全 文 ：Received 25 Dec. 2003 Accepted 9 Mar. 2004
Supported by the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry.
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Acta Botanica Sinica
植 物 学 报 2004, 46 (7): 819-828
Salicylic Acid Modulates Aluminum-induced Oxidative Stress
in Roots of Cassia tora
WANG You-Sheng, WANG Jin, YANG Zhi-Min*, WANG Qing-Ya, LÜ Bo, LI Shao-Qiong,
LU Ya-Ping, WANG Song-Hua, SUN Xin
(Department of Biochemistry and Molecular Biology, College of Life Sciences, Nanjing Agricultural
University, Nanjing 210095, China)
Abstract: Salicylic acid (SA) plays an important role in mediating some biotic and abiotic stress-induced
oxidative stresses in plants. However, it remains unknown about the role of SA in mediating oxidative
stress induced by aluminum. In this study, we investigated the changes in concentrations of H2O2 and O2-,
some antioxidative enzyme activities and several physiological parameters involved in oxidative damage to
plasma membrane in the root tips of Cassia tora L. Results indicated that 20 mmol/L aluminum (Al) caused
increases in electrolytes leakage, malondlaldehyde (MDA) content and intense staining with Evans blue in
root tips, while treatment with 5 mmol/L SA suppressed the Al-induced increase in MDA. Examination of
H2O2 and O2-, the major ROS responsible for lipid peroxidation indicated that root tips challenged with Al in
the presence of SA contained significantly lower contents of H2O2 and O2- than those with Al alone.
However, the reduced levels of H2O2 appeared not to be correlated with enhanced activities of ROS-
scavenging enzymes like catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR), but
with increased activities of guaiacol peroxidase (POD). SA-induced reduction of H2O2 was also correlated
with suppression of O2- accumulation and superoxide dismutase (SOD) activities. From the results, it is
concluded that SA might activate an H2O2-mediated pathway, which in turn initiated a POD-dependent
antioxidative mechanism for retarding lipid peroxidation or preserving membrane integrity in root tips of
C. tora. We presented the evidence of time-dependent changes in endogenous SA in root tips exposed to
20 mmol/L Al.
Key words: salicylic acid; aluminum; oxidative stress; hydrogen peroxide; Cassia tora
Aluminum ions (Al3+) are toxic to plants when they are
produced in acidic soils. The general symptoms include
inhibition of root growth, disruption of nutrient uptake and
other secondary metabolisms (Kochian, 1995). Besides
these, an early response of plants to Al is the oxidative
damage to root tips (Yamamoto et al., 2001). Due to its high
affinity for plasma membrane, Al could cause plasma mem-
branes stiffening, facilitate the iron- or other transition met-
als-mediated radical chain reactions and thereby promote
the lipid peroxidation (Cakmak and Horst, 1991; Yamamoto
et al., 2001; Kuo and Kao, 2003). The mitochondrial activity
was also sensitive to Al3+; the metal-induced depression of
respiration, ATP together with generation of ROS appeared
to be the critical events of Al toxicity in plant cells
(Yamamoto et al., 2002).
To get insight into the mechanism of Al toxicity associ-
ated with oxidative stress, some researchers have exam-
ined molecular responses of Al-treated cells or plants (Ezaki
et al., 1995; Richards et al., 1998; Sivaguru et al., 2003).
Several genes encoding anti-oxidative enzymes have been
cloned and shown to be up-regulated by Al in plant roots.
These Al-induced genes are those encoding anionic per-
oxidase (POD), glutathione S-transferase (GST), and su-
peroxide dismutase (Richards et al., 1998; Ezaki et al., 2000).
Although the biological roles of Al-induced genes are
largely unknown, evidence from transgenic plants shows
that overexpression of a tobacco GST gene (parB) and a
tobacco POX gene (NtPox) in Arabidopsis could confer
resistance to Al-induced oxidative stress (Ezaki et al., 2001).
These results suggest that in addition to the generally-
accepted tolerant mechanism of Al-induced efflux of or-
ganic acids, there may be another mechanism responsible
for the tolerance to Al-induced oxidative stress (Ezaki et
al., 2001).
Cassia tora L. is a wild annul plant which is distributed
mainly over the south and middle part of China. It has been
reported specifically to secrete a large amount of citrate in
response to Al (Ma et al., 1997; Ishikawa et al., 2000). We
previously showed that treatment with exogenous salicylic
acid (SA) could stimulate Al-responsive citrate efflux from
Acta Botanica Sinica 植物学报 Vol.46 No.7 2004820
roots, and the SA-promoted citrate release was correlated
with the reduction of Al-induced inhibition of root growth,
suggesting that SA could confer Al tolerance in plants (Yang
et al., 2003). Recently, a protective effect of SA was re-
ported in barley when exposed to toxic levels of cadmium
(Cd) (Metwally et al., 2003). Similarly, the tobacco plants
growing on the medium supplied with exogenous SA in-
creased their thermotolerance (Dat et al., 2000). Protection
of mustard seedlings from heat shock has been obtained
when seedlings were sprayed with SA solutions (Lopez-
Delgado et al., 1998). Furthermore, hydroponic treatment
with SA decreased the effects of chilling injury in maize
plants (Janda et al., 1999). In most of the cases, the SA-
induced tolerance to these stresses has been related to the
increased antioxidative capacity.
It was proposed that one of the mechanisms involved in
SA effect on biotic stress was the up-regulation of H2O2
(Chen et al., 1993). In tobacco and Arabidopsis leaves,
treatment with exogenous SA caused H2O2 accumulation
(Chen et al., 1993; Rao et al., 1997). The process was asso-
ciated with resistance to pathogen attack. In this model, SA
served as a signal molecule that could bind to catalase. The
SA-binding catalase showed no activities and therefore
resulted in H2O2 accumulation. Since H2O2 could induce
pathogen resistant gene (PR) expression, SA was proposed
to activate PR expression by increasing the levels of H2O2,
and consequently, H2O2 was proposed to serve as a sec-
ond messenger in a defense signaling transduction path-
way (Klessig et al., 2000). With regard to the effect of SA
on H2O2 under abiotic stresses, Dat et al. (1998) reported
that treatments of mustard seedlings with either heat shock
at 55 °C or exogenous SA could induce a transient increase
in endogenous H2O2 and this process was accompanied
by a reduced CAT activity. In potato micro-plants, both
exogenous SA and H2O2 induced a high tolerance of plants
to high temperature (Lopez-Delgado et al., 1998). These
results suggested that both SA and H2O2 were involved in
signal transduction leading to the acclimation during the
heat stress (Dat et al., 1998). Examination of SA effects on
Al-induced oxidative stress may be also interesting. SA
belongs to a group of plant phenolics widely distributed in
plants and is proposed to be a hormone-like substance. No
information, however, is available on the effect of SA on
H2O2 production and antioxidative enzymes capable of me-
tabolizing H2O2 under Al stress. We hypothesized that SA
might also play a major role in mediating Al-induced oxida-
tive stress in addition to the role of SA in modulation of Al-
responsive citrate efflux (Yang et al., 2003). In the present
study, we investigated physiological responses, including
production of H2O2 and O2-, activities of H2O2 and O2--
scavenging enzymes, lipid peroxidation and cell death in
the root tips of C. tora under Al stress. And we examined
the possible involvement of SA in mediating oxidative stress
under the same condition. We also provided evidence that
SA modulated the Al-induced oxidative stress in root tips
of C. tora probably through the H2O2 transduction
pathway.
1 Materials and Methods
1.1 Plant materials and treatment
Uniform commercial seeds of Cassia tora L. were se-
lected and soaked in distilled water for 12 h and germinated
on a mesh tray floating on the solutions containing 0.5
mmol/L CaCl2 (pH 4.5). After germination, seedlings grew
for 3 d at (24±1) °C, with a photosynthetic photon flux den-
sity of 200 mmol.m-2.s-1 and 14-h photoperiod. The cul-
ture solutions were changed daily. When the average root
length was about 5.5 cm, seedlings were transferred to 0.5
mmol/L CaCl2 (pH 4.5) containing 20 mmol/L Al and/or 5
mmol/L SA for combined treatments. After treatment for 12
h, root tips of 0.5 cm length were excised, immediately fro-
zen in liquid nitrogen and stored at -80 °C.
1.2 H2O2 determination
The content of H2O2 was measured according to the
method of Patterson et al. (1984) with the following
modification: 50 root tips frozen in liquid nitrogen were
ground to a fine powder and extracted in 3 mL ice-cold
acetone. The homogenate was centrifuged at 10 000g at 4
°C for 20 min. The supernatant fractions were collected.
Naught point five mL of the samples was mixed with 1.5 mL
of mixture of CHCl3 and CCl4 (1:3, V:V). Then, 2.5 mL of
distilled water was added. The mixture was centrifuged at
1 000g for 1 min and the water phase was collected for H2O2
determination. The reaction mixture contained 0.5 mL of
buffer (0.2 mol/L phosphate buffer solution, pH 7.8), 0.5 mL
water phase sample, and 20 mL of catalase (0.5 unit) (to set
controls) or the same unit of inactive catalase protein
(treatments). After incubation at 37 °C for 10 min, 0.5 mL of
200 mmol/L Ti-4-(2-pyridylazo) resorcinol (Ti-PAR) was
added. The reaction mixtures were incubated at 45 °C for 20
min. The absorbance at 508 nm was monitored.
1.3 O2- determination
O2- production was determined according to the method
of Able et al. (1998). Briefly, 120 root tips were ground with
3 mL of 50 mmol/L Tris-HCl buffer (pH 7.5). The homoge-
nate was centrifuged at 5 000g at 4 °C for 10 min. The reac-
tion mixture (1 mL) contained 50 mmol/L Tris-HCl buffer
(pH 7.5), 0.5 mmol/L XTT (sodium,3-{1-[phenylamino-
WANG You-Sheng et al.: Salicylic Acid Modulates Aluminum-induced Oxidative Stress in Roots of Cassia tora 821
carbonyl]-3,4-tetrazolium}-bis(4-methoxy-6-nitro)
benzenesulfonic acid hydrate) and 50 mL sample extracts.
Corrections were made for the background absorbance in
the presence of 50 units of SOD. O2- production rate was
calculated by an extinction coefficient of 2.16´104 mol.
m-1.cm-1.
1.4 MDA determination
The MDA content was determined by a procedure based
on the method of Heath and Packer (1968). Briefly, 50 root
tips frozen in liquid nitrogen were ground in 3 mL of 0.1%
TCA solution. The homogenate was centrifuged at 15 000g
for 10 min and 0.5 mL of the supernatant fraction was mixed
with 2 mL of 0.5% TBA in 20% TCA. The mixture was
heated at 90 °C for 20 min, chilled on ice, and then centri-
fuged at 10 000g for 5 min. The absorbance of the superna-
tant was measured at 532 nm. The value for non-specific
absorption at 600 nm was subtracted. The amount of MDA
was calculated by using extinction coefficient of 155
mmol.L-1.cm-1.
1.5 Measurement of electrolyte leakage of roots
One hundred root tips were cut off and rinsed three
times, dried with filter papers and put into a test tube. Ten
mL of deionized water was added to the tube to soak the
tips and the tubes were shaken at 80 r/min at (25 ±1) °C for
2 h. The measurement of the water conductance and calcu-
lation of leakage percentage of electrolytes were based on
the method described by Gong et al. (2001).
1.6 Enzyme activity determination
Frozen root tip tissues were homogenized in ice-cold 50
mmol/L phosphate buffer (pH 7.8) containing 1 mmol/L
EDTA. The homogenate was centrifuged at 15 000g at 4 °C
for 10 min. The supernatant was used for enzyme
measurement. The activities of ascorbate peroxidase (EC
1.11.1.11) were determined in the presence of 0.5 mmol/L
ascorbic acid and 0.5 mmol/L H2O2 by monitoring the de-
crease in absorbance at 290 nm (Janda et al., 1999). Analy-
sis of guaiacol peroxidase (EC 1.11.1.7) capacity was based
on oxidation of guaiacol using hydrogen peroxide
(Hammerschmidt et al., 1982). Activities of catalase (EC 1.
11.1.6) were determined spectrophotometrically by moni-
toring the decrease in absorbance at 240 nm (Durner and
Klessig, 1996). Activities of superoxide dismutase (EC 1.1.
5.1.1) were assayed by measuring its capacity of inhibiting
the photochemical reduction of nitro-blue tetrazolium
(Beauchamp and Fridovich, 1971).
Extracts for determination of glutathione reductase (EC
1.6.4.2) activities were prepared from 50 root tips homog-
enized under ice-cold with 2 mL of extraction buffer con-
taining 50 mmol/L Tris-HCl (pH 7.6) and 1 mmol/L EDTA.
The homogenate was centrifuged at 15 000g at 4 °C for 10
min. GR activities were determined by the changes in ab-
sorbance in 340 nm described by Foyer and Halliwell (1976).
The total protein content in enzyme extracts was deter-
mined by the method of Bradford (1976) using bovine se-
rum albumin as a standard.
1.7 Analyses of oxidative damage to cell plasma mem-
brane
Histochemical detection of the loss of plasma membrane
integrity in root tips was performed by the method described
by Yamamoto et al. (2001). Following the Al and/or SA
treatments, intact roots of seedlings were washed with 0.5
mmol/L CaCl2 several times, dried with filter papers and
immediately immersed in 5 mL of Evans blue solutions
(0.025% (W/V) Evans blue in 100 mmol/L CaCl2 (pH 5.6)) for
1 h. After that, stained roots were washed three times with
sufficient volume of 100 mmol/L CaCl2 (pH 5.6), observed
under a light microscope (model SZH-ILLD; Olympus,
Tokyo) and photographed on color film (ASA 200, Koda
Photo Film, USA).
1.8 Gel electrophoresis
The isoenzymes of PODs were separated on 7% non-
denaturating polyacrylamide gels, at a constant 100 V for 4 h.
For detection of activities, the gels were stained for 5-10 min
in 0.33 mmol/L acetate buffer (pH 6.0) with 0.02 mmol/L
benzidine, 0.6 mmol/L NH4Cl, 0.1 mmol/L EDTA, and 0.07
mmol/L H2O2. After removing the staining solution, gels were
washed with distilled water and soaked in the water for 3 d.
The water was changed 3-4 times a day, and with time the
PODs on the gels appeared to be several brown zones.
1.9 SA determination
One hundred root tips were ground with 1 mL methanol.
The homogenate was centrifuged at 20 000g and 4 °C for 5
min. The supernatant was passed through a Sep-Pak C18
cartridge (Waters). Three hundred mL of the extract was
dried under N2 steam and then dissolved in 300 mL phos-
phorus buffer solution for SA measurement. SA concentra-
tions were determined according to the method of a direct
enzyme-linked immunosorbent assay (ELISA) described by
Wang et al. (2002).
1.10 Statistical analysis
Each result shown in tables and figures was the mean of
at least three replicated treatments. The significance of dif-
ferences between treatments was statistically evaluated by
standard deviation and Student’s t-test methods.
2 Results
2.1 Al-induced oxidative damage to the root tips of C. tora
In the present study, treatment with Al at 20 mmol/L for
Acta Botanica Sinica 植物学报 Vol.46 No.7 2004822
12 h caused an oxidative damage to roots of C. tora. As
shown in Fig.1, root tips within 10 mm length exposed to
20-50 mmol/L Al exhibited intense staining of Evans blue,
an observation similar to that reported in Al-treated pea
root tips (Yamamoto et al., 2001).
To confirm the result of root oxidative damage, MDA,
an indicator of lipid peroxidation in root tips was measured.
Exposure seedlings to 20 mmol/L Al for 12 h resulted in a
36% increase in MDA content compared with Al-free con-
trols (Fig.2). In addition, a time-course experiment was con-
ducted to examine the leakage of electrolytes from roots.
Although no significant increases in leakage of electro-
lytes from 20 mmol/L Al-treated root tips were detected dur-
ing the first 9 h, the prolonged treatment with Al caused a
dramatic increase in electrolytes leakage (Fig.3). At the end
of experiment, the ion leakage from Al-treated roots doubled
that of controls (Al-free only).
2.2 Effect of SA on Al-induced oxidative damage in root
tips
Compared with root tips treated with 20 mmol/L Al alone,
application of 5 mmol/L SA and 20 mmol/L Al to root-bath-
Fig.1. Al-induced loss of plasma membrane integrity in the root
tips of Cassia tora. Seedlings were exposed to 0.5 mmol/L CaCl2
(pH 4.5) solutions containing Al at 0, 10, 20 and 50 mmol/L for 12
h. Then, the roots were stained with Evans blue for 1 h and
immediately photographed under a light microscope. Bar in the
graph indicates 1 mm.
Fig.2. Effect of SA on lipid peroxidation in the root tips of
Cassia tora. Seedlings were exposed to 0.5 mmol/L CaCl2 (pH
4.5) solutions containing combined levels of Al (0, 20 mmol/L)
and SA (0, 5 mmol/L) for 12 h. Vertical bars represent standard
deviation of the mean (n = 3). Asterisk indicates that mean values
are significantly different between +Al-SA and +Al+SA treat-
ments (P < 0.05).
Fig.3. Time course of electrolyte leakage of root tips of Cassia
tora. Seedlings were exposed to 0.5 mmol/L CaCl2 (pH 4.5) solu-
tions containing 20 mmol/L Al only (+Al-SA, ■) or 20 mmol/L
Al and 5 mmol/L SA (+Al+SA,□). Control seedlings were ex-
posed to 0.5 mmol/L CaCl2 solutions containing 0 mmol/L Al and
0 mmol/L SA (-Al-SA, ●) or 0 mmol/L Al and 5 mmol/L SA
(-Al+SA, ○). Vertical bars represent standard deviation of the
mean (n = 3). Asterisk indicates that mean values are significantly
different between +Al-SA and +Al+SA treatments (P < 0.05).
Fig.4. Effect of SA on Al-induced loss of plasma membrane
integrity in the root tips of Cassia tora. Seedlings were exposed
to 0.5 mmol/L CaCl2 (pH 4.5) solutions containing combined
levels of Al (0, 20 mmol/L) and SA (0, 5 mmol/L) for 12 h. Then,
the roots were stained with Evans blue for 1 h and immediately
photographed under a light microscope. Bar in the graph indicates
1 mm.
WANG You-Sheng et al.: Salicylic Acid Modulates Aluminum-induced Oxidative Stress in Roots of Cassia tora 823
ing medium greatly alleviated the Al-induced oxidative
stress. As shown in Fig.3, seedlings treated with SA in the
presence of Al showed a constantly low level of electrolyte
leakage, even though there was a slight increase in ion
leakage at latter stages. The beneficial effect of SA was
confirmed by the histochemical analyses. There was very
little staining with Evans blue in the SA-treated root tips
(Fig.4), indicating that SA could serve as an antioxidant
against Al-induced oxidative damage to root cells.
2.3 Effect of SA on Al-induced O2- and H2O2 accumula-
tion
Treatment of seedlings with 20 mmol/L Al alone for 12 h
resulted in a significant increase in O2- content, whereas
simultaneous treatment with 5 mmol/L SA reduced O2- ac-
cumulation by 16.8 % compared with the 20 mmol/L Al treat-
ment alone (Fig.5).
In absence of Al, additional 5 mmol/L SA caused no
significant alternations of endogenous H2O2 levels during
a 12-h experiment (Fig.6). However, there was a rapid H2O2
accumulation after the start of 20 mmol/L Al treatment
(Fig.6). The peak was found 3 h after the initial treatment.
Then, there was a slight drop, but a high level of H2O2 was
observed thereafter. When seedlings were supplied with 5
mmol/L SA, a transient and rapid increase in H2O2 produc-
tion was observed, and the maximum level of H2O2 was
detected at 1 h after the experiment. Following that time, the
concentrations of H2O2 declined to the basal level.
2.4 Effect of SA on antioxidant enzymes under Al stress
Total activities of SOD in root tips were measured and a
significant increase in the activities was observed after a 12
h of Al treatment (Fig.7). Incubation of root tips with 5
mmol/L SA lowered the Al-dependent increase in SOD
activities.
It is well known that excess levels of H2O2 in plant cells
can be diminished by the two potent H2O2-scavenging
enzymes, CAT and APX. During the initial 6 h of experiment,
there was no marked stimulation of CAT activities in Al-
treated root tips (Fig.8). The stimulation, however, was en-
hanced thereafter. Treatment of seedlings with 5 mmol/L
SA and 20 mmol/L Al resulted in increases in CAT activities.
This result was similar to that with 20 mmol/L Al treatment
alone. However, no differences in CAT activities between
+Al-SA- and +Al + SA-treated root tips were observed.
Activities of APX in Al treated-root tips fluctuated
Fig.5. Effect of SA on the production of O2- in the root tips of
Cassia tora. Seedlings were exposed to 0.5 mmol/L CaCl2 (pH
4.5) solutions containing combined levels of Al (0, 20 mmol/L)
and SA (0, 5 mmol/L) for 12 h. Vertical bars represent standard
deviation of the mean (n = 3). Asterisk indicates that mean values
are significantly different between +Al-SA and +Al+SA treat-
ments (P < 0.05).
Fig.6. Time-dependent changes in H2O2 levels in the root tips of
Cassia tora. Seedlings were exposed to 0.5 mmol/L CaCl2 (pH
4.5) solutions containing 20 mmol/L Al only (+Al-SA, ■) or 20
mmol/L Al and 5 mmol/L SA (+Al+SA,□). Control seedlings were
exposed to the 0.5 mmol/L CaCl2 solutions containing 0 mmol/L
Al and 0 mmol/L SA (-Al-SA, ●) or 0 mmol/L Al and 5 mmol/L
SA (-Al+SA, ○). Vertical bars represent standard deviation of
the mean (n = 3). Asterisks indicate that mean values are signifi-
cantly different between +Al-SA and +Al+SA treatments (P <
0.05).
Fig.7. Effect of SA on SOD activities in the root tips of Cassia.
tora. Seedlings were exposed to 0.5 mmol/L CaCl2 (pH 4.5) solu-
tions containing combined levels of Al (0, 20 mmol/L) and SA (0,
5 mmol/L) for 12 h. Vertical bars represent standard deviation of
the mean (n = 3). Asterisk indicates that mean values are significantly
different between +Al-SA and +Al+SA treatments (P < 0.05).
Acta Botanica Sinica 植物学报 Vol.46 No.7 2004824
around the values of controls, indicating that there was no
effect of Al on the enzyme activities. Addition of 5 mmol/L
SA to the medium with 20 mmol/L Al had an inhibitory effect
on APX activities during the first 6 h (Fig.8), but APX ac-
tivities began to recover after that. At the time of 12 h, the
activity exceeded the levels of controls (-Al-SA).
Treatment with 20 mmol/L Al induced a significant in-
crease in GR activities during the initial treatment, then GR
activities remained constant up to the end of experiment
(Fig.8). Although supplying seedlings with SA caused an
increased GR activity in Al-treated root tips during the ini-
tial 3 h, after prolonged treatments, the GR activities gradu-
ally declined.
Treatments with Al and Al plus SA induced a progres-
sive increase in nonspecific peroxidase activities as com-
pared with Al-free controls (Fig.8). The significant induc-
tion was found after 6 h. The induction was more pro-
nounced in root tips of seedlings exposed to 5 mmol/L SA
with 20 mmol/L Al. To confirm the increase in POD activities,
six isoforms of peroxidases were visualized in root tips of
C. tora by loading equal amounts of proteins on non-
denaturating polyacrylamide gels (Fig.9). Although there
was no additional new band induced by Al and/or SA, the
band intensity was stronger in roots with 5 mmol/L SA and
20 mmol/L Al than in roots with 20 mmol/L Al treatment
alone, suggesting the POD activities could be stimulated
by SA.
2.5 Effect of Al on endogenous SA content in root tips
To show if endogenous SA was involved in the modu-
lation of Al-induced oxidative stress, the SA content in
root tips over the time of experiment was examined. Results
showed that SA synthesis was induced 3 h after 20 mmol/L
Fig.8. Time-dependent changes in activities of CAT, APX, GR, and POD in the roots of Cassia tora. Seedlings were exposed to 0.5
mmol/L CaCl2 (pH 4.5) solutions containing 20 mmol/L Al only (+Al-SA, ■) or 20 mmol/L Al and 5 mmol/L SA (+Al+SA,□). Control
seedlings were exposed to the 0.5 mmol/L CaCl2 solutions containing 0 mmol/L Al and 0 mmol/L SA (-Al-SA, ●) or 0 mmol/L Al and 5
mmol/L SA (-Al+SA, ○). Vertical bars represent standard deviation of the mean (n = 3). Asterisks indicate that mean values are
significantly different between +Al+SA and +Al-SA treatments (P < 0.05).
Fig.9. Effect of SA on activities of peroxidase isoenzymes in
Cassia tora root tips. Seedlings were exposed to 0.5 mmol/L
CaCl2 (pH 4.5) solutions containing combined levels of Al (0, 20
mmol/L) and SA (0, 5 mmol/L) for 12 h. Total proteins were iso-
lated and subjected to non-denaturating polyacrylamide gels.
WANG You-Sheng et al.: Salicylic Acid Modulates Aluminum-induced Oxidative Stress in Roots of Cassia tora 825
Al exposure. Over the time the SA contents increased and
the peak SA content was observed at 9 h (Fig.10).
3 Discussion
Most of biotic and abiotic stresses activate a common
mechanism involving the production of reactive oxygen
species like H2O2 and O2- in plant cells. H2O2 and O2- have
been also found to be generated in Al-treated plants such
as soybean (Cakmak and Horst, 1991), pea (Yamamoto et
al., 2002) and maize (Boscolo et al., 2003). A similar result
was observed with C. tora (Figs.5, 6). It was possible that
the observed increases in H2O2 or O2- under the Al stress
accounted for the lipid peroxidation or plasma membrane
damage in root tip cells.
Our results demonstrated that seedlings of C. tora grow-
ing on the medium containing 5 mmol/L SA displayed very
little visible symptom in Al-treated root tips, as presented
after staining with Evens blue (Fig.4). This phenomenon
was supported by the results of the decreased MDA pro-
duction and electrolyte leakage (Figs.2-4). In this regard,
SA appeared to be an antioxidant against Al-induced oxi-
dative stress. Although the reason for it was not
understood, the Al-induced increase in SA contents in root
tips suggested that SA played a key role in modulation of
the Al-induced oxidative stress. Furthermore, measurement
of H2O2 and O2- concentrations showed that supplying
Al-stressed seedlings with SA largely lowered the accumu-
lation of H2O2 and O2- in root tips. We paid a particular
attention to H2O2 because recent investigations indicated
that H2O2 played a key role in the plant defense against
pathogen attack (Klessig et al., 2000; Mittler, 2002). During
the response to pathogen, ROS was produced by plant
cells via the enhanced enzymatic activity of plasma-mem-
brane-bound NADPH oxidases (Mittler, 2002). In this case,
accumulation of H2O2 in pathogen-elicited cells has been
shown to be toxic to both pathogen and plant cells, and the
death of plant cells around the infection site would prevent
pathogen from spreading. However, the role of H2O2 dur-
ing pathogen-related defense seemed to be different from
that in abiotic stress resistance, because in the latter case,
cellular concentrations H2O2 must be under fine control
(Mittler, 2002). H2O2 at moderate levels may act as a second
messenger for stress signaling and lead to activation of
resistant genes. Therefore, whether H2O2 exerts a benefi-
cial or harmful effect appear to depend on concentrations
of action.
Production of H2O2 during abiotic stresses has been
proposed as part of the signaling cascade leading to pro-
tection from stresses (Dat et al., 1998; van Camp et al.,
1998). Heat shock induced an elevation in endogenous
H2O2, thereby increasing thermotolerance in mistard seed-
lings (Dat et al., 1998). Treatments with exogenous H2O2
could confer the potato microplant resistance to heat shock
(Lopez-Delgado et al., 1998) and chilling tolerance (Prasad
et al., 1994). Our observations indicated that SA-treated
root tips under Al stress displayed a transient H2O2
elevation, and the peak occurred 1 h after the start of ex-
periment (Fig.6), suggesting that SA might induce a puta-
tive mechanism for antioxidation through an up-regulation
of H2O2 synthesis. The transient rise in H2O2 might signal
a specific pathway leading to the downstream protective
and physiological responses.
The SA-induced change in H2O2 concentrations in root
tips under Al stress would be associated with the changes
in H2O2-scavenging capacity. For this reason, we exam-
ined the activities of CAT, one of the major antioxidant
enzymes that eliminate hydrogen peroxide by converting it
into oxygen and water. Results indicated that CAT in SA-
treated (+Al+SA) root tips showed generally lower activi-
ties than those in Al-alone-treated (+Al-SA) root tips, but
no significant difference was observed. This result was not
consistent with the result in maize plants subjected to low-
temperature, in which plants showed significant decreases
in CAT activities when treated with SA (Janda et al., 1999).
The relation between CAT activity and SA has been well
defined in biotic stress (van Breusegem et al., 2001). Cata-
lase is proposed to be a SA receptor. When SA is bound to
catalase, it becomes a form of inactivation, leading to H2O2
accumulation in plant cells. Our results, however, appeared
not to be the case described above because no pronounced
Fig.10. Time-dependent changes in SA levels in the roots of
Cassia tora seedlings exposed to the 0.5 mmol/L CaCl2 (pH 4.5)
solutions containing 0 (filled squires) and 50 mmol/L (open squires)
Al for 12 h. Vertical bars represent standard deviation of the mean
(n = 3). Asterisks indicate that mean values are significantly dif-
ferent between the Al treatments and controls (P < 0.05).
Acta Botanica Sinica 植物学报 Vol.46 No.7 2004826
effect of SA on CAT activity was observed in Al-treated
root tips. We therefore turned attention to the other two
H2O2-degradation enzymes, APX and GR, which operate in
the ascorbate-glutathione cycle. Similarly, there were no
marked changes in the activities of APX and GR between
the treatments of +Al-SA and +Al+SA (Fig.8). These re-
sults indicated that the SA-induced changes in H2O2 lev-
els might not be involved in alterations in activities of CAT,
APX or GR in Al-stressed root tips of C. tora.
One possible mechanism for SA-modulated decrease in
H2O2 levels might be the elevated activities of POD (Fig.8).
The increased POD activities should contribute to the re-
moval of Al-induced H2O2. PODs have been proposed to
be present mainly in cell wall. There are a variety of isoforms
localized in different plant species and tissue cells. PODs
have been reported to catalyze the oxidation of various
organic compounds like phenolics, lignin or suberin
(Lagrimini, 1991; Quiroga et al., 2000). The reactions have
been involved in a range of physiological responses such
as pathogen defense (Cosgrove, 1997), salt stress (Lin and
Kao, 2001), and metal stress (Schützendübel et al., 2001).
H2O2 has been reported to serve as a necessary substrate
for these processes (Schoper, 1996; Lin and Kao, 2001;
Schützendübel et al., 2001). Schützendübel et al. (2001)
reported that Cd at 50 mmol/L induced increases in POD
activities, which was companied by accumulation of phe-
nolics in pine root tips, and the process was linked to re-
duction of H2O2 by the oxidation of aromatic substrates
such as phenolics or monolignols. Formation of lignin pre-
cursors catalyzed by POD has also required H2O2 for a rapid
cross-linking of polymers (Schopfer, 1996).
In the present study, SA-promoted increases in POD
activities appeared to contribute to the increased capacity
for antioxidation. Several reports also indicated that PODs
were activated in response to chilling (Janda et al., 1999),
heavy metal stress (Schützendübel et al., 2001; Chen et al.,
2002), salinity (Lin and Kao, 2001) and aluminum (Cakmak
and Horst, 1991). However, whether the activated POD rep-
resents a result of toxicity (Cakmak and Horst, 1991) or
capacity of antioxidation remains to be elucidated. A recent
study showed that overexpression of an Al-induced per-
oxidase gene (AtPox) in transgenic Arabidopsis seedlings
resulted in increases in root elongation, lower levels of Al
and lipid peroxides in root tips, suggesting that increased
POD activity could confer Al tolerance (Ezaki et al., 2000).
Another evidence in favor of our data was that phenolics
such as phenlpropanoids could serve as an antioxidant for
protecting of Al toxicity in cultured tobacco cells (Yamamoto
et al., 1998).
SOD catalyzes the conversion of superoxide radicals
(O2-) to O2 and H2O2. An increase in SOD activities should
be correlated with a decrease in O2-, but whether H2O2 is
accumulated or not largely depends on the H2O2-
scanvening capacity. SOD activities in Al-treated root tips
were stimulated by O2- (Cakmak and Horst, 1991; Boscolo
et al., 2003), suggesting that O2- would be the cause of the
increased activity of SOD. In the study, C. tora exposed to
20 mmol/L Al showed a high level of O2- and SOD activities
in root tips, while simultaneous treatment of seedlings with
5 mmol/L SA suppressed the accumulation of O2- and SOD
activities. This suggested that SA might block the produc-
tion of O2- by a protective mechanism, which in turn re-
sulted in the relatively low activities of SOD and low levels
of H2O2 in root tips.
In summary, our data provided evidence that treatment
with SA could alleviate the Al-induced oxidative damage to
root tips. This phenomenon was correlated with the ob-
served suppression of H2O2 and O2- production in SA-
treated root tips under Al stress. However, the reduction of
H2O2 levels could not be due to the increased activities of
the ROS-scavenging enzymes CAT, APX and GR, but to
increases in POD activities. These results suggested that
POD would play an important role against Al-induced oxi-
dative stress. SA-induced reduction of H2O2 might be also
attributed to the decreases in O2- content and SOD activities.
Considering all these results, we could conclude that SA
might activate an H2O2-meidated pathway, which in turn
initiated a POD-dependent antioxidative mechanism for re-
tarding lipid peroxidation or preserving membrane integrity
in root tips of C. tora.
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