Knocking out ACR2 does not affect arsenic redox status in Arabidopsis thaliana: implications for as detoxification and accumulation in plants

Abstract

Many plant species are able to reduce arsenate to arsenite efficiently, which is an important step allowing detoxification of As through either efflux of arsenite or complexation with thiol compounds. It has been suggested that this reduction is catalyzed by ACR2, a plant homologue of the yeast arsenate reductase ScACR2. Silencing of AtACR2 was reported to result in As hyperaccumulation in the shoots of Arabidopsis thaliana. However, no information of the in vivo As speciation has been reported. Here, we investigated the effect of AtACR2 knockout or overexpression on As speciation, arsenite efflux from roots and As accumulation in shoots. T-DNA insertion lines, overexpression lines and wild-type (WT) plants were exposed to different concentrations of arsenate for different periods, and As speciation in plants and arsenite efflux were determined using HPLC-ICP-MS. There were no significant differences in As speciation between different lines, with arsenite accounting for >90% of the total extractable As in both roots and shoots. Arsenite efflux to the external medium represented on average 77% of the arsenate taken up during 6 h exposure, but there were no significant differences between WT and mutants or overexpression lines. Accumulation of As in the shoots was also unaffected by AtACR2 knockout or overexpression. Additionally, after exposure to arsenate, the yeast (Saccharomyces cerevisiae) strain with ScACR2 deleted showed similar As speciation as the WT with arsenite-thiol complexes being the predominant species. Our results suggest the existence of multiple pathways of arsenate reduction in plants and yeast.

Figure 1. RT-PCR of AtACR2 in wild-type Arabidopsis thaliana and T-DNA insertion lines of AtACR2 gene.

Figure 2. Time-course of arsenate (As(V)) reduction in Arabidopsis thaliana wild-type and AtACR2 mutants.

As speciation in roots (A), shoots (B) and the ratio of shoot to root As concentration (C). Plants were exposed to 5 µM As(V) for 0.5–24 h.

Figure 3. Dose-response of arsenate (As(V)) reduction in Arabidopsis thaliana wild-type and AtACR2 mutants.

As speciation in roots (A), shoots (B) and the ratio of shoot to root As concentration (C). Plants were exposed to 5–100 µM As(V) for 24 h.

Figure 4. Arsenate (As(V)) uptake and arsenite (As(III)) efflux in wild-type and AtACR2 mutants of Arabidopsis thaliana.

Plants were exposed to 5 µM As(V) in a phosphate-free nutrient solution for 6 h.

Figure 5. Effect of AtACR2 overexpression on the percentage of As(III) in Arabidopsis thaliana.

Plants were exposed to 5 µM As(V) for 1 day or 1 week.

Figure 6. Root elongation as affected by arsenate and phosphate concentrations in wild-type, AtACR2 knockout mutant (acr2-1) and overexpression line (ACR2-OE1).

Figure 7. Arsenic speciation in yeast by X-ray absorption near edge structure (XANES).

From top to bottom: yeast wild-type, ScACR2 deletion strain (RM1), RM1 expressing PvACR2 and standard compounds (solid line, As(GS)₃ at pH 7; dashed line, arsenite pH7; dotted line, arsenate at pH 7). Fitting with linear combination of standard spectra gave essentially a single component of As(GS)₃ indicating that all three samples contained As(III) coordinated by three aliphatic thiolate ligands.