Genome-wide association mapping identifies a new arsenate reductase enzyme critical for limiting arsenic accumulation in plants

Abstract

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.

Figure 1. High leaf arsenic concentration in A. thaliana Kr-0 is controlled by a single recessive locus.

(A) The frequency distribution of leaf arsenic concentrations in 349 A. thaliana accessions. Arrows indicate leaf arsenic concentration of accessions highlighted in the text. (B) Leaf arsenic concentration in A. thaliana accession Col-0, Kr-0, and their F1 progeny. Data represent the mean leaf arsenic concentration ± SE (n = 7–12). (C) The frequency distribution of leaf arsenic concentrations in F2 progeny of a cross between Kr-0 and Col-0. Red box indicates F2 plants used to create the low arsenic pool for XAM; Green box indicates F2 plants used to create the high arsenic pool for XAM. All leaf arsenic concentration data are accessible using the digital object identifiers (DOIs) 10.4231/T9H41PBV and 10.4231/T9QN64N6 (see http://dx.doi.org/) and available in Data S1.

Figure 2. The High Arsenic Content 1 (HAC1) gene controls natural variation in leaf arsenic in A. thaliana.

(A) Genome-wide association analysis of leaf arsenic concentration at 213,497 SNPs across 377 A. thaliana accessions using a mixed model analysis with correction for population structure. (B) A detailed plot of the peak region on chromosome 2 is shown with the location of HAC1 indicated by the vertical red line. (C) DNA microarray-based bulk segregant analysis of the high leaf arsenic phenotype of Kr-0 using phenotyped F2 progeny from a Kr-0×Col-0 cross genotyped using the 256K AtSNPtilling microarray. Lines represent allele frequency differences between high and low leaf arsenic pools of F2 plants at SNPs known to be polymorphic between Kr-0 and Col-0 (Solid line = sense strand probes, dashed line = antisense strand probes). (D) The causal gene was mapped between CAPS makers CS8901K and CS9249K using 315 F2 plants. (E) Fine mapping narrowed hac1 down to a 40 kb interval between markers CS9M and CS9040 using 1,321 F2 plants. Numbers below the horizontal line in (D) and (E) represent the number of recombinants between the indicated marker and hac1. (F) Gene structure of different HAC1 alleles. Arrows indicate T-DNA insertion sites for hac1-1 (GABI_868F11) and hac1-2 (SM_3_38332). Grey boxes indicate exons, and black lines indicate introns. The causal polymorphism in Kr-0 is shown to the right. (G) Leaf arsenic concentrations of different HAC1 alleles and their F1 progenies indicate through deficiency complementation that HAC1 is the causal gene for the high leaf arsenic in Kr-0. (H) Kr-0 was transformed with the Col-0 genomic DNA fragment of HAC1 (including 1.5Kb promoter sequence) and shown to complement the high leaf arsenic of Kr-0 to Col-0 levels in five independent transgenic lines (represented by numbers above the line in the x-axis legend), confirming HAC1 is the causal gene for high leaf arsenic in Kr-0. Data in (G) and (H) represents the means ± S.E. (n = 4–12 independent plants per genotype). Letters above bars indicate statistically different groups using a one-way ANOVA followed by least significant difference (LSD) test at the probability of p<0.05. All leaf arsenic concentration data are accessible using the digital object identifiers (DOIs) 10.4231/T9H41PBV and 10.4231/T9VD6WCJ (see http://dx.doi.org/) and available in Data S2.

Figure 3. HAC1 plays a central role in limiting arsenic accumulation during arsenate exposure.

When grown in hydroponic media containing 5 µM arsenate, both Kr-0 and the two hac1 null alleles show a clear increase in arsenate accumulation in shoots (A) and roots (B), and arsenite accumulation in shoots (C) but not in roots (D). Letters above bars indicate statistically different groups using a one-way ANOVA followed by least significant difference (LSD) test at the probability of p<0.05. Data represent means ± S.E. (n = 4). Raw data available in Data S3.

Figure 4. HAC1 encodes an arsenate reductase.

HAC1 from A. thaliana suppresses the arsenate sensitivity of E. coli lacking the ArsC arsenate reductase. Strains were grown at 16°C and cell density measured at an optical density of 600 nm after 72 hr growth in different concentrations of arsenate (A). WT = E. coli wild type (W3110); ΔarsC = arsC mutant in WC3110; Vector = empty pCold-TF; HAC1 = pCold-TF vector containing the A. thaliana HAC1 gene (pCold-TF-HAC1). (B) After growth of ΔarsC transformed with pCold-TF-HAC1 in media containing 10 µM arsenate for 72 hr arsenite was detected in the culture solution. However, arsenite was not detected after growth of ΔarsC transformed with pCold-TF empty vector. EV = empty pCold-TF; n.d = not detected. (C) Arsenate reductase activity in cell free extracts of E. coli ΔarsC mutant transformed with pCold-TF (EV) or pCold-TF-HAC1 (HAC1). Arsenate reductase activity estimated as the oxidation of NADPH followed by a loss of absorbance at 340 nm. Data represents means ± S.E. (n = 3). Asterisks above bars in (C) represent a statistically significant difference (p<0.01), calculated using a Student's t-test. Raw data available in Data S4.

Figure 5. Sequence analysis of HAC and ACR2 genes from plants and yeast.

(A) A dendrogram showing the relationship among genes encoding arsenate reductase in A. thaliana, rice, yeast, and E. coli. Numbers at nodes show bootstrap values obtained from 1,000 replicate analyses. (B) Sequence alignment of ACR2 and HAC genes. Asterisk represents the Leu⁵³ converted into a Thr and followed by a stop codon in the HAC1 Kr-0 allele. Dashed box represents the conserved catalytic site in the ACR2-like arsenate reductases. Gene codes for sequences used to generate the dendrogram are as follows; OsHAC1-1 LOC_Os02g01220; OsHAC1-2 LOC_Os04g17660; OsACR2-1 LOC_Os10g39860; OsACR2-2 LOC_Os03g01770; AtACR2 AT5G03455; ScHAC1 YOR285W; PvACR2 DQ310370; arsC YP_005275964.

Figure 6. HAC1 functions in the roots to limit arsenic accumulation in shoots.

(A) Reciprocal grafting determines that the high leaf arsenic phenotype of Kr-0 is driven by the root. Col-0 NG = non-grafted Col-0; Kr-0 NG = non-grafted Kr-0; Col-0_S/Col-0_R = Col-0 self grafted; Kr-0_S/Kr-0_R = Kr-0 self grafted; Kr-0_S/Col-0_R = Kr-0 shoot grafted onto a Col-0 root; Col-0_S/Kr-0_R = Col-0 shoot grafted onto a Kr-0 root. (B) Quantitative real-time RT-PCR indicates HAC1 is predominantly expressed in A. thaliana roots. Expression of HAC1 was calculated as 2^−ΔCT relative to UBC (At5g25760). (C – E) Root specific expression of HAC1 revealed by accumulation of the HAC1-GFP fusion protein driven by expression of HAC1-GFP by the HAC1 native promoter in Col-0 wild type, imaged using a confocal microscope showing GFP fluorescence (C), bright light (D), and an overlay (E). Scale bar = 50 µm. Letters above bars in (A) indicate statistically different using a one-way ANOVA followed by least significant difference (LSD) test at the probability of p<0.05. Asterisks above bars in (B) represent a significant difference (p<0.01) using a Student's t-test. Data (A and B) represent means ± S.E. (n = 7–13 [A] and n = 4 [B]). Raw data available in Data S5.

Figure 7. HAC1 in roots is both constitutively expressed and induced by arsenate.

Wild-type Col-0 plants were grown on agar solidified medium and, seven days after germination, were transferred to agar solidified medium containing various concentrations of arsenate (A) or arsenite (B), and after an additional three days, roots were harvested and HAC1 expression level determined using quantitative real-time RT-PCR. Exposure to arsenate increased the steady state levels of HAC1 mRNA in roots above the untreated control at all arsenate concentrations tested. However, similar treatment with arsenite reduced the steady state levels of HAC1 mRNA in roots (B) at all concentrations tested. Letters above bars indicate statistically different groups using a one-way ANOVA followed by least significant difference (LSD) test at the probability of p<0.05. Data represent means ± S.E. (n = 3). Raw data available in Data S6.

Figure 8. HAC1 plays a central role in arsenic efflux.

Both Kr-0 and the two hac1 null alleles show a clear reduction in efflux of arsenite from roots compared to Col-0 after 24 and 48 hr exposure to arsenate in the hydroponic nutrient solution (A), whereas there is no difference in arsenate uptake from the same solution (B). All lines were grown hydroponically for 3 wk, and 5 µM arsenate were added for analysis thereafter. The uptake of arsenate and efflux of arsenite was calculated from changes in their concentration in the hydroponic growth media. Letters above bars indicate statistically different groups using a one-way ANOVA followed by least significant difference (LSD) test at the probability of p<0.05. Data represent means ± S.E. (n = 4). Raw data available in Data S7.

Figure 9. HAC1 is required to limit arsenic accumulation in the stele.

Synchrotron μ-XRF mapping of arsenic in root cross sections. Plants were exposed to 10 µM arsenate for 10 days in hydroponic solution. Root sections at approximately 2 cm from the tip were cut and prepared with high pressure freezing and freeze substitution and sectioned at 7 µm thickness. μ-XRF was performed at the UK Diamond Light Source with a beam size and step size = 2 µm and X-ray fluorescence detected using a silicon drift detector. (A) Both calcium (red) and arsenic (green) are imaged in wild-type Col-0 and both hac1 mutant alleles to allow the localization of arsenic to be observed in relation to the overall cellular structure of the root marked by calcium within the cell walls. (B) Quantification of arsenic accumulation across the root section in the same samples shown in (A). Ep, epidermis; Co, cortex: St, stele.

Figure 10. Loss-of-function of HAC1 confers increased sensitivity to arsenate.

Both Col-0 wild-type and the two hac1 null alleles were grown in agar solidified nutrient medium containing 0 µM (A) and 100 µM (C) arsenate, and after 12 days a representative photograph taken. Plants were also grown in the same conditions on nutrient medium containing a range of arsenate concentrations and the root length (B) and shoot fresh weight (D) determined after 12 days of growth. Letters above bars in (B, D) indicate statistically different groups within treatments using a one-way ANOVA followed by least significant difference (LSD) test at the probability of p<0.05. Data represent means ± S.E. (n = 4). Raw data available in Data S8.

Figure 11. HAC1 and ACR2 do not interact additively as part of the metabolism of arsenic.

Wild-type Col-0, single acr2-2 and hac1-2 mutants and the acr2-2 hac1-2 double mutant were grown hydroponically for 3 wk and 5 µM arsenate were added for analysis thereafter. Accumulation of arsenate and arsenite was monitored in both roots (A) and shoots (B) for all genotypes. The uptake of arsenate (C) and efflux of arsenite (D) was also monitored and calculated from changes in their concentrations in the hydroponic growth media. Letters above bars indicate statistically different groups using a one-way ANOVA followed by least significant difference (LSD) test at the probability of p<0.05. Data represent means ± S.E. (n = 4). Raw data available in Data S9.

Figure 12. HAC1 and ACR2 do not interact epistatically as part of the arsenic resistance mechanism.

Wild-type Col-0 and single acr2-2 and hac1-2 mutants and the acr2-2 hac1-2 double mutant were grown on agar solidified nutrient medium containing 0 µM (A) and 100 µM (C) arsenate and after 12 days a representative photograph taken. Plant were also grown in the same conditions on nutrient medium containing a range of arsenate concentrations and the root length (B) and shoot fresh weight (D) determined after 12 days of growth. Letters above bars in (B, D) indicate statistically different groups within treatments using a one-way ANOVA followed by least significant difference (LSD) test at the probability of p<0.05. Data represent means ± S.E. (n = 4). Raw data available in Data S10.

Figure 13. Schematic of the proposed role of HAC1 in arsenate metabolism in roots.

A model and proposed function of HAC1 in the chemical transformations and transport processes arsenic undergoes during its radial transport from the soil, across the root and into the central vascular system for transport to the shoot. Pt, phosphate transporter; E, effluxer; U, unknown arsenate reductase; PC_n-As^III, phytochelatin-arsenite complex, V, vacuole.