutamate and the enzyme. The reaction
was started by adding N-acetylornithine and stopped by
adding 600 lL of ninhydrin reagent (0.4 m citric acid ⁄ 1%
ninhydrin in 2-methoxyethanol, 1 : 2 v ⁄ v). After boiling for
10 min, 200 lL4m NaOH was added to the mixture and
light absorbance at 470 nm was measured. One unit
is defined as the amount of enzyme that forms 1 lmol
ornithineÆmin
)1
.
Purification of GAT from wild watermelon leaves
All purification steps were carried out at 0–4 °C, and col-
umn chromatographies were performed with FPLC system

(Amersham Biosciences, Uppsala, Sweden). Wild water-
melon leaves (300 g) were homogenized with a blender in
300 mL extraction medium containing 100 mm potassium
phosphate buffer pH 8.0, 1 mm EDTA, 10 mm 2-mercapto-
ethanol, 1 mm phenylmethylsulfonyl fluoride and 1% (w ⁄ v)
polyvinylpolypyrrolidone. After filtering through six layers
of gauze, the resulting homogenate was centrifuged at
12 000 g for 30 min. Unless stated otherwise the centrifuga-
tion described below was carried out in the same way.
N-Acetylornithine was added to the supernatant to give a
K. Takahara et al. Wild watermelon glutamate N-acetyltransferase
FEBS Journal 272 (2005) 5353–5364 ª 2005 FEBS 5361
final concentration of 10 m m. The mixture was then
warmed in a water bath at 70 °C for 10 min with gentle
stirring. The mixture was promptly cooled in an ice bath
until the temperature dropped below 4 °C and centrifuged.
The supernatant was brought to 20% saturation by adding
(NH
4
)
2
SO
4
powder, incubated on ice with gentle stirring
for 30 min and centrifuged. The supernatant was applied to
a Butyl-Sepharose Fast Flow column (1.6 cm i.d. · 10 cm;
Amersham Bioscience) equilibrated with buffer A contain-
ing 20 mm potassium phosphate buffer pH 7.0, 1 mm
EDTA, 10 mm 2-mercaptoethanol, 1 mm dithiothreitol
(DTT) and 20% (v ⁄ v) glycerol, and 20% saturation of

(NH
4
)
2
SO
4
. The enzyme was eluted with a linear gradient
of 30–0% saturation of (NH
4
)
2
SO
4
in buffer A. Active
fractions were collected and applied to a Sephadex G-25
column (5 cm i.d. · 20 cm) equilibrated with buffer B con-
taining 5 mm potassium phosphate buffer (pH 8.0), 1 mm
EDTA, 10 mm b-mercaptoethanol, 1 mm DTT, and 20%
(v ⁄ v) glycerol. Active fractions were pooled and applied
to a Mono Q HR column (0.5 cm i.d. · 5 cm; Amersham
Bioscience) equilibrated with buffer B. The column was
developed with a 0–200 mm linear gradient of KCl in buffer
B. Active fractions were pooled and concentrated with
Centriplus YM-10 (Amicon, Bevery, MA, USA). The
enzyme solution was applied to a Sephadex 200 column
(1.6 cm i.d. · 60 cm; Amersham Bioscience) equilibrated
with 20 mm potassium phosphate buffer pH 7.0, 1 mm
EDTA, 10 mm 2-mercaptoethanol, 1 mm DTT, 150 mm
KCl and 20% (v ⁄ v) glycerol and developed with the same
buffer. Active fractions were loaded onto a Mono Q HR

column (0.5 cm i.d. · 5 cm; Amersham Bioscience) then a
Mono S HR column (0.5 cm i.d. · 5 cm; Amersham Bio-
science). In these chromatographies GAT activity was
recovered in the flow-through fractions. Active fractions
were applied to a Sephadex G-25 column (1.6 cm
i.d. · 10 cm) equilibrated with buffer C containing 5 mm
potassium phosphate buffer pH 7.0, 10 mm 2-mercapto-
ethanol, 1 mm DTT, and 20% (v ⁄ v) glycerol. Active frac-
tions were applied to a hydroxyapatite column (0.7 cm
i.d. · 5.2 cm; Bio-Rad, Hercules, CA, USA) equilibrated
with buffer C. The column was developed with a 5–100 mm
linear gradient of potassium phosphate in buffer C. Aliqu-
ots from the active fractions were used for SDS ⁄ PAGE
on a 12.5% polyacrylamide gel, and proteins were
visualized by silver staining with a commercial kit
(Daiichikagaku Chemical, Osaka, Japan). Proteins were
measured according to the Bradford method [35], using
BSA as a standard.
For analysis of the N-terminal amino acid sequence,
CLGAT subunits were separated by SDS ⁄ PAGE and
electroblotted onto polyvinyldene difluoride membranes.
Blotted proteins were stained with Coomassie Brilliant
R-250. Stained regions were cut from the membrane and
used for sequencing with an automated protein sequencer
(Model 492, Applied Biosystems, Foster City, CA, USA).
For analysis of MALDI-TOF MS data 10 lL of purified
CLGAT ( 0.5 mgÆmL
)1
) were mixed with 1 lL matrix
solution containing 10 mgÆ mL

)1
a-cyano-4-hydroxycinnamic
acid and 50% (v ⁄ v) acetonitrile. The mixture was loaded
onto a sample plate and the solvent was removed by evapor-
ation. The molecular mass of CLGAT was determined by
MALDI-TOF MS (Autoflex II, Bruker Daltonics, Billerica,
MA, USA), calibrated with insulin (5734 Da), cyto-
chrome c (12361 Da), myoglobin (16952 and 8476 Da) and
ubiquitin (8566 Da; all Sigma) as standard proteins.
Western blotting
Proteins from SDS ⁄ PAGE were transferred to polyvinyl-
dene difluoride membranes (Sequi-Blot PVDF membrane;
Bio-Rad) using a semidry blotting apparatus (NA-1512, Ni-
hon-Eido, Tokyo, Japan). The specific antibody for DRIP-
1 [25] was used at a dilution of 1 : 500 in buffer containing
30 mm Tris ⁄ HCl buffer pH 7.5, 200 mm NaCl and 5%
(w ⁄ v) skim milk. Immunoreactive proteins were detected
with an Immunostaining HRP-1000 kit (Konica, Tokyo,
Japan) according to the manufacturer’s instructions.
cDNA cloning
Total RNA was prepared from 3 g wild watermelon leaves
subjected to drought stress for 1 day using TRIzol (Invitro-
gen, Carlsbad, CA, USA). Poly(A)
+
RNA was isolated
from the total RNA using a mRNA purification kit (Amer-
sham Bioscience) according to the manufacturer’s instruc-
tions. The sequence of the watermelon EST clone
(accession number AI563351) was used to design four
GAT-specific primers; CLGAT5a (5¢-GGCATCAACAT-

CACAAGCAACAAGTGCAAG-3¢), CLGAT5b (5¢-TCCT-
CCATCAATCTGCTTCCATGGACCATC-3¢), CLGAT3a
(5¢-GCTGTGGCTACGAATGAGGCCGCC-3) and CLGAT3b
(5¢-AAGGGAGAGAAACCTGACCTTGCACTTG-3¢). The
5¢-RACE was performed using Marathon cDNA Amplification
Kit (Clontech, Palo Alto, CA) according to the manufacturer’s
instructions, using CLGAT5a for the first PCR and CLGAT5b
for the second PCR. For 3¢-RACE, single-stranded cDNA was
synthesized using First-Strand cDNA Synthesis Kit (Amersham
Bioscience) with a NotI-d(T)
18
bifunctional primer (5¢-TAA-
CTGGAAGAATTCGCGGCCGCAGGAAT
(18)
-3¢). The single-
stranded cDNA was used for PCR w ith the primers Not1
(5¢-AACTGGAAGAATTCGCGGCCGC-3¢) and CLGAT3a.
An aliquot from t he first PCR products was subjected to the
second round of PCR using primers Not2 (5¢-GAA GAAT-
TCGCGGCCGCAGG-3¢) and CLGAT5b. Amplified products
were cloned into the plasmid vector pBC (Stratagene, La Jolla,
CA, USA).
Sequencing was carried out using the BigDye Terminator
Cycle Sequencing Kit (Applied Biosystems) with a DNA
sequencer (model 3100, Applied Biosystems).
Wild watermelon glutamate N-acetyltransferase K. Takahara et al.
5362 FEBS Journal 272 (2005) 5353–5364 ª 2005 FEBS
Isolation of genomic DNA and Southern blot
analysis
Genomic DNA was isolated from the leaves of wild water-

melon as described previously [36]. Aliquots of genomic
DNA (10 lg) were digested with the indicated restriction
enzymes. The digests were then subjected to electrophor-
esis on 0.8% agarose gel and transferred to Hybond N
+
membranes (Amersham Bioscience). For probe prepar-
ation, a 615 bp GAT fragment was amplified from the
CLGAT cDNA by PCR with the primers CLGAT3b and
CLGAT3c (5¢-GGTACTCGTATCCCCATCCACCG-3¢).
The amplified fragment was purified using the MinElute
Gel Extraction Kit (Qiagen, Valencia, CA, USA), and
used as a template for preparation of a radioactive probe
with the Prime-It II Random primer Labeling Kit (Strata-
gene). Hybridization was performed as described previ-
ously [37].
GFP transient assay
The sequence of the putative chloroplast transit peptide
(amino acids 1–26) was amplified by PCR and cloned into
the modified GFP vector pTH2XA [38]. The construct was
designed to fuse the DNA sequence of the putative chloro-
plast transit peptide to the 5¢-end of the GFP gene to be
expressed under control of the cauliflower mosaic virus 35S
promoter. The plasmid (1 lg) was introduced into tobacco
leaves by bombardment (PDS-1000 ⁄ He apparatus, Bio-
Rad) according to the manufacturer’s instructions. The
fluorescence was visualized with confocal microscopy
(LSM510, Carl Zeiss, Jena, Germany) after overnight incu-
bation.
Acknowledgements
We thank Junko Tsukamoto for technical assistance

with Edman sequencing and MALDI-TOF MS analy-
sis. This work was supported partly by a Creative
Scientific Research from the Japan Society for the Pro-
motion of Science (JSPS-13GS0023) and partly by an
Intellectual Cluster Program of the Ministry of Educa-
tion, Culture, Sports, Science and Technology, Japan.
References
1 Boyer JS (1982) Plant productivity and environment.
Science 218, 443–448.
2 Tabaeizadeh Z (1998) Drought-induced responses in
plant cells. Int Rev Cytol 182, 193–247.
3 Chen TH & Murata N (2002) Enhancement of tolerance
of abiotic stress by metabolic engineering of betaines
and other compatible solutes. Curr Opin Plant Biol 5,
250–257.
4 Hare PD, Cress WA & Van Staden J (1998) Dissecting
the roles of osmolyte accumulation during stress. Plant
Cell Environ 21, 535–553.
5 Stoop JM, Williamson JD & Pharr DM (1996) Manni-
tol metabolism in plants: a method for coping with
stress. Trends Plant Sci 1, 139–144.
6 Kawasaki S, Miyake C, Kohchi T, Fujii S, Uchida M &
Yokota A (2000) Response of wild watermelon to
drought stress: Accumulation of an ArgE homologue
and citrulline in leaves during water deficits. Plant Cell
Physiol 41, 864–873.
7 Akashi K, Miyake C & Yokota A (2001) Citrulline, a
novel compatible solute in drought-tolerant wild water-
melon leaves, is an efficient hydroxyl radical scavenger.
FEBS Lett 508, 438–442.

8 Thompson JF (1980) Arginine synthesis, proline synthe-
sis, and related processes. In The Biochemistry of Plants,
Vol. 5, pp. 375–402. Academic Press, New York.
9 Cunin R, Gransdorff N, Pierard A & Stalon V (1986)
Biosynthesis and metabolism of arginine in bacteria.
Microbiol Rev 50, 314–352.
10 Davis RH (1986) Commpartmental and regulatory
mechanisms in the arginine pathways of Neurospora
crassa and Saccharomyces cerevisiae. Microbiol Rev 50,
280–313.
11 Vogel HJ & Bonner DM (1956) Acetylornithinase of
Escherichia coli: partial purification and some proper-
ties. J Biol Chem 218, 97–106.
12 Leisinger T & Haas D (1975) N-acetylglutamate
synthase of Escherichia coli regulation of synthesis
and activity by arginine. J Biol Chem 250, 1690–1693.
13 Glansdorff N (1964) Topography of cotransducible
arginine mutations in Escherichia coli K-12. Genetics
51, 167–179.
14 Sakanyan V, Kochikyan A, Mett I, Legrain C,
Charlier D, Pierard A & Glansdorff N (1992) A
re-examination of the pathway for ornithine biosynthesis
in a thermophilic and two mesophilic Bacillus specoes.
J Gen Microbiol 138, 125–130.
15 Liu Y, Van Heeswijck R, Hoj P & Hoogenraad N
(1995) Purification and characterization of ornithine
acetyltransferase from Saccharomyces cerevisiae. Eur J
Biochem 228, 291–296.
16 Pauwels K, Abadjieva A, Hilven P, Stankiewicz A &
Crabeel M (2003) The N-acetylglutamate synthase ⁄

N-acetylglutamate kinase metabolon of Saccharomyces
cerevisiae allows co-ordinated feedback regulation of the
first two steps in arginine biosynthesis. Eur J Biochem
270, 1014–1024.
17 Baetens M, Legrain C, Boyen A & Glansdorff N (1998)
Genes and enzymes of the acetyl cycle of arginine
biosynthesis in the extreme thermophilic bacterium
Thermus thermophilus HB27. Microbiology 144,
479–492.
K. Takahara et al. Wild watermelon glutamate N-acetyltransferase
FEBS Journal 272 (2005) 5353–5364 ª 2005 FEBS 5363
18 Slocum RD (2005) Genes, enzymes and regulation of
arginine biosynthesis in plants. Plant Physiol Biochem
(in press).
19 Arabidopsis Genome Initiative (2000) Analysis of the
genome sequence of the flowering plant Arabidopsis
thaliana. Nature 408, 796–815.
20 Javid-Majd F & Blanchard JS (2000) Mechanism analy-
sis of the argE-encoded N-acetylornithine deacetylase.
Biochemistry 39, 1285–1293.
21 Nielsen H, Engelbrecht J, Brunak S & von Heijne G
(1997) Identification of prokaryotic and eukaryotic
signal peptides and prediction of their cleavage sites.
Protein Eng 10, 1–6.
22 Marc F, Weigel P, Legrain C, Glansdorff N &
Sakanyan V (2001) An invariant threonine is involved
in self-catalyzed cleavage of the precursor protein for
ornithine acetyltransferase. J Biol Chem 276, 25404–
25410.
23 Abadjieva A, Hilven P, Pauwels K & Crabeel M

(2000) The yeast ARG7 gene product is autoproteo-
lyzed to two subunit peptides, yielding active
ornithine acetyltransferase. J Biol Chem 275, 11361–
11367.
24 Mass WK (1994) The arginine repressor of Escherichia
coli. Microbiol Rev 58, 631–640.
25 Yokota A, Kawasaki S, Iwano M, Nakamura C,
Miyake C & Akashi K (2002) Citrulline and DRIP-1
protein (ArgE homologue) in drought tolerance of wild
watermelon. Ann Bot (Lond) 89, 825–832.
26 Radin JW, Lu Z, Percy RG & Zeiger E (1994) Genetic
variability for stomatal conductance in Pima cotton
and its relation to improvements of heat adaptation.
Proc Natl Acad Sci USA 91, 7217–7221.
27 Larcher W (1995) Plant under stress. In Physiological
Plant Ecology, 3rd edn, pp. 321–448. Springer-Verlag,
New York.
28 Marc F, Weigel P, Legrain C, Almeras Y, Santrot M,
Glansdorff N & Sakanyan V (2000) Characterization
and kinetic mechanism of mono- and bifunctional
ornithine acetyltransferases from thermophilic micro-
organisms. Eur J Biochem 267, 5217–5226.
29 Van de Casteele M, Demarez M, Legrain C, Glansdorff
N & Pierard A (1990) Pathways of arginine biosynthesis
in extreme thermophilic archeo- and eubacteria. J Gen
Microbiol 136, 1177–1183.
30 Crabeel M, Abadjieva A, Hilven P, Desimpelaere J &
Soetens O (1997) Characterization of the Saccharomyces
cerevisiae ARG7 gene encoding ornithine acetyltrans-
ferase, an enzyme also endowed with acetylglutamate

synthase activity. Eur J Biochem 250, 232–241.
31 Jauniaux JC, Urrestarazu A & Wiame JM (1978)
Arginine metabolism in Saccharomyces cerevisiae:
subcellular localization of the enzymes. J Bacteriol 133,
1096–1107.
32 Crabeel M, Soetens O, De Rijcke M, Pratiwi R &
Pankiewicz R (1996) The ARG11 genes of Saccharo-
myces cervisiae encodes a mitochondrial integral
membrane protein required for arginine biosynthesis.
J Biol Chem 271, 25011–25018.
33 Lee Y, Choi YA, Hwang ID, Kim S & Gv & Kwon
YM (2001) cDNA cloning of two isoforms of ornithine
carbamoyltransferase from Canavalia lineata leaves and
the effect of site-directed mutagenesis of the carbamoyl
phosphate binding site. Plant Mol Biol 46, 651–660.
34 Perler FB (1998) Breaking up is easy with esters. Nat
Struct Biol 5, 249–252.
35 Bradford MM (1976) A rapid and sensitive method for
the quantitation of microgram quantities of protein
utilizing the principle of protein-dye binding. Anal
Biochem 72, 248–254.
36 Akashi K, Nishimura N, Ishida Y & Yokota A (2004)
Potent hydroxyl radical-scavenging activity of drought-
induced type-2 metallothionein in wild watermelon.
Biochem Biophy Res Commun 323, 72–78.
37 Sambrook J, Fritsch EF & Maniatis T (1989) Molecular
cloning: A Laboratory Manual, 3rd edn. Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, New
York.
38 Kohchi T, Mukougawa K, Frankenberg N, Masuda M,

Yokota A & Lagarias JC (2001) The Arabidopsis HY2
gene encodes pytochromobilin synthase, a ferredoxin-
dependent biliverdin reductase. Plant Cell 13, 425–436.
Wild watermelon glutamate N-acetyltransferase K. Takahara et al.
5364 FEBS Journal 272 (2005) 5353–5364 ª 2005 FEBS