Botanical Studies (2006) 47: 137-144.
*
Corresponding author: E-mail: zmyang@njau.edu.cn; Tel:
86-25-84395057.
Negative regulation of aluminum-responsive citrate
efflux from roots of Cassia tora by an anion channel
antagonist
Yao Jua XUE, Fu Liang XIE, and Zhi-Min YANG*
Department of Biochemistry and Molecular Biology, College of Life Science, Nanjing Agricultural University, Nanjing,
210095, P.R. China
(Received August 19, 2005; Accepted November 4, 2005)
ABSTRACT.
In a search for the regulatory basis of citrate efflux, three anion channel antagonists,
anthracene-9-carboxylic acid (A9C), niflumic acid (NIA), and phenylglyoxal (PG) were examined for their
effects on the aluminum-responsive citrate release. Treatment with 8 £gM A9C for 9 h resulted in a 60% de-
crease in Al-responsive citrate release in Cassia tora. However, no inhibitory effects of NIA and PG on the
citrate efflux were found. Because Al-induced citrate efflux was linked to the plant tolerance to Al toxicity,
the root growth and Al accumulation were measured at the same time. Treatment of the seedlings with 20
£g
M Al alone inhibited root elongation, but simultaneous incubation with A9C resulted in an additional inhibi-
tion of root growth and Al accumulation. By contrast, NIA and PG exerted no effects on root growth. Three
antagonists examined in the present study had no effect on the activities of citrate synthase (CS, EC 4.1.3.7)
and aconitase (Aco, EC 4.2.1.3), or on the citrate accumulation in the Al-treated root tips, suggesting that
the inhibition of Al-responsive citrate efflux by the anion channel antagonists was not involved in citrate
metabolism. Further, we examined the interaction between A9C and salicylic acid (SA), which has been
found to promote the Al-responsive citrate efflux and thereby Al tolerance in C. tora. Treatment with A9C
at 8
£g
M exerted a negative effect on the SA promotion of Al-responsive citrate efflux. We also found that
the cancellation of SA effect by A9C on citrate efflux caused an additional inhibition of root growth in the
presence of Al. Taken together, we speculate that a putative A9C-sensitive anion channel may be responsible
for the mediation of Al-activated citrate efflux in the roots of Cassia tora.
Keywords: Aluminum; Anion channel; Antagonist; Citrate exudation.
INTRODUCTION
Acid soils make up about 40% of the arable land of
the world (Foy et al., 1978). In China, they cover 21%
(Wang and Lin, 1993). The solubility of aluminum in
neutral and alkaline soils is low. However, in acidic soils
aluminum becomes soluble, and the concentration of
free Al
3+
increases considerably, resulting in rapid root
growth inhibition, the most easily recognized symptom
of aluminum toxicity (Kochian, 1995; Tesfaye et al.,
2001). So far, a variety of mechanisms for Al toxicity to
plants have been proposed (Barcelo and Poschenrieder,
2002; Kochian et al., 2004). Meanwhile, mechanisms for
Al tolerance in a wide range of plant species have also
been identified (Matsumoto, 2000). Among them, the
exudation of organic acids from roots and chelation of Al
with organic acids outside cells is the most often advanced
(Barcelo and Poschenrieder, 2002). Citrate, malate, and
oxalate are organic acids commonly released in plants
upon Al exposure (Tesfaye et al., 2001; Yang et al., 2001;
Anoop et al., 2003), and these can chelate Al
3+
, thus pre-
venting it from entering the root cells. Although a number
of tolerant plant species exhibiting the exudation of
organic acids upon Al exposure have been identified, the
regulatory mechanism relating to physiological processes
remains to be elucidated.
Increasing evidence has shown that exudation of
organic acids might occur through an Al-activated
(Ryan et al., 1997; Kollmeier et al., 2001) or phosphorus
deficiency-induced (Zhang et al., 2004) anion channel
localized in the plasma membrane of root apexes. Several
lines of study have indicated that the anion channels are
closely associated with citrate or malate efflux (Ryan et
al., 1997; Kollmeier et al., 2001; Pineros and Kochian,
2001; Zhang et al., 2001). Since the relationship between
Al-activated opening of anion channels and permeable
organic acids has been established, the anion transport
systems are considered likely candidates mediating the Al-
responsive efflux of organic acids (Pineros et al., 2002).
PHYSIOLOGY
pg_0002
138
Botanical Studies, Vol. 47, 2006
However, these channels may be different in type and
nature (Ryan et al., 1997; Kollmeier et al., 2001; Pineros
et al., 2001; Zhang et al., 2001; Zhang et al., 2004). For
example, in wheat the Al-activated channels required
extracellular Al
3+
to maintain channel activity (Ryan et
al., 1997) while in maize root cortical cells, the opening
of Al-activated channels did not require a continuous Al
exposure for their active state (Kollmeier et al., 2001).
The suggestion has been made that the efflux of organic
acids may be mediated by different transporters (Pi-
neros et al., 2001). Meanwhile, some researchers have
employed pharmacological or physiological antagonists
to investigate the properties of anion transporters in plants
(Yamamoto et al., 1997; Mithofer et al., 2001). By using
antagonists of anion channels, Ryan et al. (1995) were
able to demonstrate that the Al-responsive efflux of malate
from tolerant wheat plant was blocked by niflumic acid.
However, in another Al tolerant plant species, buckwheat,
no inhibition of oxalate release by niflumic acid was
observed (Zheng et al., 1998).
Cassia tora possesses an Al tolerance mechanism
responsible for Al exclusion by citrate release in
roots (Ishikawa et al., 2000).
However, the regulatory
process of Al-responsive citrate efflux is still unknown.
Recently, we have identified several key enzymes in-
volved in the pathway of citrate synthesis (Yang et al.,
2004) and an intermediate component that is involved
in the modulation of citrate release (Yang et al., 2003).
In this report, we further investigated the physiological
effects of three different anion channel antagonists,
anthracene-9-carboxylic acid (A9C), niflumic acid (NIA)
and phenylglyoxal (PG) on the Al-responsive citrate
efflux. In addition, we have identified the responses
of citrate synthase and aconitase activities and root
citrate accumulation, which are closely linked to citrate
metabolism. The outcome of the present study may help
us in understanding the underlying mechanisms for the
regulation of Al-induced citrate efflux and their relation to
Al tolerance of the plant species.
MATERIALS AND METHODS
Plant material and treatment
Seeds of Cassia tora L. were selected and soaked in
distilled water overnight and distributed on a net tray.
When seeds were germinated, seedlings were transferred
to an aerated solution containing 0.5 mM CaCl
2
(pH 4.5)
and grown at 22¢XC for 3 days, with a light intensity of
100 £gmol m
-2
s
-1
and 14 h photoperiod. The solution was
changed daily. After that, the seedlings were transferred
to plastic containers containing 0.5 mM CaCl
2
(pH 4.5)
solution with 20 £gM Al and/or different concentrations
of anion-channel antagonists, anthracene-9-carboxylic
acid (A9C), niflumic acid (NIA), and phenylglyoxal (PG).
The concentrations of antagonists were 0, 2, 4, 8 and 12
£gM, and the SA concentration was 0.5 £gM, where the Al-
responsive citrate efflux is optimally stimulated (Yang et
al., 2003). Plants were harvested, and 30 root tips (for one
treatment) of 0.5 cm were collected. Harvested root tips
were immediately frozen in liquid nitrogen and stored at
-80
¢X
C for further analysis. Both antagonists and salicylic
acid were prepared in the 0.5 mM CaCl
2
solution.
Collection of root exudates and citrate
measurement
Following the treatments, seedlings were removed from
the treatment solutions. The remaining solutions contain-
ing root exudates were colleted, frozen, and lyophilized
(Yang et al., 2003). The dried exudates were dissolved in 5
mL distilled water and passed through a cationic exchange
resin column (16 mm ¡Ñ 25 cm) containing 4 g of Dowex
5 W ¡Ñ 8 (100-200 mesh, H
+
form). The eluates were
freeze-dried again, and the residue was finally dissolved in
1 mL distilled water for citrate measurement.
Citrate concentrations in exudates and root tips were
measured according to the modified method described by
Yang et al. (2004). Root tips were ground in 1 mL solution
of ice-cold 0.6 N perchloric acid, and the homogenate was
centrifuged at 15000 g at 4¢XC for 5 min. The supernatant
was collected and neutralized with 80 £gL of K
2
CO
3
(69%,
w/v). The neutralized solution was centrifuged at 15000 g
at 4¢XC for 5 minand then used to determine citrate content.
Usually, 300 £gL of sample solution was incubated with
120 £gL of buffer (1 M Tris-HCl, pH 7.8), 15 £gL of 10 mM
NADH, 2 £gL of a lactate dehydrogenase (1.25 units)/ma-
late dehydrogenase (6 units) mixture, and 650 £gL distilled
water. After a stable reading, 10 £gL citrate lyase (0.5 unit)
was added, and the decline in A
340
due to oxidation of
NADH was monitored.
Enzyme assay
Root tips were homogenized in iced-cold 50 mM
HEPES-NaOH buffer (pH 7.5) containing 5 mM MgCl
2
,
5 mM EDTA, 10% (v/v) glycerol, and 0.1% (v/v) Triton
X-100. The activity of citrate synthase was spectrophoto-
metrically assayed by recording a decrease in acetyl CoA
at 412 nm for 4 min. The reaction mixture contained 100
mM Tris-HCl buffer (pH 8.0), 5 mM MgCl
2
, 0.5 mM 5,
5-dithio-bis-2-nitrobenzoic acid, 0.2 mM acetyl CoA,
and 1 mM oxalacetic acid (Yang et al., 2003). Aconitase
activity was spectrophotometrically measured at 240 nm
by following the formation of cis-aconitase from isocitrate
in the solution containing 75 mM Tris-HCl (pH 7.6), 30
mM citric acid (Kennedy et al., 1983). The total protein
content in enzyme extracts was determined by the method
of Bradford using bovine serum albumin as a standard
(Bradford, 1976).
Determination of Al content in root tips
The collected root tips were placed in a 1.5 mL Eppen-
dorf tube, and to it 1 mL of 2 N HCl was added. Al in root
apexes was extracted and measured by a graphite furnace
atomic absorption spectrophotometer (180-80 Hitachi, To-
kyo).
pg_0003
XUE et al. ¡X Antagonist inhibition of Al-induced citrate efflux
139
Statistical analysis
In citrate efflux experiments, 70 seedlings were used for
a treatment. For measurement of root elongation, a mini-
mum of 15 seedlings was sampled for a treatment. Each
result shown in tables and figures was the mean of at least
three replicated treatments. The significance of differences
between treatments was statistically evaluated by standard
deviation and Student¡¦s t-test methods.
RESULTS
Effect of anion-channel antagonists on Al-
responsive citrate efflux, Al content and root
growth
Citrate in the root-bathing solution without Al was
undetectable based on the method used in the present
study (Table 1). Also, application of A9C, NIA or PG in
the absence of Al had no effect on citrate efflux (Table
1). Treatment of seedlings with 20 £gM Al for 9 h induced
a high level of citrate release from roots. However,
application of 8 £gM A9C for 9 h resulted in a 60% smaller
release. No inhibitory effects of either NIA or PG were
observed on the citrate efflux from Al-treated roots.
Since efflux of organic acids confers Al tolerance in
plants, a decrease in citrate efflux after treatment with A9C
might cause decline in Al exclusion from root tips. As
shown in Table 1, Al content in root tips treated simultane-
ously with 20 £gM Al and 8 £gM A9C increased by 1.45%
over 20 £gM Al alone. Nevertheless, neither NIF nor PG
had any effect on the accumulation of Al.
Because inhibition of root growth is the primary
response to Al toxicity, the root elongation was measured.
Although treatment with 20 £gM Al alone was able to
inhibit root growth, simultaneous treatment with A9C
caused an additional inhibition of root growth, with a 33%
decrease as compared to the control (Table 1). By contrast,
neither NIF nor PG had any significant effects on root
growth.
The significant impact of A9C on both citrate release
and root growth was displayed in a dose-dependent
manner in the presence of Al (Figure 1). For instance,
A9C (12 £gM) decreased Al-induced citrate efflux by 67%.
Likewise, A9C-induced inhibition of citrate efflux was
observed in a time-course study, in which the inhibition
Table 1. Effects of anion channel antagonists, A9C, NIA and PG on the root citrate efflux, Al content and the root elongation of
Cassia tora. Seedlings were exposed to 0.5 mM CaCl
2
(pH 4.5) solutions containing 8 £gM antagonists with 0 or 20 £gM Al for 9 h.
Values are the means ¡Ó SD (n=3).
Treatment
Citrate efflux
(nmol plant
-1
9 h
-1
)
Al content
(nmol tip
-1
)
Root elongation
(cm)
-Al
¡V
0.11¡Ó0.01
0.81¡Ó0.06
-Al+A9C
¡V
0.12¡Ó0.02
0.79¡Ó0.06
-Al+NIF
¡V
0.09¡Ó0.01
0.83¡Ó0.07
-Al+PG
¡V
0.10¡Ó0.01
0.73¡Ó0.07
+AL
1.47¡Ó0.15
1.46¡Ó0.20
0.42¡Ó0.04
+Al+A9C
0.59¡Ó0.07
2.11¡Ó0.18
0.28¡Ó0.03
+Al+NIF
1.52¡Ó0.06
1.37¡Ó0.09
0.44¡Ó0.03
+Al+PG
1.51¡Ó0.09
1.41¡Ó0.14
0.39¡Ó0.04
"
¡V
" Undetectable.
Figure 1. Effect of A9C on the citrate efflux and root elonga-
tion of C. tora in the presence of Al. Seedlings were exposed
to the 0.5 mM CaCl
2
(pH 4.5) solutions containing various
concentrations of A9C with 20 £gM Al for 9 h. Vertical bars rep-
resent standard deviation of the mean (n=3). Asterisks indicate
that mean values are significantly different between the A9C
treated and control seedlings (0 £gM A9C) (p < 0.05).
pg_0004
140
Botanical Studies, Vol. 47, 2006
in the Al-treated roots exposed to 8 £gM A9C for 9 h was
threefold greater than the controls (20 £gM Al alone) (Fig-
ure 2). In a parallel experiment, the correlation between
the Al-responsive citrate efflux and inhibition of root
growth in the presence of A9C was observed (Figures 1
and 2).
A 3-h pulse of Al treatment experiment was performed
to check the possibility that A9C interferes with Al
3+
action in the treatment solution, thus resulting in a de-
creased citrate release. The result showed that the Al-
activated citrate efflux was blocked by the late presence
of 8 £gM A9C (Table 2), indicating that the interference of
A9C with Al
3+
action in the solution was of minimal im-
portance in the experiment.
Effect of anion-channel antagonists on the
citrate content and enzyme activities in root
tips
Treatment with Al at 20 £gM stimulated the citrate
accumulation and citrate synthase activity, but caused
decreases in the aconitase activity in root tips as compared
with controls (Table 3). However, application of A9C,
NIF and PG had no marked effect on the Al-responsive
citrate content or the activities of enzymes. In another
experiment, citrate content and activities of the enzymes
in the root tips were measured as a function of A9C
concentrations in the growth medium (Figure 4). The
citrate content (Figure 3) and citrate synthase or aconitase
activities (Figure 4) underwent no marked changes even
though the concentration of A9C was raised up to 12 £gM
in the treatment medium.
Effect of A9C on SA-promoted Al-responsive
citrate efflux and root growth
In our previous study, salicylic acid was found to
promote the Al-responsive citrate efflux in C. tora (Yang et
al., 2003). This finding prompted us to investigate whether
A9C affected the SA-promotion of citrate exudation. At
first, seedlings were pretreated with 8 £gM A9C for 3 h.
Figure 2. Effect of A9C on the
pattern of
Al-induced citrate
efflux in C. tora over time. Seedlings were exposed to 0.5 mM
CaCl
2
(pH 4.5) solutions containing 20 £gM Al (filled circles)
alone or 20 £gM Al + 8 £gM A9C (open circles). Root exudates
were collected every 3 h after the initiation of treatments. Verti-
cal bars represent standard deviation of the mean (n=3). As-
terisks indicate that the mean values are significantly different
between the A9C treated and control seedlings (0 £gM A9C) (p <
0.05).
Table 2. Effect of A9C on the citrate efflux from roots of
Cassia tora after a 3-h pulse of Al treatment. Seedlings were
first exposed to 0.5 mM CaCl
2
(pH 4.5) solution containing 20
£g
M Al for 3 h, and then rinsed with 0.5 mM CaCl
2
(pH 4.5)
solution. Afterwards, the seedlings were transferred to the Al-
free solution (0.5 mM CaCl
2
, pH 4.5) or the solution containing
only 8
£g
M A9C and incubated for 7 h. Values are the means ¡Ó
SD (n=3).
Treatment
Citrate efflux
Initial 3 h Subsequent 7 h nmol plant
-1
7 h
-1
+Al
-Al
1.15¡Ó0.03 (100%)
+Al
-Al+A9C 0.42¡Ó0.05 (36.5%)
Table 3. Effects of anion channel antagonists, A9C, NIF and PG on the citrate content and citrate synthase and aconitase activities
in the root tips of Cassia tora. Seedlings were exposed to 0.5 mM CaCl
2
(pH 4.5) solutions containing 8 £gM antagonists with 0 or
20 £gM Al for 9 h. Values are the means ¡Ó SD (n=3).
Treatment
Citrate content
(nmol tip
-1
)
Citrate synthase activity
(units mg
-1
protein)
Aconitase activity
(units mg
-1
protein)
-Al
0.97¡Ó0.09
0.12¡Ó0.01
0.11¡Ó0.01
-Al+A9C
1.02¡Ó0.07
0.11¡Ó0.01
0.10¡Ó0.02
-Al+NIF
1.13¡Ó0.09
0.12¡Ó0.01
0.11¡Ó0.01
-Al+PG
0.99¡Ó0.06
0.11¡Ó0.02
0.12¡Ó0.02
+AL
1.58¡Ó0.12
0.16¡Ó0.01
0.06¡Ó0.01
+Al+A9C
1.48¡Ó0.16
0.17¡Ó0.02
0.05¡Ó0.01
+Al+NIF
1.46¡Ó0.09
0.15¡Ó0.01
0.06¡Ó0.01
+Al+PG
1.65¡Ó0.17
0.16¡Ó0.02
0.06¡Ó0.01
pg_0005
XUE et al. ¡X Antagonist inhibition of Al-induced citrate efflux
141
They were then treated with 20 £gM Al alone and 20 £gM Al
plus 5 £gM SA. We found that A9C- pretreated seedlings
excluded sufficient amounts of citrate after exposure to 20
£gM Al for 9 h (Figure 5). However, the citrate exudation
from either Al-treated or Al+SA-treated seedlings was
significantly suppressed by the pretreatment with A9C.
To confirm that A9C independently acted against the
effect of SA, a time-course experiment was performed. We
first allowed the two sets of seedlings to receive the same
treatment with both 20 £gM Al and 5 £gM SA for 9 h. Then,
one set of the seedlings was subjected to the treatment
with 8 £gM A9C only for 6 h (Figure 6). The citrate efflux
was analyzed. Control seedlings exhibited a progressive
increase in citrate efflux after activation of Al and SA
while seedlings treated with A9C for 6 h showed an im-
mediate inhibition of citrate efflux. These results indicate
a strong effect of A9C on the SA action in Al-responsive
citrate efflux.
Exudation of organic acids confers Al resistance
in many Al-tolerance plant species (Barcelo and
Poschenrieder, 2002). An increase in citrate efflux due
to SA application was observed in C. tora (Yang et al.,
2003). As a result, an increase in root elongation was
found. After demonstrating a negative effect of A9C on
the SA-regulated citrate efflux, it could be expected that
Figure. 3. Effect of A9C on the citrate content in root tips of
Cassia tora in the presence of Al. Seedlings were exposed to 0.5
mM CaCl
2
(pH 4.5) solutions containing various concentrations
of A9C and 20 £gM Al for 9 h. Values are the means ¡Ó SD (n=3).
Figure 4. Effects of A9C on the activities of citrate synthase (a)
and aconitase (b) in the root tips of Cassia tora in the presence
of Al. Seedlings were exposed to 0.5 mM CaCl
2
(pH 4.5) solu-
tions containing various concentrations of A9C and 20 £gM Al
for 9 h. Values are the means ¡Ó SD (n=3).
ƒ×
Figure 5. A9C inhibition of Al-induced citrate efflux response
to salicylic acid. Seedlings were first exposed to 0.5 mM CaCl
2
(pH 4.5) solution containing 8 £gM A9C for 3 h, and then trans-
ferred to the solution (0.5 mM CaCl
2
, pH 4.5) containing 20 £gM
Al alone and 20 £gM Al + 5 £gM SA for 9 h. Values are the means
¡Ó SD (n=3). Asterisks indicate that the mean values are signifi-
cantly different between seedlings treated with 20 £gM Al in the
presence and absence of 5 £gM SA (p < 0.05).
pg_0006
142
Botanical Studies, Vol. 47, 2006
application of A9C removed the effect of SA-stimulated
root elongation. It was found that the root growth was
strongly inhibited by the pretreatment with A9C even
though the SA was present in the growth medium (Figure
7).
DISCUSSION
In acidic soils, Al-induced exudation of organic acids
from plants protects plant roots from Al
3+
toxicity (Ko-
chian, 1995). In the present study we confirmed such
a response by testing the inhibitory effect of an anion
channel antagonist, namely anthracene-9-carboxylate,
which is frequently used for animal electrophysiology
(Chao and Mochizuki, 1992; Tamai et al., 2000; Kaya et
al., 2002; Parai and Tabrizchi, 2002). We found a strong
negative effect of A9C on the Al-responsive citrate
efflux from roots of Cassia tora. Such antagonists are
useful because they may provide a basic approach to
understanding the nature of anion transporters in plants.
Application of anion channel antagonists in identifying
different ionic transporters in plant cells has been
reported (Yamamoto et al., 1997; Mithofer et al., 2001).
For example, by using several antagonists, ion-transport
systems such as H
+
-ATPase and carriers and anion
channels associated with plasma or tonoplast membrane
were characterized (Ryan et al., 1995; Yamashita et al.,
1996). However, their function and specificity in higher
plants remains to be elucidated.
Exudation of organic acids is considered to occur
through Al-activated anion channels located on the plasma
membrane of root tip cells in some plant species such as
wheat (Ryan et al., 1997), maize (Kollmeier et al., 2001;
Pineros et al., 2001) and white lupin (Zhang et al., 2004).
The response of these plants to Al differs greatly among
species, and even genotypes within a species vary. These
characteristics may bring about great changes in the types
or the amount of released organic acids (Ryan et al.,
1997). A good understanding of regulatory mechanisms
for organic acid efflux by an anion channel requires more
evidence from a variety of plant species. We examined
the effect of anion channel antagonists on Al-responsive
citrate efflux from roots of C. tora. A9C has been shown
to be effective against citrate efflux (Table 1, Figure 2).
These results suggest that Al-responsive citrate efflux may
be associated with the activation of a putative anion chan-
nel responsible for the citrate release.
Because exudation of organic acids confers Al tolerance
in C. tora (Yang et al., 2003) and other Al-tolerant plant
species (Miyasaka et al., 1991; Zheng et al., 1998; Yang
et al., 2001), the inhibition of citrate efflux by A9C would
affect the Al exclusion from roots and thus cause an ad-
ditional inhibition of root growth. Our results showed that
the simultaneous treatment with 8 £gM A9C for 9 h led to
a more than 30% decrease in root elongation over 20 £gM
Al treatment alone (Table 1). Also, treatment with A9C
caused relatively higher accumulation of Al in root tips
(Table 1). It was noted that A9C alone was not able to
exert toxicity on the roots, evidenced by the data on roots
treated with 8 £gM A9C showing no significant difference
from the controls (Table 1). To exclude the possibility that
A9C added to the root-bathing medium might trick Al
3+
,
thus reducing its activity or toxicity to roots, a 3-h pulse
experiment was performed. Our data indicate that A9C
F igure 6. A9C inhi bi tion of Al-res pons ive c itrate effl ux
respons e to s ali cylic a cid over ti me. S eedl ings were fi rs t
incubated in 0.5 mM CaCl
2
(pH 4.5) solution containing 20 £gM
Al + 5 £gM SA for 9 h, and then transferred to the 0.5 mM CaCl
2
(pH 4.5) solution containing 0 £gM (filled circles) and 8 £gM
A9C (open circles) for the continuous exudation of citrate for 6
h. Root exudates were collected every 3 h after the initiation of
treatments. Values are the means ¡Ó SD (n=3). Asterisks indicate
that the mean values are significantly different between the two
treatments.
Figure 7. Effect of A9C on SA promotion of root growth in the
presence of Al. Seedlings were first exposed to 0.5 mM CaCl
2
(pH 4.5) solution containing 8 £gM A9C for 3 h before being
transferred to the solution (0.5 mM CaCl
2
, pH 4.5) containing
0, 20 £gM Al and/or 5 £gM SA for 9 h. Values are the means ¡Ó
SD (n=3). Asterisks indicate that the mean values of seedlings
treated with 20 £gM Al in the presence of of 5 £gM SA differ sig-
nificantly from those treated in its absence (p < 0.05).
pg_0007
XUE et al. ¡X Antagonist inhibition of Al-induced citrate efflux
143
was able to independently affect Al-responsive citrate
efflux (Table 3).
Since Al-responsive efflux of citrate in Cassia tora
(Ishikawa et al., 2000; Yang et al., 2004) and several other
plant species (Miyasaka et al., 1991; Pineros et al., 2002)
is associated with the activation of citrate metabolism,
it becomes important to determine the activities of en-
zymes closely linked to citrate synthesis and degradation.
Our results demonstrate that neither citrate synthase nor
aconitase activities were affected by the tested anion
channel antagonists in this study. We also measured the
citrate content and did not find a marked change in the
level of citrate in the root tips. Therefore, we conclude
that the action of these antagonists was not involved in
the mediation of the citrate metabolism in the presence of
aluminum.
Earlier study with in C. tora showed that exogenous ap-
plication of SA at 5 £gM promoted the Al-responsive citrate
efflux, and this effect was correlated with decreases in Al-
induced inhibition of root growth as well as in Al accu-
mulation in root tips (Yang et al., 2003). These data could
allow us to analyze whether the inhibitor A9C could block
the effect of SA on the Al-responsive citrate efflux. Nearly
70 percent of citrate efflux was inhibited by the addition
of 8 £gM A9C to the medium in the presence of Al and
SA as compared to the control (Al and SA only) (Figure
5). However, it caused no difference in citrate efflux
between the +Al+A9C- and +Al+A9C+SA-treated plants.
This result suggests that A9C can abolish the release of
citrate from both Al-induction and SA-promotion in root
tips. To confirm the A9C action, a similar experiment for
root growth was performed. The root elongation in the
presence of Al and SA decreased by about 50 percent due
to addition of 8 £gM A9C when compared with control
(Figure 7). Although the process by which A9C acted on
SA effects is not clear in the present study, A9C might
possibly block the opening of a putative citrate permeable
anion channel stimulated by SA. More detailed studies are
required to characterize the anion channels of the plasma
membrane of C. tora.
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