Engineering Arsenic Tolerance and Hyperaccumulation in Plants for Phytoremediation by a PvACR3 Transgenic Approach

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1 pubs.acs.org/est Engineering Arsenic Tolerance and Hyperaccumulation in Plants for Phytoremediation by a PvACR3 Transgenic Approach Yanshan Chen,,, Wenzhong Xu,, Hongling Shen,, Huili Yan,, Wenxiu Xu, Zhenyan He, and Mi Ma*, Key Laboratory of Plant Resources, Institute of Botany, Chinese Academy of Sciences, Beijing , China University of Chinese Academy of Sciences, Beijing , China *S Supporting Information ABSTRACT: Arsenic (As) pollution is a global problem, and the plant-based cleanup of contaminated soils, called phytoremediation, is therefore of great interest. Recently, transgenic approaches have been designed to develop As phytoremediation technologies. Here, we used a one-gene transgenic approach for As tolerance and accumulation in Arabidopsis thaliana. PvACR3, a key arsenite [As(III)] antiporter in the As hyperaccumulator fern Pteris vittata, was expressed in Arabidopsis, driven by the CaMV 35S promoter. In response to As treatment, PvACR3 transgenic plants showed greatly enhanced tolerance. PvACR3 transgenic seeds could even germinate and grow in the presence of 80 μm As(III) or 1200 μm arsenate [As(V)] treatments that were lethal to wildtype seeds. PvACR3 localizes to the plasma membrane in Arabidopsis and increases arsenite efflux into external medium in shortterm experiments. Arsenic determination showed that PvACR3 substantially reduced As concentrations in roots and simultaneously increased shoot As under 150 μm As(V). When cultivated in As(V)-containing soil (10 ppm As), transgenic plants accumulated approximately 7.5-fold more As in above-ground tissues than wild-type plants. This study provides important insights into the behavior of PvACR3 and the physiology of As metabolism in plants. Our work also provides a simple and practical PvACR3 transgenic approach for engineering As-tolerant and -hyperaccumulating plants for phytoremediation. INTRODUCTION Heavy metals and metalloids, such as cadmium (Cd), lead (Pb), mercury (Hg), and arsenic (As), are released into the environment by mining, industry, and agriculture, causing severe global environmental pollution and threatening human health. 1,2 Being a carcinogen, arsenic adversely affects the health of millions of people, particularly when exposure occurs over prolonged periods. 3 Arsenic uptake by humans mainly occurs through drinking As-contaminated water and eating plants grown in contaminated soil. 4 However, the removal of As from soil by physical remediation methods, such as soil removal or burial, is expensive and environmentally destructive. 5,6 In this context, the plant-based cleanup of contaminated soils, called phytoremediation, is of major interest. 1,7,8 Phytoremediation technologies are currently available for As through the use of naturally selected hyperaccumulating ferns In 2001, Pteris vittata (Chinese brake fern) was first reported as an As hyperaccumulator by Ma et al. 9 This plant is highly efficient in terms of extraction of As from soil and its translocation to above-ground biomass; thus, this species has great potential as a low-cost remediation method for As-contaminated soils. 9 To date, several hyperaccumulating ferns in the order Pteridales have been identified, including several Pteris species and Pityrogramma calomelanos. 12 However, the geographic and ecological distribution of these ferns is limited and their biomass is low compared to some other plants. Transgenic plants have been used in the development of phytoremediation technologies. 8 Guo et al. 13,14 found that the expression of GSH1 and AsPCS1, or AsPCS1 and YCF1, conferred As tolerance and led to As accumulation in Arabidopsis thaliana. Dhankher et al. 5,7 made important progress by overexpressing two bacterial genes, ArsC and γ- ECS, ina. thaliana to increase As tolerance and accumulation. These strategies provided new insights into As hyperaccumulation in plants. They also indicated that multiple genes may be needed in the engineering of transgenic plants for soil cleanup. However, transforming multiple genes into plants and optimizing their expression is time-consuming and costly. Arsenate [As(V)] is the dominant form of As in aerobic soils. 15,16 In plants, As(V) is taken up by phosphate transporters as an analogue of the macronutrient phosphate in the root plasmalemma; 17 most As(V) is reduced to arsenite Received: March 19, 2013 Revised: July 29, 2013 Accepted: July 30, 2013 Published: July 30, American Chemical Society 9355

2 [As(III)] in the root cytosol. 18 In most plants, a large amount of As is accumulated in the roots and only a small fraction is exported via the xylem to above-ground tissues. 19 For effective phytoremediation, it is essential to enhance As transport from roots to shoots, where it can be easily harvested. 6,19 In 2010, the As(III) antiporter PvACR3 was identified in the As-hyperaccumulating fern P. vittata. 20 PvACR3 localizes to the vacuolar membrane in gametophytes and is likely to extrude As(III) into the vacuole for sequestration. Here, we found that, in Arabidopsis, PvACR3 was localized to the plasma membrane and the expression of PvACR3 conferred As tolerance. Moreover, we also found that the expression of PvACR3 increased As translocation to plant shoots under As(V) treatment without causing severe toxicity effects. Although the mechanisms need further investigation, this PvACR3 transgenic approach proved effective in Arabidopsis and can be used to engineer As tolerance and accumulation in plants for As phytoremediation. MATERIALS AND METHODS Synthesis of the PvACR3 Gene from Pteris vittata. PvACR3 (GI ) Coding sequence (CDS) was cloned from a cdna library of the arsenic hyperaccumulating fern P. vittata preserved in our laboratory (collected from an Ascontaminated mine site in Hubei Province, China 21 ) by use of the following primers: 5 -ATG GAG AAC TCA AGC GCG GAG CGG A-3 and 5 -CTA AAC AGA AGG CCC CTT CCT CTG A-3. Generation and Selection of Transgenic Arabidopsis. Adapters were added to PvACR3 CDS by use of the following primers: 5 -acg ggg gac tct aga gga tcc ATG GAG AAC TCA AGC GCG GAG CGG A-3 and 5 -ggg aaa ttc gag ctc ggt acc CTA AAC AGA AGG CCC CTT CCT CTG A-3 (underlining indicates recombination sequences). The PCR product was then cloned into the 35S promoter cassette of psn1301 (derived from pcambia1301, CAMBIA) 13 between BamHI and KpnI restriction sites by recombination, using the CloneEZ PCR cloning kit (Genscript). The constructed binary vector was named psn1301-pvacr3. Agrobacterium strain C58 was transformed with the binary vector psn1301-pvacr3 by electroporation. The Agrobacterium culture was used to transform A. thaliana Col-0 by Agrobacterium-mediated dip floral transformation. 22 Homozygous lines were identified in the T3 generation via segregation analysis. Plant Growth Conditions and Arsenic-Tolerance Analysis. For analysis of arsenic resistance of Arabidopsis seedlings, seeds were surface-sterilized and sown on medium containing 1/2 Murashige and Skoog (MS) salts, 1% sucrose, and 0.5 g/l 2-(N-morpholino)ethanesulfonic acid (MES), ph 5.9, and solidified with 0.8% agar. After 2 days to synchronize germination at 4 C in the dark, the plates were placed in a growth chamber at 22 C with a 16-h light/8-h dark regime to facilitate germination. Arabidopsis seedlings, 3 days after germination, were transferred onto plates containing the 1/2 MS agar medium without or supplemented with various concentrations of sodium arsenite/arsenate and grown vertically at 22 C with a 16-h light/8-h dark regime for 7 or 21 days. For soil cultivation, Arabidopsis seeds were germinated and cultivated in soil spiked with 5 or 10 ppm arsenate. The pots were placed at 22 C with a 16-h light/8-h dark regime for ca. 3 months and the rosette leaves, stalks, and seeds were harvested for arsenic determination Total Arsenic Determination. Three-day-old Arabidopsis seedlings were transferred onto plates containing the 1/2 MS agar medium supplemented with different concentrations of arsenite or arsenate and grown vertically under a 16-h light/8-h dark photoperiod for 7 or 15 days. The plants ( per treatment) were washed with distilled water three times and the shoots and roots were collected. All samples for arsenic determination were dried at 80 C for 6 h and digested with a concentrated acid (guaranteed reagent) mixture of HNO 3, HClO 4, and H 2 SO 4 (volume ratio = 4:1:0.5) at 250 C for 8 h. The concentrated acid was diluted with distilled water to a final concentration of 5%. The arsenic concentration of the solution was determined on an inductively coupled plasma optical emission spectrometer (ICP-OES) (icap6300, Thermo Electron Corp.). Arsenite Efflux Assays and Arsenic Speciation Analyses. Uniform 10-day-old Arabidopsis seedlings (18 per treatment) were transferred from agar plates to vessels with 15 ml of 10 μm As(V) nutrient solution (1/2 MS salts, 1% sucrose, and 0.5 g/l MES, ph 5.9) and cultivated for 24 h, after which the As species in the solution were determined as described by Duan et al. 23 Arsenic species in the plants were determined as described by Xu et al. 18 Arsenic speciation was determined by anion-exchange chromatograms from HPLC/ ICP-MS (Agilent LC1100 series and Agilent ICP-MS 7500ce; Agilent Technologies). For other methods, see Materials and Methods section in Supporting Information. RESULTS PvACR3 Increases Arsenite Efflux in Yeast. Nucleotide BLAST showed that the cloned PvACR3 CDS (GI ) varied by 20 nt from the previously reported PvACR3 (GI ), 20 which resulted in a nine amino acid difference. A comparison of the protein between the current PvACR3 (GI ) and the former PvACR3 (GI ) 20 is presented as Figure S1 in Supporting Information. To confirm the roles of PvACR3 (GI ), full-length coding sequences were cloned into pag413gal-ccdb under control of the GAL1 promoter and expressed in the Δacr3 yeast mutant (BY4741 background), which is sensitive to As(III) due to the deletion of yeast As(III) antiporter ACR3 on the plasma membrane. 24 Growth tests were performed with 50 μm As(III). As shown in Figure S2A in Supporting Information, the expression of PvACR3 induced by galactose enhanced the tolerance of Δacr3 to As(III) and completely suppressed the As(III)-sensitive phenotype. Arsenic determination showed that PvACR3 substantially reduced As accumulation in the yeast (Figure S2B in Supporting Information). These results indicate that the obtained version of PvACR3 was a functional As(III) antiporter that played an important role in As(III) efflux into the external medium across the yeast plasma membrane. PvACR3 Confers Arsenic Tolerance on Transgenic Plants. To investigate the effect of PvACR3 on plant growth, we produced two homozygous transgenic lines (L9 and L11) that expressed PvACR3 under the control of the constitutive CaMV 35S promoter. Three-day-old seedlings were transferred onto plates with various concentrations of As(III) or As(V) and cultivated for 7 days. In the absence of As, these plants appeared similar to the wild type (WT), indicating that neither insertion nor expression of PvACR3 was deleterious (Figure 1A). In the presence of 10 μm As(III) or 300 μm As(V), the

3 Figure 1. Tolerance of transgenic Arabidopsis plants expressing PvACR3 to arsenic (As). (A C) Comparative growth of 3-day-old Arabidopsis seedlings grown vertically on plates with (A) no arsenic, (B) 10 μm arsenite [As(III)], and (C) 300 μm arsenate [As(V)] for 7 days. (D) Comparative growth of 3-day-old Arabidopsis seedlings grown vertically on 300 μm As(V) for 21 days. A single seedling each of the wild type (WT), L9, and L11 was moved to a new plate for photography. (E) RT-PCR detection of intact PvACR3 mrna transcripts in both shoots and roots in transgenic lines L9 and L11. (F, G) Statistical analyses of (F) root elongation and (G) fresh weight of 3-day-old seedlings of WT (black bars), L9 (white bars), and L11 (gray bars) after cultivation under different As(III) and As(V) concentrations for 7 days (n = 8 plants per treatment per line). Plants shown are representative of three independent experiments. Scale bars, 15 mm. Asterisks indicate significant differences from the WT detected by one-way analysis of variance (ANOVA) (*, P < 0.05). Error bars, mean ± SEM. transgenic lines had uninhibited roots and grew at approximately the same rate as unchallenged controls, while the WT was severely stunted (Figure 1B,C). In the presence of 10 μm As(III), root elongation in the WT dropped by 94% compared with the control, whereas root elongation in L9 and L11 dropped by only 1% and 5%, respectively (Figure 1F). Upon exposure to other As treatments, transgenic plants also displayed more vigorous root growth with better-developed root systems, whereas root elongation in control seedlings was severely or totally inhibited (Figure 1F). Reverse transcription polymerase chain reaction (RT-PCR) analysis showed that the PvACR3 transcript accumulated in both shoots and roots of L9 and L11, while it was not detected in WT plants (Figure 1E). In addition to vigorous root growth, the above-ground parts of the PvACR3 transgenic lines were also in better condition, with green and thriving leaves, during As stress (Figure 1B,C). Transgenic plants accumulated approximately 2-fold more fresh weight than WT plants over the 7-day growth period (Figure 1G). When grown on 300 μm As(V) plates for up to 3 weeks, the transgenic plants accumulated 10-fold more biomass (data not shown) than the WT plants, which died due to As poisoning (Figure 1D). To further evaluate the effect of As stress on seed germination and subsequent growth, seeds were sown on Ascontaining medium. The germination and growth progress of transgenic lines on As-free plates were consistent with WT plants. On 60 or 80 μm As(III) plates, WT seeds were stunted at the germination stage, while the transgenic lines produced well-developed cotyledons and roots (Figure S3A in Supporting Information). In the presence of 300 μm As(V), the transgenic plants grew at approximately the same rate as unchallenged controls, but WT Arabidopsis was initially stunted at the 9357 germination stage and finally killed during the elongation stage (Figure S3B in Supporting Information). Unexpectedly, most transgenic seeds survived on plates with up to 1200 μm As(V) and developed 2 4 green leaves (Figure S3B). In conclusion, these results demonstrate that the expression of PvACR3 in Arabidopsis enhanced As resistance and promoted growth, regardless of the presence of As(III) or As(V). PvACR3 Localizes to the Plasma Membrane in Arabidopsis. The subcellular location of the As(III) antiporter PvACR3 in Arabidopsis is critical for its physiological function. We therefore prepared (C-terminal) PvACR3 green fluorescent protein (GFP) fusions. GFP fluorescence in transgenic Arabidopsis was then visualized. As shown in Figure 2A,B, the GFP signal was localized in the periphery of root cells. After plasmolysis, fluorescence was mostly associated with the plasma membrane but not with the cell wall (Figure 2E,F). These results suggest that PvACR3 localized to the plasma membrane. To verify this, transgenic Arabidopsis root cells were stained with FM4-64, a plasma membrane marker; red fluorescence labeling of the plasma membrane was observed (Figure 2C), and the GFP and red fluorescence signals overlapped (Figure 2D). This further confirmed that PvACR3 localized to the plasma membrane in Arabidopsis. Heterologous Expression of PvACR3 Increases Arsenite Efflux. Expression of yeast ACR3 has been proven to increase arsenite efflux in rice 23 and Arabidopsis. 25 In consideration of PvACR3 being localized to the plasma membrane in Arabidopsis, arsenite efflux experiments were performed. Uniform Arabidopsis plants were treated with 10 μm As(V), and then As(V) and As(III) concentrations in both plants and the external nutrient solution were monitored after 24 h. As(III) efflux into external medium by plant roots follows

4 plates with 5 μm As(III) or 150 μm As(V), which represent moderate As concentrations and caused no severe growth inhibition in the WT, and cultivated for 7 days. Then the As concentrations in shoots (Figure 4A,B) and roots (Figure Figure 2. PvACR3 localizes to the plasma membrane in Arabidopsis. The PvACR3-GFP fusion protein was expressed in Col-0 Arabidopsis under control of the CaMV 35S promoter, and GFP fluorescence was observed in roots by confocal microscopy. (A) Bright-field image, (B) GFP green fluorescence, and (C) FM4-64 staining of an exodermis cell of the pgfp121-pvacr3 transgenic line shown at the root hair zone. (D) Merged image of B and C. (E) Bright-field image and (F) GFP fluorescence were observed after plasmolysis in root exodermis cells. Scale bars, 40 μm. As(V) uptake and reduction in the root cells. 18 After 24 h, hardly any As(III) was detected in the solution without plants, whereas As(III) concentrations in the solutions of PvACR3- expressing lines were much higher than those of the WT (Figure S4 in Supporting Information). As(III) efflux activity was then calculated from the production of As(III) in the solution according to root biomass and as a percentage of As(V) uptake. Both results showed that As(III) efflux activity of L9 and L11 was more than 2-fold higher than that of the WT (Figure 3), which suggests that PvACR3 increases As(III) efflux into external medium in Arabidopsis roots. Expressing PvACR3 Decreases Root Arsenic Accumulation and Alters the Partitioning of Arsenic in Arabidopsis. Three-day-old seedlings were transferred to Figure 4. Arsenic (As) accumulation in PvACR3 transgenic Arabidopsis plants under different As treatments. Three-day-old seedlings were transferred onto plates containing 5 μm arsenite [As(III)] or 150 μm arsenate [As(V)] and grown for 7 days, after which As accumulation was measured in the shoots and roots of the wild-type (WT), L9, and L11 seedlings. Shoot As concentrations are shown for (A) 5 μm As(III) and (B) 150 μm As(V) treatments, and root As concentrations are shown for (C) 5 μm As(III) and (D) 150 μm As(V) treatments. Shoot:root As concentration ratios (translocation factor) are given for (E) 5 μm As(III) and (F) 150 μm As(V) treatments. (G) As species as a percentage of total As in Arabidopsis roots and shoots under 150 μm As(V) treatment. The experiment was performed in triplicate. Asterisks indicate significant differences from the WT determined by one-way ANOVA (*, P < 0.05). Error bars, mean ± SEM. Figure 3. PvACR3 increases arsenite [As(III)] efflux. Uniform 10-dayold Arabidopsis seedlings (18 plants for each treatment) were transferred into 15 ml of 10 μm arsenate [As(V)] nutrient solution and cultivated for 24 h, after which the production of As(III) in the solution was determined by anion-exchange chromatograms from HPLC/ICP-MS. As(III) efflux was calculated (A) according to root biomass and (B) as a percentage of As(V) uptake. The experiment was performed in triplicate. Asterisks indicate significant differences from the WT determined by one-way ANOVA (*, P < 0.05). Error bars, mean ± SEM. 4C,D) were measured. Compared to the WT control, transgenic lines had substantially lower concentrations of As in their roots in both the As(III) and As(V) treatments. In the roots of L9 and L11, arsenic accumulation dropped by 90% under 5 μm As(III) and 75% under 150 μm As(V), compared to the WT (Figure 4C,D). The large reduction of As in the roots is the result of the increased As(III) efflux into the external medium (Figure 3) and also provides an explanation for the observed As tolerance in transgenic plants. 9358

5 As(V) is often reduced to As(III), making As(III) the dominant form in plants. 5,18,26 The As species in plants under 150 μm As(V) were determined (Figure 4G), which also supported this notion. The As species pattern was significantly different in roots of the PvACR3-expressing lines and the WT. In WT roots, 72% of total As was As(III), whereas in PvACR3 transgenic lines, 52 54% of total As was As(III) (Figure 4G). Both the decreased As concentrations and the decreased proportion of As(III) further demonstrated that PvACR3 expression enhanced As(III) efflux into external medium from Arabidopsis roots. In most plants, large amounts of As are accumulated in the roots after uptake and only small amounts are transported to shoots. 6 This is consistent with the very low translocation factors (ratio of As concentration in shoot to that in root) 27 that were calculated for WT Arabidopsis, which were 0.05 for As(III) (Figure 4E) and 0.15 for As(V) (Figure 4F) in our experiments. In transgenic lines, although shoot As concentration decreased after 5 μm As(III) treatment (Figure 4A), the translocation factors were much higher (Figure 4E), which indicates that the level of root-to-shoot As translocation in transgenic lines increased in the presence of As(III). With exposure to 150 μm As(V), the translocation factors in both transgenic lines also increased significantly (0.75 and 0.72 for L9 and L11, respectively, compared to 0.15 for the WT; Figure 4F). Furthermore, shoot As accumulation in L9 and L11 increased by 15% and 22%, respectively, compared with the WT (Figure 4B). Analysis of the As species revealed that the proportion of As(III) increased slightly in shoots in L9 and L11 compared with WT, while it decreased in roots (Figure 4G). These results indicated that PvACR3 may increase As(III) translocation to achieve increased As translocation following treatment with As(V). Increased Movement of Arsenic to Shoots Is Not Detrimental to Overall Tolerance in Arabidopsis. To protect photosynthetic systems, arsenic is prevented from entering plant shoots. 25 We investigated whether the enhanced As levels caused harm to the overall tolerance in Arabidopsis. Three-day-old seedlings were cultivated for 15 days in a 150 μm As(V) treatment (Figure 5A). Under these conditions, shoot As accumulation increased significantly, by 15% and 32% in L9 and L11, respectively, compared to the WT (Figure 5C). Note that the As levels in L9 and L11 reached 970 and 1113 μg/g dry weight ( 1 ; Figure 5C), respectively, which represents very high As levels for the As-sensitive weed Arabidopsis. However, L9 and L11 appeared dark green and vigorous, with more healthy leaves compared to the WT; in the WT, the leaves were stunted and turned yellow after long-term As exposure (Figure 5A). As a result, the transgenic lines attained 3-fold higher shoot biomass levels than the WT (Figure 5B). This indicates that the expression of PvACR3 conferred increased As translocation to shoots and simultaneously resulted in As detoxification in leaves. Our data suggest that transgenic plants retained higher As concentrations and much higher amounts of biomass and were able to accumulate 4-fold more As in their shoots (data not shown). Expression of PvACR3 Enhances Shoot Arsenic Accumulation in Arabidopsis after Long-Term Soil Cultivation. Because PvACR3 resulted in both As tolerance and As accumulation in shoots in As(V) treatments, the PvACR3 transgenic approach may be suitable for engineering As tolerance and achieving enhanced accumulation in plants for As phytoremediation. An important and crucial step in Figure 5. Expression of PvACR3 improves shoot growth and enhances both shoot biomass and arsenic (As) accumulation in transgenic Arabidopsis under arsenate [As(V)] treatment. (A) Three-day-old seedlings were transferred onto plates containing 150 μm As(V) for 15 days, after which (B) shoot fresh weight and (C) shoot As accumulation in wild-type (WT), L9, and L11 seedlings were measured. The experiment was performed in triplicate (n = plants per treatment per line). Asterisks indicate significant differences from the WT determined by one-way ANOVA (*, P < 0.05). Error bars, mean ± SEM. Scale bar, 15 mm. validating the potential of our strategy was to determine whether this single-gene transgenic approach works in soils that contain microorganisms and where As is bound to the soil and sediment, compared to being soluble in agar plates. At the same time, soil cultivation provides optimal conditions for long-term cultivation and may optimize the bioaccumulation of As in PvACR3 transgenic plants, whereas sterile agar plates are only suitable for short-term growth. We performed long-term soil cultivation experiments in which Arabidopsis seeds were sown in soil that was spiked with As(V). In artificially contaminated soil containing 10 ppm As(V), the above-ground tissues of transgenic plants were better developed, with higher biomass of rosette leaves and stalks and increased seed production compared to WT plants (Figure 6A,B). After 90 days of exposure to As(V), the transgenic plants also accumulated notably more As in their above-ground tissues compared to the WT: 4-fold more As in rosette leaves, 7-fold more in stalks, and 3-fold more in seeds (Figure 6C). These data suggest that the total amount of As stored in each transgenic plant shoot was 7.5-fold higher than in the WT. In soil containing 5 ppm As, the above-ground tissues of transgenic plants also exhibited greater As concentrations (Figure 6D). Transgenic plants also had much higher [As] shoot :[As] soil ratios, that is, the accumulation factor (AF), 28 which suggests they would be more suitable for As phytoremediation. 9359

6 Figure 6. Transgenic Arabidopsis plants accumulated more biomass and arsenic (As) in above-ground tissues after long-term cultivation in soil. (A) Seeds of wild type (WT), L9, and L11 were sown on soil spiked with 10 ppm arsenate [As(V)] and grown for 70 days. (B) Dry weights and (C) As concentrations of above-ground tissues (rosette leaves, stalks, and seeds) of WT (black bars), L9 (white bars), and L11 (gray bars) plants grown in soil spiked with 10 ppm As(V) for 3 months (n = 12 plants per treatment per line) are shown. (D) As concentrations in above-ground tissues of WT, L9, and L11 grown in soil spiked with 5 ppm As(V) for 3 months. The experiment was performed in triplicate. Asterisks indicate significant differences from the WT determined by one-way ANOVA (*, P < 0.05). Error bars, mean ± SEM. Scale bar, 40 mm. DISCUSSION Arsenite Efflux Mediated by PvACR3 Is Critical for Arsenic Detoxification in Arabidopsis. Although As(III) efflux transporters have been rarely reported, As(III) efflux by plant roots has been demonstrated. 18 Aquaporin Lsi1 facilitates both As(III) uptake and As(III) efflux in rice roots. 29,30 Some other aquaporins in plants have also been reported to transport As(III) bidirectionally and may result in As(III) efflux. 31,32 However, the aquaporin-mediated efflux of As(III) is a passive process, with the direction of flux depending on the concentration gradient. 30 In contrast, the As(III) antiporter ACR3 relies on the proton motive force for energy. 24 Thus, As(III) efflux mediated by P. vittata ACR3 may be an active process that can be more efficient than aquaporins. Moreover, the highly conserved ACR3 gene was lost in angiosperm genomes. 20 In the As-hyperaccumulating fern P. vittata, PvACR3 extrudes As(III) into vacuoles for sequestration since it localizes to the vacuolar membrane in gametophytes. 20 However, the localization of PvACR3 in the sporophyte can be complicated. If PvACR3 localizes to the vacuolar membrane in sporophyte roots, then large amounts of arsenic can be retained in roots, which contradicts previous reports that P. vittata accumulates more arsenic in fronds than in roots. 9 Moreover, arsenategrown sporophyte roots accumulated 15-fold more ACR3 transcripts than frond tissue. 20 Actually, Indriolo et al. 20 suggested that PvACR3 may also have a function in terms of loading arsenite into the xylem. This can be achieved when PvACR3 localizes to the plasma membrane in sporophyte roots in P. vittata. Thus, the localization and function of PvACR3 in P. vittata, especially in sporophyte roots, should be further investigated. Unlike in P. vittata gametophytes, PvACR3 localizes to the plasma membrane in Arabidopsis (Figure 2). Considering the different subcellular localization, we hypothesize that PvACR3 is responsible for As(III) efflux from the cytoplasm in Arabidopsis. In root exodermis cells, this was confirmed by the increased As(III) efflux into external medium in PvACR3- expressing lines (Figure 3), which also led to considerable reduction in As accumulation (Figure 4C,D) and a decreased As(III) proportion (Figure 4G) in the roots. In shoots, PvACR3-mediated As(III) efflux may result in As sequestration in apoplasts. Overall, an increased As(III) efflux would reduce both root As levels and whole-plant cytoplasmic As concentration, which would release the plant from As toxicity and promote growth (Figures 1, 5A, and 6A, Figure S3 in Supporting Information). Arsenic apoplast deposition would explain the more vigorous growth observed in transgenic plant shoots, even though As accumulation in shoots increased under As(V) (Figures 5 and 6). In root endodermis or xylem parenchyma cells, the activity of PvACR3 is thought to mediate As(III) efflux toward or into the xylem for translocation, similar to the effects of Lsi2 in rice. 25,29 This would explain the considerably higher As translocation factors in transgenic Arabidopsis (Figure 4E,F). This is also consistent with the results of heterologous expression of ScACR3 in Arabidopsis, which has been shown to increase As translocation to the shoot. 25 However, because large amounts of As(III) were extruded from the exodermis of roots into the external medium, the increased translocation ability did not lead to increased shoot As levels under As(III) in our experiments (Figure 4A). 9360

7 Expression of PvACR3 Enhances Shoot Arsenic Accumulation in Arabidopsis. Arsenic accumulation in shoots can be complicated in As(V) treatments. The increased shoot As levels in transgenic plants under 150 μm As(V) suggest that PvACR3 expression enhanced As translocation from roots to shoots (Figures 4B and 5C). In soils containing 10 ppm As, the total shoot As accumulation was 1.17 and 1.14 μg/plant in L9 and L11, respectively, markedly higher than 0.15 μg/plant in the WT (calculated from data in Figure 6B,C), which also supports this conclusion. The aforementioned PvACR3-mediated efflux of As(III) toward or into the xylem may be critical for achieving As accumulation, considering that most As(V) is reduced to As(III) in plant roots (Figure 4G). In general, large amounts of As are accumulated in plant roots after uptake and are not readily transported to plant shoots. 6,26 However, arsenic is also more easily transported into shoots in As(V) treatments. This is consistent with the higher translocation factors obtained from WT Arabidopsis (Figure 4E,F). We supposed that, in the As(III) treatment, large amounts of As(III) were restricted to the exodermis and cortex in plant roots, likely by chelation and vacuolar sequestration, 33 and only limited amounts were transported to endodermis and xylem parenchyma cells for subsequent xylem loading and translocation. In contrast, in As(V) treatments, a small proportion of As may enter the root stele in the form of As(V) and be subsequently reduced to As(III), which is more likely to achieve As(III) xylem loading and translocation to shoots. Thus, PvACR3-mediated As(III) xylem loading may be more efficient with As(V) than with As(III). In addition, As(V) that is translocated to shoots (Figure 4G) can also be reduced into As(III) in above-ground tissues and subsequently deposited in apoplasts for both detoxification and accumulation through the activity of PvACR3. Proposed Model of Arsenic Tolerance and Shoot Arsenic Accumulation in PvACR3 Transgenic Arabidopsis. Genetic engineering for phytoremediation was recently reviewed by Zhu and Rosen. 19 The key mechanisms include As(V) reduction, As sequestration, As loading to the xylem, and volatilization through leaves. Arsenic sequestration and arsenic loading to the xylem conferred by PvACR3 activity may represent crucial steps in achieving increased As translocation and accumulation in shoots during exposure to As(V) in our experiments. Here we propose a model of As tolerance and shoot As accumulation in PvACR3 transgenic Arabidopsis (Figure S5 in Supporting Information). We suggest that PvACR3 has three major effects due to its capacity for As(III) efflux: (i) increasing As(III) efflux into external medium in root exodermis cells, (ii) conferring As(III) efflux toward or into xylem for translocation in root stele cells, and (iii) extruding As(III) into apoplast for sequestration in leaf cells. As(III) efflux into external medium may decrease As uptake and negatively affect As accumulation in shoots. However, after prolonged exposure to As(V), increased As efflux into xylem and As sequestration in apoplasts may play the dominant role in As metabolism, promoting shoot As accumulation (Figures 5 and 6). In soils, arsenic is bound to the soil and sediment, whereas it is soluble in agar plates. Thus, arsenite efflux from plant roots in soils may increase the As levels in the root rhizosphere. In aerobic soils that contain microorganisms, the rhizosphere As(III) can be oxidized to As(V) by both heterotrophic and chemoautotrophic oxidizing bacteria, 34,35 and the As(V) can be 9361 reabsorbed by plant roots. As a result, PvACR3-mediated increased arsenite efflux into external medium may have limited effects of decreasing arsenic uptake in soils. Actually, when cultivated for a relatively long time, the vigorous and healthy growth of transgenic roots can promote the uptake and transport of As. Furthermore, a highly developed root system is assumed to be advantageous for the phytoremediation of heavy-metal polluted soil because more roots would be available for metal uptake. 36 Engineering Arsenic Tolerance and Hyperaccumulation in Plants for Phytoremediation. Arsenic-hyperaccumulating ferns take up large amounts of As from the soil and move it to their shoots without exhibiting any toxic effects Other plants that do not hyperaccumulate As have potential applications in areas that are not ecologically suitable for fern growth. 28 However, the biological properties of As tolerance and accumulation need to be improved in these species before they can be used as a practical application. PvACR3 is a critical arsenite transporter responsible for arsenic hyperaccumulation in P. vittata. Hence, this transporter is available for manipulation into other species with wider geographic and ecological distributions and greater biomass. Because PvACR3 expression substantially increased arsenic tolerance and significantly enhanced shoot arsenic accumulation in Arabidopsis, we are examining the effects of PvACR3 in other species to further advance arsenic tolerance and hyperaccumulation for field use. We envision that further improvements in phytoremediation technologies can be made by simply transforming PvACR3 into fast-growing, highbiomass plants. Understanding the properties of PvACR3 and the physiology of arsenic behavior in plants should also lead to the development of engineering arsenic hyperaccumulator plants. 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