Natural Variants of AtHKT1 Enhance Na(+) Accumulation in Two Wild Populations of Arabidopsis

Plants are sessile and therefore have developed mechanisms to adapt to their environment, including the soil mineral nutrient composition. Ionomics is a developing functional genomic strategy designed to rapidly identify the genes and gene networks involved in regulating how plants acquire and accumulate these mineral nutrients from the soil. Here, we report on the coupling of high-throughput elemental profiling of shoot tissue from various Arabidopsis accessions with DNA microarray-based bulk segregant analysis and reverse genetics, for the rapid identification of genes from wild populations of Arabidopsis that are involved in regulating how plants acquire and accumulate Na(+) from the soil. Elemental profiling of shoot tissue from 12 different Arabidopsis accessions revealed that two coastal populations of Arabidopsis collected from Tossa del Mar, Spain, and Tsu, Japan (Ts-1 and Tsu-1, respectively), accumulate higher shoot levels of Na(+) than do Col-0 and other accessions. We identify AtHKT1, known to encode a Na(+) transporter, as being the causal locus driving elevated shoot Na(+) in both Ts-1 and Tsu-1. Furthermore, we establish that a deletion in a tandem repeat sequence approximately 5 kb upstream of AtHKT1 is responsible for the reduced root expression of AtHKT1 observed in these accessions. Reciprocal grafting experiments establish that this loss of AtHKT1 expression in roots is responsible for elevated shoot Na(+). Interestingly, and in contrast to the hkt1–1 null mutant, under NaCl stress conditions, this novel AtHKT1 allele not only does not confer NaCl sensitivity but also cosegregates with elevated NaCl tolerance. We also present all our elemental profiling data in a new open access ionomics database, the Purdue Ionomics Information Management System (PiiMS; http://www.purdue.edu/dp/ionomics). Using DNA microarray-based genotyping has allowed us to rapidly identify AtHKT1 as the casual locus driving the natural variation in shoot Na(+) accumulation we observed in Ts-1 and Tsu-1. Such an approach overcomes the limitations imposed by a lack of established genetic markers in most Arabidopsis accessions and opens up a vast and tractable source of natural variation for the identification of gene function not only in ionomics but also in many other biological processes

Contribution of Tsu-1 <i>HKT1</i> Allele to Survival Under NaCl Stress

<div><p>(A) Relative NaCl tolerance of <i>hkt1–1</i>, Col-0, and Tsu-1. Five-week-old <i>hkt1–1</i>, Col-0, and Tsu-1 plants were treated biweekly with 100 mM NaCl. Picture was taken 6 wk after beginning of the salt treatment.</p><p>(B) NaCl tolerance of F2 plants from a Tsu-1 × Col-0 cross. Picture of one of four plant growth trays used in the experiment. Each tray includes plants from each parent (Col-0 and Tsu-1) as well as F2 plants.</p><p>(C) Distribution of the parental lines Col-0 (<i>n</i> = 11) and Tsu-1 (<i>n</i> = 14) recorded dead each week after beginning of the salt treatment. Presented data are percentage of plants dead each week after beginning of salt treatment.</p><p>(D) Distribution of F2 plants (Tsu-1 × Col-0, <i>n</i> = 95) genotyped as Col-0 homozygous or Tsu-1 homozygous or heterozygous for the <i>HKT1</i> allele and recorded dead each week after beginning of the salt treatment. Presented data are percentage of plants dead each week after beginning of salt treatment.</p></div

The Tandem Repeat Upstream of At<i>HKT1</i> Is Determinant for At<i>HKT1</i> Expression and Maintenance of Shoot Na⁺ Accumulation

<div><p>(A) Diagram of the T-DNA insertions in Line 1425. Represented are the two tandem repeats (hatched boxes), positions of the two T-DNA insertions (inverted triangles), and the arrows indicating the orientation of the left border (LB) for each T-DNA insertion. Numbering is based on the A of the At4g10310 start codon ATG as +1. The diagram is not drawn to scale.</p><p>(B) At<i>HKT1</i> expression using quantitative real-time PCR in Col-0, <i>sos3–1</i> (Col-0 <i>gl1</i>), Line 1425, and <i>hkt1–1</i>. RNA was isolated from shoot and root of 6-wk-old plants grown in soil under short-day conditions. For normalization across samples, the expression of the <i>Actin 1</i> gene was used, and relative fold induction was calculated in comparison to At<i>HKT1</i> expression in Col-0 shoot using the ΔΔ<i>C</i>_t method. Presented data are the mean of at least three biological replicates, and the error bars represent ±SD.</p><p>(C) Na⁺ accumulation in shoot tissue of Col-0, <i>sos3–1</i> (Col-0 <i>gl1</i>), Line 1425, <i>sos3–1 hkt1–1,</i> and <i>hkt1–1</i> grown for 6 wk in soil under short-day conditions (same plants used for quantitative real-time PCR above). Presented data are the mean ± SE (<i>n</i> = 12).</p></div

The Higher Na⁺ Accumulation in Shoots of Ts-1 and Tsu-1 Is a Monogenic Recessive Trait Caused by a Gene Located on Chromosome IV

<div><p>(A) Distribution of Na⁺ accumulation in shoot tissue of F2 segregating population obtained from crossing Col-0 with Ts-1. Presented data are distribution of Col-0 (<i>n</i> = 99), Ts-1 (<i>n</i> = 20), and F2 (Ts-1 × Col-0) (<i>n</i> = 158) plants.</p><p>(B) Distribution of Na⁺ accumulation in shoot tissue of F2 segregating population obtained from crossing Col-0 with Tsu-1. Presented data are distribution of Col-0 (<i>n</i> = 80), Tsu-1 (<i>n</i> = 16), and F2 (Tsu-1 × Col-0) (<i>n</i> = 143). Na⁺ contents were calculated for each plant as a percentage relative to Col-0 average Na⁺ content. The “Control pool” and “High Na⁺ pool” labels indicate the F2 plants used to prepare the corresponding DNA pools for the DNA microarray-based BSA.</p><p>(C) Hybridization of genomic DNA from Ts-1 (blue) and Tsu-1 (red) to DNA microarray (ATH1). Data are presented as a Scaled pool hybridization difference (SPHD), the difference between the hybridization of the two pools at the SFPs, scaled so that the difference between Col-0 and the accession would be equal to 1. SFPs were selected based on their <i>D</i>-statistic, which is a modified <i>t</i>-statistic that avoids spurious large values due to low hybridization levels [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020210#pgen-0020210-b012" target="_blank">12</a>]. Dashed vertical lines on Chromosome IV represent the mapping confidence interval of 0.875 Mbp on either side of the peak. Confidence intervals were calculated using algorithms derived from simulations by Borevitz et al. [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020210#pgen-0020210-b012" target="_blank">12</a>], and the scripts accessed at <a href="http://www.naturalvariation.org" target="_blank">http://www.naturalvariation.org</a>.</p></div

Genetic Analysis of the High Na⁺ Trait in Shoots of Arabidopsis thaliana Accessions Ts-1 and Tsu-1

<div><p>(A) Seven-week-old plants of Col-0, Ts-1, and Tsu-1 accessions grown under short-day conditions.</p><p>(B) Na⁺ levels in the shoot are higher in Ts-1 and Tsu-1. Na⁺ content in shoots of Cvi-0 (<i>n</i> = 12), Se-0 (<i>n</i> = 12), Van-0 (<i>n</i> = 12), Mt-0 (<i>n</i> = 12), Nd-1 (<i>n</i> = 12), Mrk-0 (<i>n</i> = 12), Col-0 (<i>n</i> = 12), Kas-2 (<i>n</i> = 12), Est-1 (<i>n</i> = 12), Ler-2 (<i>n</i> = 12), Ts-1 (<i>n</i> = 12), and Tsu-1 (<i>n</i> = 8). Presented data are the mean ± SD.</p><p>(C) The higher Na⁺ accumulation in the shoot of Ts-1 and Tsu-1 is a recessive trait. Na⁺ content in shoots of Col-0 (<i>n</i> = 40), Ts-1 (<i>n</i> = 20), Tsu-1 (<i>n</i> = 20), and F1 plants derived from the crosses Ts-1 × Col-0 (<i>n</i> = 19) and Tsu-1 × Col-0 (<i>n</i> = 13). Presented data are the mean ± SE.</p><p>(D) Ts-1 and Tsu-1 are allelic for the loci responsible for the higher Na⁺ levels in the shoot. Na⁺ content in shoots of Col-0 (<i>n</i> = 140), Ts-1 (<i>n</i> = 40), Tsu-1 (<i>n</i> = 40), and F1 hybrid plants derived from the cross Tsu-1 × Ts-1 (<i>n</i> = 10).</p><p>Presented data are the mean ± SE.</p></div