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BIOLOGICAL SCIENCES / ENVIRONMENTAL SCIENCES
Hyperaccumulation of arsenic in the shoots of Arabidopsis silenced for arsenate reductase (ACR2)
*Department of Genetics, University of Georgia, Athens, GA 30602-7223; and Department of Biochemistry and Molecular Biology, Wayne State University, Detroit, MI 48201
Communicated by Jeffrey L. Bennetzen, University of Georgia, Athens, GA, November 9, 2005 (received for review April 25, 2005)
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Escherichia coli ArsC | drinking water | CDC25 | toxicant | arsenic pollution
Phytoremediation is the use of plants to clean up environmental pollutants and is considered an important alternative to physical methods for cleaning up arsenic (4). Our objective is to develop a genetics-based arsenic phytoremediation strategy that can be used in any plant species. Plants that hyperaccumulate arsenic to high levels aboveground would be harvested and the arsenic further concentrated by incineration. In previous studies, we engineered model plants expressing a bacterial arsenate reductase (ArsC; EC 1.20.4.1
[EC]
) aboveground and constitutively expressing 
-glutamylcysteine synthetase (5). By reducing arsenate to arsenite in leaves and trapping arsenite in thiol-peptide complexes, these plants accumulate 3-fold more arsenic aboveground than wild type and are also highly tolerant to toxic levels of arsenic. The research described herein extends these observations and attacks a particular problem limiting the engineered phytoremediation of arsenic: its transport from roots to shoots.
The arsenate oxyanions HAsO42– and H2AsO4– are the most prevalent forms of arsenic in surface soil, water, and within cells, and these oxyanions contain arsenic in the pentavalent state [As(V)]. Because arsenate is chemically very similar to phosphate, an important plant nutrient, plants take up and translocate arsenate in place of phosphate (6, 7). This plant characteristic should be very useful in phytoremediation strategies, but there is a serious limitation to this system. Most plants appear to have high levels of endogenous arsenate reductase activity, which reduces >95% of the arsenate they take up to arsenite (5, 8). Arsenite, which at neutral pH contains arsenic in the trivalent oxidation state [As(III)], and probably as the acid HAsO32–, is highly reactive and readily forms As(III)-thiol complexes (9). It appears that in most plants, arsenite is sequestered in roots, preventing it from moving up into stems, leaves, and reproductive organs. Thus, the enzymatic reduction of arsenate to arsenite in roots is a major barrier to engineering plants that will efficiently translocate arsenic to aboveground tissues (5, 8).
Our research has focused on identifying and blocking arsenate reduction activity to engineer plants with enhanced phytoremediation potential. In most, if not all, organisms, arsenate reduction is enzymatically catalyzed (10). The first eukaryotic arsenate reductase to be identified was ScAcr2p from the yeast Saccharomyces cerevisiae (11). In this study, we identified an Arabidopsis thaliana arsenate reductase 2 gene (ACR2) that shares homology with ScACR2. ACR2 complemented the arsenic processing functions of Escherichia coli arsenate reductase (arsC–) mutants. We silenced ACR2 expression in Arabidopsis, and the knockdown plants hyperaccumulated large amounts of arsenic aboveground.
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25–27% identity and 32–33% similarity to a 132-aa region within the 600-aa-long sequences of yeast S. cerevisiae and Schizosaccharomyces pombe CDC25 (Fig. 1). The PTPases are modulators of signal transduction pathways that regulate numerous cell functions. Arabidopsis ACR2 has recently been described as a plant CDC25 homolog and shown to act as a PTPase: functioning in vitro by activating plant cyclin-dependent kinase activity and hydrolyzing phosphate from artificial substrates (16, 17) and functioning in vivo in fission yeast by accelerating the mitotic cell cycle leading to shortened cell length (18). However, a phylogenetic analysis (Fig. 1B) showed that Arabidopsis ACR2 and the other plant ACR2 homologs were more closely related to known arsenate reductases than to CDC25 PTPases. Parsimony and heuristic trees had nearly indistinguishable topographies (data not shown).
ACR2 Complements Arsenate Reductase Activity in E. coli.
We performed two genetic complementation tests in E. coli arsC mutants to determine whether Arabidopsis ACR2 encodes a protein with arsenate reductase activity. First, we tested the ability of the 132-residue Arabidopsis ACR2 polypeptide to complement the arsenic sensitivity of E. coli strain AW10 (19), which is defective in arsenate reductase activity. In this strain, the entire chromosomal ars operon, including the arsenate reductase gene arsC, is deleted (
ars), but the arsA and arsB genes encoding the bacterial arsenite efflux pump are carried on a plasmid (pArsAB200) (12). Expression of the pump confers resistance to arsenite, but the cells remain sensitive to arsenate, allowing complementation by heterologous arsenate reductases. The Arabidopsis ACR2 gene was cloned under control of the bacterial promoter to make pACR2/BS (Fig. 1D) (see Methods). Strain AW10 pArsAB200 was transformed with plasmid pACR2/BS and also with an empty pBS vector or pNA1 (expresses bacterial arsC) (5) as negative and positive controls, respectively. A wild-type W3110 strain with an intact ars operon was also included as a positive control. The growth kinetics of these strains grown on liquid media with a fixed concentration of arsenate (250 µM), after induction by the lac inducer isopropyl 
-D-thiogalactoside, are shown in Fig. 2. Strain AW10 with pArsAB200 and pBS was sensitive to arsenate, as expected, because it cannot enzymatically reduce arsenate and make arsenite available to the export pump (20). The Arabidopsis sequence in pACR2/BS complemented this phenotype and showed significant resistance to arsenate that was almost equivalent to that conferred by expression of the bacterial ArsC protein from pNA1. Neither strain grew quite as well as the wild-type control (W3110 + pBS). Resistance to arsenate conferred by pACR2/BS is most simply interpreted as caused by the reduction of arsenate to arsenite by ACR2, with subsequent extrusion of arsenite out of the cells by the arsenite export pump.
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ars). AW3110 is the parent strain to AW10 and does not contain a plasmid encoding the ArsA/B pump to eliminate arsenite. AW3110 was transformed with an empty pBS vector, pNA1, or pACR2/BS. Strain W3110 with an intact ars operon and pBS served as a wild-type control with a functional ars operon. Half-strength LB with increasing concentrations of arsenate (125–1,000 µM) were inoculated with equivalent amounts of each strain, and the final cell density of these strains was determined after 16 h of growth, as shown in Fig. 2B. Expression of Arabidopsis ACR2 or bacterial ArsC in the AW3110 strain resulted in a significant increase in arsenate sensitivity relative to both wild-type W3110 plus pBS and AW3110 plus pBS. The cells expressing ACR2 or arsC never reached high densities in the presence of even the lowest concentrations of arsenic (125 µM). AW3110 (pBS) was nearly as resistant to arsenate as wild type, because it lacked the endogenous ArsC and, thus, did not generate toxic arsenite. The dominant-negative phenotypes for bacterial ArsC or Arabidopsis ACR2 expression were indistinguishable and were consistent with the production of toxic arsenite, which cannot be eliminated from the AW3110 strain lacking an arsenite export pump. These data strongly suggest that the Arabidopsis ACR2 gene encodes an arsenate reductase.
Generating Plant Lines Knocked Down for ACR2 Activity.
We used RNAi to knock down ACR2 expression. The RNAi gene construct ACR2Ri (Fig. 1E) targeted 200 bp of the 3' UTR of the ACR2 transcript for degradation and was expressed under the control of a constitutive promoter in transgenic A. thaliana. Six RNAi lines with single transgene insertions were designated as Ri25, Ri27, Ri32, Ri35, Ri37, and Ri39. The T2 generation transgenic seeds and plants did not show any phenotypic differences from nontransformed Arabidopsis when grown on regular plant media or soil.
Immunoblot analysis of root tissues by using an ACR2-specific antibody showed a significant decrease in the levels of the 14.5-kDa ACR2 protein in all of the knockdown RNAi lines compared with wild-type plants (Fig. 3A). Specifically, the RNAi lines contained from 2% to 50% of wild-type protein levels. The levels of the 110-kDa phosphoenolpyruvate (PEP) carboxylase (pAbPEPC) polypeptide served as a loading and protein transfer control (Fig. 3B). ACR2 protein was also detected in shoot tissues of wild-type plants (Fig. 3C), although the levels of protein were lower than in roots. Microarray analyses of Arabidopsis transcript levels suggest that ACR2 mRNA is constitutively expressed at low levels in all plant organs (21).
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Hyperaccumulation of Arsenic in the Shoots of the ACR2 Knockdown Lines.
Our model predicts that arsenate is the most mobile form of arsenic in the majority of plant species and that arsenite stays trapped in roots. Thus, if these RNAi lines enzymatically reduced arsenate less efficiently than wild type because of lower ACR2 enzyme levels, they should transport more arsenate to shoots. The ACR2Ri knockdown lines showed significantly higher concentrations of arsenic in their shoots and retained slightly less arsenic in their roots than wild type (Fig. 5A and B), when grown on 100 µM arsenate. At this concentration of toxicant, the RNA interference (RNAi) lines were not significantly inhibited in growth relative to wild type. Quantitative assays showed that these RNAi knockdown lines accumulated 10- to 16-fold more arsenic in shoots (350–500 ppm arsenic) compared with wild-type controls, which accumulated only 30 ppm arsenic. In several repetitions of this experiment with 75 and 100 µM arsenate in the medium, all of the RNAi lines tested accumulated between 6 and 20 times higher levels of arsenic than the wild type (data not shown). Whereas wild-type plants have shoot-arsenic concentrations that are only 1% of their root levels, the RNAi lines have shoot concentrations that are 25% of their root levels (Fig. 5). Clearly, ACR2 activity plays a significant role in blocking long-distance arsenic transport and accumulation in wild-type plants.
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ACR2 Knockdown Lines Accumulated Less Phosphorus in the Presence of Arsenate, but Not Arsenite.
Competition between arsenate and phosphate has been shown in several arsenic uptake studies (7, 22, 23). We analyzed the accumulation of total phosphorus in plants grown with the normal amount of phosphate (625 µM) in half-strength MS medium, but we included 100 µM arsenate. The ACR2 knockdown plants accumulated 2- to 3-fold less phosphorus in shoots compared with wild-type plants in response to arsenate exposure (Fig. 5D), and some lines retained slightly less phosphorus in roots (Fig. 5E). When grown with 25 µM arsenite in the medium, there was no difference between phosphorus accumulation in the wild-type and RNAi lines (Fig. 5F). When no arsenate was added to growth media, there was again no difference in phosphorus accumulation between wild-type and the ACR2-deficient lines, with all lines accumulating 8,000 ppm phosphorus in shoots (data not shown).
Plant lines ACR2Ri25 and ACR2Ri32, which had only moderate reductions in ACR2 protein levels compared with the other four lines examined (Fig. 3A), showed only slight differences from wild type in the root accumulation of arsenic and phosphorus (Fig. 5 B and E). The ACR2Ri25 line also showed less accumulation of arsenic in shoots than the other lines, a finding that was consistent with its moderate RNAi phenotype (Figs. 3A and 5A). Further experimentation would be necessary to explain why line ACR2Ri32 accumulated as much arsenic in shoots as the other stronger epialleles with less ACR2 protein.
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The most parsimonious interpretation of arsenic accumulation data is that the knockdown lines were unable to reduce arsenate to arsenite efficiently, and thus, more arsenate moved into aboveground plant tissues as an analogue of phosphate. It is logical to propose that plants evolved arsenate reductase activities precisely to prevent the competition of arsenate for phosphate transporters and to allow normal translocation of phosphate on arsenic-contaminated soils.
Engineering Plants for the Phytoremediation of Arsenic.
Part of our strategy for engineering an aboveground hyperaccumulator of arsenic is based on blocking the endogenous reduction of arsenate to arsenite and thus allowing more efficient arsenate transport into aboveground organs (26). The arsenic phenotypes of the plant lines silenced for ACR2 expression are consistent with ACR2 playing a major role in the reduction of arsenate to arsenite and arsenic retention in roots. Here, we have shown that silencing the activity of ACR2 by RNAi resulted in long-distance translocation of 10- to 16-fold more arsenic aboveground. Our prior studies showed that Arabidopsis expressing E. coli ArsC and -glutamylcysteine synthetase accumulated 3-fold more arsenic aboveground and were more tolerant to arsenic than wild-type plants (5). Combining a knockdown of ACR2 with the expression of these two bacterial genes has the potential to generate a super hyperaccumulator with normal plant growth and 30- to 40-fold higher levels of aboveground arsenic. Such plants could contribute significantly to the remediation of arsenic pollution. It should be possible to silence homologues of ACR2 in field-adapted grasses, shrubs, and trees suited to the phytoremediation of arsenic-contaminated sites and water resources.
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To silence ACR2 activity, we made an RNAi construct in which the 3' UTR sequence from ACR2 (207 nt after the stop codon) was assembled in reverse and forward orientations, respectively, flanking a 1,000-nt -glucuronidase spacer region to make a stem–loop RNA transcript (Fig. 1E) by using the methods described in ref. 28. The 1.4-kb PCR fragment was cloned under control of the cauliflower mosaic virus 35S promoter and nitric-oxide synthase terminator in a binary vector described in ref. 29 to make ACR2Ri. This construct was introduced into A. thaliana (Columbia ecotype) by Agrobacterium-mediated transformation by using the vacuum infiltration procedure (30). The T1 generation seeds were screened for a linked kanamycin resistance marker, and the first six lines that segregated for a single insertion were selected for further study.
Arsenate Resistance and Sensitivity Assays in Bacteria.
Attempts to complement yeast ScAcr2 mutants with the Arabidopsis ACR2 were unsuccessful; therefore assays of the plant arsenate reductase gene activity were performed in E. coli arsC mutant backgrounds. Liquid culture growth assays for E. coli strains (Fig. 2) were performed as described in Mukhopadhyay and Rosen (24). The data reported are the average results of three replicates. Strains were grown overnight at 37°C in Luria–Bertani medium (LB) with appropriate antibiotics and isopropyl -D-thiogalactoside (IPTG). For the time-dependent liquid growth curve assays (Fig. 2), the cultures were diluted 100-fold into half-strength LB and grown for various time periods indicated in the presence of final concentrations of 100 mg/liter ampicillin, 50 mg/liter kanamycin, 1 mM IPTG, and 250 µM sodium arsenate. Cell density was measured as Klett units at 1-h intervals. Strains AW10 with pArsAB200 and W3100 are described in Liu et al. (12) and AW3110 in Carlin et al. (20).
Immunoblot Assays of ACR2 Protein. Shoot or root tissues were ground in liquid nitrogen, suspended in extraction buffer (31), and pelleted at 10,000 x g. The pellet was reextracted with sample buffer (32) and centrifuged again at 10,000 x g, and the supernatant contained the ACR2 protein. An equal amount (10 µg) of total protein from each sample was resolved on a 12% (wt/vol) polyacrylamide gel by SDS/PAGE and blotted to membrane. Equal loading of each sample was first assured by Coomassie staining of samples on a separate gel (data not shown). Western blots of plant extracts were developed as described in Bizily et al. (33), by using polyclonal sera as described for phosphoenolpyruvate carboxylase (33) and mouse antisera to ACR2 followed by horseradish peroxidase-conjugated goat anti-mouse antisera (Sigma) and enhancement by using an enhanced chemiluminescence kit from Amersham Pharmacia following the manufacturer’s instructions. The mouse antibody was prepared against a 4-fold multiple antigenic peptide containing the C-terminal 25-aa region of ACR2 by using a method described in ref. 34.
Arsenic and Phosphorus Accumulation in Plants. The ACR2 RNAi knockdown lines were grown on half-strength Murashige and Skoog (MS) medium (35) containing 100 µM sodium arsenate (7.5 ppm elemental arsenic) for 3 weeks. The shoots and roots from these plants were harvested, washed, and extracted for inductively coupled plasma mass spectrometry determination of total arsenic or phosphorus as described in refs. 5 and 36.
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Abbreviations: ACR, arsenate reductase; PTPase, protein-tyrosine phosphatase; RNAi, RNA interference.
To whom correspondence should be addressed. E-mail: meagher{at}uga.edu
Present address: Department of Plant, Soil, and Insect Sciences, University of Massachusetts, Amherst, MA 01002. 
Author contributions: O.P.D. and R.B.M. designed research; O.P.D. and E.C.M. performed research; B.P.R. contributed new reagents/analytic tools; and O.P.D. and R.B.M. wrote the paper.
Conflict of interest statement: No conflicts declared.
Data deposition: The sequence reported in this paper has been deposited in the GenBank database (accession no. NM_120425 and Arabidopsis thaliana chromosome V locus At5g03455).
© 2006 by The National Academy of Sciences of the USA
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