A mutant of the Arabidopsis phosphate transporter PHT1;1 displays enhanced arsenic accumulation

Abstract

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.

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 μM As(V) (+AsV) or without (−AsV) at different Pi concentrations. (B) Root hair elongation after 8 d of growth on media containing 30 μM Pi (−AsV) or 30 μM Pi plus 30 μM As(V) (+AsV). (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 μM Pi. Plants in (D) to (F) were grown for an additional 4 d on the same medium supplemented with 50 μM As(V) (+AsV) or without As(V) (−AsV). Plants in (C) and (G) were grown for an additional 12 d on the same medium supplemented with 50 μM 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) = 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 (+P). (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.

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 μM 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 μM As(V) (+AsV) or without As(V) (−AsV); plants in (E) were grown for an additional 12 d on the same medium supplemented with 50 μM 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) = 0.5 cm.

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)-containing 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 (PHO84^pht1;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 PHO84^pht1;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 (+P), transferred to Pi-deficient medium (−P) for 3 d, and finally transferred to −P medium supplemented with either 30 μM Pi (30P), 30 μM As(V) (AsV), 30 μM As(III) (AsIII), 50 μM Cd (Cd), or 50 μM 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.

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 (+P), transferred to Pi-deficient medium (−P) for 3 d, and finally transferred to −P medium supplemented with either 30 μM As(V) (AsV), 50 μM Cd (Cd), or 50 μM 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 μM Pi (0) and then transferred to the same medium supplemented with 15, 30, or 50 μM (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.