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Continuing Australian Journal of Plant Physiology

Volume 29, 2002

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Funct. Plant Biol., 2002, 29, 643648

Response of oxidative metabolism in watermelon plants

subjected to cold stress
Rosa M. RiveroA, Juan M. Ruiz, Pablo C. Garca, Luis R. Lpez-Lefebre, Esteban Snchez and
Luis Romero
Department of Plant Biology, Faculty of Sciences, University of Granada, 18071 Granada, Spain.
ACorresponding author; email: lromero@ugr.es
Abstract. The objective of the present work was to determine the effect of thermal stress on oxidative metabolism
in Citrullus lanatus [Thomb.] Mansf. cv. Dulce maravilla. Plants were grown for 30 d at two temperatures (10 and
35C), at which time we measured the leaf concentration of antioxidant compounds (ascorbate, dehydroascorbate,
reduced glutathione, oxidized glutathione) and enzymatic activities [superoxide dismutase (SOD), catalase,
guaiacol peroxidase, ascorbate peroxidase, dehydroascorbate reductase and glutathione reductase], as well as total
hydrogen peroxide (H2O2) concentration and shoot dry weight. Our results indicate that chilling stress occurred in
watermelon plants at 10C, while 35C is the optimal temperature for this plant. Low temperature stress caused:
(i) decreased shoot weight; (ii) accumulation of H2O2; (iii) increased SOD activity; and (iv) decreased enzyme
activities associated with detoxifying H2O2. The novelty of this study centres on the fact that so few cold-sensitive
species have been examined to date additional cold-sensitive species need to be studied to determine if there are
shared characteristics in terms of how they respond to cold stress. Most studies have examined single antioxidant
responses, whereas we conducted a comprehensive examination of many antioxidant responses.
Keywords: antioxidant compounds, antioxidant enzymes, Citrullus lanatus, cold stress, oxidative metabolism,
watermelon.
Introduction
Under natural conditions of growth and development, plants
are invariably exposed to different stresses such as drought,
heat, chilling, pollutants, and UV radiation (McKersie and
Leshem 1995; Paliyath and Fletcher 1995; Pinhero et al.
1997). Many of the injuries caused to plants by stress are
associated with oxidative damage at the cellular level, which
results in active oxygen species (AOS) such as H2O2, the
superoxide radical (O2) and the hydroxyl radical (OH)
(Allen 1995).
AOS are highly reactive and can damage membrane
lipids, proteins, chlorophyll, and nucleic acids, thus disrupting the homeostasis of the organism (Shaaltiel and Gressel
1986; Scandalios 1993). Plants have evolved several mechanisms to prevent or alleviate the damage from AOS,
including antioxidant enzymes such as SOD (EC 1.15.1.1),
which is located in the chloroplast, mitochondrion and
cytosol, and converts O2 to H2O2 (Salin 1988; Bowler et al.
1992; Scebba et al. 1998). The H2O2 generated in glyoxysomes and peroxisomes is detoxified to H2O, mainly by

catalase (CAT; EC 1.11.1.6), while in other subcellular

compartments it is converted to H2O by ascorbate peroxidase (APX; EC 1.11.1.11) (Ushimaru et al. 1992, 2000).
However, since ascorbate (AsA) is oxidized to dehydroascorbate (DHA) in this reaction, a system for the regeneration of AsA is necessary. This regeneration system is the
so-called glutathione/ascorbate cycle, in which the enzyme
dehydroascorbate reductase (DHAR; EC 1.8.5.1) catalyses
the re-reduction of DHA to AsA using glutathione (GSH),
with the resultant production of the oxidized form of
glutathione (GSSG) (Hodges et al. 1997a). Finally, NADPH
reduces GSSG again, in a reaction catalysed by glutathione
reductase (GR; EC 1.6.4.2) (reviewed by Asada and
Takahashi 1987; Salin 1988; Ushimaru et al. 2000). In
addition, guaiacol peroxidase (GPX; EC 1.11.1.7), which is
involved in various biosynthetic pathways using H2O2
including lignin synthesis, also plays an important role in
antioxidative protection (Peters et al. 1989; Takahama and
Oniki 1992).
One type of stress that gives rise to AOS production, and
therefore to the activation of antioxidant systems in plants, is

Abbreviations used: AOS, active oxygen species; APX, ascorbate peroxidase; AsA, ascorbate; CAT, catalase; DHA, dehydroascorbate; DHAR,
dehydroascorbate reductase; dw, dry weight; FeEDDHA, ethylenediamine-di(o-hydroxyphenylacetic acid); fw, fresh weight; GPX, guaiacol
peroxidase; GR, glutathione reductase; GSH, reduced glutathione; GSSG, oxidized glutathione; H2O2, hydrogen peroxide; NBT, nitroblue
tetrazolium; O2, superoxide radical; OH, hydroxyl radical; PPFD, photosynthetic photon flux density; SOD, superoxide dismutase.
CSIRO 2002

10.1071/PP01013

1445-4408/02/050643

644

the stress provoked by low temperatures (Aidun et al. 1991;

Elstner and Oswald 1994; Hodges et al. 1997a, b, c; Queiroz
et al. 1998). Previous comparisons of different species have
demonstrated that chilling-sensitive species have a lower
antioxidant capacity than tolerant species (Jahnke et al.
1991; Walker and McKersie 1993). Hodges et al. (1996,
1997a), comparing antioxidant enzyme activities and antioxidant compound concentrations in differentially chillingsensitive inbred maize lines, demonstrated that these lines
had less capacity to combat the toxic oxygen compounds
that the tolerant lines.
The watermelon is a plant that requires optimal temperatures of 3337C for growth and development (Brinen 1979;
Maroto 1995) and, hence, exposure to lower temperatures
can significantly alter its metabolism. In light of the above
facts, the aim of the present work was to examine the
enzymatic activities, as well as the concentration of antioxidant compounds, involved in the development of oxidative metabolism in watermelon plants grown at 10 and 35C,
in order to determine the capacity of these plants to
acclimatize and develop under low temperatures.
The novelty of this study centres on the fact that so few
cold-sensitive species have been examined to date
additional cold-sensitive species need to be studied to
determine if there are shared characteristics in terms of how
they respond to cold stress. Most studies have examined
single antioxidant responses, whereas we conducted a
comprehensive examination of many antioxidant responses.
Materials and methods
Plant growth
Seeds of watermelon (Citrullus lanatus [Thomb.] Mansf. cv. Dulce
maravilla) were germinated and grown for 30 d in a growth chamber at
optimal growth temperatures (3337C; Brinen 1979; Maroto 1995).
Twelve plants were transferred to a cultivation chamber set at 10C
(day/night). The thermal stress experiment was conducted for 30 d
(3060 d after sowing). After 30 d, plants displayed visual symptoms of
damge caused by low temperatures. The growth chamber was
maintained at a relative humidity of 6080%, with a 16 h photoperiod
at a photosynthetic photon flux density (PPFD) of 350 mol m2 s1
(measured at the top of the plants).
During all experiments, seedlings were grown in individual pots
(25-cm upper diameter, 17-cm lower diameter, 25-cm in height) filled
with vermiculite, and received a nutrient solution (pH 6.06.1)
containing the following: 2 mM KNO3, 4 mM Ca(NO3)2,
1.5 mM NaH2PO4, 2 mM CaCl2, 3 mM K2SO4, 1.25 mM MgSO4,
5 M Fe-EDTA, 2 M MnSO4, 1 M ZnSO4, 0.25 M CuSO4,
0.05 M (NH4)6Mo7O24 and 2.5 M H3BO3 (van Zinderen 1986).
Nutrient solution was contained in a 50-L tank, and distributed to
plants continuously via a localized drip irrigation system.

R. M. Rivero et al.

glutathione (triplicate assays for each extraction). The other half were
used to determine shoot dry weight. Leaves of these plants were dried
in a force-air oven at 70C for 24 h. Dry weight was recorded and
expressed as g dw shoot1.
Metabolite assays
The methods used for extraction of total H2O2 were those of MacNevin
and Uron (1953) and Brennan and Frenkel (1977). Hydroperoxides
form a specific complex with titanium (Ti4+), which can be measured
by colourimetry at 415 nm. The concentration of peroxide in the
extracts was determined by comparing the absorbance against a
standard curve representing a titaniumH2O2 complex from 0.1 to
1 mM. The hydroperoxides represent the total peroxides.
AsA, DHA and total ascorbate (AsA + DHA) were determined
following Gossett et al. (1994). From the same extract, AsA and total
ascorbate were assayed. Ascorbate standards of between 0.001 and
0.5 mol mL1 ascorbate in m-phosphoric acid were analysed in the
same manner as extracts. For each sample, DHA was estimated from
the difference between total ascorbate and AsA.
GSSG, GSH and total glutathione (GSSG + GSH) were determined
following Gossett et al. (1994). From the same extract, GSSG and total
glutathione were assayed. A standard curve was developed by
preparing solutions of 0.0020.0001 g mL 1 GSH in
60 mL m-phosphoric acid (pH 2.8) containing 1 m M EDTA, diluting
1:2000 with 50 mL L1 Na2PO4, and analysing in the same manner as
the extracts. Levels of GSH were estimated as the difference between
total glutathione and GSSG.
Enzyme assays
SOD activity was assayed by monitoring the inhibition of the
photochemical reduction of nitroblue tetrazolium, according to the
methods of Giannopolitis and Ries (1977) and Beyer and Fridovitch
(1987), with some modifications (Yu et al. 1998). A 5-mL reaction
mixture was used, containing 50 mM HEPES (pH 7.6), 0.1 mM EDTA,
50 mM Na2CO3 (pH 10.0), 13 mM methionine, 0.025% (v/v) Triton
X-100, 63 M NBT, 1.3 M riboflavin and an appropriate aliquot of
enzyme extract. The reaction mixtures were illuminated for 15 min at a
PPFD of 380 mol m2 s1. Identical reaction mixtures that not were
illuminated were used to correct for background absorbance. One unit
of SOD activity was defined as the amount of enzyme required to cause
50% inhibition of the reduction of NBT as monitored at 560 nm.
CAT activity was determined as described by Badiani et al. (1990),
by following the consumption of H2O2 (extinction coefficient,
39.4 mM1 cm1) at 240 nm for 3 min. GPX activity was determined as
described by Kalir et al. (1984) and Ruiz et al. (1998), by the oxidation
of guaiacol in the presence of H2O2 (extinction coefficient,
26.6 mM1 cm1) at 470 nm. APX activity was determined according to
Gossett et al. (1994), by following the decrease in the absorbance at
290 nm (A290) of an assay mixture containing 0.5 mM AsA (extinction
coefficient, 2.8 mM1 cm1). DHAR activity was determined following
Ushimaru et al. (2000), and GR activity was assayed as described in
Ushimaru et al. (1992).
In our enzyme assays, activity rates were determined at substrate
saturation. All activities were expressed as a function of the oxidized or
reduced substrate per milligram of protein per minute. The protein
concentration was determined by the method of Bradford (1976) using
bovine serum albumin as the standard.

Plant sampling
Plants were sampled 60 d after sowing, all sampled leaves being in the
mature state. The material was rinsed three times in H2O after
disinfecting with 1% non-ionic detergent (Decon 90, Bryn Mawr, PA,
USA) (Wolf 1982), and then blotted on filter paper. Of each treatment,
half the plants were used for analysis of SOD, CAT, GPX, APX,
DHAR, GR, H2O2, AsA, DHA, total ascorbate, GSH, GSSG and total

Statistical analysis
An analysis of the variance by means of a t-test was made, with the
purpose of seeing and justifying the statistically-significant differences
existing between experiments (10 and 35C). Results shown are mean
values s.e. Levels of significance are represented by the following: *,
P<0.05; **, P<0.01; ***, P<0.001; and ns, not significant by t-test at

Oxidative metabolism and cold stress

645

Units SOD mg1 protein min1

14
12

a. SOD
10C
35C

10
8
6
4
2

mol DAsA reduced mg1 protein min1

mol guaiacol oxidized mg1 protein min1

25
20

c. GPX
10C
35C

15
10
5
0

e. DHAR

0.025
0.002

10C
35C

0.015
0.010
0.005
0.000

Fig. 1.

mol AsA oxidized mg1 protein min1

The watermelon, which requires optimal temperatures of

3337C for growth and development (Brinen 1979; Maroto
1995), undergoes metabolic alterations in suboptimal temperatures. Under the experimental conditions used here, the
highest values for shoot dry weight were registered at 35C
(optimal temperature for this species) and the lowest at 10C
(Table 2), representing a 63.36% reduction. Under stress
conditions caused by low temperatures, in addition to
metabolic alteration, oxidative damage can occur, triggering
overproduction of AOS (Okuda et al. 1991). Proper functioning of the scavenging system is essential to maintain the
concentration of any AOS formed at relatively low levels
(Scebba et al. 1998). SOD catalyses the dismutation of O2

mol H2O2 reduced mg1 protein min1

Results and discussion

to H2O2 and O2 (McCord and Fridovitch 1969; Ushimaru

et al. 2000). Increased SOD activity in spinach plants was
found during exposure to low temperatures (Schner and
Krause 1990), and high SOD activity has been associated
with chilling in plants where overproduction of O2 is
involved (Bowler et al. 1992; Rao et al. 1996). In our
research, SOD activity was significantly higher (P<0.001) at
10C than at 35C (a 1.7-fold rise; Fig. 1a). In our
experiment, at 10C H2O2 concentration rose significantly
(P<0.001), to almost 4-fold higher than that found at 35C
(Table 1). This appears to confirm that greater H2O2
accumulation may be due largely to the increase in SOD
activity at 10C, given the positive relation found between
these two parameters (SOD activityH2O2, r=0.912***).
The above results could explain the dry weight reduction
in watermelon plants subjected to 10C, since one of the
initial symptoms of H2O2 accumulation is reduced foliar

mol NADPH oxidized mg1 protein min1

P=0.05. We also performed a correlation analysis for each of the

variables opposed to H2O2.

b. APX

0.25
0.20

10C
35C

0.15
0.10
0.05
0.00

d. CAT

8
7

10C
35C

6
5
4
3
2
1
0

f. GR

0.45
0.40
0.35

10C
35C

0.30
0.25
0.20
0.15
0.10
0.05
0.00

Effect of temperature on activities of antioxidant enzymes.

646

Table

R. M. Rivero et al.

1.

Effect of temperature on ascorbate and H2O2

concentrations
AsA, DHA and total ascorbate expressed as mol ascorbate g1 fw.
H2O2 expressed as mmol of H2O2 g1 fw. Data are means s.e. (n = 6)

AsA
10C
35C
Significance

16.64 0.66
4.92 0.41
***

DHA

Total
ascorbate

H 2 O2

6.17 0.08 22.81 0.71 97.54 1.23

3.48 0.001 8.41 0.21 26.32 1.01
***
***
***

biomass (Table 2; Willenkens et al. 1997). The lowest

concentration of H2O2 resulted at 35C, coinciding with the
highest foliar biomass (biomassH2O2, r=0.823***),
results that appear to corroborate an inverse relationship
between the amount of biomass of a plant and its foliar H2O2
concentration.
Many studies have shown that CAT and GPX activities
increase during exposure to low temperatures (Pinhero et al.
1997). However, in our experiment, CAT and GPX diminished at 10C with respect to 35C, by 63 and 51%,
respectively (Figs 1c, d). The lower CAT and GPX activities
in our watermelon plants subjected to 10C could also
account for the high accumulation of foliar H2O2 (CAT
activityH2O2,
r=0.935***;
GPX
activityH2O2,
r=0.907***). Finally, the reduction of CAT and GPX
activities at 10C could be due to an inactivation of these
enzymes by the low temperature. These results agree with
those of Hodges et al. (1997b), who reported lower CAT
activity in chilling-sensitive maize hybrids.
The activities of APX (Fig. 1b), DHAR (Fig. 1e) and GR
(Fig. 1f) in all cases had a behaviour similar to that found for
CAT (Fig. 1d) and GPX (Fig. 1c). At 10C, we found a
reduction of these activities of 85, 19 and 87%, respectively
compared with 35C. As with CAT and GPX, the relationship between the enzymatic activities of the glutathione/
ascorbate cycle and H2O2 concentration (APX
activityH2O2, r=0.754***; DHAR activityH2O2,
r=0.567*; GR activityH2O2, r=0.789***) could also
explain the accumulation of this compound in watermelon
plants subjected to 10C, as well as the low H2O2 concentration in plants grown at 35C, as at this temperature these
enzymes are active, thus avoiding accumulation of AOS
Table 2.

Effect of temperature on glutathione concentration and

shoot dry weight
GSH, GSSG and total glutathione expressed as mol glutathione g1 fw.
Shoot dry weight expressed as g plant1. Data are means s.e. (n = 6)

GSSG
10C
35C
Significance

GSH

Total
glutathione

Shoot dry
weight

3.87 0.02 21.69 1.99 25.56 1.96 4.53 0.10

1.08 0.008 2.24 0.64 3.32 0.92 18.215 0.08
***
***
***
***

(mainly H2O2). These results are contrary to those reported

by Hodges et al. (1997b) and Pinhero et al. (1997), since
these authors found increased activities only in plants
treated with low temperatures which were able to develop
cold tolerance.
In our experiment, the statistically significant decrease
(P<0.001) in enzymatic activities at 10C appears to imply
an enzymatic inactivation by the low temperatures and a
lack of cold tolerance in watermelon plants. In plants grown
at 35C, we found a significant increase in the activities of
all enzymes involved in eliminating H2O2, confirming that
the antioxidant defence system of the plant functions
correctly under optimal growth conditions, impeding
massive accumulation of H2O2 and maintaining correct
functioning of its cell metabolism.
The concentration of the antioxidant compounds that
participate in the glutathione/ascorbate cycle (AsA, DHA,
total ascorbate, GSH, GSSG and total glutathione), and the
concentration of H2O2 and shoot dry weight, at 10 and 35C
are shown in Tables 1 and 2. Both substrates behaved
similarly at the different temperatures, with concentrations
proving consistently lower at 35 than at 10C. These results
could be explained as follows. Under cold stress, the plant
boosts synthesis of different forms of glutathione and AsA
in the cell, in order for these to be used as substrates for the
glutathione/ascorbate cycle, and in this way aid in the
detoxification of different AOS produced, thereby prolonging plant survival as long as the stress conditions persist.
However, our results indicate that possibly, with the 10C
treatment, there is an inactivation of the principal enzymes
of this pathway due to the low temperature. This would
explain the higher concentration of glutathione and AsA at
10C, both in total as well as oxidised and reduced forms,
since these substrates were not being used by the APX,
DHAR or GR. Therefore, H2O2 would not be detoxified, in
accord with our experimental results, which showed the
highest H2O2 concentrations at 10C. At 35C, the glutathione and AsA concentrations were lower, confirming the
hypothesis that under normal growth conditions the antioxidant defence systems continue to function (Salin 1988),
although at a slower pace than that required during cold
stress. This fact supports the idea that low temperatures
inhibit these activities.
However, we found that the concentration of the oxidised
forms of both AsA (Table 1, DHA) and glutathione (Table 2,
GSSG) were less than the concentrations of their reduced
forms (AsA and GSH, respectively). This might be because,
for the detoxification of H2O2 beginning with the
glutathione/ascorbate cycle, the initial substrate is AsA. In
addition, for efficient detoxification, this AsA must be regenerated by DHAR activity, which is possible only if there are
high GSH concentrations in the medium. As indicated above,
the low temperatures could inhibit the antioxidant enzymes of
the glutathione/ascorbate cycle. Thus, it is logical that the AsA

Oxidative metabolism and cold stress

and GSH concentrations should be higher than those of the

oxidised forms (DHA and GSSG), since these substrates are
not being used by these enzymes to detoxify H2O2, and this
compound therefore accumulates in the cells of these plants.
In short, according to our experimental results, we
conclude that the watermelon is a species that requires high
temperatures for optimal development. When the plants are
grown at low temperatures (10C), H2O2 accumulates and
cannot be eliminated even by the glutathione/ascorbate cycle,
as the same low temperatures possibly inactivate its main
enzymes. This H2O2 accumulation would, firstly, reduce
foliar biomass and, afterwards, kill the plant. On the contrary,
watermelon plants grown at optimal temperatures (35C)
develop well, indicating good detoxification of AOS and
therefore a correct functioning of the antioxidant systems of
the plant. This again confirms the results of our experiment.
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Manuscript received 24 January 2001, received in revised form

29 October 2001, accepted 26 November 2001

http://www.publish.csiro.au/journals/fpb