a mutant of the arabidopsis phosphate transporter pht1;1 ...a mutant of the arabidopsis phosphate...
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A Mutant of the Arabidopsis Phosphate Transporter PHT1;1Displays Enhanced Arsenic Accumulation
Pablo Catarecha,1 Ma Dolores Segura,1 Jose Manuel Franco-Zorrilla, Berenice Garcıa-Ponce,2 Monica Lanza,Roberto Solano, Javier Paz-Ares, and Antonio Leyva3
Departamento de Genetica Molecular de Plantas, Centro Nacional de Biotecnologıa, Consejo Superior de Investigaciones
Cientıficas, Cantoblanco, E-28049 Madrid, Spain
The exceptional toxicity of arsenate [As(V)] is derived from its close chemical similarity to phosphate (Pi), which allows the
metalloid to be easily incorporated into plant cells through the high-affinity Pi transport system. In this study, we identified
an As(V)-tolerant mutant of Arabidopsis thaliana named pht1;1-3, which harbors a semidominant allele coding for the high-
affinity Pi transporter PHT1;1. pht1;1-3 displays a slow rate of As(V) uptake that ultimately enables the mutant to accumulate
double the arsenic found in wild-type plants. Overexpression of the mutant protein in wild-type plants provokes phenotypic
effects similar to pht1;1-3 with regard to As(V) uptake and accumulation. In addition, gene expression analysis of wild-type
and mutant plants revealed that, in Arabidopsis, As(V) represses the activation of genes specifically involved in Pi uptake,
while inducing others transcriptionally regulated by As(V), suggesting that converse signaling pathways are involved in plant
responses to As(V) and low Pi availability. Furthermore, the repression effect of As(V) on Pi starvation responses may reflect
a regulatory mechanism to protect plants from the extreme toxicity of arsenic.
INTRODUCTION
Arsenic, one of the most toxic metals found in soils, is derived
from both natural and anthropogenic sources (Tamaki and
Frankenberger, 1992; Fitz and Wenzel, 2002; Nordstrom, 2002;
Oremland and Stolz, 2003). Arsenic can be solubilized in ground
water, exposing animals and humans to potentially toxic effects
(Nickson et al., 1998; Meharg and Hartley-Whitaker, 2002;
Gomez et al., 2004; Katz and Salem, 2005).
In soils, the most abundant arsenic species is arsenate [As(V)]
(Tamaki and Frankenberger, 1992; Brown et al., 1999). As(V) tox-
icity is derived from its close chemical similarity to phosphate (Pi);
this mimicry enables As(V) to alter Pi metabolism (Clarkson and
Hanson, 1980; Raghothama, 1999; Fitz and Wenzel, 2002). In-
deed, the similarity between these two anions makes plants highly
sensitive to As(V) because it is easily incorporated into cells
through the high-affinity Pi transport system (Meharg and Macnair,
1990, 1991b, 1992b). Since this transport system is induced by
Pi starvation, As(V) uptake is highly dependent upon the amount
of Pi available in the soil (Bieleski, 1973; Raghothama, 1999).
Arabidopsis thaliana mutants exhibiting As(V) tolerance harbor null
alleles coding for the high affinity Pi transporters PHT1;1 or PHT1;4
(Shin et al., 2004), indicating that these transporters play a major
role in As(V) uptake. In addition, a mutation in PHOSPHATE
TRANSPORTER TRAFFIC FACILITATOR1 (PHF1), which is re-
quired for efficient trafficking of Pi transporters to the plasma
membrane, also results in a strong tolerance to As(V) (Gonzalez
et al., 2005). Moreover, tolerance to As(V) in a variety of species,
such as Holcus lanatus, is achieved through a reduction in As(V)
uptake due to a suppression of the high-affinity Pi uptake system
(Meharg and Macnair, 1990, 1991b, 1992b; Meharg and Hartley-
Whitaker, 2002; Bleeker et al., 2003). These plants also exhibit
enhanced arsenic accumulation (Meharg and Macnair, 1991a),
and genetic analysis revealed that a single dominant locus could
be responsible for both phenotypes (Macnair et al., 1992). Due to
the apparent contradiction between reduced As(V) uptake and
enhanced arsenic accumulation, it has been speculated that a
complex rather than a simple locus was responsible for both traits
(Meharg and Macnair, 1992a).
Once As(V) enters the cell, it is promptly reduced to arsenite
[As(III)] (Pickering et al., 2000; Meharg and Hartley-Whitaker,
2002), which is highly toxic but is rapidly complexed with soluble
thiols, in most cases phytochelatins (Clemens et al., 1999;
Pickering et al., 2000; Meharg and Hartley-Whitaker, 2002),
and then sequestered into vacuoles (Salt and Rauser, 1995;
Lombi et al., 2002). This strategy has been widely used by plants
to cope with arsenic and heavy metals. However, studies per-
formed with the arsenic hyperaccumulator plant Pteris vittata
indicate that there must be additional arsenic accumulation
mechanisms that have yet to be identified (Zhao et al., 2003;
Raab et al., 2004). Recently, the first screening for As(V)-tolerant
mutants in Arabidopsis yielded the identification of the mutant
arsenic resistant1 (ars1) (Lee et al., 2003), but the identity of the
ars1 gene is still unknown.
Here, we report the identification and characterization of a new
semidominant mutant allele of the high-affinity Pi transporter
1 These authors contributed equally to this work2 Current address: Laboratorio de Genetica Molecular y Evolucion,Instituto de Ecologıa, Universidad Nacional Autonoma de Mexico, Ap.Postal 70-275, Mexico D.F. 04510, Mexico.3 To whom correspondence should be addressed. E-mail [email protected]; fax 34-91-585-4506.The author responsible for distribution of materials integral to thefindings presented in this article in accordance with the policy describedin the Instructions for Authors (www.plantcell.org) is: Antonio Leyva([email protected]).www.plantcell.org/cgi/doi/10.1105/tpc.106.041871
The Plant Cell, Vol. 19: 1123–1133, March 2007, www.plantcell.org ª 2007 American Society of Plant Biologists
PHT1;1. This allele, named pht1;1-3, exhibits an enhanced ability
to accumulate arsenic while Pi and As(V) uptake rate is reduced,
suggesting that this may be the single mechanism operating in
naturally selected arsenic-tolerant plants. Additionally, we show
that As(V) suppresses the Pi starvation response while activating
other genes potentially involved in As(V) detoxification/tolerance,
suggesting that an As(V) Pi interacting pathway operates in
plants to reduce arsenic uptake.
RESULTS
Screening for As(V)-Tolerant Mutants
To identify As(V)-tolerant mutants, we first studied the pheno-
typic changes of wild-type Arabidopsis seedlings in response to
the metalloid. Since As(V) competes with Pi for the Pi uptake
system, we performed a morphological analysis of seedlings
directly sown on medium containing 30 mM As(V) supplemented
with different Pi concentrations. As expected, As(V) toxicity symp-
toms increased as Pi concentrations decreased (Figure 1A). In the
above-ground (aerial) tissues, the most emblematic symptoms
observed were growth arrest and anthocyanin accumulation. In
roots, growth arrest was also characteristic of As(V) toxicity,
and root hair elongation appeared to be completely inhibited
(Figure 1B). Arabidopsis seedlings grown in the presence of 30
mM As(V) and 30 mM Pi demonstrated intermediate toxicity symp-
toms, indicating that this concentration range may be suitable for
screening As(V)-tolerant mutants. Under these conditions, we
screened 100,000 M2 seedlings from a population of ethyl
methanesulfonate–mutagenized Columbia lines and ultimately
identified nine mutants. One of the selected mutants developed
a larger aerial part, with less growth arrest than that observed in
wild-type plants when grown in the presence of 30 mM As(V)
(Figure 1A). In addition, this mutant was able to elongate root hairs
when grown on Pi-lacking medium supplemented with 30 mM
As(V) (Figure 1B). Moreover, after an extended exposure to As(V),
the mutant clearly accumulated less anthocyanins in the aerial
portion and exhibited longer roots than wild-type plants (Figure
1C). We named this mutant pht1;1-3 in accordance with its mo-
lecular characterization (described below).
pht1;1-3 Shows Enhanced Arsenic Accumulation
Genetic analysis revealed that the tolerant phenotype displayed
by pht1;1-3 is caused by a single mutation and that heterozygous
plants showed an intermediate As(V) tolerance phenotype (Fig-
ure 1C). When plants were exposed to As(V) for a shorter time
(Figure 1D), quantification of root length (Figure 1E) and antho-
cyanin accumulation (Figure 1F) confirmed the intermediate
tolerant phenotype of the heterozygotes. Therefore, in the con-
ditions used here, the mutation behaved as semidominant. To
further characterize the pht1;1-3 tolerance phenotype and to
establish its potential for arsenic phytoremediation, we deter-
mined the arsenic concentration in mutant and wild-type plants.
As shown in Figure 1G, pht1;1-3 plants accumulate at least twice
the arsenic than that accumulated by wild-type plants after 12 d
of growth on As(V)-containing medium. Based on these pheno-
types, pht1;1-3 was chosen for further characterization.
pht1;1-3 Harbors a Missense Mutation in the Pi
Transporter PHT1;1
To identify the pht1;1-3 mutant locus, we first selected plants
that were able to elongate root hairs after 8 d of growth on 30 mM
As(V) and Pi-lacking medium from an F2 mapping population
obtained from crosses with the ecotype Landsberg erecta (Ler).
Due to the semidominant phenotype displayed by pht1;1-3, we
selected homozygous F2 plants at the PHT1;1 locus based on
the segregation of the As(V) tolerance phenotype in their respec-
tive F3 offspring. Using 62 of those selected F2 plants, we
mapped the PHT1;1 locus to chromosome V, close to the marker
DFR. There is a cluster of four genes encoding Pi transporters
linked to this marker, which represent potential candidate genes.
Direct sequencing of the PHT1;1 locus revealed that this gene is
mutated in pht1;1-3 . This mutation results from a single nucle-
otide exchange, which encodes a nonconservative amino acid
substitution (Gly-378 to Glu) at the predicted ninth transmem-
brane domain of the transporter. Therefore, pht1;1-3 is a new
semidominant allele for the Pi transporter PHT1;1 (Shin et al., 2004).
To evaluate the effect of the pht1;1-3 allele on the Pi starvation
response, we first took advantage of the fact that the mutagenized
collection from where pht1;1-3 was isolated harbors the Pi star-
vation responsive reporter gene IPS1:b-glucuronidase (GUS)
(Martın et al., 2000; Rubio et al., 2001). Hence, the Pi starvation
response can be easily monitored in these plants through histo-
chemical GUS staining. As shown in Figure 2A, either wild-type or
pht1;1-3 plants exhibit GUS staining when grown on Pi lacking
(�P) medium. However, in contrast with what was observed for
wild-type plants, GUS staining was also present in pht1;1-3 plants
grown on medium containing 1 mM Pi (þP). This result was con-
firmed by RNA gel blot analysis of the IPS1 expression in plants
grown under high Pi (Figure 2B). In this experiment, we also eval-
uated the expression of the Pi transporter gene PHT1;1, which is
responsive to Pi starvation. As shown in Figure 2B, transcript ac-
cumulation for any of these genes was higher in the ph1;1-3
mutant than in wild-type plants when grown both in complete
(1 mM) and intermediate (0.1 mM) concentrations of Pi. In line with
this, quantification of soluble Pi on either Pi condition revealed that
pht1;1-3 accumulates less than half the Pi accumulated by wild-
type plants (Figure 2C). Therefore, pht1;1-3 exhibited a reduction
in Pi content while arsenic accumulation was enhanced.
Overexpression of pht1;1-3 Results in Decreased Pi
Content and Enhanced Arsenic Accumulation
To confirm whether the pht1;1-3 allele is responsible for the
observed semidominant mutant phenotypes, we obtained trans-
genic Arabidopsis lines in which either pht1;1-3 or PHT1;1 alleles
were expressed in wild-type plants under the control of the
constitutive 35S promoter (Figure 3A). No obvious phenotypic
differences were observed between wild-type and any of the
expressor lines in medium without As(V). However, in the pres-
ence of As(V), wild-type plants expressing pht1;1-3 displayed an
As(V)-tolerant phenotype, while expression of the wild-type allele
conferred hypersensitivity to the metalloid (Figure 3A). Quantifi-
cation of root length and anthocyanin accumulation in these
plants confirmed that the As(V) tolerance phenotypes were
1124 The Plant Cell
Figure 1. As(V) Tolerance Phenotype Displayed by pht1;1-3.
(A) Above-ground phenotype of plants grown for 8 d on media with 30 mM As(V) (þAsV) or without (�AsV) at different Pi concentrations.
(B) Root hair elongation after 8 d of growth on media containing 30 mM Pi (�AsV) or 30 mM Pi plus 30 mM As(V) (þAsV).
Arsenic Hyperaccumulator Mutant 1125
enhanced in the pht1;1-3 expressor line, while plants expressing
the wild-type allele exhibited hypersensitivity to As(V) (Figures 3B
and 3C). Analysis of soluble Pi and arsenic content in these lines
showed that the expression of the mutant protein results in Pi
content reduction and increased arsenic accumulation (Figures
3D and 3E, respectively). Pi and As(V) uptake experiments
revealed that both Pi and As(V) influx were reduced in the mutant
and in the pht1;1-3 expressor line, indicating that differential Pi
versus As(V) uptake rates were not the cause of the opposite
behavior in Pi and As(V) accumulation displayed by the mutant
(Figure 3F). Moreover, competition Pi uptake experiments per-
formed in wild-type and mutant plants showed that Pi uptake rate
decreases in a similar proportion both in wild-type and mutant
plants when exposed to increasing As(V) concentrations (Figure
3G). Therefore, differential affinity in Pi and As(V) transport was
not the cause for the tolerance phenotypes observed in pht1;1-3.
These results also indicated that the expression of the mutated
protein in wild-type plants accurately mimics the mutant phenotype
with regard to As(V) tolerance and both Pi and As(V) accumulation.
Mimicry of the pht1;1-3 Mutation in the Yeast
Pho84p Transporter
The Gly residue mutated in the semidominant mutation pht1;1-3
is highly conserved not only in all the high-affinity Pi transporters
from Arabidopsis, but also in other plants and from yeast. Based
on this information, we decided to evaluate whether the expres-
sion of the native yeast Pi transporter carrying an equivalent
mutation to that of pht1;1-3 might result in similar phenotypes to
the ones observed in plants. In yeast, the Pi starvation response
is easily monitored through staining for acid phosphatase activ-
ity, which drives the production of a dark-red precipitate and is
highly induced when grown on low Pi medium. In this experi-
ment, we used as the wild type the Saccharomyces cerevisae
strain PAM1 (Martinez and Persson, 1998), which harbors a native
copy of the high-affinity Pi transporter gene PHO84, a PHT1;1
yeast homolog. This strain was transformed with PHO84pht1;1-3, a
mutagenized version of the PHO84 cDNA, encoding a Gly-to-Glu
mutation identical to the one present in the pht1;1-3 allele. In the
presence of 550 mM Pi, cells expressing PHO84pht1;1-3 exhibited
more phosphatase activity than cells transformed with either
PHO84 cDNA or the empty vector PAM1 (Figure 4A, top panel).
Furthermore, Pi and As(V) uptake experiments showed that the
rate of Pi and As(V) transport in cells expressing PHO84pht1;1-3
was significantly lower than in the original PAM1 (Figure 4B). No
significant differences in Pi and As(V) uptake rates were observed
between PAM1 and the cells expressing the wild-type PHO84.
Expression of PHO84pht1;1-3 also conferred a slight increase in
the tolerance to As(V) (Figure 4A, bottom panel). This increase is
not as noteworthy as the one seen in pht1;1-3 overexpressors,
Figure 1. (continued).
(C) to (G) As(V) tolerance phenotype ([C] and [D]), root length (n $ 12; P < 0.01) (E), anthocyanin accumulation (n > 3; P < 0.01) (F), and total arsenic
accumulation (n $ 3; P < 0.01) (G) of plants grown for 7 d on 30 mM Pi. Plants in (D) to (F) were grown for an additional 4 d on the same medium
supplemented with 50 mM As(V) (þAsV) or without As(V) (�AsV). Plants in (C) and (G) were grown for an additional 12 d on the same medium
supplemented with 50 mM As(V). Wild-type (white bars), pht1;1-3 (black bars), and heterozygous PHT1;1/pht1;1-3 (gray bars). Error bars represent SD.
Bars in (C) and (D) ¼ 0.5 cm.
Figure 2. pht1;1-3 Exhibits a Constitutive Pi Starvation Response.
(A) Histochemical GUS analysis of IPS1:GUS expression in wild-type and pht1;1-3 seedlings grown on Pi-lacking medium (�P) and on medium
supplemented with 1 mM Pi (þP).
(B) and (C) RNA gel blot analysis of PHT1;1 and IPS1 (B) and soluble Pi content (n $ 3; P < 0.01) (C) in wild-type (white bars) and pht1;1-3 (black bars)
seedlings grown in the presence of 1 mM or 0.1 mM Pi. Error bars represent SD.
1126 The Plant Cell
probably due to the fact that wild-type S. cerevisiae itself exhibits
a high degree of intrinsic tolerance to As(V). Therefore, we can
conclude that the introduction of an equivalent pht1;1-3 Gly-to-
Glu substitution in Pho84p provokes, in yeast, effects similar to
those seen in Arabidopsis overexpressing pht1;1-3.
As(V) Represses the Pi Starvation Response while
Activating Arsenic-Responsive Genes
To further characterize the pht1;1-3 mutant phenotype, and
because of the similarity between Pi and As(V), we next investi-
gated the effect of As(V) on the Pi starvation response in pht1;1-3
and in wild-type plants. We performed RNA gel blot analysis of
the Pi-responsive genes PHT1;1, SQD1, IPS1, and PHF1 in wild-
type and pht1;1-3 plants grown in the presence of As(V). Addition-
ally, we included in this experiment plants treated with Pi, As(III),
cadmium (Cd), and nickel (Ni). As expected, all Pi-responsive
genes analyzed were induced by Pi starvation (Figure 5A). When
wild-type plants were then transferred for 8 h to the same me-
dium supplemented with either 30 mM As(V) or Pi, the amount of
transcript corresponding to each of the Pi-responsive genes was
reduced (Figure 5B). As(V) was less efficient than Pi in the
Figure 3. Phenotypic Characterization of Wild-Type Plants Overexpressing pht1;1-3.
As(V) tolerance phenotype (A), root length (n $ 15; P < 0.01) (B), anthocyanin accumulation (n $ 4; P < 0.01) (C), soluble Pi content (n $ 3; P < 0.1) (D),
arsenic accumulation (n $ 3; P < 0.01) (E), Pi and As(V) uptake rates (n $ 3; P < 0.1) (F), and Pi uptake rates in competition with increasing As(V)
concentrations (n $ 3; P < 0.01) (G). All plants were grown for 7 d on 30 mM Pi; plants in (D), (F), and (G) were analyzed at that point; plants in (A) to (C)
were grown for an additional 4 d on the same medium supplemented with 50 mM As(V) (þAsV) or without As(V) (�AsV); plants in (E) were grown for an
additional 12 d on the same medium supplemented with 50 mM As(V). Wild-type (white bars), pht1;1-3 (black bars), pht1;1-3 overexpressor (35S:pht1;1-3;
blue bars), and PHT1;1 overexpressor (35S:PHT1;1; orange bars). Error bars represent SD. Bars in (A) ¼ 0.5 cm.
Arsenic Hyperaccumulator Mutant 1127
repression of SQD1, IPS1, and PHF1. By contrast, As(V) was
more efficient than Pi in the repression of the Pi transporter
PHT1;1. In pht1;1-3 plants, gene responsiveness to Pi starvation
was reduced with respect to that in wild-type plants (Figure 5B).
The reason for this behavior remains to be elucidated, but re-
duced gene responsiveness to Pi starvation was also observed in
other mutants displaying partially constitutive Pi starvation re-
sponse, such as phf1 and siz1 (Gonzalez et al., 2005; Miura et al.,
2005). Despite reduced responsiveness of SQD1 and IPS1 in Pi-
starved mutant plants, a reduced repression of these genes was
Figure 4. Expression in Yeast of the Pi Transporter Pho84p Carrying an
Equivalent Mutation to pht1;1-3.
(A) Phosphatase activity (top panel) and growth assay on As(V)-contain-
ing medium (bottom panel) of yeast PAM1 mutant cells transformed with
the expression vector harboring either no insert (PAM1), PHO84 cDNA
(PHO84), or the PHO84 cDNA carrying an equivalent mutation to pht1;1-3
(PHO84pht1;1-3).
(B) Pi uptake (left) and As(V) uptake (right) determined in the S. cerevisiae
strains described in (A) (n $ 4; P < 0.01 for PHO84pht1;1-3). See Methods.
Bars represent SD.
Figure 5. Expression Analysis of Pi Starvation–Responsive Genes in
Wild-Type and pht1;1-3 Plants.
RNA gel blot analysis (A) and densitometry analysis (B) of the expression
of Pi-responsive marker genes PHT1;1, SQD1, IPS1, and PHF1. Plants
were grown for 7 d on medium containing 1 mM Pi (þP), transferred to Pi-
deficient medium (�P) for 3 d, and finally transferred to �P medium
supplemented with either 30 mM Pi (30P), 30 mM As(V) (AsV), 30 mM As(III)
(AsIII), 50 mM Cd (Cd), or 50 mM Ni (Ni) for 8 h. All intensity levels in (B) are
represented as relative to �P levels in wild-type plants. White bars, wild-
type plants; black bars, pht1;1-3 plants.
1128 The Plant Cell
observed by Pi and particularly by As(V), for which repression was
almost negligible. By contrast, PHT1;1 was completely down-
regulated by As(V) but not by Pi (Figure 5B). Therefore, each gene
exhibits a different sensitivity to the repression by As(V) or Pi. We
also examined the significance of Pi/As(V) uptake in the As(V)
response. In the laboratory we have identified two As(V)-inducible
genes named ASI3 and ASI4 that encode a short-chain dehy-
drogenase/reductase (At4g13180) and glyoxalase II (At4g33540),
respectively. Regardless of its putative role in arsenic detoxifica-
tion, both genes were induced in response to As(V) but not by Cd
or Ni (Figure 6A), suggesting that the induction is not part of a
general stress response. Actually, both genes respond to As(V)
and As(III) in a dose-dependent manner (Figure 6B), although the
amount of transcript detected in response to As(V) was higher in
wild-type plants than in pht1;1-3 (Figure 6C). By contrast, no
differences were observed in the response to As(III) of both genes
between the wild-type and the mutant backgrounds (Figure 6C).
These observations indicate that As(V) requires the Pi transport
system to induce ASI3 and ASI4, whereas As(III) may use an
independent pathway.
Therefore, we conclude that As(V) downregulates genes tran-
scriptionally regulated by Pi starvation, being particularly efficient
in the repression of the Pi/As(V) uptake system. The repression
occurs conversely to the activation of As(V)-responsive genes.
DISCUSSION
In plants, restriction of As(V) uptake is the major strategy used by
naturally selected As(V)-hypertolerant ecotypes. Actually, As(V)
toxicity is based on the similarity between Pi and As(V), which
allows the metalloid to be easily incorporated into plants cells
through the high-affinity Pi transport system (Meharg and Macnair,
1990, 1991b, 1992b; Meharg and Hartley-Whitaker, 2002; Bleeker
et al., 2003). Therefore, it is reasonable that plant adaptation to
As(V)-contaminated soils has been achieved through alteration
of the Pi/As(V) uptake system.
In this study, we identified an arsenic-tolerant mutant in
Arabidopsis, named pht1;1-3, which harbors a new allele coding
for the high-affinity Pi transporter PHT1;1. Characterization of
pht1;1-3 revealed that decreased As(V) uptake contributes to
enhanced arsenic content. Our investigations also uncovered
the existence of an integrated Pi/As(V) signaling pathway, which
modulates As(V) uptake.
The constitutive Pi starvation response displayed by pht1;1-3
is consistent with the observed reduction in Pi content, indicating
that pht1;1-3 may be functionally impaired. However, despite
As(V) uptake reduction, pht1;1-3 accumulates at least twice the
amount of arsenic found in wild-type plants. The semidominant
nature of the pht1;1-3 mutation allowed the confirmation of this
association between reduced arsenic uptake and increased
arsenic accumulation using transgenic plants overexpressing
pht1;1-3. Indeed, pht1;1-3 overexpression affected both traits.
These two phenotypes conferred by the pht1;1-3 semidomi-
nant mutation help to explain the genetic data on arsenic
tolerance of naturally selected variants of H. lanatus involving a
single chromosomal region. In fact, it is unnecessary to invoke
that complex loci are responsible for both apparently contradic-
tory phenotypes.
Figure 6. Expression Analysis of the As(V)-Responsive Genes ASI3 and
ASI4.
(A) RNA gel blot analysis of plants grown for 7 d on medium containing
1 mM Pi (þP), transferred to Pi-deficient medium (�P) for 3 d, and finally
transferred to �P medium supplemented with either 30 mM As(V) (AsV),
50 mM Cd (Cd), or 50 mM Ni (Ni) for 8 h.
(B) and (C) Gel blot (B) and densitometry analysis (C) of dose-dependent
expression of ASI3 (continuous line) and ASI4 (dashed line) in wild-type
(white circles) and pht1;1-3 (black squares) plants. Seedlings were grown
for 5 d on medium with 30 mM Pi (0) and then transferred to the same
medium supplemented with 15, 30, or 50 mM (increasing slope) As(V)
(AsV) or As(III) (AsIII) for 8 h. All intensity levels in (C) are represented as
relative to 0 levels for each gene and genotype.
Arsenic Hyperaccumulator Mutant 1129
One possible explanation for the apparent paradox repre-
sented by the association between decreased As(V) uptake and
enhanced arsenic accumulation is that lowering As(V) content in
the cytoplasm may allow the arsenic detoxification machinery to
cope more efficiently with the metalloid, allowing for greater
accumulation of arsenic into the vacuole. As(V) reduction to
As(III) is a prerequisite for compartmentalization into the vacuole,
and it has been recently shown that enhanced As(V) reductase
activity is also a major determinant in As(V) hypertolerance
(Bleeker et al., 2006). Overexpression of the As(V) reductase
gene in Arabidopsis confers tolerance to low As(V). Therefore,
decreasing As(V) uptake may allow As(V) reductase to process
most of the As(V) present in the cytoplasm of pht1;1-3, thus
enhancing As(III) sequestration into the vacuole. The fact that the
response of ASI3 and ASI4 to As(V) is reduced in pht1;1-3, while
As(III) responsiveness appears to be unaffected, provides further
evidence that the increased tolerance of pht1;1-3 to the metalloid
is due to reduced As(V) uptake in the mutant rather than en-
hanced arsenic responses.
The semidominant character of the pht1;1-3 mutation also has
mechanistic implications. Dominant negative mutations have
been reported in a wide variety of transporters (Zhou and Christie,
1997; Brockmann et al., 2001; Elumalai et al., 2002; Hahn et al.,
2003; Rungroj et al., 2004), suggesting that transporters may
work as dimers or higher-order oligomers (Monahan et al., 2002;
Reinders et al., 2002; Ludewig et al., 2003). In fact, there is evi-
dence that Pi transporters in Arabidopsis and Medicago truncatula
may form functional dimers (Chiou et al., 2001; Shin et al., 2004).
Our finding that a pht1;1-3 equivalent allele of the PHO84 yeast Pi
transporter interferes with Pi/As(V) uptake in yeast suggests that
oligomer formation in Pi transporters is universal.
The results presented here indicate that As(V) rapidly repressed
genes involved in the Pi starvation response and induced the
expression of other As(V)-responsive genes. While the repressor
function on Pi starvation–responsive genes is specific for As(V),
induction of arsenic-responsive genes is also mediated by As(III).
This leads us to propose a model whereby arsenic acts through
two different signaling pathways. Based on the analogy between
Pi and As(V), we propose that As(V) could mislead the Pi sensor,
thus triggering the repression of the Pi starvation–responsive
genes. In that case, As(V) should act as a nonmetabolizable
Pi analog as it was seen for phosphite (Ticconi et al., 2001;
Varadarajan et al., 2002; Giots et al., 2003; Pratt et al., 2004). It
is noticeable that repression by As(V) is not equally efficient for all
Pi starvation–responsive genes, being most efficient in the re-
pression of the Pi transporter even more than Pi itself, which is
incorporated faster than As(V). The outstanding sensitivity of the
Pi transporter to As(V) prompts us to speculate that the repres-
sion of Pi starvation responses by arsenic is not only an obvious
consequence of the chemical similarity between As(V) and Pi but
also reflects a natural selection process that capitalizes on this
situation to ensure efficient repression of Pi starvation responses,
particularly of the Pi transporter. On the other hand, given the fact
that once As(V) enters the cell it is rapidly reduced to As(III)
(Pickering et al., 2000; Meharg and Hartley-Whitaker, 2002),
which has no chemical similarity to Pi and therefore no chance to
confound the Pi sensor, we propose that activation of ASI3 and
ASI4 occurs via an As(III) signaling pathway.
In conclusion, we propose that, due to the chemical similarity
between As(V) and Pi, plants have evolved an integrated sensing
mechanism in which As(V) and Pi signaling pathways act in
opposition to preserve plant integrity from arsenic toxicity. Our
data open the possibility of further evaluating the As(V) response
to identify exclusive, or overlapping, elements in As(V), As(III),
and Pi starvation responses that may be relevant to arsenic
perception and accumulation.
METHODS
Plant Material and Growth Conditions
Arabidopsis thaliana ecotypes used in this study were Columbia (Col-0)
and Ler. Seeds were surface-sterilized and plated onto Bates and Lynch
medium (Bates and Lynch, 1996) and solidified with 0.6% bacto-agar
(Difco), which was supplemented with appropriate amounts of Pi
(KH2PO4) or As(V) (NaH2AsO4�7H2O), unless otherwise specified. Growth
conditions were the same as described previously (Franco-Zorrilla et al.,
2002). The mutant pht1;1-3 was selected from an ethyl methanesulfonate–
mutagenized population derived from a Col transgenic line harboring
the Pi starvation reporter construct IPS1:GUS (Rubio et al., 2001). asi
(arsenic-induced) lines were identified in the laboratory from an F3 prog-
eny of a transposant line collection generated in the Exon trapping insert
consortium (EXOTIC QLG2-CT-1999-000351; http://www.jic.bbsrc.ac.
uk/science/cdb/exotic/). Flanking DNA from asi lines was cloned by
thermal asymmetric interlaced PCR (Liu et al., 1995).
Plant Measurements and Histochemical Staining
The method by Ames (1966) was used to quantify soluble Pi, and histo-
chemical GUS stainings were performed as described by Martın et al.
(2000). Roots were analyzed after 7 d (soluble Pi) or 8 d (GUS and root
hairs) of culture in the desired Pi concentration. All P values were obtained
through a Student’s t test.
For measuring root length, anthocyanin accumulation, and As(V) ac-
cumulation, plants were cultured for 7 d on 30 mM Pi medium and then
transferred to fresh medium supplemented with 50 mM As(V). Samples
were collected 4 d (root length and anthocyanin accumulation) or 12 d
(arsenic content) later. To measure As content, plants were dried at 608C
for 5 d, mineralized with HNO4-H2O2 in a pressure digester, and analyzed
for total arsenic content through inductively coupled plasma–mass
spectrometry (ICP-MS) at the Centro de Espectrometrıa Atomica of the
Universidad Complutense de Madrid. Anthocyanins were measured ac-
cording to Swain and Hillis (1959).
Plant Uptake Experiments
Plates used in preculture for both Pi and As(V) uptake experiments
contained half-strength Bates and Lynch medium solidified with 0.4%
bacto-agar supplemented with 30 mM Pi and were covered with 0.4-mm-
pore nylon mesh. This medium permits solid culture, long root production,
and easy root cleaning. Plants were sown onto the mesh and cultured for
7 d before being carefully extracted and washed with sterile water. Plants
were then treated with PI and PII buffers as by Narang et al. (2000). Pi
uptake experiments were performed in 3-mL wells at 5 mM KH232PO4
(ICN) and competing As(V) when suitable. As(V) uptake experiments were
performed in 400-mL pots at 5 mM As(V). The duration of each uptake
experiment was 1 h. Plants were introduced in either scintillation vials for32Pi measurements or in 50-mL tubes for As(V) measurements and dried
at 608C for 5 d. 32Pi was measured in a scintillation counter with 10 mL of
scintillation liquid (ICN), and arsenic content was analyzed trough ICP-MS
as described above.
1130 The Plant Cell
Genetic Analysis and Positional Cloning of pht1;1-3
pht1;1-3 plants were backcrossed three times to wild-type plants.
Backcrossed seeds were crossed to the Ler ecotype. Positional cloning
was performed by selecting F2 seedlings displaying the mutant pheno-
type and homozygous for the PHT1 locus according to the segregation of
the mutant phenotype in their respective F3 offspring. DNA from these F2
selected plants was prepared (Dellaporta et al., 1983) and used to analyze
the linkage of the pht1;1-3 mutation to previously described genetic
markers (Konieczny and Ausubel, 1993; Bell and Ecker, 1994). To identify
the mutant gene, we used the Cereon collection of Col-Ler genetic
polymorphisms (http://www.Arabidopsis.org/cereon/index.html).
cDNA Isolation and Overexpression
pht1;1-3 and PHT1;1 cDNA were amplified by RT-PCR using total RNA
extracted from Pi-starved plants using primers 59-CCTCAACTCTCCA-
GAGAAGTTC-39 and 59-ACATCATAACTTAAGGTCAACGAG-39. These
cDNAs were cloned into plasmid pBIBA7, a 35S promoter–containing
vector derived from pBIB (Becker, 1990). The second intron of PHT1;1,
amplified from genomic DNA using primers 59-CAAGTTGTTCTATGGTC-
CAATGTTCG-39 and 59-CTGTTTTCAATCTTCTACGTACGA-39, was in-
serted in its place in both cDNAs. This construct was necessary to avoid
toxicity to Agrobacterium tumefaciens. Plants were transformed as by
Bechtold et al. (1993).
RNA Extraction and Gel Blot Analysis
Total RNA was extracted with RNAwiz (Ambion), and 20 mg were loaded
per sample and blotted onto Hybond Nþ membranes (Amersham) as
indicated by the manufacturer. Hybridizations were performed as by
Church and Gilbert (1984), and membranes were washed with 0.53 SSC
before exposing. Probes corresponding to IPS1 (Martın et al., 2000),
SQD1 (Essigmann et al., 1998), PHF1 (Gonzalez et al., 2005), and PHT1;1
(Smith et al., 1997) were obtained as by Franco-Zorrilla et al. (2005);
probes corresponding to ASI3 and ASI4 were obtained by PCR ampli-
fication of the coding region on plant DNA.
Yeast Transformation and Constructs
The PHO84pht1;1-3 point mutation was introduced in PHO84 cDNA (kindly
provided by Georg Leggewie) through directed mutagenesis using
primers 59-CAAGATGATGAATTCGGCTAATTGAATTG-39 and 59-TCAA-
TTAGCCGAATTCATCATCTTGAC-39 containing the equivalent point
mutation found in pht1;1-3 (highlighted in boldface). Both PHO84pht1;1-3
and PHO84 cDNAs were cloned into the yeast expression vector
p181A1NE (Leggewie et al., 1997). Both constructs were transformed
into Saccharomyces cerevisiae PAM1 (kindly provided by Bengt Persson)
(Martinez and Persson, 1998), according to Gietz and Woods (2002).
Yeast was grown on YNB medium (Difco) supplemented with 2% sucrose
(Calbiochem), unless stated otherwise. For acid phosphatase activity, we
designed a Pi-deficient medium (SW medium) following the Wicherham
formula (Sigma-Aldrich), in which appropriate amounts of KCl were sub-
stituted for KH2PO4, as done by Lau et al. (1998).
Yeast Acid Phosphatase Activity and Tolerance Experiments
Acid phosphatase activity was assayed in growing yeast cells in liquid
YNB medium overnight, which was then diluted to OD660 ¼ 0.1. Droplets
(3 mL) of the diluted culture were spotted onto SW plates with 550 mM
KH2PO4, in which sucrose was replaced by 3% glycerol (Bun-ya et al.,
1991). Acid phosphatase activity was assayed as by Toh-e et al. (1973).
Tolerance to As(V) was assayed in growing yeast cells in liquid YNB
medium overnight. Droplets (3 mL) of media diluted to OD660 ¼ 0.1 were
spotted onto YPD media supplemented with 3.5 mM As(V) as described
by Bun-ya et al. (1996).
Yeast Uptake Experiments
Pi uptake experiments were performed as by Daram et al. (1999) using
carrier-free H332PO4 (ICN). Uptake took 4 minutes at 308C in a 40 mL mix;
Pi final concentration was 500 mM, and pH was adjusted to 6.5. Samples
were then diluted to 10 mL with chilled water and filtered trough Me25
membranes held by 3-cm polysulphone holders (both from Schleicher
and Schuell). Membranes were washed twice with chilled water before
being dried on 3M paper and introduced into scintillation vials for
counting. As(V) uptake experiments were scaled to a 40-mL uptake
mixture in 50-mL screw cap tubes (Falcon), and uptake was stopped by
immersing tubes in chilled water, followed quickly by centrifugation. Cells
were then washed twice with chilled water, and pellets were dried at 608C
for 5 d. Total As was measured through ICP-MS as described above.
Accession Numbers
Arabidopsis Genome Initiative locus identifiers for the genes mentioned in
this article are At5g43350 (PHT1;1), At3g52190 (PHF1), At3g09922 (IPS1),
At4g33030 (SQD1), At4g13180 (ASI3), and At33540 (ASI4).
ACKNOWLEDGMENTS
We thank Georg Leggewie for kindly providing the PHO84 cDNA in the
yeast expression vector p181A1NE and Bengt Persson for providing the
S. cerevisiae strain PAM1. We also thank C.L. Toran for critical reading
of the manuscript. The excellent technical assistance of Marıa Jesus
Benito and Yolanda Leo del Puerto is also acknowledged. P.C. and M.L.
were supported by Spanish Ministry of Education Grants BIO99-0229
and BIO2001-1204, respectively; B.G.-P. was supported by a postdoc-
toral contract of the Consejo Nacional de Ciencia y Tecnologıa (010116).
This research was supported by the EXOTIC grant (Contract QLG2-CT-
1999-000351) funded by the 5th European Framework Program, by the
Comunidad de Madrid (Contract 07B/0037/2002), and by the Spanish
Comision Interministerial de Ciencia y Tecnologıa (Contract BIO2001-1204).
Received February 13, 2006; revised February 27, 2007; accepted March
9, 2007; published March 30, 2007.
REFERENCES
Ames, B.N. (1966). Assay of inorganic phosphate, total phosphate and
phosphatases. Methods Enzymol. 8: 115–118.
Bates, T.R., and Lynch, J.P. (1996). Stimulation of root hair elongation
in Arabidopsis thaliana by low phosphorus availability. Plant Cell
Environ. 19: 529–538.
Bechtold, N., Ellis, J., and Pelletier, G. (1993). In planta Agrobacterium
mediated gene transfer by infiltration of adult Arabidopsis thaliana
plants. C. R. Acad. Sci. III, Sci Vie 316: 1194–1199.
Becker, D. (1990). Binary vectors which allow the exchange of plant
selectable markers and reporter genes. Nucleic Acids Res. 18: 203.
Bell, C.J., and Ecker, J.R. (1994). Assignment of 30 microsatellite loci
to the linkage map of Arabidopsis. Genomics 19: 137–144.
Bieleski, R.L. (1973). Phosphate pools, phosphate transport, and
phosphate availability. Annu. Rev. Plant Physiol. 27: 225–252.
Bleeker, P.M., Hakvoort, H.W., Bliek, M., Souer, E., and Schat,
H. (2006). Enhanced arsenate reduction by a CDC25-like tyrosine
Arsenic Hyperaccumulator Mutant 1131
phosphatase explains increased phytochelatin accumulation in
arsenate-tolerant Holcus lanatus. Plant J. 45: 917–929.
Bleeker, P.M., Schat, H., Vooijs, R., Verkleij, J.A.C., and Ernst,
W.H.O. (2003). Mechanisms of arsenate tolerance in Cytisus striatus.
New Phytol. 157: 33–38.
Brockmann, K., et al. (2001). Autosomal dominant glut-1 deficiency
syndrome and familial epilepsy. Ann. Neurol. 50: 476–485.
Brown, G.E., Jr., Foster, A.L., and Ostergren, J.D. (1999). Mineral
surfaces and bioavailability of heavy metals: A molecular-scale per-
spective. Proc. Natl. Acad. Sci. USA 96: 3388–3395.
Bun-ya, M., Nishimura, M., Harashima, S., and Oshima, Y. (1991).
The PHO84 gene of Saccharomyces cerevisiae encodes an inorganic
phosphate transporter. Mol. Cell. Biol. 11: 3229–3238.
Bun-ya, M., Shikata, K., Nakade, S., Yompakdee, C., Harashima, S.,
and Oshima, Y. (1996). Two new genes, PHO86 and PHO87, involved
in inorganic phosphate uptake in Saccharomyces cerevisiae. Curr.
Genet. 29: 344–351.
Chiou, T.J., Liu, H., and Harrison, M.J. (2001). The spatial expression
patterns of a phosphate transporter (MtPT1) from Medicago trunca-
tula indicate a role in phosphate transport at the root/soil interface.
Plant J. 25: 281–293.
Church, G.M., and Gilbert, W. (1984). Genomic sequencing. Proc. Natl.
Acad. Sci. USA 81: 1991–1995.
Clarkson, D.T., and Hanson, J.B. (1980). The mineral nutrition of higher
plants. Annu. Rev. Plant Physiol. 31: 239–298.
Clemens, S., Kim, E.J., Neumann, D., and Schroeder, J.I. (1999).
Tolerance to toxic metals by a gene family of phytochelatin synthases
from plants and yeast. EMBO J. 18: 3325–3333.
Daram, P., Brunner, S., Rausch, C., Steiner, C., Amrhein, N., and
Bucher, M. (1999). Pht2;1 encodes a low-affinity phosphate trans-
porter from Arabidopsis. Plant Cell 11: 2153–2166.
Dellaporta, S.L., Wood, J., and Hicks, J.B. (1983). A plant DNA
minipreparation: Version II. Plant Mol. Biol. R1: 19–21.
Elumalai, R.P., Nagpal, P., and Reed, J.W. (2002). A mutation in the
Arabidopsis KT2/KUP2 potassium transporter gene affects shoot cell
expansion. Plant Cell 14: 119–131.
Essigmann, B., Guler, S., Narang, R.A., Linke, D., and Benning, C. (1998).
Phosphate availability affects the thylakoid lipid composition and the
expression of SQD1, a gene required for sulfolipid biosynthesis in
Arabidopsis thaliana. Proc. Natl. Acad. Sci. USA 95: 1950–1955.
Fitz, W.J., and Wenzel, W.W. (2002). Arsenic transformations in the
soil-rhizosphere-plant system: Fundamentals and potential applica-
tion to phytoremediation. J. Biotechnol. 99: 259–278.
Franco-Zorrilla, J.M., Martın, A.C., Leyva, A., and Paz-Ares, J.
(2005). Interaction between phosphate-starvation, sugar, and cytoki-
nin signaling in Arabidopsis and the roles of cytokinin receptors CRE1/
AHK4 and AHK3. Plant Physiol. 138: 847–857.
Franco-Zorrilla, J.M., Martın, A.C., Solano, R., Rubio, V., Leyva, A.,
and Paz-Ares, J. (2002). Mutations at CRE1 impair cytokinin-induced
repression of phosphate starvation responses in Arabidopsis. Plant J.
32: 353–360.
Gietz, R.D., and Woods, R.A. (2002). Transformation of yeast by the
LiAc/SS carrier DNA/PEG method. Methods Enzymol. 350: 87–96.
Giots, F., Donaton, M.C., and Thevelein, J.M. (2003). Inorganic
phosphate is sensed by specific phosphate carriers and acts in
concert with glucose as a nutrient signal for activation of the protein
kinase A pathway in the yeast Saccharomyces cerevisiae. Mol.
Microbiol. 47: 1163–1181.
Gomez, G., Baos, R., Gomara, B., Jimenez, B., Benito, V., Montoro,
R., Hiraldo, F., and Gonzalez, M.J. (2004). Influence of a mine tailing
accident near Donana National Park (Spain) on heavy metals and
arsenic accumulation in 14 species of waterfowl (1998 to 2000). Arch.
Environ. Contam. Toxicol. 47: 521–529.
Gonzalez, E., Solano, R., Rubio, V., Leyva, A., and Paz-Ares, J.
(2005). PHOSPHATE TRANSPORTER TRAFFIC FACILITATOR1 is a
plant-specific SEC12-related protein that enables the endoplasmic
reticulum exit of a high-affinity phosphate transporter in Arabidopsis.
Plant Cell 17: 3500–3512.
Hahn, M.K., Robertson, D., and Blakely, R.D. (2003). A mutation in the
human norepinephrine transporter gene (SLC6A2) associated with
orthostatic intolerance disrupts surface expression of mutant and
wild-type transporters. J. Neurosci. 23: 4470–4478.
Katz, S.A., and Salem, H. (2005). Chemistry and toxicology of building
timbers pressure-treated with chromated copper arsenate: A review.
J. Appl. Toxicol. 25: 1–7.
Konieczny, A., and Ausubel, F.M. (1993). A procedure for mapping
Arabidopsis mutations using co-dominant ecotype-specific PCR-
based markers. Plant J. 4: 403–410.
Lau, W.W., Schneider, K.R., and O’Shea, E.K. (1998). A genetic study
of signaling processes for repression of PHO5 transcription in Sac-
charomyces cerevisiae. Genetics 150: 1349–1359.
Lee, D.A., Chen, A., and Schroeder, J.I. (2003). ars1, an Arabidopsis
mutant exhibiting increased tolerance to arsenate and increased
phosphate uptake. Plant J. 35: 637–646.
Leggewie, G., Willmitzer, L., and Riesmeier, J.W. (1997). Two cDNAs
from potato are able to complement a phosphate uptake-deficient
yeast mutant: Identification of phosphate transporters from higher
plants. Plant Cell 9: 381–392.
Liu, Y.G., Mitsukawa, N., Oosumi, T., and Whittier, R.F. (1995).
Efficient isolation and mapping of Arabidopsis thaliana T-DNA insert
junctions by thermal asymmetric interlaced PCR. Plant J. 8: 457–463.
Lombi, E., Zhao, F.J., Fuhrmann, M., Ma, L.Q., and McGrath, S.P.
(2002). Arsenic distribution and speciation in the fronds of the hyper-
accumulator Pteris vittata. New Phytol. 156: 195–203.
Ludewig, U., Wilken, S., Wu, B., Jost, W., Obrdlik, P., El Bakkoury,
M., Marini, A.M., Andre, B., Hamacher, T., Boles, E., von Wiren,
N., and Frommer, W.B. (2003). Homo- and hetero-oligomerization
of ammonium transporter-1 NH4þ uniporters. J. Biol. Chem. 278:
45603–45610.
Macnair, M.R., Cumbes, Q.J., and Meharg, A.A. (1992). The genetics
of arsenate tolerance in Yorkshire fog Holcus lanatus L. Heredity 69:
325–335.
Martın, A.C., del Pozo, J.C., Iglesias, J., Rubio, V., Solano, R., de La
Pena, A., Leyva, A., and Paz-Ares, J. (2000). Influence of cytokinins
on the expression of phosphate starvation responsive genes in
Arabidopsis. Plant J. 24: 559–567.
Martinez, P., and Persson, B.L. (1998). Identification, cloning and
characterization of a derepressible Naþ-coupled phosphate trans-
porter in Saccharomyces cerevisiae. Mol. Gen. Genet. 258: 628–638.
Meharg, A.A., and Hartley-Whitaker, J. (2002). Arsenic uptake and
metabolism in arsenic resistant and nonresistant plant species. New
Phytol. 154: 29–43.
Meharg, A.A., and Macnair, M.R. (1990). An altered phosphate uptake
system in arsenate-tolerant Holcus lanatus L. New Phytol. 116: 29–35.
Meharg, A.A., and Macnair, M.R. (1991a). Uptake, accumulation and
translocation of arsenate in arsenate-tolerant and non-tolerant Holcus
lanatus L. New Phytol. 117: 225–231.
Meharg, A.A., and Macnair, M.R. (1991b). The mechanism of arsenate
tolerance in Deschampsia cespitosa (L.) Beauv. and Agrostis capillaris
L. New Phytol. 119: 291–297.
Meharg, A.A., and Macnair, M.R. (1992a). Genetic correlation between
arsenate tolerance and the rate of influx of arsenate and phosphate in
Holcus lanatus L. Heredity 69: 336–341.
Meharg, A.A., and Macnair, M.R. (1992b). Suppression of the high
affinity phosphate uptake system: A mechanism of arsenate tolerance
in Holcus lanatus L. J. Exp. Bot. 43: 519–524.
1132 The Plant Cell
Miura, K., Rus, A., Sharkhuu, A., Yokoi, S., Karthikeyan, A.S.,
Raghothama, K.G., Baek, D., Koo, Y.D., Jin, J.B., Bressan, R.A.,
Yun, D.J., and Hasegawa, P.M. (2005). The Arabidopsis SUMO E3
ligase SIZ1 controls phosphate deficiency responses. Proc. Natl.
Acad. Sci. USA 102: 7760–7765.
Monahan, B.J., Unkles, S.E., Tsing, I.T., Kinghorn, J.R., Hynes,
M.J., and Davis, M.A. (2002). Mutation and functional analysis
of the Aspergillus nidulans ammonium permease MeaA and evi-
dence for interaction with itself and MepA. Fungal Genet. Biol. 36:
35–46.
Narang, R.A., Bruene, A., and Altmann, T. (2000). Analysis of phos-
phate acquisition efficiency in different Arabidopsis accessions. Plant
Physiol. 124: 1786–1799.
Nickson, R., McArthur, J., Burgess, W., Ahmed, K.M., Ravenscroft,
P., and Rahman, M. (1998). Arsenic poisoning of Bangladesh
groundwater. Nature 395: 338.
Nordstrom, D.K. (2002). Public health. Worldwide occurrences of
arsenic in ground water. Science 296: 2143–2145.
Oremland, R.S., and Stolz, J.F. (2003). The ecology of arsenic. Science
300: 939–944.
Pickering, I.J., Prince, R.C., George, M.J., Smith, R.D., George, G.N.,
and Salt, D.E. (2000). Reduction and coordination of arsenic in Indian
mustard. Plant Physiol. 122: 1171–1177.
Pratt, J.R., Mouillon, J.M., Lagerstedt, J.O., Pattison-Granberg, J.,
Lundh, K.I., and Persson, B.L. (2004). Effects of methylphosphonate,
a phosphate analogue, on the expression and degradation of the
high-affinity phosphate transporter Pho84, in Saccharomyces cerevi-
siae. Biochemistry 43: 14444–14453.
Raab, A., Feldmann, J., and Meharg, A.A. (2004). The nature of
arsenic-phytochelatin complexes in Holcus lanatus and Pteris cretica.
Plant Physiol. 134: 1113–1122.
Raghothama, K.G. (1999). Phosphate acquisition. Annu. Rev. Plant
Physiol. Plant Mol. Biol. 50: 665–693.
Reinders, A., Schulze, W., Kuhn, C., Barker, L., Schulz, A., Ward,
J.M., and Frommer, W.B. (2002). Protein-protein interactions be-
tween sucrose transporters of different affinities colocalized in the
same enucleate sieve element. Plant Cell 14: 1567–1577.
Rubio, V., Linhares, F., Solano, R., Martın, A.C., Iglesias, J., Leyva,
A., and Paz-Ares, J. (2001). A conserved MYB transcription factor
involved in phosphate starvation signaling both in vascular plants and
in unicellular algae. Genes Dev. 15: 2122–2133.
Rungroj, N., Devonald, M.A., Cuthbert, A.W., Reimann, F.,
Akkarapatumwong, V., Yenchitsomanus, P.T., Bennett, W.M.,
and Karet, F.E. (2004). A novel missense mutation in AE1 causing
autosomal dominant distal renal tubular acidosis retains normal
transport function but is mistargeted in polarized epithelial cells.
J. Biol. Chem. 279: 13833–13838.
Salt, D.E., and Rauser, W.E. (1995). MgATP-dependent transport of
phytochelatins across the tonoplast of oat roots. Plant Physiol. 107:
1293–1301.
Shin, H., Shin, H.S., Dewbre, G.R., and Harrison, M.J. (2004). Phos-
phate transport in Arabidopsis: Pht1;1 and Pht1;4 play a major role in
phosphate acquisition from both low- and high-phosphate environ-
ments. Plant J. 39: 629–642.
Smith, F.W., Ealing, P.M., Dong, B., and Delhaize, E. (1997). The
cloning of two Arabidopsis genes belonging to a phosphate trans-
porter family. Plant J. 11: 83–92.
Swain, T., and Hillis, H.E. (1959). Phenolic constituents of Prunus
domestica. I. Quantitative analysis of phenolic constituents. J. Sci.
Food Agric. 10: 63–68.
Tamaki, S., and Frankenberger, W.T., Jr. (1992). Environmental bio-
chemistry of arsenic. Rev. Environ. Contam. Toxicol. 124: 79–110.
Ticconi, C.A., Delatorre, C.A., and Abel, S. (2001). Attenuation of
phosphate starvation responses by phosphite in Arabidopsis. Plant
Physiol. 127: 963–972.
Toh-e, A., Ueda, Y., Kakimoto, S.I., and Oshima, Y. (1973). Isolation
and characterization of acid phosphatase mutants in Saccharomyces
cerevisiae. J. Bacteriol. 113: 727–738.
Varadarajan, D.K., Karthikeyan, A.S., Matilda, P.D., and Raghothama,
K.G. (2002). Phosphite, an analog of phosphate, suppresses the co-
ordinated expression of genes under phosphate starvation. Plant
Physiol. 129: 1232–1240.
Zhao, F.J., Wang, J.R., Barker, J.H.A., Schat, H., Bleeker, P.M., and
McGrath, S.P. (2003). The role of phytochelatins in arsenic tolerance
in the hyperaccumulator Pteris vittata. New Phytol. 159: 403–410.
Zhou, X.R., and Christie, P.J. (1997). Suppression of mutant pheno-
types of the Agrobacterium tumefaciens VirB11 ATPase by overpro-
duction of VirB proteins. J. Bacteriol. 179: 5835–5842.
Arsenic Hyperaccumulator Mutant 1133
DOI 10.1105/tpc.106.041871; originally published online March 30, 2007; 2007;19;1123-1133Plant Cell
Lanza, Roberto Solano, Javier Paz-Ares and Antonio LeyvaPablo Catarecha, Ma Dolores Segura, José Manuel Franco-Zorrilla, Berenice García-Ponce, Mónica
Accumulation Phosphate Transporter PHT1;1 Displays Enhanced ArsenicArabidopsisA Mutant of the
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