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Link to original content: https://pubmed.ncbi.nlm.nih.gov/22969436/
Genome-wide association studies identify heavy metal ATPase3 as the primary determinant of natural variation in leaf cadmium in Arabidopsis thaliana - PubMed Skip to main page content
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. 2012 Sep;8(9):e1002923.
doi: 10.1371/journal.pgen.1002923. Epub 2012 Sep 6.

Genome-wide association studies identify heavy metal ATPase3 as the primary determinant of natural variation in leaf cadmium in Arabidopsis thaliana

Dai-Yin Chao  1 , Adriano Silva, Ivan Baxter, Yu S Huang, Magnus Nordborg, John Danku, Brett Lahner, Elena Yakubova, David E Salt
Affiliations

Genome-wide association studies identify heavy metal ATPase3 as the primary determinant of natural variation in leaf cadmium in Arabidopsis thaliana

Dai-Yin Chao et al. PLoS Genet. 2012 Sep.

Abstract

Understanding the mechanism of cadmium (Cd) accumulation in plants is important to help reduce its potential toxicity to both plants and humans through dietary and environmental exposure. Here, we report on a study to uncover the genetic basis underlying natural variation in Cd accumulation in a world-wide collection of 349 wild collected Arabidopsis thaliana accessions. We identified a 4-fold variation (0.5-2 µg Cd g(-1) dry weight) in leaf Cd accumulation when these accessions were grown in a controlled common garden. By combining genome-wide association mapping, linkage mapping in an experimental F2 population, and transgenic complementation, we reveal that HMA3 is the sole major locus responsible for the variation in leaf Cd accumulation we observe in this diverse population of A. thaliana accessions. Analysis of the predicted amino acid sequence of HMA3 from 149 A. thaliana accessions reveals the existence of 10 major natural protein haplotypes. Association of these haplotypes with leaf Cd accumulation and genetics complementation experiments indicate that 5 of these haplotypes are active and 5 are inactive, and that elevated leaf Cd accumulation is associated with the reduced function of HMA3 caused by a nonsense mutation and polymorphisms that change two specific amino acids.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Genome-wide association analysis of leaf Cd accumulation in a worldwide collection of A. thaliana accession grown in a common garden.
A. The frequency distribution of leaf Cd concentration in 349 A. thaliana accessions grown in a common garden. Horizontal bars represent the standard deviations of five accessions grown in all experimental blocks. B. Genome-wide association mapping of leaf Cd at 213,497 SNPs across 337 A. thaliana accessions using a mixed model analysis implemented in EMMA . Horizontal dashed line indicates a genome-wide significance threshold of −log10 = 5. C. Detailed plot of the region shown in the red box in B.
Figure 2
Figure 2. High leaf Cd in A. thaliana Col-0 is recessive to the low leaf Cd in the CS28181 accession.
A. Leaf Cd concentration in A. thaliana accession CS28181, Col-0 and their F1 progeny. Data represent the mean leaf Cd concentration ± standard errors (n = 7–12 independent plants per genotype). B. The frequency distribution of leaf Cd concentration in F2 progeny of a cross between CS28181 and Col-0. Arrows indicate leaf Cd concentration of the parent accessions. Letters above each bar in (A) indicate statistically significant groups using a one-way ANOVA with groupings by Tukey's HSD using a 95% confidence interval.
Figure 3
Figure 3. Genetic linkage mapping of the low leaf Cd locus in A. thaliana accession CS28181.
A. DNA microarray-based bulk segregant analysis of the low leaf Cd phenotype of CS28181 using phenotyped F2 progeny from a CS28181×Col-0 cross genotyped using the 256K AtSNPtilling microarray. Lines represent allele frequency differences between high and low leaf Cd pools of F2 plants at SNPs known to be polymorphic between CS28181 and Col-0 (black line = sense strand probes, blue line = antisense strand probes). B. Fine mapping localizes the causal gene to a 500 kb interval between markers Fo14.5M and Fo15 indicated by the red vertical lines. Black bars represent the CS28181 genotype, grey bars represent heterozygous genotypes and white bars represent Col-0 genotype. Recombinants were selected from 317 CS28181×Col-0 F2 plants. Leaf Cd concentration was determined in the F2 and/or the F3 generation. C. Localization and gene structure of HMA2 and HMA3 in the mapping interval. Black bars indicate exons and black lines indicate introns.
Figure 4
Figure 4. Transgenic complementation of the high leaf Cd phenotype of A. thaliana Col-0.
The A. thaliana Col-0 accession was transformed with either CS28181 HMA3 (A) or HMA2 (B) and leaf Cd concentration determined. Transgenic complementation lines were made by introducing the CS28181 genomic DNA fragments of HMA3 and HMA2 (including promoter sequences) into the Col-0 accession. Data represents the mean leaf Cd concentration ± standard errors (n = 6–12 independent plants per genotype). Letters above bars indicate statistically different groups using a one-way ANOVA with groupings by Tukey's HSD using a 95% confidence interval.
Figure 5
Figure 5. Reciprocal grafting determines that the low leaf Cd phenotype of CS28181 is driven by the root.
Bars represent the leaf Cd concentration of reciprocally grafted A. thaliana CS28181 and Col-0 accessions. NG = Non-grafted plants; SG = Self grafted plants; CS28181/Col-0 = CS28181 shoot grafted onto a Col-0 root; Col-0/CS28181 = Col-0 shoot grafted onto a CS28181 root. Data represent means of leaf Cd concentration ± standard errors (n = 5–14 independent plants per grafting type). Letters above bars indicate statistically different groups using a one-way ANOVA with groupings by Tukey's HSD using a 95% confidence interval.
Figure 6
Figure 6. Quantification of expression of HMA3 in various A. thaliana accessions by quantitative real-time RT–PCR.
A. Expression of HMA3 in shoot and root of A. thaliana accession Col-0 and CS28181. B. Correlation between root expression of HMA3 and leaf Cd accumulation in 14 A. thaliana accessions. For the analysis UBC (AT5G25760) was used as an internal normalization standard across all samples. The expression of HMA3 was calculated as 2−ΔCT relative to UBC. Data represent means ± standard error (n = 4 independent biological replicates per accession and tissue).
Figure 7
Figure 7. Natural HMA3 protein coding haplotypes and their association with leaf Cd concentration across 149 A. thaliana accessions.
A. Ten main HMA3 protein coding haplotypes. B. Leaf Cd concentration of A. thaliana accessions grouped by HMA3 protein coding haplotype. C. Predicted structural model of HMA3 with the position of the amino acid substitutions in (A) indicated in the model. H1–H8, transmembrane helixes. A-Domain = actuator domain; ATP-BD = ATP binding domain; Metal-BD = C-terminal metal binding domain. D. Leaf Cd concentration of F1 progenies of Duk×Col-0, Col-0×Van-0 and Col-0×Ler-0 and their parents in the same experiment. Data represents the means leaf Cd concentration ± standard errors (n = 5–19 in B, 12–20 in C). Letters right of the bars in (B) or above bars in (D) indicate statistically significant groups using one-way ANOVA with groupings by Tukey's HSD using a 95% confidence interval.
Figure 8
Figure 8. The effect of HMA3 function on A. thaliana leaf Zn and Co.
The A. thaliana Col-0 accession was transformed with CS28181 HMA3 and leaf Zn (A) and Co (B) concentration determined. Transgenic lines were made by introducing the CS28181 genomic DNA fragment of HMA3 (including promoter sequence) into the Col-0 accession. Data represents the mean leaf Zn and Co concentrations ± standard errors (n = 7–12 independent plants per genotype). Letters above bars indicate statistically significant groups using a one-way ANOVA with groupings by Tukey's HSD using a 95% confidence interval.
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