The rice aquaporin Lsi1 mediates uptake of methylated arsenic species

Abstract

Pentavalent methylated arsenic (As) species such as monomethylarsonic acid [MMA(V)] and dimethylarsinic acid [DMA(V)] are used as herbicides or pesticides, and can also be synthesized by soil microorganisms or algae through As methylation. The mechanism of MMA(V) and DMA(V) uptake remains unknown. Recent studies have shown that arsenite is taken up by rice (Oryza sativa) roots through two silicon transporters, Lsi1 (the aquaporin NIP2;1) and Lsi2 (an efflux carrier). Here we investigated whether these two transporters also mediate the uptake of MMA(V) and DMA(V). MMA(V) was partly reduced to trivalent MMA(III) in rice roots, but only MMA(V) was translocated to shoots. DMA(V) was stable in plants. The rice lsi1 mutant lost about 80% and 50% of the uptake capacity for MMA(V) and DMA(V), respectively, compared with the wild-type rice, whereas Lsi2 mutation had little effect. The short-term uptake kinetics of MMA(V) can be described by a Michaelis-Menten plus linear model, with the wild type having 3.5-fold higher V(max) than the lsi1 mutant. The uptake kinetics of DMA(V) were linear with the slope being 2.8-fold higher in the wild type than the lsi1 mutant. Heterologous expression of Lsi1 in Xenopus laevis oocytes significantly increased the uptake of MMA(V) but not DMA(V), possibly because of a very limited uptake of the latter. Uptake of MMA(V) and DMA(V) by wild-type rice was increased as the pH of the medium decreased, consistent with an increasing proportion of the undissociated species. The results demonstrate that Lsi1 mediates the uptake of undissociated methylated As in rice roots.

Figure 1.

As speciation using HPLC-ICP-MS. Rice plants were exposed to 5 μm MMA(V) or DMA(V) for 24 h. A, Chromatogram of standards containing 25 ng As L⁻¹ of As(III), MMA(V), and DMA(V) and 7.5 ng As L⁻¹ MMA(III). B, Nutrient solutions after 24 h exposure. C and D, Root extracts of the wild type and lsi1 mutant exposed to MMA(V) (C) or DMA(V) (D). E and F, Xylem sap collected from the wild type and lsi1 mutant exposed to MMA(V) (E) or DMA(V) (F). G and H, Shoot extracts of the wild type and lsi1 mutant exposed to MMA(V) (G) or DMA(V) (H). ⁷⁵As signal was normalized by the signal of the internal standard ⁷²Ge.

Figure 2.

Concentrations of MMA(V + III) in roots (A), MMA(V) in shoots (B), and xylem sap (C) of the lsi1, lsi2, and lsi1si2 (double mutant [DM]) mutants and their wild-type rice. Plants were exposed to 5 μm MMA(V) for 24 h at pH 5.5. Error bars are +ses (n = 4). FW, Fresh weight.

Figure 3.

Kinetics of MMA(V) uptake into the roots of the wild-type rice and the lsi1 mutant. Plants were exposed to 0 to 50 μm MMA(V) for 30 min at pH 5.5. Lines are the fitted curves of the Michaelis-Menten plus linear model. Error bars are ±ses (n = 4). FW, Fresh weight.

Figure 4.

Concentrations of DMA(V) in roots (A), shoots (B), and xylem sap (C) of the lsi1, lsi2, and lsi1lsi2 (double mutant [DM]) mutants and their wild-type rice. Plants were exposed to 5 μm DMA(V) for 24 h at pH 5.5. Error bars are +ses (n = 4). FW, Fresh weight.

Figure 5.

Kinetics of DMA(V) uptake into the roots of the wild-type rice and the lsi1 mutant. Plants were exposed to 0 to 50 μm DMA(V) for 30 min at pH 5.5. Lines are the fitted curves of a linear model. Error bars are ±ses (n = 4). FW, Fresh weight.

Figure 6.

Effect of medium pH on the proportion of undissociated MMA(V) in the medium and the uptake of MMA(V) by wild-type rice (WT1). Plants were exposed to 5 μm MMA(V) for 24 h at different pHs. Total As concentrations in roots (A) and shoots (B), and MMA(V) concentration in xylem sap (C). Error bars are +ses (n = 4). FW, Fresh weight.

Figure 7.

Effect of medium pH on the proportion of undissociated DMA(V) in the medium and the uptake of DMA(V) by wild-type rice (WT1). Plants were exposed to 5 μm DMA(V) for 24 h at different pHs. Total As concentrations in roots (A) and shoots (B), and DMA(V) concentration in xylem sap (C). Error bars are +ses (n = 4). FW, Fresh weight.

Figure 8.

Transport activity of the rice Lsi1 expressed in X. laevis oocytes. Oocytes were incubated with 100 μm MMA(V) (A) or DMA(V) (B) for 30 min. Injection of water serves as the control. Error bars are +ses (n = 3).