Arsenic tolerance in Arabidopsis is mediated by two ABCC-type phytochelatin transporters Won-Yong Songa,b,1, Jiyoung Parka,1, David G. Mendoza-Cózatlc,1, Marianne Suter-Grotemeyerd, Donghwan Shima, Stefan Hörtensteinerb, Markus Geislerb, Barbara Wederb, Philip A. Reae, Doris Rentschd, Julian I. Schroederc,2,3, Youngsook Leea,2,3, and Enrico Martinoiaa,b,2,3 aPohang University of Science and Technology–University of Zurich Cooperative Laboratory, Department of Integrative Bioscience and Biotechnology, World Class University Program, Pohang University of Science and Technology, Pohang 790-784, Korea; bInstitute of Plant Biology, University of Zurich, 8008 Zurich, Switzerland; cDivision of Biological Sciences, Cell and Developmental Biology Section, University of California, San Diego, La Jolla, CA 92093-0116; dInstitute of Plant Sciences, University of Bern, 3013 Bern, Switzerland; and ePlant Science Institute, Department of Biology, University of Pennsylvania, Philadelphia, PA 19104-6018 Edited by Maarten Koornneef, The Max Planck Institute for Plant Breeding Research, Cologne, Germany, and approved October 19, 2010 (received for review September 20, 2010) Arsenic is an extremely toxic metalloid causing serious health pro- million people are estimated to face a risk of death from cancer blems. In Southeast Asia, aquifers providing drinking and agricul- caused by arsenic (3–5). tural water for tens of millions of people are contaminated with To reduce nutritional arsenic intake, identification of the arsenic. To reduce nutritional arsenic intake through the consump- mechanisms implicated in arsenic accumulation and detoxification tion of contaminated plants, identification of the mechanisms for of plants is a prerequisite. Considerable progress has been made arsenic accumulation and detoxification in plants is a prerequisite. during the last years in the identification of the molecular mecha- Phytochelatins (PCs) are glutathione-derived peptides that chelate nisms of arsenic (As) uptake, metabolism, and translocation (for heavy metals and metalloids such as arsenic, thereby functioning as reviews see refs. 6–8). After entering into roots through high- the first step in their detoxification. Plant vacuoles act as final detox- affinity phosphate transporters, arsenate [As(V)] is readily reduced ification stores for heavy metals and arsenic. The essential PC–metal to arsenite [As(III)]. Alternatively, under reducing conditions, As (loid) transporters that sequester toxic metal(loid)s in plant vacuoles (III) is taken up by membrane proteins belonging to the aquaporin have long been sought but remain unidentified in plants. Here we family (9, 10). In rice, it has been shown that As(III) is exported PLANT BIOLOGY show that in the absence of two ABCC-type transporters, AtABCC1 subsequently to the xylem by the silicon efflux transporter Lsi2, and AtABCC2, Arabidopsis thaliana is extremely sensitive to arsenic resulting in root-to-shoot transport of arsenic (11). The final step of and arsenic-based herbicides. Heterologous expression of these arsenic detoxification in the cell is the sequestration into vacuoles, ABCC transporters in phytochelatin-producing Saccharomyces cere- which depends mainly on glutathione (GSH) and phytochelatins visiae enhanced arsenic tolerance and accumulation. Furthermore, (PCs). PCs are GSH-derived metal-binding peptides whose syn- membrane vesicles isolated from these yeasts exhibited a pro- thesis is induced by arsenic as well as other toxic metals (12, 13). nounced arsenite [As(III)]–PC2 transport activity. Vacuoles isolated PCs chelate As(III) and heavy metals with their thiol groups, and from atabcc1 atabcc2 double knockout plants exhibited a very low the metal(loid)–PC complexes are sequestered into vacuoles, pro- residual As(III)–PC2 transport activity, and interestingly, less PC was tecting cellular components from the reactive metal(loid)s. produced in mutant plants when exposed to arsenic. Overexpres- PC-mediated detoxification is unique to plants and a few other sion of AtPCS1 and AtABCC1 resulted in plants exhibiting increased PC-producing organisms but different from that of budding yeast arsenic tolerance. Our findings demonstrate that AtABCC1 and or humans, in which PCs or PC synthase (PCS) do not exist. The AtABCC2 are the long-sought and major vacuolar PC transporters. transporters responsible for active transport of PC-conjugated Modulation of vacuolar PC transporters in other plants may allow As(III) as well as for transport of PC-conjugated heavy metals engineering of plants suited either for phytoremediation or reduced into plant vacuoles (14–17) are yet to be identified but have been accumulation of arsenic in edible organs. proposed to be ABC transporters, as is the case with glutathione (GS)-conjugated As(III) (As(GS)3) in other organisms (18, 19). ABC transporter | vacuolar sequestration | multidrug resistance-associated For example, in Saccharomyces cerevisiae it has been shown that protein arsenic is detoxified by YCF1, an ABC protein transporting As (GS)3 into vacuoles (18). In humans, it has been shown that fi rsenic has been widely used in medicine, industry, and ag- HsABCC1 and HsABCC2 are involved in arsenic detoxi cation Ariculture. In medicine, it was used to treat diseases such as by transporting As(III) conjugated to GSH (19). Another ABC syphilis, trypanosomiasis, or amoebic dysentery. In agriculture, transporter, HMT1, has been proposed to act as a vacuolar PC Schizosaccharomyces pombe hmt1 arsenic-based herbicides, such as disodium methanearsonate transporter in , although an de- (DSMA), continue to be applied for weed and pest control. letion mutant continued to exhibit PC accumulation in vacuoles, However, arsenic is a highly toxic environmental pollutant that causes global health problems. In Southeast Asia as well as in regions with extensive mining, such as China, Thailand, and the Author contributions: W.-Y.S., J.P., D.G.M.-C., J.I.S., Y.L., and E.M. designed research; W.-Y.S., J.P., D.G.M.-C., M.S.-G., D.S., S.H., M.G., B.W., P.A.R., and D.R. performed research; United States, arsenic concentrations in water can be far above and J.P., J.I.S., Y.L., and E.M. wrote the paper. the World Health Organization limit of 10 μg/L (133 nM), con- Conflict of interest statement: Y.L., E.M., J.I.S., W.-Y.S., J.P., and D.G.M.-C. have filed a centrations that are known to cause health problems. People are patent on the reduction of arsenic in crops and the use of ABCCs for phytoremediation exposed to arsenic poisoning both by drinking contaminated based on the discovery reported in the manuscript. water and by ingesting crops cultivated in soils irrigated with This article is a PNAS Direct Submission. polluted water. It has been shown that arsenic in rice paddy fields 1W.-Y.S., J.P., and D.G.M.-C. contributed equally to this work. is readily taken up by rice plants and translocated to the grains, 2J.I.S., Y.L., and E.M. contributed equally to this work. constituting a danger for populations that rely mostly on rice for 3To whom correspondence may be addressed. E-mail: [email protected], ylee@postech. their diet (1, 2). In Bangladesh alone an estimated 25 million ac.kr, or [email protected]. people are exposed to water contaminated with arsenic exceeding This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 50 μg/L, the Bangladesh government standard, and more than 2 1073/pnas.1013964107/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1013964107 PNAS Early Edition | 1of6 Downloaded by guest on September 29, 2021 and HMT1 did not confer arsenic resistance (20, 21). In plants, no ortholog for HMT1 has been identified. Here we report the identification of two ABC transporters re- quired for arsenic detoxification. These transporters are plant PC transporters that have been sought since the discovery of PCs (12, 22). Thus this finding provides a key to understanding the detoxifi- cation of many xenobiotic molecules, heavy metals, and metalloids, including arsenic, that are conjugated with PCs for detoxification. Results atabcc1 atabcc2 Double Knockout Mutant Is Hypersensitive to Arsenic and Arsenic-Based Herbicide. To identify candidate transporters likely involved in arsenic detoxification in plants, we focused on screening the ABCC subfamily of ABC transporters, also known as the multidrug resistance-associated proteins (MRPs). This subfamily includes ABC transporters implicated in heavy metal resistance, such as ScYCF1, the yeast vacuolar As(GS)3 trans- porter (18), and the two human arsenic-detoxifying ABC trans- porters (19). Members of this family have also been shown to transport GSH conjugates and to confer cadmium resistance in plants and humans (23–26). In addition, for many ABCC proteins in Arabidopsis, vacuolar localization has been demonstrated or proposed on the basis of data obtained using either Western blotting of membrane fractions, GFP fusion proteins, or vacu- olar proteomics approaches (27–30). We have therefore isolated homozygous knockout lines for all 15 Arabidopsis ABCC genes and have grown these mutants in the presence of arsenic and arsenic- containing herbicides. Two different forms of arsenic were used because they have different entry pathways to the cell, as well as Fig. 1. Hypersensitivity of atabcc1 atabcc2 double knockout mutants to ar- differential metabolism. Whereas As(V) is taken up by the high- senic [As(V)] and DSMA, an arsenic-based herbicide. (A and B) Wild-type, affinity phosphate transporter (31, 32), DSMA is much more hy- AtABCC1 knockout (abcc1-3), AtABCC2 knockout (abcc2-2), and double drophobic and is rapidly absorbed by plant roots. Furthermore, As knockout (abcc1 abcc2) seedlings grown in 100 mg/L of DSMA (A) and 50 μM (V) is reduced to As(III) in the cell, whereas in DSMA arsenic is of As(V) (B) containing half-strength MS media for 16 d. Note that single already present in the As(III) form. Although no As(V)-sensitive knockout mutants of AtABCC1 or AtABCC2 are also sensitive to the arsenic atabcc knockout mutant was found, the growth of two deletion herbicide (A) but not to As(V) (B).