Fission yeast HMT1 lowers seed cadmium through phytochelatin-dependent vacuolar sequestration in Arabidopsis

Abstract

Much of our dietary uptake of heavy metals is through the consumption of plants. A long-sought strategy to reduce chronic exposure to heavy metals is to develop plant varieties with reduced accumulation in edible tissues. Here, we describe that the fission yeast (Schizosaccharomyces pombe) phytochelatin (PC)-cadmium (Cd) transporter SpHMT1 produced in Arabidopsis (Arabidopsis thaliana) was localized to tonoplast, and enhanced tolerance to and accumulation of Cd2+, copper, arsenic, and zinc. The action of SpHMT1 requires PC substrates, and failed to confer Cd2+ tolerance and accumulation when glutathione and PC synthesis was blocked by L-buthionine sulfoximine, or only PC synthesis is blocked in the cad1-3 mutant, which is deficient in PC synthase. SpHMT1 expression enhanced vacuolar Cd2+ accumulation in wild-type Columbia-0, but not in cad1-3, where only approximately 35% of the Cd2+ in protoplasts was localized in vacuoles, in contrast to the near 100% found in wild-type vacuoles and approximately 25% in those of cad2-1 that synthesizes very low amounts of glutathione and PCs. Interestingly, constitutive SpHMT1 expression delayed root-to-shoot metal transport, and root-targeted expression confirmed that roots can serve as a sink to reduce metal contents in shoots and seeds. These findings suggest that SpHMT1 function requires PCs in Arabidopsis, and it is feasible to promote food safety by engineering plants using SpHMT1 to decrease metal accumulation in edible tissues.

Figure 1.

Rescue of Cd²⁺ sensitivity in hmt1^- mutant LK100 and subcellular localization analysis. A, LK100 transformed with empty vector pART1 (V/LK100) or c-myc-tagged SpHMT1 (H/LK100). Wild-type strain Sp223 transformed with pART1 (V/Sp223) was used as positive control. B, Subcellular localization of SpHMT1. Left, bright field; middle, fluorescence; and right, merged images of Enhanced Yellow Fluorescent Protein (EYFP; top) and EYFP:SpHMT1 (bottom). Red color represents autofluorescence of chloroplasts. Bars = 20 μm.

Figure 2.

Enhanced metal tolerance and accumulation in SpHMT1 plants. Surface sterile seeds plated onto one-fourth Murashige and Skoog medium supplemented with 50 μm CdCl₂ (A), 150 μm KH₂AsO₄ (B), 40 μm CuSO₄ (C), and 150 μm ZnSO₄ (D), and grown vertically for 4 weeks. W2-11 and W7-3 are independent lines transgenic for pBI121-35S/SpHMT1:cmyc in wild-type Col-0 background. Fresh weight (FW; E) and metal content (F) were determined in plants grown under conditions in A to D. Values are mean ± sd, n = 12 for E, or n = 4 for F, each containing greater than five plants. Asterisks indicate significant difference between wild type and the transgenic lines at P < 0.05 (*) and 0.01 (**) by t tests.

Figure 3.

Cd²⁺ sensitivity and accumulation in cad1-3 expressing SpHMT1. Plants were germinated and grown vertically on one-fourth Murashige and Skoog plates under control condition (A) or with 5 μm CdCl₂ (B). M10-8 and M1-12 represent cad1-3 plants transformed with pBI121-35S/SpHMT1:cmyc. Photographs taken at day 7 (A) and day 20 (B) post germination. C, Fresh weights of plants grown under control condition (CK) in A or with 5 μm CdCl₂ (Cd5) in B. D, Cd²⁺ concentration in seedlings grown under conditions in A and B. Values are mean ± sd, n = 12 for C, or n = 4 for D, each containing greater than five plants. Lowercase letters indicate whether averages were statistically different at P < 0.05 by t tests. [See online article for color version of this figure.]

Figure 4.

Subcellular localization of Cd²⁺. Protoplasts and vacuoles isolated from leaves of soil-grown plants (Col-0, cad1-3, and transgenic W2-11, W7-3, M1-12, and M10-8) were measured for Cd²⁺ content. Values above the bars represent the percentage of vacuolar Cd²⁺ relative to the total in protoplasts. Acid phosphatase (ACP) activity specific to vacuole was determined and used to normalize Cd²⁺ accumulation; data are mean ± se from three independent experiments.

Figure 5.

Distribution of GSH and PCs. Protoplasts and vacuoles were extracted from soil-grown plant leaves of Col-0 (A), cad1-3 (B), and cad2-1 (C). GSH and PCs measured by fluorescence HPLC. D, Synthesized GSH, PC2, PC3, and PC4 used as standards. E, Blank control. Peaks are indicated by arrows. [See online article for color version of this figure.]

Figure 6.

Increased vacuolar PCs in transgenic SpHMT1 plants. Soil-grown plants were used for protoplasts and vacuoles isolation. PC contents in vacuoles from Col-0 and the transgenic plants W2-11 and W7-3 were determined as described in “Materials and Methods.” Acid phosphatase (ACP) activity specific to vacuoles was determined and used to normalize PC accumulation. Data are mean ± se from three independent experiments. Asterisks indicate significant difference between wild type and the transgenic lines at P < 0.05 (*) and 0.01 (**) by t tests.

Figure 7.

Long-distance Cd²⁺ transport from roots to shoots. Plants were grown hydroponically for 4 weeks before treatment with 10 μm CdCl₂ for 24 h (A), 3 d (B), and 7 d (C). Cd²⁺ concentrations in stems (S), rosette leaves (L), and roots (R) of wild-type Col-0 and transgenic lines (W2-11, W7-3, and W3-13) were determined. Values are mean ± sd, n = 4, each containing greater than eight plants. Asterisks indicate significant difference between wild type and the transgenic lines at P < 0.05 (*) and 0.01 (**) by t tests.

Figure 8.

Targeted SpHMT1 expression in roots decreased metal accumulation in leaves and seeds. Metal content in rosette leaves (L), roots (R), and seeds (D–F) measured in plants grown hydroponically to 4 weeks of age and exposed to 10 μm CdCl₂ (A), 100 μm KH₂AsO₄ (B), or 20 μm CuSO₄ (C) for 3 d before sampling, or in 2-week-old hydroponically grown plants supplemented with 5 μm CdCl₂ (D), KH₂AsO₄ (E), or CuSO₄ (F) until maturation of seeds. A5-9 and A26-4 represent independent pBI121-Adh/SpHMT1:cmyc transformed lines, while W2-11 represents a pBI121-35S/SpHMT1:cmyc transgenic line. Values are mean ± sd, n = 4, each containing greater than eight plants. Asterisks indicate significant difference between wild type and the transgenic lines at P < 0.05 (*) and 0.01 (**) by t tests.