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

These findings suggest that CLGAT can effectively participate in the biosynthesis of citrulline in wild watermelon leaves during drought⁄ strong-light stress.. In addition to citruline, c

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: yokota@bs.naist.jp

(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.

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

7 mm 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, N-acetyl-ornithine carbamoyltransferase; ASS, argininosuccinate synthase and ASL, argininosuccinate lyase.

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)1Æmg protein)1;

Fig 2A) Although bacterial AODs are activated by a

divalent metal ion such as Co2+ or Zn2+ [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)1mg 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)

Hydroxyapatite < 0.02 0.94 > 4.7 > 7000

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· 106primary 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 elution volumes of the enzyme and marker

proteins in Superdex 200 gel chromatography Thy, thyroglobulin;

Fer, ferritin; Ald, aldolase; Ova, ovalbumin.

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.

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 Km 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 H2OÆm)2Æs)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 H2OÆm)2Æs)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

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).

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 (Ki ¼ 1.75 mm [29]); and that from S cerevisiae is moderately inhibited by arginine (10% inhibition with

5 mm 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 Ki 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 m M 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.

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

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

a

[15, 30].b[28].cExpressed as a percentage of the maximum activity.

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 Co2+

and Zn2+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)2Æs)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 photonsÆm)2Æs)1at 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 (CoCl2 or ZnCl2) 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 lL 4 m 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

final concentration of 10 mm 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

(NH4)2SO4 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

(NH4)2SO4 The enzyme was eluted with a linear gradient

of 30–0% saturation of (NH4)2SO4 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 mgÆmL)1a-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 DRIP-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 with the primers Not1 (5¢-AACTGGAAGAATTCGCGGCCGC-3¢) and CLGAT3a

An aliquot from the first PCR products was subjected to the second round of PCR using primers Not2 (5¢-GAAGAAT-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)