Chao, Dai-Yin and Chen, Yi and Chen, Jiugeng and Shi, Shulin and Chen, Ziru and Wang, Chengcheng and Danku, John and Zhao, Fang-Jie and Salt, David E. (2014) Genome-wide association mapping identifies a new arsenate reductase enzyme critical for limiting

Inorganic arsenic is a carcinogen, and its ingestion through foods such as rice presents a significant risk to human health. Plants chemically reduce arsenate to arsenite. Using genome-wide association (GWA) mapping of loci controlling natural variation in arsenic accumulation in Arabidopsis thaliana allowed us to identify the arsenate reductase required for this reduction, which we named High Arsenic Content 1 (HAC1). Complementation verified the identity of HAC1, and expression in Escherichia coli lacking a functional arsenate reductase confirmed the arsenate reductase activity of HAC1. The HAC1 protein accumulates in the epidermis, the outer cell layer of the root, and also in the pericycle cells surrounding the central vascular tissue. Plants lacking HAC1 lose their ability to efflux arsenite from roots, leading to both increased transport of arsenic into the central vascular tissue and on into the shoot. HAC1 therefore functions to reduce arsenate to arsenite in the outer cell layer of the root, facilitating efflux of arsenic as arsenite back into the soil to limit both its accumulation in the root and transport to the shoot. Arsenate reduction by HAC1 in the pericycle may play a role in limiting arsenic loading into the xylem. Loss of HAC1-encoded arsenic reduction leads to a significant increase in arsenic accumulation in shoots, causing an increased sensitivity to arsenate toxicity. We also confirmed the previous observation that the ACR2 arsenate reductase in A. thaliana plays no detectable role in arsenic metabolism. Furthermore, ACR2 does not interact epistatically with HAC1, since arsenic metabolism in the acr2 hac1 double mutant is disrupted in an identical manner to that described for the hac1 single mutant. Our identification of HAC1 and its associated natural variation provides an important new resource for the development of low arsenic-containing food such as rice. Citation: Chao D-Y, Chen Y, Chen J, Shi S, Chen Z, et al. (2014) Genome-wide Association Mapping Identifies a New Arsenate Reductase Enzyme Critical for Limiting Arsenic Accumulation in Plants. PLoS Biol 12(12): e1002009. doi:10.1371/journal.pbio. 1002009 Academic Editor: Julin N. Maloof, University of California Davis, United States of America Received August 1, 2014; Accepted October 21, 2014; Published December 2, 2014 Copyright: 2014 Chao et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. Raw leaf arsenic concentrations used in Figure 1 and Figure 2 along with other ionomics data are available using the digital object identifiers (DOIs) 10.4231/T9H41PBV, 10.4231/T9QN64N6 and 10.4231/ T9VD6WCJ (see http://dx.doi.org/). Further, all raw data used to create figures in the paper is provided as additional supplementary files Data S1, S2, S3, S4, S5, S6, S7, S8, S9, S10, S11, S12, S13. Funding: We acknowledge support from the US National Institutes of Health (http://www.nih.gov/) (grant 2R01GM078536 and 2P4ES007373-19A1 to DES), European Commission (http://ec.europa.eu/index_en.htm) (grant PCIG9-GA-2011-291798 to DES) and UK Biotechnology and Biological Sciences Research Council (http://www.bbsrc.ac.uk/home/home.aspx) (grants BB/L000113/1 to DES, and BB/H006303/1 to FJZ) and the Ministry of Education of China (http://www.moe.edu. cn/publicfiles/business/htmlfiles/moe/moe_2792/) (grant IRT1256 to FJZ), start-up funds from the Shanghai Institutes for Biological Sciences (http://english.sibs. cas.cn/) to DYC, the ‘1000 youth talents program’ from the Chinese Central Government (http://www.1000plan.org/) to DYC, and the Diamond Light Source (http://www.diamond.ac.uk/Home.html) for the provision of synchrotron beamtime (grant SP9505 to FJZ). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * Email: dychao@sibs.ac. cn (DYC); fangjie.zhao@njau.edu. cn (FJZ); david.salt@abdn.ac.uk (DES) Abbreviations: m-XRF, synchrotron mX-ray fluorescence; ACR2, arsenic compounds resistance; Ann-1, Annecy-1; App1-16, Applerydvägen1-16; ArsC, arsenical resistanceC; ATQ1, arsenate tolerance QTL; B, boron; Benk-1, Bennekom-1; Bur-0, Burren-0; C, cytosine; CAPS, cleaved-amplified polymorphic sequence; Col-0, Columbia-0; DW, dry weight; EDTA, ethylenediaminetetraacetic acid; EMMA, efficient mixed-model association; GEO, gene expression omnibus; GFP, green fluorescent protein; GWA, genome-wide association; HAC1, high arsenic content 1; Hn-0, Hennetalsperre-0; HPLC-ICP-MS, high performance liquid chromatography–inductively coupled plasma–mass spectrometry; ICP-MS, inductively coupled plasma–mass spectrometry; iHub, ionomics hub; In, Indium; IPTG, isopropyl b-D-1-thiogalactopyranoside; Kas-1, Kashmir; Kr-0, Krefeld; MES, 2-(N-morpholino)ethanesulfonic acid; MOPS, 3-(N-morpholino)propanesulfonic acid; mRNA, messenger ribonucleic acid; NADPH, nicotinamide adenine dinucleotide phosphate; NASC, Nottingham Arabidopsis stock centre; PHO1, phosphate1; PHT1;1, phosphate transporter1;1; PiiMS, Purdue ionomics information management system; QTL, quantitative trait loci; RT-PCR, Reverse Transcription Polymerase Chain Reaction; RILs, recombinant inbred lines; RNAi, ribonucleic acid interference; SDS, sodium dodecyl sulfate; SNP, single nucleotide polymorphism; T, thymine; TAIR9, The Arabidopsis information resource 9 genome release; T-DNA, transfer deoxyribonucleic acid; WRKY6, WRKY transcription factor 6; XAM, extreme array mapping. PLOS Biology | www.plosbiology. org 1 December 2014 | Volume 12 | Issue 12 | e1002009