Plant Cell Physiol. 46(12): 1915–1923 (2005)
doi:10.1093/pcp/pci202, available online at www.pcp.oupjournals.org
JSPP © 2005
Nitric Oxide Reduces Aluminum Toxicity by Preventing Oxidative Stress in
the Roots of Cassia tora L.
You-Sheng Wang and Zhi-Min Yang *
Department of Biochemistry and Molecular Biology, College of Life Science, Nanjing Agricultural University, Nanjing, 210095, PR China
;
Nitric oxide (NO) as a key signaling molecule has been
involved in mediation of various biotic and abiotic stressinduced physiological responses in plants. In the present
study, we investigated the effect of NO on Cassia tora L.
plants exposed to aluminum (Al). Plants pre-treated for
12 h with 0.4 mM sodium nitroprusside (SNP), an NO
donor, and subsequently exposed to 10 µM Al treatment for
24 h exhibited significantly greater root elongation as compared with the plants without SNP treatment. The NO-promoted root elongation was correlated with a decrease in Al
accumulation in root apexes. Furthermore, oxidative stress
associated with Al treatment increased lipid peroxidation
and reactive oxygen species, and the activation of lipoxygenase and antioxidant enzymes was reduced by NO. Such
effects were confirmed by the histochemical staining for the
detection of peroxidation of lipids and loss of membrane
integrity in roots. The ameliorating effect of NO was specific, because the NO scavenger cPTIO [2-(4-carboxy-2phenyl)-4,4,5,5-tetramethylinidazoline-1-oxyl-3-oxide] completely reversed the effect of NO on root growth in the presence of Al. These results indicate that NO plays an
important role in protecting the plant against Al-induced
oxidative stress.
Keywords: Aluminum toxicity — Cassia tora L. — Nitric
oxide – Oxidative stress.
Abbreviations: CAT, catalase; cPTIO, 2-(4-carboxy-2-phenyl)4,4,5,5-tetramethylinidazoline-1-oxyl-3-oxide; LOX, lipoxygenase; NBT,
nitroblue tetrazolium; NO, nitric oxide; NOS, nitric oxide synthetase;
POD, peroxidase; ROS, reactive oxygen species; SA, salicylic acid;
SNP, sodium nitroprusside; SOD, superoxide dismutase; TBARS, thiobarbituric acid-reactive substances.
Introduction
One of the responses of plants to aluminum (Al) toxicity
is oxidative stress that results in lipid peroxidation of the
plasma membrane in plants (Cakmak and Horst 1991, Ikegawa
et al. 2000, Yamamoto et al. 2001). The excess formation of
reactive oxygen species (ROS) such as O2– and H2O2 in plant
cells is one of the primary responses to Al exposure
(Yamamoto et al. 2002, Devi et al. 2003, Kuo and Kao 2003).
*
Although it is not fully understood whether Al-induced oxidative damage to plant cells is due to the increase in ROS levels,
it has been shown that the increased ROS levels activate
expression of genes for antioxidative enzymes such as superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC
1.11.1.6) and peroxidase (POD, EC 1.11.1.7) (Ezaki et al.
2000). The activated antioxidative systems are beneficial for
plant performance under Al stress, because adequate capacity
of antioxidative enzymes and other antioxidant metabolites
may help in the removal of excess ROS and inhibit lipid
peroxidation (Mittler 2002, Wang et al. 2004). For example,
Arabidopsis plants overexpressing a tobacco POD gene
(NtPox) exhibit enhanced resistance to Al-induced oxidative
stress and increased tolerance to Al toxicity (Ezaki et al. 2000).
Although there is no evidence showing that Al alone directly
triggers the oxidative stress, there are some reports indicating
that activities of some antioxidative enzymes increase in Alexposed plants (Cakmak and Horst 1991, Boscolo et al. 2003,
Devi et al. 2003). This process is usually accompanied by
enhanced lipid peroxidation (Cakmak and Horst 1991). Under
this condition, the Al-induced increase in activities of antioxidative enzymes appears to be the result of oxidative stress.
Nitric oxide (NO) is a biologically active gaseous molecule and has received increasing attention due to its association with plant responses to pathogen attack (Klessig et al.
2000, Neill et al. 2003), the hypersensitive response and programmed cell death (Delledonne et al. 1998, de Pinto et al.
2002). Also, it is shown to be involved in responses to abiotic
stresses such as low and high temperatures (Neill et al. 2002,
Uchida et al. 2002), salt (Zhao et al. 2004) and drought (Zhao
et al. 2001) stresses. Thus, NO is proposed to be one of the
important second messengers in plant cells (Beligni et al.
2002). In animals, the biosynthesis of NO is regulated by nitric
oxide synthase (NOS, EC 1.14.13.39). In plants, however, the
enzyme has not yet been isolated, but the activity of NOS for
NO synthesis can be detected (Neill et al. 2003). Application of
exogenous NO can also mediate various physiological processes to abiotic stresses. Application of the NO donor sodium
nitroprusside (SNP) confers resistance to salt (Uchida et al.
2002), drought (Mata and Lamattina 2001), heavy metals (Hsu
and Kao 2004) and chilling (Neill et al. 2003) stresses. Several
lines of study have shown that the protective effect of NO
against abiotic stresses is closely related to the NO-mediated
reduction of ROS in plants (Kopyra and Gwózdz 2003, Zhang
Corresponding author: E-mail, [email protected]; Fax, +86-25-84396248.
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NO reduction of Al toxicity and peroxidation
Fig. 1 Effect of the concentrations
of the NO donor SNP on the Alinduced inhibition of C. tora root
growth. Seedlings were pre-treated
with SNP at the indicated concentrations (A) or at 0.4 mM (B) for 12 h
and then exposed to 10 µM Al for
24 h. Vertical bars represent the SD of
the mean (n = 30). Asterisks indicate
that mean values are significantly different between the treatments of Al +
SNP and Al alone (P < 0.05).
et al. 2003, Hsu and Kao 2004). Under these conditions, NO
appears to serve as an antioxidant agent able to scavenge ROS
to protect plant cells from ROS damage (Laxalt et al. 1997).
NO may also function as a signaling molecule and indirectly
mediate ROS levels in the cascade of events leading to alterations of antioxidative gene expression (Leshem 1996).
It has been known that Al is able to cause oxidative stress
in plants. However, the process of Al-induced oxidative damage to plant cells and the regulatory mechanism for antioxidation in plants are unknown. Understanding of these
physiological processes in which Al triggers peroxidationrelated responses and a self-amelioration mechanism is of critical importance. The aim of this investigation is to examine the
role of NO in mediating Al-induced oxidative stress in plants.
This work may increase our understanding of the mechanisms
of NO amelioration of Al toxicity in plants.
Results
Effect of NO donor on Al-induced inhibition of root growth
Seedlings of Cassia tora exposed to 0–50 µM Al showed
inhibition of root growth with increasing Al concentration (data
not shown). Treatment with Al at 10 µM resulted in approximately 50% inhibition of root elongation during the first 24 h
after the start of Al exposure (Fig. 1). Therefore, this concentration of Al was used for estimation of the role of NO in mediating root elongation under Al stress. A preliminary experiment
with SNP at 0, 0.1, 0.2, 0.3, 0.4, 0.6 and 0.8 mM was performed to determine the point where SNP showed the most significant effect. As shown in Fig. 1, pre-treatment with the NO
donor SNP at 0.4 mM had the greatest effect on the Al-induced
inhibition of root elongation. The root growth increased by
>30% in seedlings pre-treated with 0.4 mM SNP (followed by
10 µM Al treatment) as compared with the control (10 µM Al
alone). However, a further increase in SNP concentration up to
0.8 mM had a detrimental effect on the root growth. Thus, an
SNP concentration-dependent change in root elongation under
Al stress was observed (Fig. 1). The improved root growth with
SNP pre-treatment was shown to be time dependent. During
the experiment, seedlings pre-treated with the NO donor began
to exhibit an increase in root growth compared with seedlings
treated with only Al at 12 h after the Al treatment (Fig. 1).
However, a significant improvement of root elongation with
SNP pre-treatment occurred at 24 h. In order to confirm the
role of SNP in reducing Al-inhibited root growth, a root growth
recovery experiment was carried out. Roots pre-treated with
0.4 mM SNP for 12 h followed by the 20 µM Al treatment for
another 12 h were found to be less inhibited and recovered
more rapidly than the roots without SNP pre-treatment (Fig. 2).
After a 72 h period of recovery, the SNP-pre-treated root elongation reached 60% of the control (–Al treatment), whereas the
root elongation without SNP pre-treatment was only 22% of
the control.
To confirm the protective effect of NO, other than the
other components in SNP, on the root growth under Al stress,
an NO-specific scavenger, 2-(4-carboxy-2-phenyl)-4,4,5,5tetramethylinidazoline-1-oxyl-3-oxide (cPTIO), was simultaneously incubated with SNP. The effect of 0.4 mM SNP on the
Al-induced root growth inhibition could be reversed by the
Fig. 2 Recovery of root growth of C. tora after SNP and Al treatments. Seedlings were pre-treated with 0.4 mM SNP for 12 h and then
exposed to 20 µM Al for another 12 h. Afterwards, they were transferred to the control solutions (–Al) (0.5 mM CaCl2, pH 4.5) for a 72 h
recovery. Control seedlings were incubated in the solution containing
0.5 mM CaCl2 only (pH 4.5). Vertical bars represent the SD of the
mean (n = 30). Asterisks indicate that mean values are significantly
different between the treatments of Al + SNP and Al (P < 0.05).
NO reduction of Al toxicity and peroxidation
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Fig. 3 Effect of the NO and NO scavengers on the Al-induced inhibition of root growth of C. tora. Seedlings were pre-treated with the NO
donor SNP at 0.4 mM with or without cPTIO (0.4 mM) for 12 h and
then exposed to 10 µM Al for 24 h. Vertical bars represent the SD of
the mean (n = 30). Asterisks indicate that mean values are significantly
different between the treatments of SNP + Al and SNP + cPTIO + Al
(P < 0.05).
Fig. 5 Histochemical detection of Al accumulation in the root apexes
of C. tora as affected by the NO donor SNP. Seedlings were pretreated with 0.4 mM SNP for 12 h and then exposed to 20 µM Al for
24 h. Afterward, the root was stained with hematoxylin. The scale bar
in the graph indicates 1 mm.
addition of 0.4 mM cPTIO (Fig. 3). This result indicates that
NO was involved in modulation of Al toxicity to root growth.
root apical region (0–2 mm), while less intense staining
occurred at the 15–20 mm region.
Effect of NO donor on Al accumulation in root apexes
Since Al accumulation in root apexes of C. tora is correlated with the Al-induced inhibition of root elongation (Yang et
al. 2003), the Al content in plant roots was measured. Addition
of 20 µM Al to the treatment medium caused a rapid uptake of
Al in root apexes during the initial 6 h. After that, they maintained a gradual accumulation of Al uptake (Fig. 4). The uptake
of Al in the SNP-pre-treated roots exhibits a similar pattern to
that in non-SNP-pre-treated roots within the first 12 h. Following that time, however, the Al accumulation rate remained
unchanged. At 24 h, the Al content in the SNP-treated root
apexes was 20% lower than that without SNP treatment. The
inhibition of Al accumulation in root apexes by the NO donor
was supported by histochemical staining (Fig. 5). Compared
with the control root, the SNP-pre-treated root apex was
stained less by hematoxylin, an indicator of Al. More intense
staining, indicating a higher level of Al, was detected at the
Effect of NO donor on Al-induced oxidative stress
To evaluate the role of NO in mediating the Al-induced
oxidative stress, we first treated roots of C. tora with 20 µM Al
for 24 h and measured the content of thiobarbituric acid-reactive substances (TBARS) as an indicator of lipid peroxidation
in the root apexes. Production of the oxidation product TBARS
in roots during the first 9 h was minimal (Fig. 6), but the production of TBARS increased thereafter. Compared with the Al
treatment alone, pre-treatment with the NO donor (plus the
same Al treatment) caused a significant decrease in levels of
TBARS at 12 h after the Al treatment, and this effect remained
until the end of the experiment. The effect of NO on the Alinduced oxidative injury of plasma membranes was also investigated by histochemical staining. The roots of C. tora treated
with Al alone were stained extensively with Schiff’s reagent
(Fig. 7A) and Evans blue (Fig. 7B), whereas those pre-treated
with SNP had only light staining. These results indicate that
Fig. 4 Effect of the NO donor SNP on the Al content in the root tips of C. tora under Al stress. Seedlings were pre-treated with SNP at 0, 0.1, 0.2, 0.3, 0.4,
0.6 and 0.8 mM for 12 h and then exposed to 20 µM
Al only for 24 h (A). In the experiment in (B), seedlings were pre-treated with SNP at 0.4 mM for 12 h.
Afterwards, they were exposed to 20 µM Al only for
24 h. Vertical bars represent the SD of the mean (n =
3). Asterisks indicate that mean values are significantly different between the treatments of Al + SNP
and Al alone (P < 0.05).
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NO reduction of Al toxicity and peroxidation
Fig. 6 Effect of the NO donor SNP on the contents of TBARS (A), lipoxygenase (LOX) activity (B) and the in-gel activity of LOX (C)
isoforms in the root apices of C. tora under Al
stress. Seedlings were pre-treated with 0.4 mM
SNP for 12 h and then exposed to 20 µM Al for
0–24 h for time course experiments (A and B).
Vertical bars represent the SD of the mean (n = 3).
Asterisks indicate that mean values are significantly different between the treatments of Al +
SNP and Al alone (P < 0.05). For the determination of the in-gel activity of LOX isoforms (C),
seedlings were pre-treated with 0.4 mM SNP for
12 h and then exposed to 20 µM Al for 12 h.
Afterwards, extracts of root apices containing
200 µg of protein were loaded onto non-denaturing polyacrylamide gels and, following electrophoresis, the gels were stained.
application of exogenous NO to the culture medium provided
protection against Al-induced oxidative injury in C. tora.
Lipoxygenase (LOX, EC 1.13.11.12) is a ubiquitously
occurring enzyme that catalyzes the peroxidation of unsaturated fatty acids of biomembranes to produce hydroperoxides
and oxy-free radicals (Gardner 1991). Thereby, the increase in
LOX activity may increase the formation of oxidation products (Axelrod et al. 1981). In the Al-stressed root, there was a
similar pattern between LOX activity and TBARS production
(Fig. 6). The effect of the NO donor on the activity of LOX
under Al stress was investigated further using non-denaturing
PAGE. The decrease in LOX activity due to the NO pre-treatment was clearly seen on the gel (Fig. 6). In addition to this,
there was a band (band 1) that was strongly induced by the
presence of Al, but could hardly be detected in the control or
NO-pre-treated roots. We assume that the appearance of this
new band induced by Al might be responsible for the Alinduced oxidative stress. Since it was possible that the lipid
peroxidation was caused by the increased ROS, the contents of
O2– and H2O2 in the root apexes were measured. Our results
show that treatment with 20 µM Al induced significant
increases in the levels of O2– and H2O2 (Fig. 8), but the production of both O2– and H2O2 was significantly inhibited in the
roots pre-treated with 0.4 mM SNP.
Effect of NO donor on activities of antioxidative enzymes and
ascorbate content in root apexes
Activities of SOD, CAT and POD in C. tora roots
increased after Al exposure, being 44.5, 56.5 and 41.4% higher
than the controls, respectively (–Al–SNP) (Table 1). The NO
donor SNP plus Al treatment (+Al+SNP) caused decreases in
SOD, CAT and POD activities of 31.9, 26.1 and 34.5, respectively as compared with the Al treatment alone (+Al–SNP).
Analysis of SOD activity by non-denaturing PAGE was per-
Fig. 7 Effect of the NO donor SNP on the Al-induced lipid peroxidation (A) and loss of plasma membrane integrity (B) in the root tips of
C. tora. Seedlings were pre-treated with 0.4 mM SNP for 12 h and
then exposed to 20 µM Al for 12 h. Afterwards, the roots were rinsed
with 0.5 mM CaCl2 (pH 4.5) solution and were stained with Schiff’s
reagent (A) or Evans blue (B), and immediately photographed under a
light microscope. The scale bar in the graph indicates 1 mm.
NO reduction of Al toxicity and peroxidation
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Fig. 8 Effect of the NO donor SNP on the
contents of O2– (A) and H2O2 (B) in the root
apices of C. tora under Al stress. Seedlings
were pre-treated with 0.4 mM SNP for 12 h
and then exposed to 20 µM Al for 12 or
24 h. Vertical bars represent the SD of the
mean (n = 3). Asterisks indicate that mean
values are significantly different between
the treatments of +Al–SNP and +Al+SNP
(P < 0.05).
Fig. 9 Effect of the NO donor SNP on
the in-gel activities of superoxide
dismutase (SOD), catalase (CAT) and
peroxidase (POD) isoforms in the root
tips of C. tora under Al stress. Seedlings
were pre-treated with 0.4 mM SNP for
12 h and then exposed to 20 µM Al for
12 h. After that, the extracts of root
apexes containing 80 µg of proteins were
loaded onto the non-denaturing polyacrylamide gels. Following the electrophoresis, the gels were stained by the
methods given in Materials and Methods.
formed. Five clear SOD isoforms were detected in the root
apex (Fig. 9). Al treatment generally induced increases in band
size. However, the increased amount of SOD isoforms was
largely inhibited by the SNP pre-treatment. Concerning the
H2O2-scavenging enzymes, only one achromatic band corresponding to CAT in root apexes of C. tora could be visualized
on a non-denaturing gel (Fig. 9). However, the band intensity
of CAT was greatly stimulated in the roots exposed to Al. In
contrast, a decreased amount of CAT with NO donor pretreatment was detected.
Table 1 Effect of the NO donor SNP on the activities of
superoxide dismutase (SOD), catalase (CAT) and peroxidase
(POD) and the content of ascorbate (ASC) in the root apexes of
C. tora under Al stress
Treatment
SOD
(U mg–1
protein)
CAT
(U mg–1
protein)
POD
(U mg–1
protein)
ASC
(nmol
apex–1)
–Al–SNP
–Al+SNP
+Al–SNP
+Al+SNP
13.2 b
14.5 b
23.8 a
16.2 b
0.10 c
0.13 c
0.23 a
0.17 b
1.7 b
1.6 b
2.9 a
1.9 b
2.5 a
2.8 a
1.3 b
2.4 a
Seedlings were pre-treated with 0.4 mM SNP for 12 h and then
exposed to 20 µM Al for 12 h. Means with different superscript letters
in the columns are significantly different (P < 0.05).
Four bands of POD isoforms in the root apexes of C. tora
can be detected (Fig. 9). POD-I isoform shows a pronounced
decrease in activity with Al treatment alone as compared with
the controls. However, pre-treatment with SNP completely
restored the Al-inhibited accumulation of the isoform. On the
other hand, POD-II and POD-III in the Al-treated roots exhibit
higher accumulation than those in the control roots, and
pre-treatment with SNP induced only a slightly increased
accumulation. It is noted that a new POD isoform (IV) was
induced when seedling roots were exposed to Al at 20 µM. Pretreatment of seedlings with SNP inhibited the accumulation of
this isoform.
Due to the role of ascorbate in mediating the root elongation (Citterio et al. 1994, Córdoba-Pedregosa et al. 1996) and
response to oxidative stress (Horemans et al. 2000), the ascorbate content in root apexes was measured. Plants under control
conditions maintained a stable level of around 2.5 nmol apex–1
in root apexes (Table 1). Treatment of plants with NO donor
alone caused a slight but not significant increase in ascorbate
content. When exposed to 20 µM Al, plants exhibited a sharp
decrease in the content of ascorbate. However, ascorbate
increased by 85.7% in root apexes pre-treated with the NO
donor as compared with in root tissues without the NO pretreatment (Table 1).
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NO reduction of Al toxicity and peroxidation
Discussion
In this study, we present evidence of the beneficial effect
of NO on the growth of C. tora under Al stress. Our results
indicate that plants pre-treated with 0.3–0.4 mM SNP, an NO
donor, for 12 h and subsequently exposed to 10 µM Al treatment for 24 h exhibit a significant promotion of root elongation as compared with the control plants (Fig. 1). The NOpromoted root elongation is correlated with the decrease in Al
accumulation in root apexes (Fig. 4, 5). Furthermore, NO can
counteract Al-induced oxidative stresses, including lipid peroxidation, increased ROS and accumulation of LOX as well as
antioxidant enzymes. Such effects were confirmed by the histochemical staining for the detection of peroxidation of lipids and
injury of membrane integrity in root apexes (Fig. 7). The
ameliorating effect of NO is specific because the NO scavenger cPTIO could reverse the effect of SNP on the root growth
(Fig. 3). These results indicate that NO is able to play important roles in promoting plant tolerance to Al toxicity. NO has
been shown to be involved in plant pathogen resistance
including signaling transduction leading to the hypersensitive
response and systemic acquired resistance (Neill et al. 2003,
Wendehenne et al. 2004). Moreover, NO has been implicated in
plant response to salt (Zhao et al. 2004), drought (Mata and
Lamattina 2001), chilling or heat (Neill et al. 2002) stresses.
Exogenous NO has been also reported to confer increased
resistance of plants to heavy metals such as cadmium (Hsu and
Kao 2004, Laspina et al. 2005) and copper (Yu et al. 2005).
Therefore, NO appears to be a molecule of multi-functions in
mediating plant responses to various stresses.
Our previous observations show that pre-exposure of C.
tora to salicylic acid (SA) reduced Al toxicity (Yang et al.
2003). The result supports a connection between the SA-promoted citrate efflux and improved elongation of roots under Al
stress. To determine whether NO plays a similar role to SA,
experiments to measure citrate efflux were performed. Results
from the analysis of the culture medium show that the SNPpre-treated plants during the Al treatment failed to secrete additional amounts of citrate as compared with the controls (Al
treatment alone) and, also, no additional accumulation of citrate in root apexes was observed (data not shown). These
results indicate that NO-promoted root elongation under Al
stress is not through the process of increasing citrate efflux
from roots.
Considering such observations, we next focused on the
examination of the relationship between NO and Al-induced
oxidative stress. Our results indicate that pre-exposure of plants
to the NO donor suppressed the Al-induced production of ROS
(Fig. 8) and peroxidation of lipids (Fig. 6). These results suggest that the NO-activated antioxidation in the plant species
might be responsible for the promoted root growth under the Al
stress. The reason for this is that the NO-mediated responses
(or events) such as the decrease in TBARS levels (Fig. 6) and
oxidative damage of roots (Fig. 7) as well as the decline of
antioxidant enzyme activities (Table 1) occurred before the
responses of the improvement of root elongation (Fig. 1) or the
decrease in Al accumulation (Fig. 4, 5). Therefore, it was possible that NO-activated antioxidative systems protected membrane lipids against peroxidation by interacting with lipid
peroxyl radicals or by blocking LOX activity (Beligni et al.
2002). It is possible that NO prevents ROS-induced cytotoxicity of the plasma membrane of roots (Mittler 2002), thus
slowing Al3+ permeability into the cells and ameliorating the
Al-inhibited root growth. However, our observations are not
consistent with the data from a study with peas (Pisum sativum), where the Al-induced lipid peroxidation was found to be
an early symptom, but not the cause of the inhibition of root
growth (Yamamoto et al. 2002).
The content of ascorbate in root apexes was measured
because several lines of study have shown that root elongation
of plants depends on the redox status in the apical region
(including meristems and elongation zones) (CórdobaPedregosa et al. 2003b, Córdoba-Pedregosa et al. 2005). In
onion (Allium cepa L.) roots, the apical region has been demonstrated to contain a high level of ascorbate (CórdobaPedregosa et al. 2005). Treatment with exogenous ascorbate
could also stimulate the root elongation of onion (CórdobaPedregosa et al. 1996) and pea (Citterio et al. 1994). On the
other hand, some evidence has shown that the apoplastic POD
is involved in enhancement of cross-linking of hydroxyprolinerich glycoproteins with phenolic acids (Takahama and Oniki
1992, Sanchez et al. 1997, Horemans et al. 2000). This process
contributes to cell wall secondary thickening (stiffening) and is
negatively correlated with cell growth and root elongation
(Zheng and Van Huystee 1992, Andrews et al. 2002, CórdobaPedregosa et al. 2003a). It should be noted that for a rapid
cross-linking of cell wall polymers, H2O2 is required for the
formation of lignin precursors catalyzed by POD (Schopfer
1996). It is interesting to find that there is very little POD activity in the meristems and elongation zones, because ascorbate is
a strong inhibitor of POD, although it does not directly inhibit
the cell wall POD (Takahama and Oniki 1992, Takahama 1993,
Córdoba-Pedregosa et al. 1996). In this study, we observed
simultaneously a decreased ascorbate level and increased POD
activity in Al-treated root apexes (Table 1). However, the
response could be reversed by the pre-exposure to the NO
donor. These results may provide an explanation for the role of
NO in mediating Al-induced oxidative stress as follows: NO
stimulates the production or maintains a high level of ascorbate in Al-treated root apexes. The NO-promoted ascorbate
level exerts a stimulating effect on the Al-inhibited root elongation possibly by blocking the POD-catalyzed cross-linking
among the structural components in cell walls. Meanwhile,
because these reactions are accompanied by the consumption
of H2O2 by ascorbate (Takahama and Oniki 1992, Sanchez et
al. 1997, Horemans et al. 2000), NO and ascorbate may not
only mediate the root elongation, but also serve as an antioxidant system for preventing oxidative damage. More experi-
NO reduction of Al toxicity and peroxidation
ments are required to elucidate the connection between the
NO- and ascorbate-dependent antioxidation and root elongation
under Al stress.
Although the ROS initiate several oxidatively destructive
processes, they also trigger various signaling pathways, and
maintenance of appropriate ROS levels might represent a survival response (Neill et al. 2003). NO can interact with ROS in
various ways, inhibiting lipid peroxidation and serving an antioxidant function during various stresses (Caro and Puctarulo
1998, Boveris et al. 2000, Neill et al. 2003). Our results with C.
tora under Al stress clearly show that application of the NO
donor reduced Al stress-induced increases in O2– and H2O2
(Fig. 8) and this process was associated with the low level of
lipid peroxidation (Fig. 6, 7). One possibility is that NO might
activate antioxidative systems to scavenge ROS or directly
abrogate O2·–-mediated cytotoxic effects through the conversion of O2·– into ONOO– (Neill et al. 2003), thus protecting
plants against Al-induced oxidative stress. The effect may represent the antioxidant property of NO for suppression of the
high levels of Al-triggered ROS.
To prevent the chemical interaction between SNP or
cPTIO and Al3+, thus reducing the toxic effect of Al ions on
plants, we separately treated the plants with SNP (or cPTIO)
and Al (see Materials and Methods). Therefore, it was impossible for the interaction of SNP (or cPTIO) with Al3+ to occur in
the culture medium. Another concern may be that NO in plants
interacts with Al3+. As a signal molecule, NO regulates a range
of different targets (Crawford and Guo 2005). Whether Al3+ is
one of these targets is unknown. However, because NO is an
active and unstable free radical in organisms (Neill et al. 2003),
it is unlikely that the active molecule NO could bind Al3+ to
form a stable complex.
In conclusion, we obtained the observation indicating that
NO is involved in amelioration of Al-induced oxidative stress
in C. tora. In order to gain an insight into the function of NO in
mediating Al-induced oxidative damage in plants, further
research is needed to determine endogenous NO and NOS
activity that is associated with Al-induced physiological
responses.
Materials and Methods
Plant culture and treatment
Uniform seeds of C. tora L. were selected, soaked in distilled
water and germinated on a mesh tray floating on 1.5 liters of a solution containing 0.5 mM CaCl2 (pH 4.5). After germination, seedlings
grew for 3 d at 22°C, with a photosynthetic photon flux density of
100 µmol m–2 s–1 and 14 h photoperiod. The solution was changed
daily. When the average root length was about 5.5 cm, the seedlings
were exposed to various treatment solutions.
SNP was used as an NO donor and cPTIO as an NO scavenger.
Both chemicals were purchased from Sigma Co. (St Louis, MO, USA).
Seedlings were incubated in the solution of 0.5 mM CaCl2 (pH 4.5)
containing SNP and/or cPTIO for pre-treatment for 12 h. Afterwards,
the root was rinsed with 0.5 mM CaCl2 (pH 4.5) several times and
exposed to various concentrations of AlCl3. After that, the root was
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rinsed again with 0.5 mM CaCl2 solution (pH 4.5) and root apexes
(5 mm) were excised from seedlings. The samples were used immediately or frozen in liquid nitrogen and stored at –80°C for later analysis. Root elongation was measured with a ruler.
Determination of Al content in root apexes
The collected root apexes were placed in a 1.5 ml Eppendorf
tube and 1 ml of 2 N HCl was added. Al in root apexes was extracted
and measured by graphite furnace atomic absorption spectrophotometry (180–80 Hitachi, Tokyo, Japan).
H2O2 determination
The H2O2 content of root apexes was measured according to the
method of Patterson (1984) with the following modification: 60 root
apexes frozen in liquid nitrogen were ground to a fine powder and
extracted in 3 ml of ice-cold acetone. The homogenate was centrifuged at 10,000×g at 4°C for 20 min. The supernatant fractions were
collected. Aliquots of 0.5 ml of the samples were mixed with 1.5 ml of
a mixture of CHCl3 and CCl4 (1 : 3, v : v). Then, 2.5 ml of distilled
water was added. The mixture was centrifuged at 1,000×g for 1 min
and the water phase was collected for H2O2 determination. The reaction mixture contained 0.5 ml of buffer (0.2 M potassium phosphate
buffer solution, pH 7.8), 0.5 ml of water phase sample and 20 µl of
CAT (0.5 U) (to set controls) or the same units of inactive CAT protein (treatments). After incubation at 37°C for 10 min, 0.5 ml of
200 mM 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.
O2– determination
O2– production was determined according to the method of Able
et al. (1998). Briefly, 120 root apexes were ground with 3 ml of
50 mM Tris–HCl buffer (pH 7.5). The homogenate was centrifuged at
5,000×g at 4°C for 10 min. The reaction mixture (1 ml) contained
50 mM Tris–HCl buffer (pH 7.5), 0.5 mM XTT (sodium, 3′-{1-[phenylamino-carbonyl]-3,4-tetrazolium-bis(4-methoxy-6-nitro)
benzenesulfonic acid hydrate) and 50 µl of sample extracts. Corrections
were made for the background absorbance in the presence of 50 U of
SOD. The O2– production rate was calculated by an extinction coefficient of 2.16×104 M–1 cm–1.
Determination of lipid peroxidation
The level of lipid peroxidation in root tissues was determined in
terms of TBARS produced based on the method of Heath and Packer
(1968). Briefly, 60 root apexes frozen in liquid nitrogen were ground
in 3 ml of 0.1% trichloroacetic acid (TCA) solution. The homogenate
was centrifuged at 15,000×g for 10 min and 0.5 ml of the supernatant
fraction was mixed with 2 ml of 0.5% thiobarbituric acid (TBA) in
20% TCA. The mixture was heated at 90°C for 20 min, chilled on ice,
then centrifuged at 10,000×g for 5 min. The absorbance of the supernatant was measured at 532 nm. The value for non-specific absorption
at 600 nm was subtracted. The amount of TBARS was calculated by
using the extinction coefficient of 155 mM cm–1.
Ascorbate assay
Frozen root apexes were homogenized with 2 vols (w/v) of cold
5% (w/v) phosphoric acid at 4°C. The homogenate was centrifuged at
20,000×g for 15 min at 4°C. The supernatant was used for analysis of
ascorbate. The ascorbate pool was measured according to Zhang and
Kirkham (1996).
1922
NO reduction of Al toxicity and peroxidation
Enzyme assays
Frozen root apex tissues were homogenized with 3 vols (w/v) of
an ice-cold extraction buffer [50 mM Tris–HCl, pH 7.8, 1 mM EDTA,
1 mM MgCl2 and 1.5% (w/w) polyvinylpyrrolidone]. The homogenate was centrifuged at 15,000×g at 4°C for 20 min. The supernatant
was used as the crude extract for the assay of enzyme activities
Analysis of guaiacol POD capacity was based on the oxidation of
guaiacol using hydrogen peroxide (Upadhyaya et al. 1985). The reaction mixture contained 2.5 ml of 50 mM potassium phosphate buffer
(pH 6.1), 1 ml of 1% hydrogen peroxide, 1 ml of 1% guaiacol and 10–
20 µl of enzyme extract. The increase in absorbance at 420 nm was
read.
Analysis of CAT activity was determined spectrophotometrically
by the method described by Beers and Sizer (1952). The reaction mixture in a total volume of 3 ml contained 1 ml of 100 mM phosphate
buffer (pH 7.0), 0.4 ml of 200 mM H2O2 and 0.1–0.2 ml of enzyme
extract. The decrease in H2O2 was monitored at 240 nm.
Activities of SOD were assayed by measurement of its capacity
to inhibit the photochemical reduction of nitroblue tetrazolium (NBT)
(Beauchamp and Fridovich 1971). A 3 ml aliquot of reaction mixture
contained 50 mM phosphate buffer (pH 7.8), 10 mM methionine,
1.17 mM riboflavin, 56 mM NBT and 30 µl of enzyme extract. The
absorbance of the solution was measured at 560 nm.
For the LOX assay, root apexes were homogenized with 3 vols
(w/v) of 50 mM phosphate buffer (pH 6.0) (Surrey 1963). The
homogenate was centrifuged at 2,000×g for 15 min at 4°C. The supernatant was used for the enzyme assay. The assay mixture in a total volume of 1.5 ml contained 200 mM borate buffer (pH 6.0), 0.25%
linoleic acid, 0.25% Tween-20 and 50 µl of enzyme extract. The reaction was carried out at 25°C for 5 min and immediately stopped by
addition of 2 ml of absolute alcohol. The reaction mixture was centrifuged to make the solution optically clear. The absorbance of solution
was measured at 234 nm.
Protein in the samples was quantified by the method of Bradford
(1976), using bovine serum albumin.
Histochemical analyses
After treatments, intact roots of seedlings were rinsed with
0.5 mM CaCl2 (pH 4.5) several times, dried with filter papers and
immediately immersed in the following specific reagents. Histochemical detection of loss of plasma membrane integrity in root apexes was
performed by the method described by Yamamoto et al. (2001). For
analysis of root plasma membrane integrity, the roots were incubated
in 5 ml of Evans blue solution [0.025% (w/v) in 100 µM CaCl2, pH
5.6] for 30 min. Histochemical detection of lipid peroxidation was performed with Schiff’s reagent (Pompella et al. 1987). The roots were
incubated in Schiff’s reagent for 60 min. After that, the stained roots
were rinsed with a solution containing 0.5% (w/v) K2S2O5 (prepared in
0.05 M HCl) until the root color became light red. Al accumulation in
root apexes was detected by hematoxylin staining as described by
Polle et al. (1978). All of the roots stained with the specific reagents
indicated above were washed three times with a sufficient volume of
distilled water, observed under a light microscope (model SZH-ILLD;
Olympus, Tokyo, Japan) and photographed on color film (ASA 200,
Kodak Photo Film, USA).
Gel electrophoresis
The isoenzymes of SOD, CAT, POD and LOX were separated on
discontinuous polyacrylamide gels (stacking gel 5% and separating gel
10%) under non-denaturing conditions. Proteins were electrophoresed
at 4°C at 10 mA in the stacking gel followed by 15 mA in the separating gel.
SOD activity was determined in the gel as described by Pereira et
al. (2002). The gels were rinsed in water and incubated in the dark for
30 min at room temperature in an assay mixture containing 50 mM
potassium phosphate buffer (pH 7.8), 1 mM EDTA, 0.05 mM riboflavin, 0.1 mM NBT and 0.3% N,N,N′′,N′′-tetramethylethylenediamine
(TEMED). After that, the gels were rinsed with water and exposed on
a light box for 10 min at room temperature and the colorless bands of
SOD activity in a purple-stained gel were visible.
To visualize CAT activity, the gels were first incubated in 0.3%
H2O2 for 20 min. After 1 min of gentle washing in water, they were
developed in a 1% (w/v) ferricyanide and 1% ferric chloride solution
(w/v) for 15 min (Woodbury et al. 1971). The gels with peroxidase isoforms were stained for 20 min in 0.2 M acetate buffer (pH 5.5) with
0.68 mM benzidine, 5.5 mM guaiacol, 0.63 mM MnCl2 and 5 mM
H2O2 (Janda et al. 1999). For detection of LOX isoenzymes, the gels
were stained for at least 6 h in a 50 mM potassium phosphate buffer
(pH 6.0) solution containing 0.1% linoleic acid, 0.02% o-dianisidine
and 6.6% ethanol (Funk et al. 1985).
Statistical analysis
Each result shown in the tables and figures was the mean of at
least three replicated treatments. The significance of differences
between treatments was statistically evaluated by SD and Student’s ttest methods.
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(Received June 12, 2005; Accepted September 11, 2005)