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<p><b>Shoot (A-C) and root (D-F) ion content for <i>HKT1;1</i> native overexpression lines.</b> Statistical significance was determined using Tukey’s HSD test between each line within treatments. Bars with the same letters indicate no significant difference (<i>p</i> < 0.05). Error bars represent standard error of the mean where n = 12–18 plants.</p

Characterization of HKT1;1 and HKT1;4 expression.

<p>Expression profiles of <i>HKT1;1</i> (A) and <i>HKT1;4</i> (B) in seeding and mature plants. Numbers below each of the bars indicate different tissues as follows: 1: seedling root, 2: seedling shoot, 3: blade of newest fully expanded leaf in seedling (leaf 3), 4: sheath of newest fully expanded leaf in seedling (leaf 3), 5: penultimate leaf sheath in mature plant, 6: penultimate leaf blade in mature plant, 7: flag leaf sheath in mature plant, 8: flag leaf blade in mature plant, 9: culm of mature plant and 10: panicle of mature plant. DAA: days after anthesis. (C, D) Dot plots comparing the expression of <i>HKT1;1</i> (C) and <i>HKT1;4</i> (D) between allelic groups at SNP-4-30535352 in control and saline conditions. The minor allele genotype, which displays higher root Na⁺ content, is indicated by red text. A Mann-Whitney test was performed within each treatment to determine differences between the two groups with asterisks indicating significance as determined using a one-way ANOVA: ***: <i>p</i> < 0.001; *: <i>p</i> < 0.05.</p

Phenotypic and genetic correlation of root and shoot ion traits in RDP1.

<p>Phenotypic and genetic correlation of root and shoot ion traits in RDP1.</p

Genome-wide association analysis of Na⁺ content and Na⁺:K⁺ in root tissue.

<p>(A) Root Na⁺ content, (B) root Na⁺:K⁺. Genome-wide association (GWA) was performed using a mixed model that accounted for population structure and relatedness between accessions of RDP1 using 365 accessions of RDP1 and 397,812 SNPs. For each trait the least squares mean was used as the dependent variable The red horizontal line indicates a statistical significance threshold of <i>p</i> < 10⁻⁵, and was determined using the M_eff method with an experiment-wise error rate of 0.05 [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006823#pgen.1006823.ref063" target="_blank">63</a>].</p

Characterization of high and low root Na⁺ isoforms of HKT1;1 in <i>Xenopus</i> oocytes.

<p>(A) Genetic variants within the ORF of <i>HKT1;1</i>. “Minor Freq” and “Major Freq” indicate the frequency of the alternate allele in the major (<i>n</i> = 19) and minor (<i>n</i> = 13) allelic groups at SNP-4-30535352. “AA change” indicates the resulting changes in protein sequence. Synonymous mutations are indicated by “-”. The grey bars represent exons while the white bars represent the 3’ and 5’ UTRs. (B) Secondary structure of OsHKT1;1 polypeptide showing the position of AA changes, as exemplified between ‘Nipponbare’ and ‘Zhenshan 2’ variants. (C, D) Comparison of OsHKT1;1-Ni and–Zh targeting to the oocyte membrane by confocal imaging of GFP-tagged transporters. (C) Representative images of oocytes expressing Ni (top) or Zh (bottom) transporters. Emitted fluorescence was collected between 505 and 510 nm. Scale bar: 100 μm. (D) Comparison of fluorescence intensity spectra at the membrane in water-injected oocytes (control) and in oocytes expressing either of the HKT1;1 variants. Data are means ± SE. (E) Voltage-clamp protocol and corresponding representative current traces recorded in control oocytes or oocytes expressing the HKT1;1 variants, in 50 mM Na-glutamate-containing bath solution. (F) Current-voltage (I-V) relationships in control oocytes (left) and in HKT1;1-Ni or -Zh-expressing oocytes (right), in either 10 or 50 mM Na-glutamate-containing bath solutions. Data are means ± SE. Insert: Activation potential of HKT1;1-Ni or -Zh currents. Asterisks indicate significant difference in activation potential as determined using Student’s t test: **: <i>p</i> < 0.005. (G) HKT1;1-Zh to HKT1;1-Ni mean current ratio at varying membrane potentials, determined from I-V data shown in (F). Shown data in (E to G) were obtained in a single oocyte batch and are representative of three experiments performed in different oocyte batches.</p

Phenotypic variation and distribution of root and shoot Na⁺ content and Na⁺:K⁺ among the five subpopulations of RDP1.

<p>(A) Root Na⁺ content, (B) root Na⁺:K⁺, (C) shoot Na⁺ content, and (D) shoot Na⁺:K⁺. Accessions were assigned to each subpopulation according to Famoso <i>et al</i> [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006823#pgen.1006823.ref029" target="_blank">29</a>]. Boxplots with the same letter indicate no significant difference as determined using ANOVA (<i>p</i> < 0.05). <i>Aromatic</i> accessions were excluded from the analysis due to low <i>n</i>.</p

Genetic characterization of <i>RNC4</i>.

<p>(A) Regional manhattan plot summarizing GWA analysis of root Na⁺ and root Na^+:K⁺. The region defining <i>RNC4</i> is indicated with the cyan bar. (B) LD plots for a subset of five haplotype blocks within <i>RNC4</i>. The vertical broken gray lines in A indicate the region characterized by haplotype blocks. The genes present in this region are illustrated in C. Genes encoding transposable elements are highlighted in the gene track in gray, while those encoding expressed proteins are highlighted in white. The regions defined by each block are indicated in gray.</p