Wenju Liu

Methylated arsenic species in plants originate from soil microorganisms

• Inorganic arsenic (iAs) is a ubiquitous human carcinogen, and rice (Oryza sativa) is the main contributor to iAs in the diet. Methylated pentavalent As species are less toxic and are routinely found in plants; however, it is currently unknown whether plants are able to methylate As. • Rice, tomato (Solanum lycopersicum) and red clover (Trifolium pratense) were exposed to iAs, monomethylarsonic acid (MMA(V)), or dimethylarsinic acid (DMA(V)), under axenic conditions. Rice seedlings were also grown in two soils under nonsterile flooded conditions, and rice plants exposed to arsenite or DMA(V) were grown to maturity in nonsterile hydroponic culture. Arsenic speciation in samples was determined by HPLC-ICP-MS. • Methylated arsenicals were not found in the three plant species exposed to iAs under axenic conditions. Axenically grown rice was able to take up MMA(V) or DMA(V), and reduce MMA(V) to MMA(III) but not convert it to DMA(V). Methylated As was detected in the shoots of soil-grown rice, and in rice grain from nonsterile hydroponic culture. GeoChip analysis of microbial genes in a Bangladeshi paddy soil showed the presence of the microbial As methyltransferase gene arsM. • Our results suggest that plants are unable to methylate iAs, and instead take up methylated As produced by microorganisms.

Complexation of Arsenite with Phytochelatins Reduces Arsenite Efflux and Translocation from Roots to Shoots in Arabidopsis

Complexation of arsenite [As(III)] with phytochelatins (PCs) is an important mechanism employed by plants to detoxify As; how this complexation affects As mobility was little known. We used high-resolution inductively coupled plasma-mass spectrometry and accurate mass electrospray ionization-mass spectrometry coupled to HPLC to identify and quantify As(III)-thiol complexes and free thiol compounds in Arabidopsis (Arabidopsis thaliana) exposed to arsenate [As(V)]. As(V) was efficiently reduced to As(III) in roots. In wild-type roots, 69% of As was complexed as As(III)-PC4, As(III)-PC3, and As(III)-(PC2)2. Both the glutathione (GSH)-deficient mutant cad2-1 and the PC-deficient mutant cad1-3 were approximately 20 times more sensitive to As(V) than the wild type. In cad1-3 roots, only 8% of As was complexed with GSH as As(III)-(GS)3 and no As(III)-PCs were detected, while in cad2-1 roots, As(III)-PCs accounted for only 25% of the total As. The two mutants had a greater As mobility, with a significantly higher accumulation of As(III) in shoots and 4.5 to 12 times higher shoot-to-root As concentration ratio than the wild type. Roots also effluxed a substantial proportion of the As(V) taken up as As(III) to the external medium, and this efflux was larger in the two mutants. Furthermore, when wild-type plants were exposed to l-buthionine sulfoximine or deprived of sulfur, both As(III) efflux and root-to-shoot translocation were enhanced. The results indicate that complexation of As(III) with PCs in Arabidopsis roots decreases its mobility for both efflux to the external medium and for root-to-shoot translocation. Enhancing PC synthesis in roots may be an effective strategy to reduce As translocation to the edible organs of food crops.

Combined NanoSIMS and synchrotron X-ray fluorescence reveal distinct cellular and subcellular distribution patterns of trace elements in rice tissues

The cellular and subcellular distributions of trace elements can provide important clues to understanding how the elements are transported and stored in plant cells, but mapping their distributions is a challenging task. The distributions of arsenic, iron, zinc, manganese and copper, as well as physiologically related macro-elements, were mapped in the node, internode and leaf sheath of rice (Oryza sativa) using synchrotron X-ray fluorescence (S-XRF) and high-resolution secondary ion mass spectrometry (NanoSIMS). Although copper and silicon generally showed cell wall localization, arsenic, iron and zinc were strongly localized in the vacuoles of specific cell types. Arsenic was highly localized in the companion cell vacuoles of the phloem in all vascular bundles, showing a strong co-localization with sulfur, consistent with As(III)-thiol complexation. Within the node, zinc was localized in the vacuoles of the parenchyma cell bridge bordering the enlarged and diffuse vascular bundles, whereas iron and manganese were localized in the fundamental parenchyma cells, with iron being strongly co-localized with phosphorus in the vacuoles. The highly heterogeneous and contrasting distribution patterns of these elements imply different transport activities and/or storage capacities among different cell types. Sequestration of arsenic in companion cell vacuoles may explain the limited phloem mobility of arsenite.

Arsenic Methylation in Soils and Its Relationship with Microbial arsM Abundance and Diversity, and As Speciation in Rice

Methylation of arsenic in soil influences its environmental behavior and accumulation by plants, but little is known about the factors affecting As methylation. As speciation was determined in the pore waters of six soils from diverse geographical locations over 54 days of incubation under flooded conditions. The concentration of methylated As (monomethylarsonic acid, MMA, and dimethylarsinic acid, DMA) varied from 0 to 85 μg L(-1) (0 - 69% of the total As in pore water). Two Bangladeshi paddy soils contaminated by irrigation of As-laden groundwater produced large concentrations of inorganic As but relatively little methylated As. Two contaminated paddy soils from China produced a transient peak of DMA during the early phase of incubation. Methylated As represented considerable proportions of the total soluble As in the two uncontaminated soils from the UK and U.S. The copy number of the microbial arsenite methyltransferase gene (arsM) correlated positively with soil pH. However, pore-water methylated As correlated negatively with pH or arsM copy number, and positively with dissolved organic C. GeoChip assay revealed considerable arsM diversity among the six soils, with 27-35 out of 66 sequences in the microarray being detected. As speciation in rice plants grown in the soils generally mirrored that in the pore water. The results suggest that methylated As species in plants originated from the soil and As methylation in soil was influenced strongly by the soil conditions.

Silicon has opposite effects on the accumulation of inorganic and methylated arsenic species in rice

Background and aimsRice (Oryza sativa) is a main source of human exposure to inorganic arsenic and mitigation measures are needed to decrease As accumulation in this staple crop. It has been shown that silicon decreases the accumulation of arsenite but, unexpectedly, increases the accumulation of dimethylarsinic acid (DMA) in rice grain. The aim of this study was to investigate why Si increases DMA accumulation.MethodsPot and incubation experiments were conducted to investigate how the addition of sparingly soluble silicate gel affected As speciation in the soil solution and the accumulation of different As species in rice tissues.ResultsSilicon addition significantly decreased the concentration of inorganic As (mainly arsenite) but increased the concentration of DMA in both the vegetative and reproductive tissues of rice. Silicon increased the concentration of DMA in the soil solution, whereas autoclaving soil decreased DMA concentration. Less DMA was adsorbed by the soil than arsenate and Si addition significantly inhibited DMA adsorption.ConclusionsSilicon increased DMA accumulation and decreased arsenite accumulation in rice through different mechanisms. Silicic acid released from the silicate gel increased the availability of DMA for rice uptake by inhibiting DMA adsorption on the soil solid phase or by displacing adsorbed DMA. Although silicic acid also increased the concentration of inorganic As in the soil solution, this effect was much smaller than the inhibitory effect of Si on arsenite uptake by rice roots.

Heterologous expression of the yeast arsenite efflux system ACR3 improves Arabidopsis thaliana tolerance to arsenic stress

• Arsenic contamination has a negative impact on crop cultivation and on human health. As yet, no proteins have been identified in plants that mediate the extrusion of arsenic. Here, we heterologously expressed the yeast (Saccharomyces cerevisiae) arsenite efflux transporter ACR3 into Arabidopsis to evaluate how this affects plant tolerance and tissue arsenic contents. • ACR3 was cloned from yeast and transformed into wild-type and nip7;1 Arabidopsis. Arsenic tolerance was determined at the cellular level using vitality stains in protoplasts, in intact seedlings grown on agar plates and in mature plants grown hydroponically. Arsenic efflux was measured from protoplasts and from intact plants, and arsenic levels were measured in roots and shoots of plants exposed to arsenate. • At the cellular level, all transgenic lines showed increased tolerance to arsenite and arsenate and a greater capacity for arsenate efflux. With intact plants, three of four stably transformed lines showed improved growth, whereas only transgenic lines in the wild-type background showed increased efflux of arsenite into the external medium. The presence of ACR3 hardly affected tissue arsenic levels, but increased arsenic translocation to the shoot. • Heterologous expression of yeast ACR3 endows plants with greater arsenic resistance, but does not lower significantly arsenic tissue levels.

Knocking Out ACR2 Does Not Affect Arsenic Redox Status in Arabidopsis thaliana: Implications for As Detoxification and Accumulation in Plants

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.