Download (Vigna umbellata) by Restoring Plasma

Plant Cell Physiol. 48(1): 66–73 (2007)
doi:10.1093/pcp/pcl038, available online at www.pcp.oxfordjournals.org
ß The Author 2006. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.
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Magnesium Enhances Aluminum-Induced Citrate Secretion in Rice Bean Roots
(Vigna umbellata) by Restoring Plasma Membrane Hþ-ATPase Activity
Jian Li Yang
1, 4
, Jiang Feng You
2, 1, 4
, Ya Ying Li 1, Ping Wu
3
and Shao Jian Zheng
3,
*
1
College of Environmental and Resource Sciences, Zhejiang University, Hangzhou 310029, PR China
2
College of Plant Science, Jilin University, Changchun 130062, PR China
3
State Key Laboratory of Plant Biochemistry and Physiology, College of Life Sciences, Zhejiang University, Hangzhou 310058, PR China
relies on the efflux of organic anions (malate, citrate and
oxalate) from the roots (Ryan et al. 2001).
The alleviation of Al toxicity by cations has been
reported many times. In general, the effectiveness with
which cations alleviate Al stress depends on their concentration and valency, with trivalent cations being more
effective than divalent cations, which are more effective
than monovalent cations (Kinraide et al. 1994). A number
of detailed studies have demonstrated that several factors
are likely to be contributing to this response, including
changes to the ionic strength of the solution and changes
to the surface charge density of the root cell membranes
which affect the electrostatic interaction between free ions
and the membrane surfaces (Kinraide 2003, Kinraide et al.
2004). One cation that has been shown to interact with
Al toxicity in a range of different ways is the essential
macronutrient magnesium (Mg). For example, Al can
induce Mg deficiency in many plant species including oats
(Grimme 1983), sorghum (Keltjens 1988) and ryegrass
(Rengel and Robinson 1989). Increasing the external Mg
concentration alleviates the Al-induced Mg deficiency as
well as alleviating the detrimental effects of Al on plant
growth (Keltjens and Tan 1993, Kinraide et al. 2004).
Furthermore, the overexpression of genes encoding Mg
transporter proteins enhances the Al resistance in yeast
cells by overcoming the Al-induced inhibition of Mg uptake
(MacDiarmid and Gardner 1996, MacDiarmid and
Gardner 1998).
The mechanism by which Mg ameliorates Al toxicity
in plants will, in part, rely on changes to the electrostatic
interactions occurring at the root cell surfaces as explained
above, but other biochemical and physiological mechanisms
also appear to be operating in some species. For instance,
Silva et al. (2001a, 2001b, 2001c) demonstrated that
micromolar concentrations of Mg in the Al treatment
solution could stimulate citrate efflux from soybean roots
and thus alleviate the inhibition of root elongation by Al.
How Mg enhances the Al-induced citrate efflux in soybean
remains unclear.
In our previous report, we found that Al could induce
citrate release from the roots of rice bean [Vigna umbellata
We demonstrated that magnesium (Mg) can alleviate
aluminum (Al) toxicity in rice bean [Vigna umbellata
(Thunb.) Ohwi & Ohashi] more effectively than is expected
from a non-specific cation response. Micromolar concentrations of Mg alleviated the inhibition of root growth by Al but
not by lanthanum, and neither strontium nor barium at the
micromolar level alleviates Al toxicity. Aluminum also
induced citrate efflux from rice bean roots, and this response
was stimulated by inclusion of 10 mM Mg in the treatment
solution. The increase in the Al-induced citrate efflux by Mg
paralleled the improvement in root growth, suggesting that
the ameliorative effect of Mg might be related to greater
citrate efflux. Vanadate (an effective Hþ-ATPase inhibitor)
decreased the Al-induced citrate efflux, while addition of Mg
partly restored the efflux. Mg addition also increased the
activity of Al-reduced plasma membrane Hþ-ATPase, as well
as helping to maintain the Mg and calcium contents in root
apices. We propose that the addition of Mg to the toxic
Al treatment helps maintain the tissue Mg content and the
activity of the plasma membrane Hþ-ATPase. These changes
enhanced the Al-dependent efflux of citrate which provided
extra protection from Al stress.
Keywords: Aluminum resistance — Citrate secretion —
Magnesium — Plasma membrane Hþ-ATPase.
Abbreviations: Al, aluminum; Ba, barium; BTP,
1,3-bis(tris[hydroxymethyl]-methylamino) propane; Ca, calcium;
La, lanthanum; Mg, magnesium; Sr, strontium.
Introduction
Aluminum (Al) toxicity is one of the most serious
yield-limiting factors in acid soils (Kochian et al. 2004).
At micromolar concentrations, Al causes rapid inhibition of
root elongation in many species, which ultimately results
in the decrease of water and nutrient uptake in plants.
Many plants are able to resist or tolerate the harmful effects
of Al stress better than others. The mechanism of resistance
that has now been reported in a wide range of plant species
4
These authors contributed equally to this work.
*Corresponding author: E-mail, [email protected]; Fax, þ 86-571-88206438.
66
Magnesium enhances citrate efflux by restoring plasma membrane Hþ-ATPase activity
Ameliorative effect of Mg on Al and La toxicity
Exposure of rice bean seedlings to 50 mM AlCl3 in a
simple Ca solution for 24 h inhibited root elongation by
68% (Fig. 1). Addition of 10 mM MgCl2 to this toxic Al
solution improved root growth by approximately 60%.
Increasing the concentration to 20 and 50 mM Mg gave no
further amelioration of growth. However, in the absence
of Al, the 10 mM MgCl2 treatment did not increase root
growth above that of controls (Fig. 1).
The amelioration of Al toxicity in rice bean by Mg was
confirmed in a time course experiment. Roots of Al- and
AlþMg-treated seedlings grew significantly faster and,
after 24 h, the roots were 42-fold longer than those of the
Al-treated seedlings (Fig. 2).
Root elongation of rice bean was also inhibited by
50 mM LaCl3. However, the addition of micromolar
concentrations of MgCl2 to these toxic solutions was
unable to alleviate the inhibition of root growth, indicating
that the amelioration of Al toxicity by Mg is not a general
ameliorative response but is specific to Al stress (Fig. 3).
Effect of Sr and Ba on Al toxicity
Addition of either 10 mM SrCl2 or 10 mM BaCl2 had no
significant effect on root elongation of rice bean irrespective
of the absence or presence of Al in the 0.5 mM CaCl2
solution (Fig. 4).
Relative root elongation (%)
Results
120
Accumulation of Al, Ca and Mg in root apices
Exposure of rice bean seedlings to 50 mM Al for 24 h
resulted in Al uptake by the roots and a decrease in the Mg
and Ca contents of the root apices by 42 and 20%,
−AI
+AI
80
b
b
b
60
c
40
20
10
0
0
10
20
Mg concentration (µM)
50
Fig. 1 Mg dose effect on Al-induced root elongation inhibition.
Two-day-old seedlings were subjected to 0.5 mM CaCl2 solution
(pH 4.5) containing either 0 or 50 mM AlCl3 with the addition of
different concentrations of MgCl2. The tap root in each seedling
was measured with a ruler before and after the 24 h treatment. Data
are means SD (n ¼ 6). Different letters indicate that the values are
significantly different at P50.05.
8
+AI
6
+AI+Mg
4
*
2
0
Effect of Mg on Al-induced citrate efflux
Addition of 50 mM Al to the test solution induced
citrate efflux from the roots of rice bean seedlings after
a delay of 4 h (Fig. 5). Citrate efflux was significantly
enhanced when 10 mM MgCl2 was added with the Al
treatment, but raising the MgCl2 concentration up to 50 mM
resulted in no further increases (Figs. 5, 6). No citrate efflux
was detected in the absence of Al whether MgCl2 was
present or not (data not shown). Twenty-four hours were
required for amelioration of Al-related inhibition of root
growth (Fig. 2) and an increase in citrate efflux (Fig. 5).
a
a
100
0
Root elongation (mm)
(Thunb.) Ohwi & Ohashi] after a lag of several hours
between Al addition and the initiation of citrate efflux
(Yang et al. 2006). We have investigated this response
further and demonstrate that micromolar concentrations
of Mg enhance the Al-induced citrate efflux in rice bean
roots. This response is associated with Mg-dependent
up-regulation of plasma membrane Hþ-ATPase activity.
67
0
4
8
12
16
Treatment duration (h)
20
24
Fig. 2 Time course of Mg alleviation of Al-induced root
elongation inhibition. Two-day-old seedlings were subjected to
0.5 mM CaCl2 solution (pH 4.5) containing 50 mM AlCl3 with or
without 10 mM MgCl2. The tap root in each seedling was measured
with a ruler periodically. Data are means SD (n ¼ 6). The asterisk
represents a statistically significant difference between þAl and
þAl þ Mg treatments (P50.05).
respectively (Table 1). Including 10 mM Mg in the Al
treatment lowered Al accumulation by 10% and reduced
the Al-dependent decrease in Mg content from 42 to 17%.
The addition of Mg treatment to the Al treatment also
prevented the decrease in Ca content of the root tissues
(Table 1).
Magnesium enhances citrate efflux by restoring plasma membrane Hþ-ATPase activity
68
25
a
a
−La
100
Citrate efflux
(µmol g−1 dry weight)
Relative root elongation (%)
120
+La
80
60
40
b
b
10
20
0
0
Mg concentration (µM)
50
b
20
0
b
+AI
20
+AI+Mg
*
15
10
5
0
10
Fig. 3 Mg dose effect on La-induced root elongation inhibition.
Two-day-old seedlings were subjected to 0.5 mM CaCl2 solution
(pH 4.5) containing either 0 or 50 mM LaCl3 with the addition of
different concentrations of MgCl2. The tap root in each seedling
was measured with a ruler before and after the 24 h treatment. Data
are means SD (n ¼ 6). Different letters indicate that the values are
significantly different at P50.05.
0
4
8
12
16
Time (h)
20
24
Fig. 5 Time course of Mg enhancement of Al-induced citrate
efflux from rice bean roots. Two-week-old seedlings were exposed
to 0.5 mM CaCl2 solution (pH 4.5) containing 50 mM AlCl3 with or
without 10 mM MgCl2. Root exudates were collected periodically
and organic anions were analyzed by HPLC. Data are means SD
(n ¼ 3). The asterisk represents statistically siginificant difference
at P50.05.
50
100
a
a
a
80
60
40
b
b
b
20
c
+AI+Ba
+AI+Sr
+AI
+Ba
+Sr
Treatments
Fig. 4 Effects of Sr and Ba on Al toxicity in rice bean. Two-day-old
seedlings were subjected to 0.5 mM CaCl2 solution (pH 4.5)
containing either 0 (control) or 50 mM AlCl3 (þAl) with or without
either 10 mM SrCl2 (þSr) or 10 mM BaCl2 (þBa). The tap root in each
seedling was measured with a ruler before and after the 24 h
treatment. Data are means SD (n ¼ 6). Different letters indicate
that the values are significantly different at P50.05.
Effect of vanadate on the Al-induced citrate efflux in rice
bean
We tested whether the plasma membrane Hþ-ATPase
influenced the Al-dependent citrate efflux response with the
specific inhibitor of this Hþ-ATPase pump, vanadate.
c
40
b
30
20
a
10
0
Control
0
Citrate efflux
(µmol (24 h)−1 g−1 dry weight)
Relative root elongation (%)
120
0
10
20
50
Mg concentration (µM)
Fig. 6 Effect of Mg on Al-induced citrate efflux from rice bean
roots. Two-week-old seedlings were subjected to 0.5 mM CaCl2
solution (pH 4.5) containing 50 mM AlCl3 with the addition of
different concentrations of MgCl2. Root exudates were collected
after 24 h exposure and organic anions were analyzed by HPLC.
Data are means SD (n ¼ 3). Different letters indicate that the
values are significantly different at P50.05.
At 25 mM, vanadate inhibited the Al-dependent citrate
efflux by approximately 58%. Vanadate itself did not
reduce Al activity and monomeric Al concentration as
predicted by GEOCHEM-PC software and the pyrocatechol violet method (data not shown). Addition of 10 mM
Mg significantly increased the citrate efflux in the presence
and absence of vanadate, suggesting that micromolar
Magnesium enhances citrate efflux by restoring plasma membrane Hþ-ATPase activity
Table 1
69
Al, Ca and Mg contents in root apices
Treatments
Al (ng root tip1)
Ca (ng root tip1)
Mg (ng root tip1)
Control
þMg
þAl
þAlþMg
ND
ND
248 2a
222 21b
529 59a
530 24a
423 42c
511 46ab
185 3a
185 17a
108 9c
153 5b
Two-day-old seedlings each with a 4–5 cm tap root length were exposed to the following treatments: no Al and Mg (control), 10 mM MgCl2
(þMg), 50 mM AlCl3 (þAl) and 50 mM AlCl3 plus 10 mM MgCl2 (þAlþMg). After 24 h exposure, ten 0–1 cm root apices from each
treatment were excised. The root apices were digested in a mixture of HNO3 and HClO4 (4 : 1, v/v), and the content of Al, Ca and Mg was
detected by ICP. Data are means SD (n ¼ 3). Different letters in the same column indicate that the values are significantly different at
P50.05. ND indicates not detectable.
*
1.2
−Mg
+Mg
20
15
*
10
5
ATPase activity
(µmol Pi mg−1 protein min−1)
Citrate efflux
(µmol (24 h)−1 g−1 dry weight)
25
0.9
0.6
0.3
0.0
0
+AI
+AI+Vanadate
Treatments
Fig. 7 Effect of vanadate on Al-induced citrate efflux from rice
bean roots in the presence or absence of Mg. Two-week-old
seedlings were exposed to 50 mM AlCl3 in 0.5 mM CaCl2 solution
(pH 4.5) containing 25 mM vanadate in the presence or absence of
10 mM MgCl2. Root exudates were collected after 24 h exposure,
concentrated and analyzed as described in Materials and Methods.
Data are means SD (n ¼ 3). Asterisks indicate that values are
significantly different between Mg and þMg treatments
(P50.05).
concentrations of Mg can partly restore the inhibitory
effects of vanadate (Fig. 7).
Control
Nitrate
Azide
Molybdate Vanadate
Treatments
Fig. 8 Specific activity of membrane-bound proteins in plasma
membrane vesicles isolated from rice bean roots. Membranes were
isolated from the 2-day-old rice bean roots. Assays were conducted
at 308C. Specific inhibitors were used as markers of tonoplast
(50 mM nitrate), mitochondrial (1 mM azide), acid phosphatase
(1 mM molybdate) and plasma membrane (0.1 mM vanadate)
origins. Data are means SD (n ¼ 3). The asterisk represents a
statistically significant difference at P50.05.
also added to the solution. Addition of Mg alone had no
effect on Hþ-ATPase activity (Fig. 9).
Discussion
Plasma membrane Hþ-ATPase activity
To determine the purity of the plasma membrane
fraction isolated from rice bean roots, the ATP hydrolytic
activity was analyzed in the presence of nitrate, azide,
molybdate and vanadate, which are inhibitors of tonoplast
Hþ-ATPase, mitochondrial Hþ-ATPase, unspecific phosphatases and plasma membrane Hþ-ATPase, respectively.
Vanadate-sensitive ATPase occupied 90.5% of the total
activity in the plasma membrane fraction, and other
inhibitor-sensitive enzyme activity was negligible (Fig. 8).
Exposure to 50 mM Al for 24 h decreased the activity
of the plasma membrane Hþ-ATPase in roots by 37%. This
inhibition was significantly reduced when 10 mM Mg was
Alleviation of Al toxicity by Mg has been reported in
several plant species including sorghum (Tan et al. 1991,
Tan et al. 1992), wheat (Ryan et al. 1994), soybean (Silva
et al. 2001a, Silva et al. 2001b, Silva et al. 2001c) and rice
(Watanabe and Okada 2005). In the present study,
micromolar concentrations of MgCl2 but not SrCl2 and
BaCl2 significantly alleviated Al toxicity (Figs. 1, 2, 4). This
alleviation does not appear to be the typical non-specific
cation response reported previously (Kinraide et al. 1992,
Ryan et al. 1994, Kinraide et al. 2004) for two main reasons,
First, millimolar concentrations of divalent cations are
usually required to protect plants from Al stress and here
Magnesium enhances citrate efflux by restoring plasma membrane Hþ-ATPase activity
70
0.8
Activity of H+-ATPase
(µmol Pi mg−1 protein min−1)
a
a
b
0.6
c
0.4
0.2
0
Control
+Mg
+AI
+AI+Mg
Treatments
Fig. 9 Effect of Al and Mg on activities of plasma membrane
Hþ-ATPase of rice bean roots. Two-day-old rice bean seedlings
were exposed to the following treatments: no Al and Mg (control),
10 mM MgCl2 (þMg), 50 mM AlCl3 (þAl) and 50 mM AlCl3 plus
10 mM MgCl2 (þAlþMg). After 24 h treatment, roots were excised
to analyze the activities of Hþ-ATPase. Data are means SD
(n ¼ 3). Different letters indicate that the values are significantly
different at P50.05.
it was found that micromolar concentrations are occurring.
Secondly, Mg could not provide similar protection from La
toxicity which demonstrates that the response is specific
for Al stress (Fig. 3). Furthermore, addition of micromolar
concentrations of Mg did not affect Al activity in the
treatment solution as calculated by GEOCHEM-PC software and the monomeric Al concentration determined by
the pyrocatechol violet method (data not shown).
We also demonstrated that Mg can increase the
Al-dependent release of citrate from rice bean roots with
the same concentration and time dependence that was
observed for the amelioration of growth (Figs. 5, 6). These
results suggest that the Mg-dependent enhancement of
citrate plays a critical role in alleviating Al toxicity in rice
bean. Silva et al. (2001b) showed that low concentrations
of Mg were able to alleviate Al toxicity in soybean in
a similar way, but the cause of the response was unclear.
The enhanced release of citrate might also contribute partly
to the decreased Al content in root tips undergoing Al þ Mg
treatment as compared with Al treatment (Table 1). When
the results from Figs. 2 and 5 were compared, we also
noticed that both root elongation and citrate efflux
increased due to the addition of Mg. However, the effect
was more significant on root elongation than on citrate
efflux. The explanation for this phenomenon is that the
increase in the efflux of organic anions is not linearly
correlated with Al resistance as manifested by the experiments on bioassay of toxicity of different ratios of
Al–organic anion complexes. For instance, when the ratio
of oxalate to Al increased from 0 to 1/2, the root elongation was almost the same but, when it increased from 1/2
to 1, the root elongation was significantly improved
(Zheng et al. 1998).
Since the plasma membrane Hþ-ATPase plays a critical
role in energizing and regulating an array of secondary
transporters (Arango et al. 2003, Sondergaard et al. 2004),
we examined the interaction between Al toxicity, citrate
efflux and the activity of the plasma membrane Hþ-ATPase
in rice bean roots. We found that Al treatment inhibited the
plasma membrane Hþ-ATPase (Figs. 8, 9) which is similar
to previous reports in barley (Matsumoto 1988), squash
(Ahn et al. 2001) and wheat (Ahn et al. 2004). Other studies
have shown that Al could both inhibit and stimulate
the plasma membrane Hþ-ATPase depending on the Al
concentration and duration of treatment (Facanha and
Okorokova-Facanha 2002, Shen et al. 2005). A specific
inhibitor of plasma membrane Hþ-ATPase, vanadate,
inhibited Al-induced citrate efflux by 58% (Fig. 7),
suggesting that plasma membrane Hþ-ATPase is involved
in the Al-induced citrate efflux from rice bean roots. Other
workers have demonstrated that the plasma membrane
Hþ-ATPase is also involved in the Al-dependent efflux of
malate from wheat (Ahn et al. 2004) and citrate from lupine
and soybean (Ligaba et al. 2004, Shen et al. 2005). Citrate
exudation from the cluster roots of white lupin (Neumann
et al. 1999, Yan et al. 2002) and from a mutant carrot cell
line (Ohno et al. 2003) is associated with enhanced proton
release. Ohno et al. (2003, 2004) showed that the Hþ release
from the mutant carrot cells was dependent upon the
up-regulation of the plasma membrane Hþ-ATPase because
the activity of plasma membrane Hþ-ATPase correlated
with citrate exudation and antisense inhibition of the
Hþ-ATPase reduced citrate release. Therefore, it is likely
that a functional Hþ-ATPase is required for the sustained
efflux of organic acid from cells to maintain membrane
potential and perhaps cytoplasmic pH, and our results in
rice bean are consistent with this hypothesis.
Mg is pivotal to the function of most ATPase proteins
and essential for maintaining the Hþ-ATPase activity on the
plasma membrane (Brooker and Slayman 1983). Exposure
of rice bean roots to 50 mM AlCl3 for 24 h significantly
decreased the Mg content in root cells (Table 1) and
depressed the activity of plasma membrane Hþ-ATPase
(Fig. 9). Since Mg was absent from the Al treatment
solution, Al may have interfered with Mg translocation to
the root tip from other tissues, or even stimulated Mg efflux
from the roots as suggested for soybean (Silva et al. 2001c).
Addition of micromolar concentrations of Mg to the Al
treatment solution increased the Mg content in root apices
(Table 1) and up-regulated the activity of plasma membrane
Hþ-ATPase (Fig. 9). Therefore, the stimulation of the
Al-dependent citrate efflux by low concentrations of Mg
Magnesium enhances citrate efflux by restoring plasma membrane Hþ-ATPase activity
could be related to the up-regulation of the plasma
membrane Hþ-ATPase activity. The greater citrate efflux,
in turn, would provide extra protection from the toxic Al
cations which enhances root growth. The stimulation of
citrate efflux by Mg reached saturation at approximately
10 mM Mg, and the decreases observed at higher concentrations may be related to Mg starting to mask the interactions
between Al and the root cells by electrostatic interactions.
The stimulation of citrate efflux by Mg was observed after
12–24 h (Figs. 2, 5) which indicates that the root cells
required some time to respond to the Mg treatment before
they could alter their physiology.
Al is a potential inhibitor of Ca uptake, and Al toxicity
often induces Ca deficiency in plants (Rengel 1992, Rengel
and Elliott 1992, Piñeros and Tester 1993, Rengel and
Zhang 2003, Zheng and Yang 2005). Yet some studies have
demonstrated that Al can inhibit root growth without
reducing Ca uptake by wheat (Ryan and Kochian 1993,
Kinraide et al. 1994, Ryan et al. 1994, Ryan et al. 1997) and
rice (Watanabe and Okada 2005). In the present study, 24 h
exposure of rice bean roots to 5 mM Al decreased the Ca
concentration at the root tip by 20%, whereas when 10 mM
Mg was included in the Al solution as well, the Ca content
remained unchanged (Table 1). While it is possible that part
of the protection afforded by Mg relies on maintaining the
Ca concentration in the root tissues, there is no evidence
that increasing Ca can enhance Al-induced organic acid
anion efflux in plant roots.
In conclusion, we have demonstrated that Mg can
alleviate Al toxicity in rice bean roots by enhancing the
Al-dependent efflux of citrate. This enhancement of citrate
efflux may be related to the restoration of plasma
membrane Hþ-ATPase activity by Mg.
Materials and Methods
Plant material and culture conditions
Seeds of rice bean were collected from Quzhou (acid soil
region, Zhejiang Province, China). Seeds were fully imbibed in
de-ionized water and then germinated at 268C in the dark. The
germinated seeds were transferred to a net tray floating on a
container filled with 5 l of 0.5 mM CaCl2 solution at pH 4.5
(Ca solution). On day 3, similar sized seedlings with tap roots
4–5 cm long were selected to assay Al sensitivity and mineral
accumulation. After 4 d growth in Ca solution, seedlings were
transplanted to 1.1 l plastic pots (16 seedlings in each pot) each
filled with 1 l of one-fifth strength Hoagland nutrient solution
containing (mM): KNO3 (1,000), Ca (NO3)2 (1,000), MgSO4 (400),
(NH4)H2PO4 (200), NaFeEDTA (20), H3BO3 (3.0), MnCl2 (0.5),
CuSO4 (0.2), ZnSO4 (0.4) and (NH4)6Mo7O24 (1.0). The pH of the
solution was adjusted to 4.5 by 0.1 M HCl and was renewed every
other day. The plants were grown in a greenhouse for 12 d and then
moved to a controlled-environment room with a 14 h/268C day and
a 10 h/228C night regime, a light intensity of 250 mmol m2 s1 and
a relative humidity of 65% for 2 d before the collection of root
exudates.
71
Root growth experiments
Two-day-old seedlings were transplanted to a compartmental
hydroponic screening system (Yang et al. 2005). To assay the
ameliorative effect of different concentrations of Mg on Al or La
toxicity, roots were exposed to 0.5 mM CaCl2 solution containing
either 50 mM AlCl3 or LaCl3 with different concentrations of
MgCl2 (0, 10, 20 or 50 mM) at pH 4.5. The length of the tap root
was measured with a ruler before and after 24 h treatment. For the
time course experiment, roots were exposed to 0.5 mM CaCl2
solution containing 50 mM AlCl3 with or without 10 mM MgCl2,
and the root length was measured at 0, 4, 8, 12 and 24 h. In order to
test whether other divalent cations can alleviate Al toxicity, roots
were exposed to 0.5 mM CaCl2 solution containing 0 or 50 mM
AlCl3 in the presence or absence of either 10 mM SrCl2 or 10 mM
BaCl2 at pH 4.5. The length of the tap root was measured with
a ruler before and after 24 h of treatment. Al resistance was
estimated by relative root elongation which was calculated as
(root elongated in various treatments/root elongated without
AlCl3) 100.
Collection of root exudates
After 2-week culture in nutrient solution (see above), the
roots were placed in 0.5 mM CaCl2 solution (pH 4.5) overnight
(14 h), and then transferred to 0.5 mM CaCl2 solution containing
50 mM AlCl3 with different concentrations of MgCl2 (0, 10, 20 or
50 mM) at pH 4.5. Root exudates were collected after 24 h. For the
time course experiment, roots were exposed to 0.5 mM CaCl2
solution containing 50 mM AlCl3 with or without 10 mM MgCl2.
Root exudates were collected at 4, 8, 12 and 24 h.
Effect of vanadate on organic acid anions efflux
After 2-week culture in nutrient solution, the roots were
washed in Ca solution as described above and exposed to 50 mM
AlCl3 in 0.5 mM CaCl2 solution (pH 4.5) containing 25 mM
vanadate with or without 10 mM MgCl2. The Al3þ activities or
monomeric Al concentrations in treatment solutions were analyzed
using the GEOCHEM-PC speciation software (Parker et al. 1995)
and the pyrocatechol violet method (Kerven et al. 1989),
respectively. Root exudates were collected after 24 h. As vanadate
was dissolved in NaOH solution, solutions containing vanadate
were prepared as follows: the pH of the 0.5 mM CaCl2 solution
containing 25 mM vanadate was pre-adjusted to about 5.0, then the
stock solutions of AlCl3 and MgCl2 were added to the required
concentrations. All the treatment solutions were finally adjusted
to pH 4.5 by 0.1 M HCl.
Analysis of organic acid anions in root exudates
Organic acid anions released from the roots were concentrated and purified according to Zheng et al. (1998). The organic
acid anions were analyzed by HPLC (Beckman) equipped with an
ion-exclusion column (Shodex RSpak KC-811, 300 8 mm) and
a guard column (50 8 mm). The mobile phase was diluted with
HClO4 solution at pH 2.1 with a flow rate of 0.8 ml min1 at 408C.
The detection wavelength was 210 nm.
Al, Ca and Mg content in root apices
Two-day-old seedlings were transferred to a compartmental
hydroponic screening system (Yang et al. 2005). The basic solution
was always 0.5 mM CaCl2 in all treatments. Seedlings were grown
in the following treatment solutions: no Al or Mg (control), 10 mM
MgCl2 (þMg), 50 mM AlCl3 (þAl) and 50 mM AlCl3 plus 10 mM
MgCl2 (þAlþMg). After 24 h treatment, 10 root apices
72
Magnesium enhances citrate efflux by restoring plasma membrane Hþ-ATPase activity
(1 cm in length) were excised from seedlings in each treatment.
The root apices were dried in an oven at 708C for 2 d, then
digested in 4 ml of the mixed acids of HClO4 and HNO3 at a ratio
of 1 : 4 (v/v). The concentrations of Al, Mg and Ca in the digestion
were determined by inductively coupled plasma atomic emission
spectrometry (IRIS/AP optical emission spectrometer, Thermo
Jarrel Ash, San Jose, CA, USA) after appropriate dilution with
high purity water.
Plasma membrane isolation
Two-day-old rice bean seedlings were grown in the four
treatment solutions as described above. After 24 h treatment, the
roots were excised to isolate the plasma membrane according to
Yan et al. (2002). The collected root samples were ground in icecold homogenization buffer in a mortar. The homogenization
buffer contained 250 mM sucrose, 250 mM KI, 2 mM EGTA, 10%
(v/v) glycerol, 0.5% (w/v) bovine serum albumin, 2 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 5 mM 2-mercaptoethanol and 50 mM 1,3-bis(tris[hydroxymethyl]-methylamino)
propane (BTP), adjusted to pH 7.8 with MES. The homogenate,
adjusted to a grinding medium/tissue ratio of 4 ml (g FW)1,
was filtered through two layers of miracloth and centrifuged at
11,500g for 10 min at 08C. The supernatants were centrifuged at
100,000g for 35 min. The microsomal pellets were resuspended in
phase buffer (250 mM sucrose, 3 mM KCl and 5 mM KH2PO4).
The microsomal membrane preparation was fractionated by twophase partitioning in aqueous dextran T500 and polyethylene
glycol according to the method of Widell et al. (1982). Phase
separation was achieved by centrifugation at 48C and 1,000g for
23 min followed by three washing steps in identical phases.
Centrifugation times for the second to fourth separation were 15,
10 and 5 min, respectively. The upper phases obtained after four
separations were diluted with phase buffer and centrifuged at
100,000g for 1 h. The pellets were washed with resuspension
buffer (250 mM sucrose, 3 mM KCl, 5 mM BTP/MES, pH 7.8) and
pelleted again. The pellets were resuspended in a buffer (250 mM
sucrose, 3 mM KCl, 5 mM BTP/MES, pH 7.8) and immediately
stored in liquid nitrogen. Protein was quantified according to
the method of Bradford (1976) using bovine serum albumin
as a standard.
Enzyme assay
The purity of the plasma membrane fraction isolated from
rice bean roots was examined in 0.5 ml of 30 mM BTP/MES buffer
(pH 6.5) containing 5 mM MgSO4, 0.02% (w/v) Brij 58 (Sigma)
and 5 mM disodium-ATP. Specific inhibitors were used as markers
of tonoplast (50 mM nitrate), mitochondrial (1 mM azide),
acid phosphatase (1 mM molybdate) and plasma membrane
(0.1 mM vanadate) origins.
Hydrolytic ATPase activity was determined in 0.5 ml of
30 mM BTP/MES buffer containing 5 mM MgSO4, 50 mM KCl,
50 mM KNO3, 1 mM Na2MoO4, 1 mM NaN3, 0.02% (w/v) Brij 58
(Sigma) and 5 mM disodium-ATP. The reaction was initiated by
the addition of 1–2 mg of membrane protein at 308C and stopped
after 30 min with 1 ml of stopping reagent [2% (v/v) concentrated
H2SO4, 5% (w/v) SDS, and 0.7% (w/v) Na2MoO4] followed
immediately by adding 50 ml of 10% (w/v) ascorbic acid. Color
development was completed after 30 min, and A700 was measured
spectrophotometrically. ATPase activity was calculated as phosphate liberated in excess of a boiled membrane control. The
difference in the samples with and without 0.1 mM vanadate was
expressed as the activities of plasma membrane Hþ-ATPase.
Statistics
The experiments were arranged in a randomized complete
design and the data were statistically evaluated by standard
deviation and Student’s t-test methods.
Acknowledgments
We thank Dr. Peter R. Ryan (Commonwealth Scientific and
Industrial Research Organization Plant Industry, Australia) for
his critical reviewing of the manuscript.This work was financially
supported by the National Natural Science Foundation of China
(No. 30571113, 30625026), Program for New Century Excellent
Talent in University (NCET-04-0554) from the Chinese Ministry
of Education.
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(Received August 31, 2006; Accepted November 15, 2006)