Plant Physiol. (1999) 120: 779-786
Properties of Enhanced Tonoplast Zinc Transport in Naturally
Selected Zinc-Tolerant Silene vulgaris
Agnes N. Chardonnens*,
Paul L.M. Koevoets,
Alisa van Zanten,
Henk Schat, and
Jos A.C. Verkleij
Department of Ecology and Ecotoxicology of Plants, Faculty of
Biology, Vrije Universiteit, De Boelelaan 1087, 1081 HV, The
Netherlands
 |
ABSTRACT |
It was demonstrated recently that
isolated tonoplast vesicles derived from plants of a Zn-tolerant
ecotype of Silene vulgaris accumulate more Zn than
vesicles derived from a Zn-sensitive ecotype. We have now characterized
the tonoplast-transport system that causes this uptake difference and
demonstrated its genetic correlation to Zn tolerance using plant
crosses. We conclude that the tonoplast Zn uptake system of the
tolerant ecotype differs greatly in its characteristics from that of
the sensitive one, with the most prominent differences being its
insensitivity to protonophores and ortho-vanadate and its stimulation
by Mg-GTP. These differences in characteristics are most likely due to
the fact that Zn can be taken up by two or more parallel pathways,
which are not present in the same proportions in both ecotypes. In both
ecotypes, Zn is actively transported across the tonoplast (temperature
coefficient > 1.6), most likely as a free ion, since citrate does
not accumulate in vesicles. Most importantly, the uptake difference
found using the ecotypes was also found between homozygous Zn-tolerant
and Zn-sensitive F3 plants, proving the genetic correlation
between increased tonoplast Zn transport and naturally selected Zn
tolerance in S. vulgaris.
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INTRODUCTION |
Silene vulgaris has many natural populations
(ecotypes), some of which grow on soils that are enriched in various
heavy metals (Ernst, 1974
). These populations have evolved increased
resistance to the metals present in the soil (Verkleij and Schat,
1990). Although the details of the genetic basis for the tolerance
mechanisms are not yet clear (Schat and Vooijs, 1997), much is known
about the physiological mechanisms of tolerance, especially in the case of Zn and Cd.
It has been demonstrated that enhanced Zn tolerance is not due to
reduced uptake. Uptake studies have shown that roots of tolerant plants
accumulate more Zn than those of sensitive plants (Mathys, 1975;
Harmens et al., 1993b). Alternatively, detoxification of heavy metals
could be achieved by intracellular binding to phytochelatins (Grill et
al., 1987). However, this mechanism has not only been rejected as the
mechanism underlying differential Cd tolerance in S. vulgaris (De Knecht et al., 1992), but its significance in the
Zn-tolerance mechanism is also rebutted, since Zn is a poor inducer of
phytochelatin sythase (Grill et al., 1989), and only low amounts of
phytochelatins are produced upon Zn exposure in S. vulgaris
(Harmens et al., 1993a). Moreover, the accumulation of phytochelatins
upon exposure to Zn is higher in the roots of sensitive plants than in
those of tolerant plants (Harmens et al., 1993a). These findings
support the hypothesis that naturally selected Zn tolerance might be
based on enhanced compartmentation in the vacuole (Ernst, 1969; Mathys,
1975), as was suggested for Cd tolerance in S. vulgaris by
De Knecht et al. (1995) and Chardonnens et al. (1998). This hypothesis
is further supported by other reports showing elevated levels of heavy
metals in vacuoles upon exposure of intact plants (Vögeli-Lange
and Wagner, 1990; Brune et al., 1995).
As demonstrated by means of a split root experiment (Harmens et al.,
1993b), the Zn-tolerance mechanism operates in root cells. Recently, we
demonstrated that isolated tonoplast vesicles derived from roots of a
Zn-tolerant ecotype of S. vulgaris take up 2 to 3 times more
Zn than vesicles derived from Zn-sensitive plants when Zn is supplied
as Zn citrate in the presence of Mg-ATP (Verkleij et al., 1998). In the
present paper, this tonoplast transport of tolerant plants is further
characterized using both direct filtration assays and fluorescence
spectroscopy. The effect of incubation temperature, several known
inhibitors of transport proteins, and Mg-GTP on the Zn uptake rate are
studied in ecotypes with different Zn sensitivity. Additionally, a
comparison is made between the uptake of Zn and the uptake of citrate
to determine whether Zn is taken up as a cation or as a Zn citrate
complex.
To demonstrate the significance of increased tonoplast Zn uptake in the
mechanism of naturally selected Zn tolerance, we attempted to
genetically link tonoplast Zn uptake with Zn tolerance. Previous experiments using crosses between Zn-sensitive and Zn-tolerant ecotypes
have shown that tolerance in S. vulgaris is based on two
major genes (Schat et al., 1996); therefore, it is possible to select
homozygous tolerant and sensitive plants derived from crosses of a
tolerant and sensitive ecotype. Replicating results obtained in uptake
experiments using these selected lines would not only eliminate the
possibility that any differences found were due to variations in the
purity or protein content of the vesicle preparations of the different
ecotypes, but, more importantly, it would very strongly link these
results to Zn tolerance.
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MATERIALS AND METHODS |
Plant Material
Seeds of Silene vulgaris (Moench) Garcke were collected
from a Zn-sensitive population at the campus of the Vrije Universiteit (Amsterdam, The Netherlands) and from a Zn-tolerant population at a
mine spoil near Plombières, Belgium. Plants were
germinated and grown hydroponically as described by Verkleij et al.
(1998). After 2 weeks, plants were used for tonoplast vesicle
isolation.
Tonoplast Vesicle Isolation
Tonoplast vesicles were isolated from roots of nonexposed plants
according to the method of Schumaker and Sze (1986), with slight
modifications as described by Verkleij et al. (1998).
Assays of ATP Hydrolysis
Several assays of ATP hydrolysis were performed to determine the
purity of the vesicle preparations and the percentage of right-side-out
vesicles using a spectrophotometric measurement of phosphate, as
described by Murphy and Riley (1962). The standard incubation was
conducted as follows: 130 µL of a buffer containing 130 mM mannitol, 0.5 mM DTT, 1.3 mM
Hepes/bis Tris propane, pH 7.4, and 7 mM KCl; 20 µL of a
tonoplast protein sample (±5 µg of vesicle protein) was added,
followed by 150 µL of Mg-ATP to a final concentration of 2 mM. The vesicles were incubated for 30 min at 37°C, after
which time the reaction was terminated by adding 4 mL of a reagent
containing 2% (w/v) SDS, molybdate, and ascorbate (Murphy and Riley,
1962).
To determine the percentage of right-side-out vesicles, an incubation
with 0.03% (w/v) Triton X-100 was compared with a standard incubation
as described above.
To assess the purity of the vesicle preparation, the standard
incubation was compared with incubation in a medium to which sodium
molybdate and sodium vanadate were added to a final concentration of
0.26 mM, and NaN3 was added to a
final concentration of 2.63 mM.
The inhibition of V-type proton ATPases was assayed by adding 5 mM KNO3 and 0.03% (w/v) Triton X-100
to the incubation medium and comparing this incubation with a standard
incubation without KNO3 but with Triton X-100.
Differences between the sensitive and the tolerant ecotype were tested
using one-way analysis of variance.
Determination of 
pH and 
Prior to all direct filtration experiments, the effect of the
concentration of chemicals used was tested with fluorescence spectroscopy. It was assessed that 5 mM
NH4Cl and 0.4 µg mL
1
gramicidin-D effectively dissipated the pH of vesicles.
Vesicles incubated with 5 mM KNO3
were not able to generate a proton gradient upon the addition of
Mg-ATP.
The effect of 0.5 mM Zn citrate, 50 µM
ortho-vanadate, 5 mM NH4Cl, or 0.4 µg mL1 gramicidin-D on the maintenance of the
proton gradient of isolated tonoplast vesicles was monitored using
acridine orange quenching in fluorescence spectroscopy, as described by
Verkleij et al. (1998). The effect of substances was expressed as the
percentage of maximal proton gradient formation. Additionally, the
effect of 0.5 mM Zn citrate on was monitored in
similar experiments using 3 µM oxonol-V quenching
(Scherman and Henry, 1980). Mg-ATP was added immediately after adding
the vesicles to the incubation medium, and Zn citrate was added after
400 s. After another 100 s, gramicidin was added to dissipate
the gradient. The effect of Zn was expressed as the percentage of
change in fluorescence.
All experiments using fluorescence spectroscopy consisted of at least
three replicates.
Uptake of Zn
All uptake experiments were performed according to the method of
Verkleij et al. (1998), with one exception. Instead of loading the
vesicles with K+ to form an artificial proton
gradient using nigericine, the vesicles were washed with resuspension
buffer. A proton gradient was allowed to form in the presence of 3 mM Mg-ATP. Zn was supplied as Zn citrate. The uptake assay
was started by the addition of Zn. In a pilot experiment, Zn uptake was
shown to be optimal after 90 s, and was therefore measured after
90 s in all experiments. In direct filtration assays, the
correction for aspecific binding of Zn to the outer side of the
membrane was made by subtracting the Zn concentration measured in a
simultaneous incubation of vesicles without ATP (Verkleij et al.,
1998). In all experiments, Zn was measured using a flame atomic
absorption spectrophotometer (model 1100B, Perkin-Elmer). All
experiments were performed several times; each replicate is the average
of a number of measurements made from a single vesicle isolation.
By adding 0.05% Triton X-100 to a vesicle incubation, it was tested
whether Zn was actually transported into the lumen of the vesicles.
The proton gradient dependence of Zn uptake was measured by adding a
protonophore to the incubation medium in direct filtration assays.
NH4Cl (5 mM) or gramicidin-D (0.4 µg mL1) was provided to the vesicles after
100 s, just prior to the addition of Mg-ATP. In other experiments,
50 µM ortho-vanadate was supplied to the vesicles. Zn was
added 400 s after the addition of tonoplast vesicles, at which
time the formation of the proton gradient was maximal. After 90 s
of incubation, the vesicles were filtered and Zn uptake was measured
using atomic absorption spectrophotometry. The effect of substances was
expressed as the percentage of uptake in a reference situation,
which was a simultaneous incubation with Zn in the presence of
Mg-ATP.
The effect of substitution of ATP by GTP and the omission of Mg from
the incubation medium were measured in similar experiments. The effect
of each change in incubation circumstances was again expressed as a
percentage of the reference situation.
In some experiments the incubation temperature was lowered from 24°C
to 4°C, and the temperature coefficient
(Q10) for both ecotypes was calculated.
Uptake of Citrate
The uptake of citrate was measured using GC (model 5890 chromatograph, Hewlett-Packard) to determine the chemical species of Zn
taken up by the tonoplast vesicles. Vesicles were incubated with either
0.1 or 0.5 mM Zn citrate, both in the presence and in the
absence of 3 mM Mg-ATP.
After filtration over a 0.45-µm nitrocellulose filter (Schleicher & Schuell), vesicles were disrupted with 2 mL of 0.1% (v/v) trifluoroacetic acid. After measuring the Zn concentration of the
samples using atomic absorption spectrophotometry, the volume was
determined and 30 µL of 15 mM glutaric acid was added as
an internal standard, followed by lyophilization of the samples.
The lyophilized samples were dissolved in 1.5 mL of water and mixed
with 50 mg of Dowex anion-exchange resin (200-400 mesh, formate form;
AG 1-X8, Bio-Rad). After sedimentation of the resin, the organic acids
were released by adding 1.5 mL of 50% (v/v) HCOOH. Subsequently, 1.3 mL of the solution was supplied to 50 mg of Dowex cation-exchange resin
(200-400 mesh, hydrogen form; AG 50W-X8, Bio-Rad). One milliliter of
the supernatant was air-dried overnight in a vial (Chrompack, Raritan,
NJ). The dried samples were resuspended in 1 mL of 96% ethanol, and
this solution was evaporated with N2 for 1.5 h. The dried samples were derivatized in two steps. First, they were
oximated by adding 0.2 mL of CHCl3 and 0.2 mL of
Stox reagent (49805, Pierce), followed by a 30-min incubation at
75°C. Second, samples were sililated by adding 0.2 mL of
bis-(trimethylsilyl)-trifluoroacetamide containing 1%
trimethylchlorosilane (8251, Chrompack) and incubating for 5 min at
75°C. The concentration of organic acids in the samples was
determined using the GC method described by Harmens et al. (1994).
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RESULTS |
Properties of Vesicles
The results of the assays of ATP hydrolysis (Table
I) show that the vesicle preparations of
different ecotypes have identical purity and inhibition rates by
NO3, although the
tolerant ecotype has a slightly lower percentage of right-side-out
vesicles than the sensitive ecotype (36.2% and 44.5%, respectively).
Differences between ecotypes in the percentages of right-side-out
vesicles, inhibition by nitrate, and purity were tested using one-way
analysis of variance and found not to be significant (P > 0.05)
in all cases.
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Table I.
Assays of ATP hydrolysis performed with tonoplast
vesicles derived from a tolerant and a sensitive ecotype of S. vulgaris
Values are means ± SE of four to five replicates.
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Effect of Zn on pH and
The formation and maintenance of the proton gradient by isolated
tonoplast vesicles of both ecotypes were monitored using acridine
orange quenching. Upon addition of Mg-ATP the formation of a proton
gradient was observed in both ecotypes (Fig.
1A). The addition of 0.5 mM
Zn citrate led to a slight decrease in pH, which was similar in both
ecotypes. Upon addition of either NH4Cl or
gramicidin, the proton gradient of the vesicles diminished (Fig. 1A).
The addition of ortho-vanadate did not affect the proton gradient in
either ecotype.
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| Figure 1.
The effect of 0.5 mM Zn citrate on the
pH and of S. vulgaris tonoplast vesicles.
Fluorescence (F) of acridine orange and oxonol-V were measured in
arbitrary units. A, Effect of 0.5 mM Zn citrate on the
pH of tonoplast vesicles of a sensitive and tolerant ecotype of
S. vulgaris, measured using acridine orange quenching
(this graph is similar for both ecotypes). B and C, Effect of 0.5 mM Zn citrate on the of tonoplast vesicles of a
Zn-sensitive (B) and a Zn-tolerant (C) ecotype was measured using
oxonol-V fluorescence. In all cases, Mg-ATP was added immediately after
adding the vesicles, and Zn citrate was added after 400 s. After
another 100 s, NH4Cl or gramicidin was added to
disrupt the gradient.
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In both ecotypes the formation of an electrical gradient was observed
upon addition of Mg-ATP using oxonol-V fluorescence quenching (Fig. 1,
B and C). When Zn citrate was added, fluorescence in the sensitive
ecotype recovered by 19% (Fig. 1B), indicating that net positive
charge was moving out of the vesicles. The fluorescence in the tolerant
ecotype did not recover at all, but was increased by 8% (Fig. 1C),
indicating that net positive charge was moving into the vesicles.
Differential Sensitivities of Zn Transport to Inhibitors and
Ionophores
The results of the Zn-uptake experiments are shown in Table
II. At a concentration of 0.5 mM Zn citrate in the incubation medium, and in the presence
of Mg-ATP, isolated tonoplast vesicles from the sensitive ecotype took
up 1.85 ± 0.12 µmol mg1 vesicle
protein. Uptake by vesicles from the tolerant ecotype was 3.68 ± 0.79 µmol mg1 vesicle protein. In the
presence of 0.05% Triton 0.70 ± 0.08 and 0.69 ± 0.21 µmol mg1 vesicle protein was measured in
sensitive and tolerant plants, respectively, which proves that Zn is
indeed taken up by the vesicles.
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Table II.
Properties of Zn uptake. The uptake of Zn in the
presence of test compounds is expressed as a percentage ± SE of up to 12 replicates of the Zn uptake in the reference
situation at 90 s, incubation of vesicles at room temperature
(24°C), with 0.5 mM Zn citrate, in the presence of MgATP
The average net uptake for vesicles in the reference situation was
1.85 ± 0.12 and 3.68 ± 0.73 µmol mg1
vesicle protein for the sensitive and the tolerant ecotype,
respectively. All values were corrected for the amount of Zn measured
without the addition of Mg-ATP (0.61 ± 0.03 µmol
mg1 vesicle protein for both ecotypes) prior to the
calculation of percentages.
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The uptake of Zn by isolated tonoplast vesicles derived from tolerant
plants was not influenced by the addition of a protonophore, whereas
the uptake of vesicles from sensitive plants was strongly reduced in
all experiments: by 52% with NH4Cl and by 22%
with gramicidin. The addition of ortho-vanadate did not affect the Zn
uptake in the tolerant ecotype, but the uptake of the sensitive ecotype
was reduced to 42%.
When ATP was replaced by GTP, a 59% increase in Zn uptake was found in
the tolerant ecotype, whereas the sensitive ecotype was inhibited by
39%. The omission of Mg diminished the Zn uptake in both
ecotypes by over 80%.
Lowering the incubation temperature from 24°C to 4°C eliminated the
Zn uptake in vesicles of both ecotypes. From the uptake rates found
at both temperatures the Q10 value for
the Zn uptake process was calculated. The
Q10 of the tolerant ecotype was 2.4, and
that of the sensitive ecotype was 1.6.
Uptake of Citrate
Tonoplast vesicles were incubated with either 0.1 or 0.5 mM Zn citrate in the presence or in the absence of Mg-ATP.
After measuring Zn with flame atomic absorption spectrophotometry, the citrate concentration of the samples was determined using GC. The Zn
concentration in the vesicles increased with the external Zn
concentration, and was higher in the presence of Mg-ATP for both
ecotypes (Fig. 2). At 0.5 mM
Zn citrate in the presence of Mg-ATP, vesicles derived from tolerant
plants took up significantly more Zn than vesicles derived from
sensitive plants (2.9 versus 0.9 µmol mg1
vesicle protein). However, the uptake of citrate by the vesicles of
either ecotype was independent of both the amount of citrate present in
the incubation medium and the presence of Mg-ATP. The discrepancy
between Zn uptake and citrate accumulation was demonstrated most
clearly by vesicles of tolerant plants incubated with 0.5 mM Zn citrate in the presence of Mg-ATP (Fig. 2B). In these
plants the amount of Zn in the vesicles was strongly increased (2.9 µmol mg1 vesicle protein), whereas the amount
of citrate remained very low (0.6. µmol mg1
vesicle protein).
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| Figure 2.
The concentration of Zn and citrate in isolated
tonoplast vesicles derived from sensitive (A) and tolerant (B) plants.
Tonoplast vesicles were incubated with either 0.1 or 0.5 mM
Zn citrate in the presence or absence of Mg-ATP. After measuring Zn
with flame atomic absorption spectrophotometry, the citrate
concentration of the samples was determined using GC (see ``Materials and Methods''). Bars represent means of three to six samples. Error
bars represent SE values. White bars, Zinc (µmol/mg
vesicle protein); shaded bars, citrate (µmol/mg vesicle protein).
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Plant Crosses and Selection for Zn Tolerance
To unambiguously link Zn tolerance to differences observed in
tonoplast vesicle Zn uptake, plants of the tolerant and the sensitive
ecotype were crossed. To obtain homozygous Zn-sensitive and Zn-tolerant
genotypes of Amsterdam × Plombières plants, sets of
F1 plants were numbered and crossed in pairs,
resulting in F2 families designated as 1 × 2, 3 × 4, and 7 × 8. Of these, 200 plants per family were tested for Zn
tolerance according to the method of Schat et al. (1996).
F2 plants showing complete inhibition of root
growth below 700 µM ZnSO4 in the
test solution (<6% of the amount of plants tested) were qualified as
homozygous sensitive; plants that continued growing at concentrations
over 4000 µM ZnSO4 in the test
solution (also <6% of the amount of plants tested) were qualified as
homozygous tolerant. The homozygous plants were transferred to fresh
nutrient solution (without additional Zn) to form new roots, put on
soil, and intercrossed to obtain enough seeds to preculture plants for tonoplast vesicle isolation and Zn uptake experiments, as described above.
The results of the uptake experiments using selected lines are shown in
Table III. Increased uptake of Zn by
tonoplast vesicles cosegregated with Zn tolerance. Vesicles derived
from tolerant F3 plants took up more Zn than
those derived from sensitive plants at all concentrations of Zn
citrate. These results were found using different sensitive and
tolerant F3 lines. This cosegregation of Zn
uptake with tolerance proves that enhanced uptake of Zn across the
tonoplast is genetically correlated with naturally selected Zn
tolerance.
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Table III.
Zn uptake by homozygous sensitive and tolerant
F3 plants of Amsterdam × Plombières
Values are means ± SE of three to five replicates for
F3 family 3 × 4, and the results of a single
isolation for the other lines. Results were corrected for nonspecific
binding of Zn to the vesicles by subtracting the Zn concentration (0.3, 0.5, or 0.8 mM) measured in a reference incubation without
Mg-ATP.
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DISCUSSION |
Our results provide the first direct evidence, to our knowledge,
for an important role of tonoplast transport in naturally selected
heavy metal tolerance. Although other authors have reported transport
of heavy metals into isolated tonoplast vesicles (Salt and Wagner,
1993; Salt and Rauser, 1995; Gries and Wagner, 1998), we were to our
knowledge the first to report differences in heavy metal uptake rate
between tonoplast vesicles derived from ecotypes of one species showing
differential heavy metal tolerance (Verkleij et al., 1998). This
finding strongly supported the hypothesis that the tonoplast plays an
essential role in naturally selected Zn tolerance, as was recently also
strongly suggested for Zn hyperaccumulation in Thlaspi
caerulescens (Lasat et al., 1998). However, this is the first time
(again, to our knowledge) that enhanced tonoplast transport has been
found to be genetically correlated with tolerance. Therefore, it is of
great interest to characterize the transport system that causes this
uptake difference in S. vulgaris.
The uptake of Cd by tonoplast vesicles derived from oat roots
demonstrated by Salt and Wagner (1993) and Gries and Wagner (1998) is
due to H+-coupled antiport activity. In our
experiments a very small decrease in the pH of vesicles of both
ecotypes was observed upon addition of Zn (Fig. 1A), suggesting that
one pathway for Zn uptake in both sensitive and tolerant plants is via
a H+-coupled Zn antiport. Although this is a
major pathway in vesicles from sensitive plants (Table II), most of the
Zn uptake in tolerant plants was not sensitive to the pH. These
results suggest that in tolerant plants an additional uptake system
must be present. This is further supported by measurements of the
across the tonoplast (Fig. 1, B and C). In contrast to the
sensitive ecotype, the tolerant ecotype did not show efflux of positive
ions upon addition of 0.5 mM Zn (Fig. 1B) and was not
reconcilable with high H+-coupled Zn antiport
activity in the tolerant ecotype. We conclude that
H+-coupled Zn antiport activity is most certainly
not the major mechanism underlying increased Zn tolerance in S. vulgaris.
It is highly unlikely that Zn is associated with the membrane, as is
the case for Ni in oat (Gries and Wagner, 1998), since almost no Zn is
found in vesicles incubated without Mg-ATP (Verkleij et al., 1998). The
dependence of Zn transport on the presence of Mg-ATP could be due to
binding of ATP to the transporter or to ATP hydrolysis. Since we found
that pH is not responsible for Zn transport, ATP hydrolysis by
the transporting protein is likely to be the driving force of Zn
transport. Indeed, Zn uptake was strongly reduced, both when the
cofactor Mg was omitted, and when the incubation temperature was
reduced from 24°C to 4°C. The Q10
values of the Zn uptake process calculated for both the sensitive and
the tolerant ecotype (1.6 and 2.4, respectively) suggest biological
activity rather than an nonspecific physical process as the cause of Zn
accumulation.
Citrate concentrations in vesicles incubated with Zn citrate did not
increase with the Zn concentration (Fig. 2), which strongly indicates
that Zn is most likely taken up as Zn2+. Further
support for this hypothesis was obtained in experiments in which Zn and
citrate were supplied to vesicles in a ratio of 1:2. In these
experiments, Zn uptake was strongly reduced, whereas the replacement of
citrate by malate in another experiment increased the uptake rate (data
not shown).
It is plausible that the tonoplast of both ecotypes contains many
cation transporters that are able to transport Zn when it is the most
abundant substrate, as was the case in our assays. These transporters
probably have a variety of characteristics that might account for some
of the variation found in transport, especially in the sensitive
ecotype. In the tolerant ecotype this variation was smaller, possibly
because transport may have been dominated by one specific Zn
transporter that might effectuate Zn tolerance. It is possible that the
Zn-tolerant ecotype shows increased Zn uptake because it contains more
units of a Zn-transport system constitutively present in all ecotypes.
However, the transport system responsible for the increased uptake in
vesicles of tolerant plants may also be due to an additional or a
modified system not present on the tonoplast of sensitive plants.
The transport protein responsible for enhanced Zn transport across the
vacuolar membrane of Zn-tolerant S. vulgaris might belong to
the ABC superfamily of membrane transporters, which are directly
energized by Mg-ATP and are able to transport a large number of
substances, such as sugars, peptides, and inorganic ions (Rea et al.,
1998). For instance, the YCF1 (yeast Cd
factor) protein from Saccharomyces cerevisiae,
which confers Cd resistance in this species, is a Mg-ATP-dependent,
uncoupler-insensitive ABC protein (Li et al., 1996). However, this
protein is inhibited by ortho-vanadate, as is the transport of
chlorophyll catabolites in oilseed rape (Hinder et al.,
1996), in which ATP could be partially replaced by GTP.
ABC-protein-mediated transport with specificity for GTP hydrolysis is
known from Escherichia coli (Zhong and Tai, 1998). Hwang et
al. (1997) described two types of Ca2+-pumping
ATPases in carrot, one of which can hydrolyze GTP nearly as well as
ATP, and is present on vacuolar membranes. In our experiments, however,
Zn uptake by vesicles from the tolerant ecotype was strongly stimulated
(59%) by the replacement of ATP by GTP. The latter finding, together
with the insensitivity of Zn uptake to vanadate, suggests that
transport in this case is not mediated by an ABC protein.
Alternatively, the Zn-transport system in Zn-tolerant S. vulgaris might be related to one of several GTP-binding proteins present on the tonoplast of spinach, which have a molecular mass of
20 to 55 kD (Perroud et al., 1997).
We conclude that tonoplast vesicles derived from Zn-tolerant
S. vulgaris possess a transport system that probably
actively transports ionic Zn into the vesicles. The fact that increased Zn uptake by isolated tonoplast vesicles cosegregates with Zn tolerance
in crosses very clearly demonstrates the importance of root tonoplast
Zn transport in naturally selected Zn tolerance. The transport process
in the tolerant ecotype is dependent on incubation temperature and the
presence of Mg-ATP or Mg-GTP, and is not due to
Zn2+/H+ antiport activity.
The nature of the protein responsible for increased Zn transport,
however, remains to be investigated. In vivo, this transport system
most likely detoxifies Zn by transporting cytosolic Zn into the
vacuole, a process that takes place far more efficiently in the
Zn-tolerant than in the Zn-sensitive ecotype.
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FOOTNOTES |
*
Corresponding author; e-mail agnesch{at}bio.vu.nl; fax
31-20-447123.
Received January 25, 1999;
accepted April 14, 1999.
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ABBREVIATIONS |
Abbreviation:
ABC, ATP-binding cassette.
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ACKNOWLEDGMENTS |
The authors would like to thank Dr. M.M.A. Blake-Kalff
(Rothampsted Experimental Station, Harpenden, UK) and Prof. W.H.O. Ernst (Vrije Universiteit, Amsterdam) for their many useful
suggestions. F.E.C. Sneller and A.H.W. Toebes are kindly thanked for
their assistance in producing the plant crosses.
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LITERATURE CITED |
Brune A,
Urbach W,
Dietz KJ
(1995)
Differential toxicity of heavy metals is partly related to a loss of preferential extraplasmatic compartmentation: a comparison of Cd-, Mo-, Ni- and Zn-stress.
New Phytol
129:
403-409
Chardonnens AN,
Ten Bookum WM,
Kuijper LDJ,
Verkleij JAC,
Ernst WHO
(1998)
Distribution of Cd in leaves of Cd tolerant and sensitive ecotypes of Silene vulgaris.
Physiol Plant
104:
75-80
[CrossRef]
De Knecht JA,
Koevoets PLM,
Verkleij JAC,
Ernst WHO
(1992)
Evidence against a role for phytochelatins in naturally selected increased Cd tolerance in Silene vulgaris (Moench.) Garcke.
New Phytol
122:
681-688
De Knecht JA,
Van Baren N,
Ten Bookum WM,
Wong Fong Sang HW,
Koevoets PLM,
Schat H,
Verkleij JAC
(1995)
Synthesis and degradation of phytochelatins in Cd-sensitive and Cd-tolerant Silene vulgaris.
Plant Sci
106:
9-18
Ernst WHO
(1969)
Zur Physiologie der Schwermetallpflanzen: Subzelluläre Speicherungs-ortedes Zinks.
Ber Dtsch Bot Ges
82:
161-164
Ernst WHO (1974) Schwermetallvegetation der Erde. Fischer-Verlag,
Stuttgart, Germany
Gries GE,
Wagner GJ
(1998)
Association of nickel versus transport of Cd and calcium in tonoplast vesicles of oat roots.
Planta
204:
390-396
[CrossRef][ISI][Medline]
Grill E,
Löffler S,
Winnacker EL,
Zenk MH
(1989)
Phytochelatins, the heavy metal binding peptides of plants, are synthesized from glutathione by a specific 
-glutamylcysteinedipeptyl transpeptidase (phytochelatin synthase).
Proc Natl Acad Sci USA
86:
6838-6842
[Abstract/Free Full Text]
Grill E,
Winnacker EL,
Zenk MH
(1987)
Phytochelatins, a class of heavy-metal-binding peptides from plants are functionally analogous to metallothioneins.
Proc Natl Acad Sci USA
84:
439-443
[Abstract/Free Full Text]
Harmens H,
Den Hartog PR,
Ten Bookum WM,
Verkleij JAC
(1993a)
Increased Zn tolerance in Silene vulgaris (Moench.) Garcke is not due to increased production of phytochelatins.
Plant Physiol
103:
1305-1309
[Abstract]
Harmens H,
Gusmao NGCPB,
Den Hartog PR,
Verkleij JAC,
Ernst WHO
(1993b)
Uptake and transport of Zn in Zn-sensitive and Zn-tolerant Silene vulgaris.
J Plant Physiol
141:
309-315
Harmens H,
Koevoets PLM,
Verkleij JAC,
Ernst WHO
(1994)
The role of low molecular weight organic acids in the mechanism of increased Zn tolerance in Silene vulgaris (Moench.) Garcke.
New Phytol
126:
615-621
[CrossRef]
Hinder B,
Schellenberg M,
Rodon S,
Ginsburg S,
Vogt E,
Martinoia E,
Matile P,
Hortensteiner S
(1996)
How plants dispose of chlorophyll catabolites: directly energized uptake of tetrapyrrolic breakdown products into isolated vacuoles.
J Biol Chem
271:
27233-27236
[Abstract/Free Full Text]
Hwang I,
Ratterman DM,
Sze H
(1997)
Distinction between endoplasmic reticulum-type and plasma membrane-type Ca2+ pumps.
Plant Physiol
113:
535-548
[Abstract]
Lasat MM,
Baker AJM,
Kochian LV
(1998)
Altered Zn compartmentation in the root symplasm and stimulated Zn absorption into the leaf as mechanisms involved in Zn hyperaccumulation in Thlaspi caerulescens.
Plant Physiol
118:
875-883
[Abstract/Free Full Text]
Li ZS,
Szcypka M,
Lu YP,
Thiele DJ,
Rea PA
(1996)
The yeast Cd factor protein (YFC1) is a vacuolar glutathione S-conjugate pump.
J Biol Chem
271:
6509-6517
[Abstract/Free Full Text]
Mathys W
(1975)
Enzymes of heavy-metal-resistant and non-resistant populations of Silene cucubalus and their interaction with some heavy metals in vitro and in vivo.
Physiol Plant
33:
161-165
[CrossRef]
Murphy J,
Riley JP
(1962)
A modified single solution method for the determination of phosphate in natural waters.
Anal Chim Acta
27:
31-36
[CrossRef]
Perroud PF,
Crespi P,
Crèvecoeur M,
Fink A,
Tacchini P,
Greppi H
(1997)
Detection and characterization of GTP-binding proteins on tonoplast of Spinacia oleracea.
Plant Sci
122:
23-33
[CrossRef]
Rea PA,
Li ZS,
Lu YP,
Drozdowicz YM,
Martinoia E
(1998)
From vacuolar GS-X pumps to multispecific ABC transporters.
Annu Rev Plant Physiol Plant Mol Biol
49:
727-760
[CrossRef][ISI]
Salt DE,
Rauser WE
(1995)
Mg-ATP-dependent transport of phytochelatins across the tonoplast of oat roots.
Plant Physiol
107:
1293-1301
[Abstract]
Salt DE,
Wagner GE
(1993)
Cadmium transport across tonoplast vesicles from oat roots: evidence for a Cd2+/H+ antiport activity.
J Biol Chem
268:
12297-12302
[Abstract/Free Full Text]
Schat H,
Vooijs R
(1997)
Multiple tolerance and co-tolerance to heavy metals in Silene vulgaris: a co-segregation analysis.
New Phytol
136:
489-496
[CrossRef]
Schat H,
Vooijs R,
Kuiper E
(1996)
Identical major gene loci for heavy metal tolerances that have evolved in different local populations and subspecies of Silene vulgaris.
Evolution
50:
1888-1895
[CrossRef]
Scherman D,
Henry JP
(1980)
Oxonol-V as a probe of chromaffin granule membrane potentials.
Biochim Biophys Acta
599:
150-166
[Medline]
Schumaker KS,
Sze H
(1986)
Calcium transport into the vacuole of oat roots: characterization of H+/Ca2+ exchange activity.
J Biol Chem
261:
12172-12178
[Abstract/Free Full Text]
Verkleij JAC,
Koevoets PLM,
Blake-Kalff MMA,
Chardonnens AN
(1998)
Evidence for an important role of the tonoplast in the mechanism of naturally selected Zn tolerance in Silene vulgaris.
J Plant Physiol
153:
188-191
Verkleij JAC,
Schat H
(1990)
Mechanisms of metal tolerance in higher plants.
In
AJ Shaw,
eds, Heavy Metal Tolerance in Plants: Evolutionary Aspects.
CRC Press, Boca Raton, FL, pp 179-193
Vögeli-Lange R,
Wagner GJ
(1990)
Subcellular localization of Cd and Cd-binding peptides in tobacco leaves. Implication of a transport function for Cd-binding peptides.
Plant Physiol
92:
1086-1093
[Abstract/Free Full Text]
Zhong XT,
Tai PC
(1998)
When an ATPase is not an ATPase
at low temperature the C-terminal domain of the ABC transporter CVAB is a GTPase.
J Bacteriol
180:
1347-1353
[Abstract/Free Full Text]
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Abstract
Introduction
