Journal of Integrative Plant Biology 2008
Differential Responses of the Activities of Antioxidant
Enzymes to Thermal Stresses between Two Invasive
Eupatorium Species in China
∗
Ping Lu1,2 , Wei-Guo Sang1 and Ke-Ping Ma1
(1 Key Laboratory of Vegetation and Environment Change, Institute of Botany, the Chinese Academy of Sciences, Beijing 100093, China;
2
Resources and Environmental Sciences College, Northeast Agricultural University, Harbin 150030, China)
Abstract
The effect of thermal stress on the antioxidant system was investigated in two invasive plants, Eupatorium adenophorum
Spreng. and E. odoratum L. The former is sensitive to high temperature, whereas the latter is sensitive to low temperature.
Our aim was to explore the relationship between the response of antioxidant enzymes and temperature in the two invasive
weeds with different distribution patterns in China. Plants were transferred from glasshouse to growth chambers at a
constant 25 ◦ C for 1 week to acclimatize to the environment. For the heat treatments, temperature was increased stepwise
to 30, 35, 38 and finally to 42 ◦ C. For the cold treatments, temperature was decreased stepwise to 20, 15, 10 and finally to 5 ◦ C.
Plants were kept in the growth chambers for 24 h at each temperature step. In E. adenophorum, the coordinated increase of
the activities of antioxidant enzymes was effective in protecting the plant from the accumulation of active oxygen species
(AOS) at low temperature, but the activities of catalase (CAT), guaiacol peroxidase (POD), ascorbate peroxidase (APX),
glutathione reductase (GR), and monodehydroascorbate reductase (MDAR) were not accompanied by the increase of superoxide dismutase (SOD) during the heat treatments. As a result, the level of lipid peroxidation in E. adenophorum was higher
under heat stress than under cold stress. In E. odoratum, however, the lesser degree of membrane damage, as indicated by
low monodehydroascorbate content, and the coordinated increase of the oxygen. Detoxifying enzymes were observed in
heat-treated plants, but the antioxidant enzymes were unable to operate in cold stress. This indicates that the plants have
a higher capacity for scavenging oxygen radicals in heat stress than in cold stress. The different responses of antioxidant
enzymes may be one of the possible mechanisms of the differences in temperature sensitivities of the two plant species.
Key words: antioxidant enzyme system; cold stress; Eupatorium adenophorum; Eupatorium odoratum; heat stress; invasive plants.
Lu P, Sang WG, Ma KP (2008). Differential responses of the activities of antioxidant enzymes to thermal stresses between two invasive Eupatorium
species in China. J. Integr. Plant Biol. doi: 10.1111/j.1744-7909.2007.00583.x.
Available online at www.jipb.net
Eupatorium adenophorum Spreng. and Eupatorium odoratum
L. are perennial members of the Compositae family. Since their
introduction to China in the early 20th century, both species
have become invasive pests (Ding 2002; Li 2002), but their
geographical distributions in China are different (Figure 1).
Received 15 Jan. 2007 Accepted 20 Mar. 2007
Supported by National Natural Science Foundation of China (30470337),
Knowledge Innovation Project of Chinese Academy of Sciences (KSCX1SW-13-03) and K C Wong Fellowship, Royal Society of UK.
∗
Author for correspondence.
Tel: +86 (0)10 6283 6278;
Fax: +86 (0)10 8259 9519;
E-mail: <[email protected]>.
C 2008 Institute of Botany, the Chinese Academy of Sciences
doi: 10.1111/j.1744-7909.2007.00583.x
E. odoratum, native to Central and South America, is coldsensitive and dies at temperatures below 0 ◦ C (Wu and Xu
1992). Hence, its distribution has been restricted to southern
China, and its northward spread is slower than E. adenophorum.
In contrast, E. adenophorum, native to Central America, could
survive in the temperature range (−11.5–35 ◦ C) (Zhao and
Ma 1989), and expanded to northern China much faster than
E. odoratum. A case study indicated that the rate of spread was
about 60 km per year from south to north in China (Xiang 1991).
However, E. odoratum is better able than E. adenophorum to
tolerate high temperatures. E. odoratum invaded the hot, dry
valleys of Yunnan and Sichuan provinces where temperatures
are high (maximum temperature 40.4 ◦ C, Cao 1993) and which
are relatively arid. E. adenophorum is rarely found in the hot, dry
valleys. The differences of the two alien species in distribution
and invasive potential pose interesting questions concerning
the mechanisms that determine their eventual distribution.
2
Journal of Integrative Plant Biology
2008
Figure 1. Distributions of Eupatorium adenophorum and E. odoratum in China.
E. adenophorum (); E. odoratum ().
However, there has been little research on their physiological
and biochemical mechanisms that confer different heat and/or
cold resistance. A better understanding of these mechanisms
is very important for controlling and predicting the potential
distributions of the two species.
It has been proven that the response of antioxidant enzyme
systems is one of the most important mechanisms of environmental acclimatization for plants. The role of these enzyme
systems for making plants tolerant to extreme environments
has already been demonstrated (Dhindsa and Matowe 1981;
Schner and Krause 1990; Hernández et al. 2000). It is currently
assumed that the negative effect of various environmental
stresses is at least partially due to the generation of active
oxygen species (AOS) and/or the inhibition of the system
that defends against them (Asada 1997; Alscher et al. 1997).
AOS are highly reactive and, in the absence of any protective
mechanism, can seriously disrupt normal metabolism through
oxidating membrane lipids, protein and nucleic acids (Smirnoff
1993; Foyer et al. 1994a, 1994b). However, to mitigate and
repair damage initiated by AOS, plants evolved cellular adaptive
responses such as upregulation of oxidative stress protectors
and accumulation of protective solutes (Horling et al. 2003).
Antioxidant defense enzymes such as superoxide dismutase
(SOD), catalase (CAT), ascorbate peroxidase (APX), guaiacol
peroxidase (POD), glutathione reductase (GR), monodehydroascorbate reductase (MDAR) and dehydroascorbate reductase (DHAR) are systems that minimize the concentrations of
superoxide and hydrogen peroxide.
Therefore, in this paper, we compared the response of
the antioxidant enzyme system to heat and cold stresses in
E. adenophorum and E. odoratum. Specifically, we studied
changes in lipid peroxidation and the antioxidant enzymes SOD,
CAT, POD, APX, GR, MDAR and DHAR under heat and cold
stress.
Results and Discussion
Superoxide dismutase catalyses the dismutation of O 2 − to
H 2 O 2 and O 2 (Bowler et al. 1992). Both species exhibited
an increase in SOD activity in both heat and cold stresses
(Figure 2A, D). In leaves of E. adenophorum, the 38 ◦ C and
42 ◦ C treatments caused a 61% and 138% increase in total
SOD activity in comparison to the control plants, respectively,
and the highest total SOD activity was observed at 42 ◦ C. In
leaves of E. odoratum, however, the highest total SOD activity
was observed at 5 ◦ C. SOD has also been reported to increase
on exposure to heat stress in mulberry (Chaitanya et al. 2002),
and to cold stress in leafy spurge (Davis and Swanson 2001).
Catalase eliminates H 2 O 2 by breaking it down directly to
form water and oxygen. In leaves of E. adenophorum, CAT
activity was depressed by high temperature, and increased
to a high level in the low temperature treatment (Figure 2B).
In leaves of E. odoratum, however, the response pattern of
CAT activity was reversed, CAT activity was enhanced by high
temperature and the plants were unable to maintain normal
activity when subjected to low temperature, such as 10 ◦ C and
5 ◦ C (Figure 2E). An increase in SOD activity with a decrease
in CAT activity has been reported when the plants were subjected to abiotic stress (Saruyama and Tanida 1995; Fu and
Huang 2001; Jung 2003). Others have found that CAT activity
Antioxidant Enzymes Respond to Heat in Eupatorium Species
E. adenophorum
E. odoratum
30
d
210
c
bc
140
c
abc ab
ab
a
70
0
B
80
f
60
e
e
e
d
c
40
b
a
a
20
0
C
480
e
e
d
360
d
240
c
c
c
b
a
120
0
5
10
15
20
25
30
35
38
42
Temperature (°C )
(Units/mg protein)
e
Superoxide dismutase
D
A
280
Peroxidase activity
Catalase activity
(µmol H2O2/min per mg protein) (µmol H2O2/min per mg protein)
Catalase activity
(µmol H2O2/min per mg protein) (µmol H2O2/min per mg protein)
Superoxide dismutase
(Units/mg protein)
350
Peroxidase activity
3
24
f
e
18
d
12
cd
b
c
cd
cd
d
d
a
6
0
E
40
e
30
c
c
c
c
20
10
a
b
0
F
e
1000
750
d
500
b
b
b
b
10
15
20
25
b
30
c
a
250
0
5
35
38
42
Temperature (°C )
Figure 2. Effect of temperature on superoxide dismutase (SOD), catalase (CAT) and guaiacol peroxidase (POD) activities of Eupatorium adenophorum
(A–C) and E. odoratum (D–F).
Values are the means of three replicates with standard errors. Means with the same letters are significantly different at P ≤ 0.05 using Duncan’s
multiple range test.
is photoinactivated under low and high temperature stresses
and CAT activity varies with plant species (Feierabend et al.
1992).
Guaiacol peroxidase catalyses H 2 O 2 -dependent oxidation of
substrate. In leaves of E. adenophorum, POD activity increased
remarkably with decreasing temperatures from 25 ◦ C to 5 ◦ C,
enabling the plants to protect themselves against the oxidative
stress, but in heat stress, POD activity was almost the same until
the temperature was at 38 ◦ C, and then declined (Figure 2C). In
contrast, in leaves of E. odoratum, POD activity was significantly
enhanced by the heat stress, but in cold stress, POD activity
remained unchanged until the temperature was at 5 ◦ C, and then
decreased (Figure 2F). These results were consistent with those
of Rivero et al. (2001), who reported differential responses of
POD activity to thermal stress between tomato and watermelon
plants. In watermelon plants, POD activity increased under high
temperature, but decreased under low temperature. In tomato
plants, POD activity decreased under high and low temperature,
and the lowest POD activity was found under high temperature
stress.
Ascorbate peroxidase uses ascorbate as the electron donor
for the reduction of H 2 O 2 and is well known to be important
in the detoxification of H 2 O 2 . In leaves of E. adenophorum,
the activity of APX increased with decreasing temperature from
4
Journal of Integrative Plant Biology
2008
E. adenophorum
E. odoratum
1
bc
bc
b
bc
bc
2
a
a
1
0
B
200
f
f
ef
de
cd
150
c
b
ab
a
100
50
C
160
d
d
d
d
d
120
c
a
80
b
a
40
0
5
10
15
20
25
30
35
38
42
Temperature (°C )
D
0.8
0.6
0.4
e
e
bc
cd
bc
cd
d
bc
a
0.2
0
E
40
d
c
b
30
a
a
a
a
a
a
20
10
0
(µmol NADH/min per mg protein)
cd
Ascorbate peroxidase activity
(µmol ascorbate/
min per mg protein)
d
3
Glutathione reductase activity
(µmol NADPH/
min per mg protein)
4
Monodehydroascorbate reductase
Glutathione reductase activity
A
0
(µmol NADH/min per mg protein)
Monodehydroascorbate reductase
(µmol NADPH/min per mg protein)
Ascorbate peroxidase activity
(µmol ascorbate/
min per mg protein)
5
F
80
g
f
60
40
b
c
c
15
20
d
e
e
30
35
a
20
0
5
10
25
38
42
Temperature (°C )
Figure 3. Effect of temperature on ascorbate peroxidase (APX), glutathione reductase (GR) and monodehydroascorbate reductase (MDAR) activities
of Eupatorium adenophorum (A–C) and E. odoratum (D–F).
Values are the means of three replicates with standard errors. Means with the same letters are significantly different at P ≤ 0.05 using Duncan’s
multiple range test.
25 ◦ C to 5 ◦ C, but in heat stress, the activity of APX reached a
peak value at 30 ◦ C and 35 ◦ C and then declined (Figure 3A).
In leaves of E. odoratum, however, the activity of APX was
increased by increasing the temperature from 25 ◦ C to 42 ◦ C,
but in cold stress, the activity of APX reached a peak value at
15 ◦ C and then declined (Figure 3D). The drastic decreases of
APX activities at 38 ◦ C or 42 ◦ C in leaves of E. adenophorum,
suggested that APX can not compensate for the loss of CAT and
POD activities at high temperatures, which seems to severely
reduce the H 2 O 2 -scavenging capacity. However, during cold
treatment, crofton weed exhibited an increased activity of H 2 O 2
detoxifying enzyme APX, CAT and POD. Thus, the levels of
membrane lipid peroxidation were relatively lower under cold
stress, which seems to be related to its poor heat tolerance. The
restriction of crofton weed to temperate climates (mainly on the
Yunnan-Guizhou Plateau) (Lu and Ma 2004) suggests that poor
heat-stress resistance may be a factor limiting the distribution of
the plants. While in E. odoratum, the drastic decreases of APX
Antioxidant Enzymes Respond to Heat in Eupatorium Species
activities were found at 10 ◦ C or 5 ◦ C, which seems to be related
to its poor cold tolerance.
Glutathione reductase, another enzyme in the Asada-Halliwell
pathway, can also remove cytosolic hydroperoxides such as
lipid hydroperoxide, using reduced glutathione as a co-substrate
(Ye and Gressel 2000). In leaves of E. adenophorum, the
activity of GR was increased significantly by decreasing the
temperature from 25 ◦ C to 5 ◦ C, but in heat stress, the GR activity
was depressed (Figure 3B). In leaves of E. odoratum, however,
the activity of GR was increased by increasing the temperature
from 25 ◦ C to 42 ◦ C, but in cold stress, the activity of GR was
unchanged (Figure 3E). According to Foyer and Halliwell (1976),
H 2 O 2 is mainly eliminated by the ascorbate-glutathione cycle.
Besides the scavenging effect of CAT and POD, cytotoxicity of
H 2 O 2, which might be produced due to constitutive and induced
levels of SOD in both species, might be disposed of with the aid
of both APX and GR.
The ascorbate-glutathione cycle needs MDAR and DHAR
in addition to APX and GR. MDAR and DHAR are enzymes
responsible for ascorbate regeneration in plant tissues. The
activity of MDAR decreased significantly in heat stress, whereas
MDAR activity in leaves of the cold-treated E. adenophorum was
not significantly different from the controls (Figure 3C). In leaves
of E. odoratum, however, the activity of MDAR was increased
by increasing the temperature from 25 ◦ C to 42 ◦ C, but in cold
stress, the activity of MDAR was decreased (Figure 3F).
Dehydroascorbate reductase activity in leaves of the two
species increased with both heat and cold stresses (Figure 4A,
C). In both species, a greater increase in DHAR activity was
found with heat treatment than in cold treatment, and the highest
DHAR activity was recorded at 42 ◦ C. DHAR is thought to play
an important role in the oxidative stress tolerance of plants
by regenerating ascorbate from dehydroascorbate (Foyer and
Mullineaux 1998). The upregulation of DHAR activity in both
cold-sensitive and heat-sensitive, and cold-tolerant and heattolerant invasive plants rather than MDHAR, suggests that in E.
adenophorum and/or E. odoratum subjected to thermal stress
conditions, ascorbate is regenerated via glutathione. It may be
that DHAR activity could participate in ascorbate regeneration
under conditions of severe stress when MDHAR activity is
A
f
3600
2700
e
1800
d
b
ab
a
a
a
900
0
12
9
B
c
c
d
b
ab
a
ab
e
ab
6
3
0
Dehydroascorbate reductase
4500
(µmol ascorbate/min per mg protein)
E. odoratum
800
MDA content (nmol/g FW)
Dehydroascorbate reductase
MDA content (nmol/g FW) (µmol ascorbate/min per mg protein)
E. adenophorum
c
5
36
C
g
640
480
320
f
e
d
c
160
c
b
c
a
0
D
i
27
h
g
f
18
e
d
9
a
b
25
30
c
0
5
10
15
20
25
30
Temperature (°C )
35
38
42
5
10
15
20
35
38
42
Temperature (°C )
Figure 4. Effect of temperature on dehydroascorbate reductase (DHAR) activity and Lipid peroxidation of Eupatorium adenophorum (A, B) and E.
odoratum (C, D).
Values are the means of three replicates with standard errors. Means with the same letters are significantly different at P ≤ 0.05 using Duncan’s
multiple range test.
6
Journal of Integrative Plant Biology
2008
limited by the availability of NADH (Asada and Takahashi
1987; Hernndez et al. 2000). This is in contrast to the situation
described by Jahnke et al. (1991) who reported the DHAR losing
almost all of its activity in cold stress of two Zea genotypes
differing in cold tolerance, Jin et al. (2003) who reported the
activities of DHAR were not sensitive to changes in temperature
of five conifers with different cold tolerance.
Heat and cold stresses led to a significant increase in MDA
content in both species (Figure 4B, D). MDA accumulation in
E. adenophorum leaves was greater with heat treatment than
with cold treatment, with greater increase at 42 ◦ C than at 5 ◦ C,
indicating that a higher degree of lipid peroxidation occurred
at higher temperatures. In leaves of E. odoratum, however,
the MDA accumulation was greater with cold treatment than
with heat treatment, with greater increase at 5 ◦ C than at 42 ◦ C,
indicating a higher degree of lipid peroxidation induced by cold
stress. Exposure to low temperature may increase the amount
of active oxygen species not only in cold-sensitive E. odoratum,
but also in cold-tolerant plants like E. adenophorum, although E.
adenophorum showed elevated activities of SOD, CAT, POD,
APX, GR, MDAR and DHAR on exposure to cold treatment, the
capability of detoxification of AOS is limited. This is similar to
some reports that have demonstrated the elevated activities of
antioxidant enzymes in salt-tolerant rice varieties while the level
of lipid peroxidation was increased under conditions of salinity
stress (Vaidyanathan et al. 2003).
The interactions of APX, CAT, SOD and their involvement in
scavenging AOS are very complex, and also involve other peroxidases (Scandalios 1997a, 1997b; Noctor and Foyer 1998).
The extent of oxidative stress causing membrane and cellular
damage might possibly differ depending on the stress imposed.
Plants may respond to different environmental stresses in
different ways, as shown in our experiment (Figures 2–4). SOD
catalyzes the dismutation of superoxide into oxygen and hydrogen peroxide, so the increase of SOD activity suggested an increased production of H 2 O 2 . H 2 O 2 is eliminated by catalase and
peroxidases, which include both enzymatic and non-enzymatic
H 2 O 2 degradation (Peltzer et al. 2002). Catalase decomposes
H 2 O 2 into water, whereas POD decomposes H 2 O 2 by oxidation
of co-substrates such as phenolic compounds and/or antioxidants (Blokhina et al. 2003). The antioxidants such as ascorbate
and glutathione are involved in scavenging H 2 O 2 primarily via
the Halliwell-Asada pathway (Horemans et al. 2000). It has
been suggested that hydrogen peroxide must be effectively
scavenged in order to minimize cytotoxicity by activated oxygen
in the presence of elevated SOD activity (Finazzi-Agro et al.
1986; Scot et al. 1987).
In E. adenophorum, the coordinated increase of oxygendetoxifying enzymes was effective in protecting the plant from
the accumulation of AOS at low temperatures, thus averting
cellular damage under unfavorable conditions. However, the
activities of CAT, POD, APX, GR and MDAR were not accompanied with increased SOD during heat stress; thus in
heat-treated E. adenophorum, cytotoxicity was likely due to the
accumulation of H 2 O 2 , which can react with O 2 − to produce
hydroxyl free radicals via the Herbert-Weiss reaction (Bowler
et al. 1992). As a result, the level of lipid peroxidation in E.
adenophorum was higher under heat stress than under cold
stress. In E. odoratum, however, the lesser degree of membrane
damage (as indicated by low MDA content) and the coordinated
increase of the oxygen-detoxifying enzymes was observed in
heat-treated plants, but the antioxidant enzymes were unable
to do this with cold stress. This indicates that the plant has a
higher capacity for scavenging oxygen radicals in heat stress
than in cold stress.
The diverse response of antioxidant enzymes such as SOD
and other enzymes (CAT, POD, APX, MDAR, GR) due to thermal stress suggests the role of oxidative stress as a component
of environmental stress on the two invasive species. It appears
that the differences in SOD and other enzymes (CAT, POD,
APX, MDAR, GR) has a direct relation to the sensitivity of the
two plants to temperature. In rice, increased activity of SOD
and decreased activities of CAT and POD were detected in
cold-sensitive cultivars, whereas increased activities of these
enzymes have been found in resistant cultivars exposed to
chilling stress, while GR in both cultivars was stable to low temperature stress (Saruyama and Tanida 1995). Clear differences
were found in the cold sensitivity of CAT and APX between
the cold-sensitive and cold-tolerant rice cultivars. In maize, the
activity of SOD and APX of the resistant variety of plants grown
at low temperatures were higher than in controls. In contrast,
in the sensitive variety of plants grown at low temperature, the
activity of SOD was higher than in the controls and the activity of
APX was lower than in the controls (Massacci et al. 1995). Our
results suggested that a coordinated increase of the activity of
oxygen-detoxifying enzymes could be necessary to protect E.
adenophorum leaves from the accumulation of oxygen radicals
at low temperatures, or protect E. odoratum leaves from the
accumulation of oxygen radicals at high temperatures.
Although several environmental stresses in plants are known
to induce the expression and/or increasing levels of antioxidative enzymes and their mRNAs (Pan and Yau 1992; Kliebenstein
et al. 1998; Morita et al. 1999), the mechanism by which these
activated oxygen-scavenging enzymes can work co-operatively,
in response to temperature stress is not yet known. Further
analysis of the regulation of gene expression in these enzymes should elucidate the mechanism of different temperature
tolerances. Identifying the genes that regulate some of the
enzymes involved in stress and finding ways to interfere with
gene regulation may prove useful to aiding an economically
viable control of persistent perennial weeds in difficult-to-reach
locations or along waterways where the use of herbicides is
undesirable or illegal (Davis and Swanson 2001). Our future
goal also involves examining the long-term responses of the
antioxidative system, to further elucidate their function in thermal
tolerance mechanisms of the two invasive species.
Antioxidant Enzymes Respond to Heat in Eupatorium Species
Considering the different distribution patterns of E. adenophorum and E. odoratum in China, our data suggest that the
differences of antioxidant enzyme activities and MDA content in
the two species may be ascribed to differences in mechanisms
underlying oxidative stress injury and subsequent tolerance to
temperature stress.
Materials and Methods
Plant material and culture conditions
Eupatorium adenophorum Spreng. seedlings were collected
from the Kunming Institute of Botany, the Chinese Academy
of Sciences and E. odoratum L. seedlings were collected from
the Xishuangbanna Tropical Botanical Garden, the Chinese
Academy of Sciences. Seedlings were planted in plastic pots
(19 cm diameter and 16 cm high) filled with a 1:1:1 mixture of
soil:sand:peat. They were grown in a glasshouse with day/night
temperatures of 30 ◦ C/20 ◦ C. Maximal photosynthetic photo flux
density was approximately 800 µmol/m2 per s. Plants were
watered daily and fertilized on a regular basis. After 4weeks,
when the plants of both species had achieved a steady state in
their growth and reached heights of about 30–40 cm, they were
subjected to the temperature treatments.
Heat and cold stress treatments
Plants of of each invasive species were divided into two groups.
For the heat stress treatment, one half of the plants were
moved from the glasshouse and placed in growth chambers at
a constant temperature of 25 ◦ C and relative humidity of 70%, a
12/12 h light/dark photoperiod regime, and photon flux density
of 250 µmol/m2 per s for 7 days to acclimatize the plants to the
growth chambers. After acclimatization, the temperature was
increased stepwise to 30 ◦ C, 35 ◦ C, 38 ◦ C and finally to 42 ◦ C and
exposed to each temperature at 24 h intervals. Simultaneously,
for the cold stress treatment, the same acclimatization period of
7 days was used. Another group of plants were then exposed
to progressively decreasing temperatures of 20 ◦ C, 15 ◦ C, 10 ◦ C
and 5 ◦ C at 24 h intervals. Each treatment was repeated in three
separate growth chambers. The soil was kept saturated at field
capacity during both treatments to avoid possible drought. The
plants that were kept at 25 ◦ C for 24 h were used as controls.
After each 24 h temperature treatment, fully expanded leaf
material was collected from three plants per treatment, frozen,
stored in liquid nitrogen and maintained at −70 ◦ C. The tissue
was ground and the enzymes extracted for assay.
Lipid peroxidation
Lipid peroxidation in E. adenophorum and E. odoratum was
determined by measuring the malondialdehyde (MDA) content,
7
a product of lipid peroxidation, in 1 g leaf fresh weight following
Madhava Rao and Sresty (2000).
Enzyme extraction
One gram of frozen leaf material was homogenized in 8 mL of
50 mM phosphate buffer (pH 7.0) that included 1 mM ethylenediaminetetraacetic acid (EDTA) and 2% (w/v) polyvinylpolypyrrolidone (PVP). The homogenate was centrifuged at 13 000g for
40 min at 4 ◦ C. In the cases of APX and DHAR activities
determination, 1 mM ascorbate and 2 mM 2-mercaptoethanol,
respectively, were added to the homogenizing buffer to prevent
enzyme inactivation. The supernatant was used for assays of
enzyme activity and protein content. Total soluble protein contents of the enzyme extracts were determined following Bradford
(1976), using bovine serum albumin (BSA) as a standard.
Enzyme assays
Superoxide dismutase activity was determined using the
method of Beauchamp and Fridovich (1971) that measures the
inhibition in the photochemical reduction of nitroblue tetrazolium
(NBT) spectrophotometrically at 560 nm. Measuring the activity
of CAT followed Knöraer et al. (1996) by monitoring the rate of
decomposition of H 2 O 2 at 240 nm for 2 min. Measuring the activity of POD followed Chance and Maehly (1955) by monitoring
the rate of the increase in the absorbance at 470 nm for 2 min
due to guaiacol oxidation. The activity of APX was measured by
following Nakano and Asada (1981), and monitoring the rate of
ascorbate oxidation at 290 nm for 3 min. The activity of GR was
assayed according to the method of Halliwell and Foyer (1978)
by following the decrease in absorbance at 340 nm for 3 min
due to NADPH oxidation. The enzyme activity of MDAR was
measured in the supernatant at 25 ◦ C as described by Hossain
et al. (1984). MDAR was assayed by following the decrease in
absorbance at 340 nm for 5 min due to NADH oxidation. The
activity of DHAR was measured in the supernatant at 25 ◦ C
as described by Hossain and Asada (1984) by monitoring the
increase in absorbance at 265 nm for 3 min due to ascorbate
formation.
Statistical analysis
The experiment was arranged in a completely randomized block
design with three replicates. Duncan’s multiple range test for
multiple comparisons was used for the analysis of statistical
significance. Comparisons with P values ≤ 0.05 were considered significantly different. All values reported are means ± SE
of three replicates.
Acknowledgements
Many thanks to Mark Williamson, Yulong Feng and F. Andrew
Smith for their suggestions about this paper and for giving much
8
Journal of Integrative Plant Biology
2008
constructive advice. The authors also thank Nan Lu, Ping Lin,
Lingfeng Chen, Nianwei Qiu, Junfeng Wang, Yanhong Bing, Li
Zhu and Fan Bai for their assistance in this research.
catalase, and glutathione peroxidase. Photochem. Photobiol. 43,
409–412.
Foyer CH, Halliwell B (1976). Presence of glutathione and glutathione reductase in chloroplasts: a proposed role in ascorbic acid
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(Handling editor: Jin-Zhong Cui)