Download Responses of Wild Watermelon to Drought Stress: Accumulation of

Plant Cell Physiol. 41(7): 864–873 (2000)
JSPP © 2000
Responses of Wild Watermelon to Drought Stress: Accumulation of an ArgE
Homologue and Citrulline in Leaves during Water Deficits
Shinji Kawasaki 1, 3, Chikahiro Miyake 1, Takayuki Kohchi 1, Shinichiro Fujii 2, Masato Uchida 2 and Akiho
Yokota 1, 4
1
2
Graduate School of Biological Sciences, Nara Institute of Science Technology, 8916–5 Takayama, Ikoma, Nara, 630-0101 Japan
Tottori Horticultural Experiment Station, 2048 Yurashuku, Daiei, Tohaku-gun, Tottori, 689-2221 Japan
;
to other in the energy cost for CO2 fixation but inferior in the
water use efficiency (Edwards and Walker 1983). Inversely, although C4-plants prevail over C3-plants in the CO2-fixation efficiency, the energy cost of C4-plants in photosynthesis is worse
than that of C3-plants (Furbank and Foyer 1988, Hatch 1992,
Dai et al. 1993). CAM-plants are superior to others in the water-use efficiency but their productivity and crop value are less
than those of the others (Osmond 1978, Nobel 1991). Although the photosynthetic mechanisms of C4- and CAM-plants
arouse ones’ interests in improving the water-use efficiency of
our C3-crop plants (Hudspeth et al. 1992, Gallardo et al. 1995,
Ku et al. 1999), the genes for these superior mechanisms are
too many even to describe (Brown and Bouton 1993, Furbank
and Taylor 1995, Cushman and Bohnert 1999). Another possible approach to fortify the water-use efficiency or drought responses of the important crop plants is to collect information as
to how the C3-plants bear drought. However, these plants are,
generally speaking, drought-intolerant compared to C4- and
CAM-plants and these studies would reach an only partial success. If there exists a plant that can survive longer than C4plants under drought conditions in the light, it will be an interesting example from which to learn. The wild species of crop
plants often show high tolerance to various environmental
stresses (Crawford 1989). It can be considered that these plants
have attained the genetic systems for stress tolerance while
growing in the natural field without artificial breeding.
Wild watermelon, Citrullus lanatus sp. belongs to the
gourd family and inhabits in Botswana. Watermelon carries out
C3-type photosynthesis (Miyake and Yokota 2000). Wild watermelon survives in the spring to summer annually in the desert
field (Gibson 1996). The climate of the Kalahari desert is very
severe for plant growth (Lovegrove 1993). Less rain fall causes accumulation of salts on the soil surface, and strong sun
light and less available water derive plants into oxidative and
heat damages. We considered that wild watermelon must conserve highly developed systems for growing under these severe environmental conditions.
In order to survive under the water-deficit conditions,
plants have to maintain their water status to keep homeostasis.
The abilities of drought-tolerant plants to prevent them to lose
water are divided into some categories; (1) reduction of water
Wild watermelon from the Botswana desert had an
ability to survive under severe drought conditions by maintaining its water status (water content and water potential). In the analysis by two-dimensional electrophoresis of
leaf proteins, seven spots were newly induced after watering stopped. One with the molecular mass of 40 kilodaltons
of the spots was accumulated abundantly. The cDNA encoding for the protein was cloned based on its amino-terminal sequence and the amino acid sequence deduced from
the determined nucleotide sequences of the cDNA exhibited homology to the enzymes belong to the ArgE/DapE/
Acy1/Cpg2/YscS protein family (including acetylornithine
deacetylase, carboxypeptidase and aminoacylase-1). This
suggests that the protein is involved in the release of free
amino acid by hydrolyzing a peptidic bond. As the drought
stress progressed, citrulline became one of the major components in the total free amino acids. Eight days after withholding watering, although the lower leaves wilted significantly, the upper leaves still maintained their water status
and the content of citrulline reached about 50% in the total
free amino acids. The accumulation of citrulline during the
drought stress in wild watermelon is an unique phenomenon in C3-plants. These results suggest that the drought tolerance of wild watermelon is related to (1) the maintenance
of the water status and (2) a metabolic change to accumulate citrulline.
Key words: Citrulline — 2-Dimensional electrophoresis —
Drought tolerance — Gas exchange — Wild watermelon.
Abbreviations: 2-D, 2-dimensional; DRIP, drought-induced polypeptide.
The nucleotide sequence in this paper has been submitted to the
DDBJ and registered under the accession number AB036420 (DRIP-1).
Introduction
Plants are divided into three groups depending on their
photosynthetic CO2 fixation mechanisms; C3-, C4- and CAMplants. Many crops belong to C3-plants. C3-plants are superior
3
4
Present address: Department of Biochemistry, University of Arizona, Biological Science West #513, Tucson, Arizona 85721, U.S.A.
Corresponding author: E-mail, [email protected]; Fax, +81-743-72-5569.
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Responses of Wild Watermelon to Drought
loss from the plant surface by cuticular and cell wall development and lignification, (2) osmotic adjustment by production of
compatible solutes and osmoprotectants, and (3) development
of water-storage tissues in roots, stems, and leaves (Gibson
1996, Ingram and Bartels 1996, Bray 1993, Bray 1997, Tabaeizadeh 1998). Drought-intolerant model plants like Arabidopsis
and tobacco do not have these developed systems and suffer
from irreversible damage from cell dehydration.
Wild watermelon showed unique tolerant responses to severe drought conditions used in this study, compared to other
plants. Wild watermelon plants were grown under well-watered
conditions at a high temperature and a high light intensity until
the fourth leaf was fully expanded. Photosynthetic gas exchange rate and water status were analysed before and after
watering was withheld. Surprisingly, wild watermelon retained
water in the leaves for several days after stopping watering and
even after the soil water content decreases to 5 to 7%. Several
polypeptides were newly induced while the soil water content
was decreasing. One of the polypeptides, which were accumulated abundantly during the drought stress, was deduced to be
related to the massive production of citrulline from glutamate.
Physiological responses of the wild watermelon plant to
drought are discussed.
Materials and Methods
Plant materials
Wild watermelon from the Kalahari desert in Botswana (Citrullus lanatus sp. No101117–1), domesticated watermelon (Citrullus
lanatus L. cv. Sanki), cucumber (Cucumis sp. cv. Shinsokusei), maize
(Zea mays L. cv, Honeybantam) were grown in the same growth chamber (16/8 h light/dark regime at temperatures of 35/25C, 50/60% humidities and 1,000 mol photons m–2 s–1) with 500-ml size of paper
pots lightly filled with standard soil for horticulture. For experiments
in this paper, 2-week-old wild watermelon plants with the fourth leaf
had fully expanded were used. Domesticated watermelon, cucumber,
and maize were grown for 2 weeks under the same conditions. Plants
were watered daily at 9 a.m. (3 h after the start of lightening) and given the nutrient solution two times a week.
Analyses of photosynthesis and other physiological parameters
Net CO2 assimilation rates, stomatal conductance and transpiration were measured with attached leaves at the saturating light intensity (1,500 mol photons m–2 s–1) at 35C using an infrared gas analysing system (Li-6400, Li-Cor, Lincoln, U.S.A.). Data were collected
twice or more around 1 p.m. (7 h after the start of lightning). Soil and
leaf tissues were dried in an oven at 70C for 24 h (Marshall and
Dumbroff 1999). The soil water content was calculated as [(soil
weight) (dried soil weight)]/(soil weight) 100 (%). The leaf water
content was calculated by [(fresh leaf weight) (dried leaf weight)]/
(fresh leaf weight) 100 (%). The leaf water potential was measured
with a Tru Psi Model sc10 (Decagon Devices Inc., U.S.A.) water potential measurement system.
Protein extraction and two-dimensional electrophoresis
Leaves detached at 1 p.m. (7 h after the start of lightning) were
promptly frozen in liquid nitrogen and homogenised in acetone containing 0.1% (v/v) 2-mercaptoethanol. Proteins were allowed to aggregate for 1 h at 20C and were centrifuged at 13,000 g for 5 min.
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The pellet was dried in vacuo and solubilized in 0.1 M Tris-HCl (pH
7) containing 2% SDS, 0.2% 2-mercaptoethanol and one tablet of protease inhibitors (CompleteTM, Boehringer Mannheim, Germany) and
then was boiled for 5 min. SDS was removed by treating the solution
with triethylamine according to the method of Konigsberg and Henderson (1983). After the final washing with acetone, the pellet was dried
in vacuo and dissolved in two-dimensional (2-D) electrophoresis sample buffer containing 8 M urea, 30 mM dithiothreitol, 2% (v/v) Pharmalyte 3–10 (Amersham Pharmacia Biotech, Tokyo) and 0.5% Triton X100.
Both isoelectrofocusing and SDS-PAGE were performed horizontally with Maltiphor II (Amersham Pharmacia Biotech) according
to the manufacturer’s instructions. Isoelectrofocusing was done with
the immobilised pH 4 to 7 gradient gel (Amersham Pharmacia Biotech) with 10 g of total proteins. The gel strip was then transferred
onto the top surface of the horizontal ExcelGelTM 8 to 18% acrylamide gradient gel (Amersham Pharmacia Biotech), and SDS-PAGE
was carried out according to the manual. The gel was silver-stained
with the silver-staining kit (Amersham Pharmacia Biotech).
The protein content was determined by the dye-binding assay
(Bradford 1976).
Analysis of N-terminal amino acid sequences
For protein sequencing, 100 g of total leaf proteins were used
for 2-D electrophoresis. Drought induced spots were identified by the
computer analysing system (2-D Analyser, Amersham Pharmacia Biotech). The 2-D gels from three independent electrophoreses were electroblotted onto polyvinylidene difluoride membranes (Bio-Rad,
U.S.A.). Spots in question were collected and sequenced by the
Edoman degradation method with a peptide sequencer (model 610A,
Perkin Elmer Applied Biosystems, Foster City, CA).
Isolation of RNA and construction of a cDNA library
To prepare cDNA library of drought-induced genes, wild watermelon leaves suffering from drought stress for one to three days were
collected and total RNA was extracted with Isogen (Nippongene, Japan). Poly(A)+ mRNA was isolated from the mixed total RNA solution
with an oligo(dT) cellulose spun column (Clontech, U.S.A.) and used
to generate a lambdaZAPII library with a Uni-ZAP XR vector system
(Stratagene). Synthesis of the first cDNA strand was primed with the
oligo(dT)-oligonucleotides and cDNA was extended with the Moloney murine leukemia virus reverse transcriptase. All steps for cDNA
synthesis and in vitro packing were performed according to the manufacturer’s instructions (cDNA synthesis kit, ZAP-cDNA synthesis kit,
and Gigapack III Gold Packing Extracts, Stratagene). Escherichia coli
strain XL1-Blue MRF was used as the bacterial host. The complexity
of the library obtained was 106 pfu.
Cloning of drought-induced polypeptide-1 cDNA
We first isolated the partial drought-induced polypeptide (DRIP)1 cDNA from the library prepared from wild watermelon leaves by
PCR amplification with gene-specific sense primers and antisense
primers that hybridized to lambdaZAPII vector. The degenerated sense
primer for the N-terminal sequence of DRIP-1 peptides (N-5–20: 5ATHAARGARATHATHGGIGG-3, where H is A or T or C, R is A or
G, and I is inosine) and a vector primer (M13-forward: 5-GTAAAACGACGGCCAGT-3) were used for the first PCR reaction. By using the
first PCR products as template, the second PCR was performed with
the nested sense primer (N13–20: 5-CARAARGARWSITAYATHCC3, where R is A G, W is A or T, S is G or C, and Y is C or T) and another vector primer (T7: 5-GTAATACGACTCACTATAGGGC-3).
The primers, N5-20 and N13-20, were designed for amino acid sequences IKEIIGG and QKESYIP, respectively. The second PCR prod-
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Responses of Wild Watermelon to Drought
Fig. 1 Typical example of wild watermelon subjected to drought for 8 d (A). Domesticated watermelon (B) and cucumber plants (C) treated for
3 and 2 d, respectively, are also shown as controls.
uct was cloned into pT7Blue vector (Novagene, Germnay). To isolate
the full-length cDNA, approximately fifty thousand plaques were
screened using the partial cDNA as the radiolabeled probe. Plaque hybridization was performed according to a standard protocol (Sambrook et al. 1989). DNA sequencing was performed by using standard
dye-terminater sequence procedure and automated sequencer (model
373S, PE Biosystems, U.S.A.).
Amino acid analysis
Total free amino acids in leaves were extracted by homogenizing
6.5 cm2 (about 300 mg) of leaves in 2.5 ml of methanol-chloroformwater (12 : 5 : 3 in volume). After centrifugation, the supernatant was
removed and the extraction repeated three times for completing extraction. Three ml of H2O and 2 ml of chloroform were added to the
pooled extracts and mixed vigorously. The aqueous phase was removed and the organic phase was re-extracted with an additional 5 ml
of H2O. The pooled aqueous extracts were evaporated to dryness at
40C under a stream of compressed air and then dissolved in 1 ml of
H2O. Individual amino acids were measured with an amino acid-analyzing system (model 835, Hitachi).
Results
Responses of wild watermelon to drought
Wild and domesticated watermelon and other plants were
grown in 500-ml soil in paper pots with enough water until the
fourth leaf was fully expanded. The growth chamber was adjusted to give the light intensity of 1,000 mol photons m–2 s–1,
the relative humidity of 50%, and the temperature of 35C in
the daytime. Drought treatment was started by stopping watering under the same conditions for others as those during
growth. Figure 1 shows wild watermelon subjected to the
drought stress for 8 d, domesticated watermelon treated for 3 d
and cucumber kept under the stress for 2 d. Cucumber was
most sensitive to the drought stress used in this study and domesticated watermelon was the second in the sensitivity. Although wild watermelon lost the lower leaves during the stress
for 8 d, other leaves looked healthy.
The water content in the soil was decreased from 48 to
55% 4 h after watering to 8 to 10% and 5 to 7% 52 and 76 h after watering, respectively, irrespective of the plant species in
the pot (Fig. 2). The rate of photosynthetic CO2 fixation with
Responses of Wild Watermelon to Drought
wild watermelon leaves was 24 mol CO2 m–2 s–1 4 h after watering (before stress) and decreased to 20 mol CO2 m–2 s–1 in
the next 24 h. The decrease of the photosynthetic rate was accompanied by the decrease in transpiration and the decrease in
stomatal conductance (data not shown). After 52 h from watering when the soil water content decreased less than 10%, wild
watermelon closed the stomata and no photosynthesis could be
observed. Rewatering of the wild plant subjected to the drought
stress for 72 h caused a gradual recovery of photosynthesis to
about 80% of the original rate in the next few days (Fig. 2).
The profile of photosynthetic responses to drought of the second leaf was almost the same as that of the third leaf shown in
Fig. 2. The domesticated watermelon and cucumber leaves
showed no photosynthetic activity even after 24 h under experimental conditions used here. Rewatering of the domesticated
watermelon at the third day caused a very slow recovery of
photosynthesis to 50% of the original in the subsequent several
days.
Changes in the leaf water content and the water potential
under drought stress are shown in Table 1. The water potential
of wild watermelon leaves were 0.7 to 0.8 MPa before stress
and decreased to 1.1 to 1.3 MPa in the next 3 d without any
decrease in the water content. This suggests that some solutes
may be accumulated to decrease the water potential, but the accumulation is not significant since turgor pressure decreases
with a decrease in the leaf water content (Boyer 1995). Eight
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Fig. 2 Changes in the rate of the net photosynthetic CO2 assimilation of the third leaf of wild watermelon (circles) and the soil water
content (squares) in the course of drought. Some plants subjected to
drought for 72 h were rewaterted thereafter (closed circles). The points
are the averages of the data from three or more independent experiments and the vertical bars are standard deviations.
days after the start of the stress, the fourth leaf still kept the water content at 81% with older leaves heavily wilted. Under
these experimental conditions, domesticated watermelon ini-
Table 1 Changes in the water potential and the water content of individual leaves of various
species of plants along with the progress of the drought stress
Plant species
Wild watermelon
Domesticated watermelon
Cucumber
Maize
Leaf number
1
2
3
4
1
2
3
4
1
2
3
4
2
3
4
Water potential and water content in parentheses
stressed for
0d
3d
8d
(MPa) (water content in %)
0.81 (843)
0.69 (863)
0.71 (864)
0.75 (852)
1.12 (793)
0.97 (804)
0.94 (824)
0.89 (793)
1.20 (833)
0.89 (862)
0.82 (832)
0.92 (853)
0.81 (832)
0.82 (862)
0.85 (891)
1.29 (805)
1.08 (852)
1.15 (833)
1.30 (833)
wilted
2.30 (745)
1.85 (773)
1.70 (774)
wilted
7.26 (352)
4.61 (362)
4.22 (523)
5.62 (775)
4.09 (762)
3.61 (762)
wilted
wilted
wilted
2.00 (813)
wilted
wilted
wilted
wilted
wilted
wilted
wilted
wilted
wilted
wilted
wilted
The water potential values are the averages from at least two independent experiments. The water content is
the mean of the values of more than two experiments (SD). Wilted leaves could not be used for measurements. The first leaf of maize got senile.
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Responses of Wild Watermelon to Drought
Fig. 3 Two-dimensional electrophoresis of polypeptides extracted from the leaves of wild watermelon well-watered or subjected to drought for
1 and 3 d. Total proteins were extracted from the second leaves and 10 g of them were subjected to electrophoresis. The first isoelectrofocusing
was done between pH 4 and 6 and the second SDS-PAGE was done with 12.5% acrylamide gel.
tially showed slight resistance to drought stress, but the whole
plant gradually lost water and began to wilt 3 d after withholding of watering (Table 1) Cucumber leaves were strongly wilting with a decrease in the water content even 1 d after stopping
of watering. Maize also showed a significant decrease of the
water potential (Table 1), and suffered from irreversible damages 3 d after the final watering.
Analysis of the polypeptide composition
The above results showed that wild watermelon could resist to the drought stress by retaining its water status of the
leaves. One may expect that the changes in metabolism and the
protein composition might contribute to the resistance to the
drought stress in wild watermelon leaves. Then, we analyzed
the change in the polypeptide composition in the leaves in the
course of the drought stress by 2-D electrophoresis.
The present 2-D electrophoresis detected approximately
one thousand spots with the third leaves of well-watered watermelon. After 28 h from watering, when the wild plant still kept
its high rate of photosynthesis (Fig. 2), seven polypeptides
newly appeared and were named as DRIPs-1 to 6 (Fig. 3). The
isoelectric points and molecular masses of these polypeptides
Responses of Wild Watermelon to Drought
Table 2
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N-terminal amino acid sequences of drought-induced polypeptides in wild watermelon leaves
Polypeptide N-Terminal sequence
DRIP-1a
DRIP-1b
DRIP-2
DRIP-3
DRIP-4
DRIP-5
DRIP-6
SVPSIKEIIGGLEKE?YIPL
SVPSIKEIIGGLEKE?YIPL
N-terminal blocked
GMDAFIFDVDETLLSNLPY
GMDAFIFDVD?TLL?NL?Y
contaminated
?VVGIDLGTTNSAV
Molecular mass
Isoelectric point
45 kDa
45 kDa
22 kDa
22 kDa
19 kDa
22 kDa
75 kDa
4.7
4.8
6.6
5.9
5.9
5.5
4.6
are listed in Table 2. Their accumulations reached the maxima
after 3 d (Fig. 3). Especially, DRIP-1a and DRIP-1b were accumulated abundantly, and their amounts were second only to ribulose bisphosphate carboxylase large subunits from the first
through third day. All of these seven polypeptides were induced extending over the first and fourth leaves, and the accumulation was slightly higher in the older leaves at the same
stage of the drought stress. These polypeptides were not detected in the well-watered 3 week-old plants indicating that the induction of the polypeptides is not due to natural leaf senescence.
Determination of the N-terminal amino acid sequences of
DRIPs
Polypeptides in the 2-D gel were transferred onto polyvinylidene difluoride membranes and N-terminal amino acid sequences of the seven DRIPs were determined. The determined
sequences are shown in Table 2. The N-terminal amino acid sequences of DRIP-1a and DRIP-1b and DRIP-3 and DRIP-4
were identical with each other. The N-terminal end of DRIP-2
was blocked. The spot of DRIP-5 located closely to another
spot and the sequence of the polypeptide could not be determined. DRIP-1 did not show any significant homology to the
reported sequences in the protein databases of PIR and SwissProt. The N-terminal amino acid sequences of DRIP-3 and
DRIP-4 showed high homology to a conserved motif of phosphohydrolase superfamily reported for various organisms
(Thaller et al. 1998), including acid phosphatase I (Tanaka et
al. 1990) and vegetative storage proteins from some plants (DeWald et al. 1992). DRIP-6 showed homology with the N-terminal region of the heat shock protein-70 protein from some organisms (Wimmer et al. 1997, Strzalka et al. 1994).
Cloning of DRIP-1 cDNA
To know the molecular entity of the most highly droughtinduced DRIP-1 polypeptide, cDNA clones for DRIP-1 were
isolated from the cDNA library using oligonucleotide designed
from its N-terminal sequence, and these nucleotide sequences
were determined (Fig. 4). The cloned cDNA for DRIP-1 was
1,578-bp long and encoded a polypeptide composed of 438
amino acid residues. The two amino acid residues of the N-ter-
Homologous protein
unknown
unknown
acid phosphatase, vegetative storage protein
acid phosphatase, vegetative storage protein
heat shock protein 70
minal end (methionine and serine) deduced from the cloned
DRIP-1 cDNA were not present in the N-terminus sequence of
DRIP-1 polypeptide indicating that these two residues should
be removed by post-translational processing.
The primary structure of the polypeptide deduced from the
DRIP-1 cDNA exhibited homology to those of the proteins belonging to the ArgE/DapE/Acy1/CPG2/YscS family (Boyen et
al. 1992, Vongerichen et al. 1994) (Fig. 5). DRIP-1 had two
consensus motifs for this family (two-arrowhead lines a and b);
the former contained a histidine residue for metal ion-binding.
The enzymes belonging to this family are acetylornithine
deacetylase (ArgE) (Meinnel et al. 1992), diaminopimeric acid
deacetylase (DapE) (Bouvier et al. 1992), amino acylase-1
(Acy1) (Mitta et al. 1993), carboxy peptidase G2 (Cpg2) (Minton et al. 1986), and carboxy peptidase S (YscS) (Spormann et
al. 1991). The common characteristics of these enzymes are to
hydrolyse the peptidic or N-acetyl bond and produce a free
amino acid as the product. DRIP-1 was 49.7%, 20.4%, 19.6%
and 14.5% homologous to an ArgE-like protein from Dictyostelium discoideum (GenBank accession number, P54638),
ArgE from E. coli (P23908), DapE from E. coli (P24176), and
Acy1 from human (Q03154), respectively. The similarity was
79.7%, 54.6%, 56.0%, and 47.8%, respectively. None of these
proteins belonging to this family have been reported in plants.
Analysis of free amino acids in leaves of wild watermelon
Wild watermelon leaves accumulated high levels of
DRIP-1, which was thought to relate to the formation of free
amino acids during the stress, as shown above. Then, we analyzed the changes in the amino acid composition in wild watermelon leaves under the drought conditions. The total amino
acid content was gradually increased during the drought stress.
Glutamate and glutamine were the major amino acids and citrulline was a minor component before the stress (Table 3).
Three days after withholding of watering, citrulline became
one of the major amino acids in the free amino acids. The relative content of citrulline reached about 50% in the total free
amino acids in the fourth leaf 8 d after stopping of watering
(Table 3).
Among the other amino acids involved in the urea cycle,
the increase in the arginine content was observed while the
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Responses of Wild Watermelon to Drought
Fig. 4 Nucleotide and deduced amino acid sequences of the cloned DRIP-1 cDNA (DDBJ accession number, AB036420). The determined Nterminal sequence of DRIP-1 is shown with bold characters. The stop codon is marked by the asterisk.
content of ornithine was not affected. The contents of stress-related amino acids such as proline, -alanine, glutamine and asparagine (Stewart and Larher 1980) did not significantly
change during drought stress, although the glutamate content
increase 2- to 3-fold (Table 3).
Discussion
The wild watermelon plant is a C3-plant (Miyake and
Yokota 2000) but showed strong resistance to the severe conditions of multi-stresses combining water deficit, high-light intensity and high temperature (Fig. 1). These strongly suggest that
Responses of Wild Watermelon to Drought
871
Table 3 Effect of the drought stress on the amino acid composition in the third leaves of wild
watermelon
Amino acid
Well-watered
Content mol (g FW)–1
3d
RWa
8 db
Citrulline
Arginine
Ornithine
Glutamate
Glutamine
Proline
Asparagine
-Alanine
Alanine
Glycine
Serine
Aspartate
Lysine
0.530.04
0.23
NDc – 0.05
2.050.36
2.061.11
ND – 0.16
ND – 0.18
ND
1.960.44
0.500.06
1.000.08
1.530.23
0.04
2.150.78
0.880.32
ND – 0.11
4.741.37
1.860.97
0.510.04
ND – 0.35
ND
0.510.18
0.230.03
0.730.29
0.740.32
0.180.08
0.830.16
0.190.09
ND
2.200.02
1.470.90
ND – 0.19
ND – 0.15
ND
1.180.85
0.260.05
0.450.05
1.620.98
0.040.01
23.629.87
5.162.75
0.080.03
6.010.63
2.370.43
0.170.04
0.110.04
ND
0.260.05
0.130.02
0.240.05
1.670.16
0.160.12
Total
14.922.96
17.204.60
11.663.36
47.7315.50
a
4 d after watering wild watermelon plants that had been stressed for 3 d.
Fourth leaf (the first to the third leaves were severely wilted).
c
Non-detectable amount.
b
wild watermelon has mechanisms to protect the photosynthetic
apparatus and cell components from irreversible damage with
its stomata completely closed. We are interested in how the C3plant copes with the severe circumstances used in this study.
Preservation of water in the plant is essential for surviving water-deficit. This long-term preservation of water in
younger leaves of wild watermelon (Table 1) may have been
accomplished by prevention of evaporation of water through
the leaf surface and salvaging water in the older leaves to
youngers. In fact, the leaf surfaces became glossy gradually
along with the progress of the drought stress. This was not observed with the domesticated watermelon plant. Accumulation
of cuticular lipids may be responsible for the preservation of
water in the wild plant. This possibility is being examined in
this laboratory.
In contrast, cucumber was most drought-sensitive among
the plants examined in this study (Fig. 1 and Table 1). The loss
of leaf water was the cause of the observed decrease in water
potential. While maize leaves preserved water for the initial 3 d,
the leaf water potential decreased. This decrease must be mainly due to an accumulation of some solutes as reported (Foyer et
al. 1998). However, these plants, including domesticated watermelon, could not survive as long as the wild watermelon.
Since the water potential equals to the sum of the osmotic
and turgor pressures, the decrease of the leaf water content
causes a decrease of the turgor pressure and consequently the
water potential (Boyer 1995). In wild watermelon leaves, the
water content decreased very slightly during drought stress
(Table 1). The decrease may cause a small decrease in the tur-
gor pressure. The decrease in the water potential of the fourth
leaf was 0.55 MPa for the initial 3 d and 1.25 MPa for the
total 8 d of the drought stress (Table 1). An accumulation of
solutes of up to 0.1–0.2 and 0.4 M, respectively, may be expected, although the contribution of the minor decrease in the
turgor pressure to the water potential was not taken into account in this experiment. This was observed in the wild watermelon leaves, as discussed below.
With enough water in the leaves, wild watermelon showed
many responses for coping with the drought stress used in this
study. The analysis of leaf proteins by 2-D electrophoresis revealed seven newly synthesized peptides during the drought
stress. DRIP-1 accounted for 3.3% of the total polypeptides
stained 3 d after the onset of drought and was the second most
abundant peptide, after the large subunits of ribulose bisphosphate carboxylase. DRIP-1 was also accumulated in the leaves
of domesticated watermelon (unpublished). However, the
amount of the DRIP-1 polypeptide in the 2-D electrophoresis of
domesticated watermelon stressed for 3 d was less than one-fifth
of that with wild watermelon. The observed difference between
wild and domesticated watermelons may be due to a difference
in preservation of the water status in these plants (Fig. 1).
In addition to the induction of DRIP-1 during the drought
stress in wild watermelon, dramatic changes in the amino acid
content and in its composition were observed (Table 3). The
observed decrease of the water potential from 0.75 to 2.0
MPa corresponds to the increase in the solute concentration of
up to 0.4 M, as discussed above. The massive accumulation of
citrulline, arginine and glutamate during the drought stress can
872
Responses of Wild Watermelon to Drought
Fig. 5 Alignments of the deduced amino acid sequence of the DRIP-1 polypeptide and those of ArgE of D. discoideum (Dic-ArgE, GenBank
accession number P54638) and E. coli (Eco-ArgE, P23908) included in the ArgE/DapE/Acy1/CPG2/YscS family. Identical residues conserved
among at least two ArgE homologues are displayed in white characters in black boxes, and similar residues are in gray boxes. Gaps introduced to
improve the alignment are indicated by bars. The conserved histidine residue is marked by the asterisk. The partial sequences covered by twoarrowhead lines a and b are Arg-E consensus sequences (Vongerichen et al. 1994).
be a cause of the decrease in the water potential. In the preliminary experiments, we found that the content of free sugars also
increased from 130 to 250 mol (g FW)–1 during the drought
stress for 5 d (Nakamura, Miyake and Yokota, unpublished).
Accumulation both of amino acids and of free sugars contributed to the decrease in the water potential. Citrulline most effectively contributed to the decrease in the water potential of the
fourth leaf during the long-term stress among the free amino
acids, while older leaves were suffering from severe wilting. It
may be suggested that nitrogen in the older leaves was translocated to the younger leaves and accumulated there as citrulline
and some other amino acids as nitrogen storage compounds. If
accumulated citrulline is localized in the cytoplasm whose volume is 10% of the total volume of a leaf mesophyll cell, its
concentration would reach about 0.3 M. A possibility that the
amino acid also functions as a compatible solute in wild watermelon leaves is being examined in this laboratory.
Citrulline is an intermediate of the ornithine/urea cycle
(Reinbothe and Mothes 1962). The reported citrulline content in
plant tissues is relatively low except watermelon fruits (Tedesco
et al. 1984) and some plants where the amino acid is a nitrogen
storage metabolite or a nitrogen carrier for the long-distance
translocation (Bollard 1957, Reinbothe and Mothes 1962). In
general, the ornithine/urea cycle functions to transfer nitrogen of
glutamine and asparate to urea (Reinbothe and Mothes 1962).
For this end, only low levels of citrulline and arginine are
enough to operate the cycle since all of the carbon atoms of ornithine return to it with a net gain of urea. To keep the levels of the
intermediates in the cycle constant, arginine has an ability to inhibit 2-N-acetylglutamate kinase (Wolf and Weiss 1980). Arginine is used for protein synthesis, and this amino acid and ornithine are precursors of polyamines and alkaloids. The depleted
carbon skeletons in the cycle are replenished from glutamate
through 2-N-acetylglutamate and then 2-N-acetylornithine,
which gives rise to ornithine by transferring the acetyl group to
glutamate. Accumulated 2-N-acetylglutamate would relieve the
inhibition of 2-N-acetylglutamate kinase by arginine. Wild watermelon leaves accumulated an extremely large amount of citrulline and a lesser amount of arginine under the drought stress.
The accumulation of these intermediates of the cycle could be
accomplished only by the operation of a pathway or an enzyme(s) that is out of such feedback control by arginine (Cybis
and Davis 1975, Tompson 1980, Shargool et al. 1988). DRIP-1
may be a polypeptide component of one of the enzymes involved in this massive accumulation of citrulline and arginine.
Since DRIP-1 is a homologue of AgrE or 2-N-acetylornithine
Responses of Wild Watermelon to Drought
deacetylase (Fig. 4, 5), this enzyme may function for massive
production of ornithine. In this case, 2-N-acetylglutamate needs
to be synthesized by 2-N-acetylglutamate synthase and the possible inhibition of 2-N-acetylglutamate kinase by arginine must
be decreased.
Acknowledgements
This study was supported by the “Research for the Future” program (JSPS-RFTF96R16001) of the Japan Society for the Promotion
of Science.
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(Received January 28, 2000; Accepted May 1, 2000)