Arsenic tolerance in Arabidopsis is mediated by two ABCC-type phytochelatin transporters

Abstract

Arsenic is an extremely toxic metalloid causing serious health problems. In Southeast Asia, aquifers providing drinking and agricultural water for tens of millions of people are contaminated with arsenic. To reduce nutritional arsenic intake through the consumption of contaminated plants, identification of the mechanisms for arsenic accumulation and detoxification in plants is a prerequisite. Phytochelatins (PCs) are glutathione-derived peptides that chelate heavy metals and metalloids such as arsenic, thereby functioning as the first step in their detoxification. Plant vacuoles act as final detoxification stores for heavy metals and arsenic. The essential PC-metal(loid) transporters that sequester toxic metal(loid)s in plant vacuoles have long been sought but remain unidentified in plants. Here we show that in the absence of two ABCC-type transporters, AtABCC1 and AtABCC2, Arabidopsis thaliana is extremely sensitive to arsenic and arsenic-based herbicides. Heterologous expression of these ABCC transporters in phytochelatin-producing Saccharomyces cerevisiae enhanced arsenic tolerance and accumulation. Furthermore, membrane vesicles isolated from these yeasts exhibited a pronounced arsenite [As(III)]-PC(2) transport activity. Vacuoles isolated from atabcc1 atabcc2 double knockout plants exhibited a very low residual As(III)-PC(2) transport activity, and interestingly, less PC was produced in mutant plants when exposed to arsenic. Overexpression of AtPCS1 and AtABCC1 resulted in plants exhibiting increased arsenic tolerance. Our findings demonstrate that AtABCC1 and AtABCC2 are the long-sought and major vacuolar PC transporters. Modulation of vacuolar PC transporters in other plants may allow engineering of plants suited either for phytoremediation or reduced accumulation of arsenic in edible organs.

Fig. 1.

Hypersensitivity of atabcc1 atabcc2 double knockout mutants to arsenic [As(V)] and DSMA, an arsenic-based herbicide. (A and B) Wild-type, AtABCC1 knockout (abcc1-3), AtABCC2 knockout (abcc2-2), and double knockout (abcc1 abcc2) seedlings grown in 100 mg/L of DSMA (A) and 50 μM of As(V) (B) containing half-strength MS media for 16 d. Note that single knockout mutants of AtABCC1 or AtABCC2 are also sensitive to the arsenic herbicide (A) but not to As(V) (B). (Scale bar, 1 cm.) (C and D) Fresh weight (C) and root length (D) of each genotype of plants grown on 0–50 μM As(V)-containing 1/2 MS media. Mean ± SEM (for fresh weights, n = 16, from four independent experiments; for root lengths, n = 332–336, from three independent experiments). *P < 0.04, **P < 0.01 (Student's t test). (E) Arsenic-sensitive phenotype of atabcc1 atabcc2 double knockout plants grown in soil. Three-week-old plants were watered with 133.5 μM As(V). Four representative plants out of 12; each was photographed 8 d after arsenic treatment.

Fig. 2.

AtABCC1 and AtABCC2 enhance arsenic tolerance and accumulation in PCS-expressing budding yeasts. (A) Expression of AtABCC1 (ABCC1) or AtABCC2 (ABCC2) in PC-producing yeast (SM7) enhanced tolerance to 100 μM of As(III). EV, empty vector control. In SM4 strain without PCS expression, no growth difference between genotypes was apparent. Cells were grown in half-strength SD-Ura (synthetic dextrose lacking uracil) media supplemented with 100 μM As(III) for 2 d. O.D.₆₀₀, optical density at 600 nm of the yeast suspension that was initially spotted on the plates. (B) Arsenic content of SM7 yeast cells expressing AtABCC1 or AtABCC2 was higher than that of cells transformed with the empty vector. Cells were grown in half-strength SD-Ura media supplemented with 100 μM As(III) for 12 h. Mean ± SEM (n = 21, from seven independent experiments). *P < 0.03, **P < 0.005 (Student's t test).

Fig. 3.

AtABCC1 and AtABCC2 mediate PC transport in microsomes isolated from yeast transformed with AtABCC1 and AtABCC2 transporters. (A) Time-dependent uptake of PC₂–As in yeast microsomes isolated from S. cerevisiae transformed with the empty vector (EV), AtABCC1, and AtABCC2. PC concentration was 25 μM. (B) Comparison of transport activities for apoPC₂ and PC₂–As, and inhibition of PC₂–As transport by NH₄⁺ and vanadate. PC concentration was 25 μM. (C) Relationship between arsenic (Left) and PC (Right) transport activity. PC₂ (500 μM) with 1 mM As(III) or 1 mM As(III) alone was used, and the concentrations of transported PC₂ and arsenic were measured. (D) Concentration dependence of PC₂–As (solid line) and apoPC₂ (dotted line) transport activity of vesicles isolated from AtABCC1 (filled squares) and AtABCC2 (filled triangles) expressing SM7 S. cerevisiae. Inset: Enlarged version at low concentrations of the substrate. Mean ± SEM (from three independent experiments with four replicates each).

Fig. 4.

Vacuoles isolated from the atabcc1 atabcc2 double knockout exhibit only a very low residual PC transport activity, and atabcc1 atabcc2 double knockouts show decreased content of PCs at the seedling stage. (A) Vacuoles were isolated from wild-type and the atabcc1 atabcc2 double knockout plants and tested for transport activities for PC₂–As and apoPC₂. As a negative control, ATP was replaced by ADP. PC concentration was 250 μM. Mean ± SEM (from two independent experiments with four replicates each). (B) Twelve-day-old seedlings were exposed to different concentrations of potassium arsenate for 96 h, and the integrated accumulation of PCs was determined by HPLC-MS. Mean of two replicates (0 μM of arsenic) or three replicates ± SEM (50 and 100 μM of arsenic).

Fig. 5.

Co-overexpression of PC synthase (AtPCS1) and AtABCC1 results in increased arsenic tolerance. (A) Wild-type plants, plants of a representative AtPCS1-overexpressing line (PCS1), and plants overexpressing AtPCS1 and AtABCC1 (MP-1 and MP-2) grown either on 110 μM As(V)-containing or control half-strength MS plates. (B) Transcript levels of AtABCC1 and AtPCS1 in the plants used in A, determined by qPCR. (C) Shoot fresh weight of wild-type and transgenic plants grown under control conditions or in the presence of 70 μM As(V). Mean ± SEM (from two independent experiments with four replicates each). *P < 0.02 by Student's t test.