Mean and variance effects for five loci in the <i>MOT1</i> region associated with either mean molybdenum concentration levels (GWA) or variance (vGWA).

<p>Mean and variance effects for five loci in the <i>MOT1</i> region associated with either mean molybdenum concentration levels (GWA) or variance (vGWA).</p

The genetic variance-heterogeneity across vBLOCK emerges from a multi-locus, multi-allelic genetic architecture.

<p><b>(A)</b> The vGWA analysis using the alternative DGLM approach also detects a strong association near <i>MOT1</i> on chromosome 2 (blue dots). The genetic variance-heterogeneity at this locus is, however, cancelled when the mean effects of the <i>DEL</i>^<i>53</i>, <i>DUP</i>^<i>326</i> and <i>SNP</i>_<i>1</i>^<i>+</i> alleles are included in the DGLM model (yellow dots). The variance in the mean leaf molybdenum concentrations is lower for the group of accessions carrying the low-variance associated variant of vBLOCK (<i>vBLOCK</i>^<i>lv</i>) (<b>B</b>) than for the group of accessions carrying the high-variance associated variant (<i>vBLOCK</i>^<i>hv</i>) <b>(C)</b>. Separate colors are used for the accessions carrying the <i>DEL</i>^<i>53</i> (purple), <i>DUP</i>^<i>326</i> (red) and <i>SNP</i>_<i>1</i>^<i>+</i> (grey) alleles in <b>(C)</b> to illustrate how these alleles generate the high variance in mean leaf molybdenum concentrations associated with <i>vBLOCK</i>^<i>hv</i>.</p

T-DNA analyses to identify candidate genes for the associations to mean leaf molybdenum concentrations.

<p>Included in the figure are the genes (colored boxes) in the region surrounding <i>SNP</i>_<i>2</i> that were bounded by the furthest up- and downstream SNPs with r² > 0.4. We measured the mean leaf molybdenum concentrations for available T-DNA insertional alleles and compared them to the wild-type Col-0. Yellow box = significant difference in leaf molybdenum concentration, deep blue box = no significant difference, light blue = no T-DNA insertion line tested. The T-DNA lines with insertions in <i>AT2G26975</i> and between <i>AT2G27020/AT2G27030</i> had altered mean leaf molybdenum concentrations.</p

Schematic illustration of the complex locus on chromosome 2 associated with leaf molybdenum concentrations.

<p><b>(A)</b> Multiple GWA and vGWA signals were detected to a complex locus around <i>MOT1</i>. There was a strong LD (D’) between three of the associated loci (SNP₁, DEL and DUP) and the high-variance associated variant of vBLOCK (<i>vBLOCK</i>^<i>hv</i>) that led to the extended vGWA signal (Red/blue arrow indicate leading vGWA SNP in the DGLM analysis). A fourth independent GWA association (SNP₂) was also detected upstream of vBLOCK. The direction of the effects for the minor alleles at the significantly associated loci (<i>SNP1</i>^<i>+</i>, <i>SNP2</i>^<i>+</i>, <i>DEL</i>^<i>53</i> and <i>DUP</i>^<i>326</i>) relative to that of the major, reference allele are illustrated with + (increased) and—(decreased), respectively. In <b>(B)</b> we illustrate the differences between the reference allele at DUP (<i>DUP</i>^<i>R</i>) and the two variants of the 330 bp duplication (<i>DUP</i>^<i>326</i> and <i>DUP</i>^<i>322</i>) in the transposable element <i>AT2TE47050</i> in the promoter region of <i>MOT1</i>.</p

LD^c between the loci altering mean leaf molybdenum concentrations.

<p>LD<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005648#t002fn003" target="_blank">^c</a> between the loci altering mean leaf molybdenum concentrations.</p

GWA and vGWA analyses for mean leaf molybdenum concentration.

<p>(A) Genome-wide results from single-locus vGWA (blue) and GWA (red) analyses across the <i>A</i>. <i>thaliana</i> genome. (B) Region on chromosome 2 where a highly significant genetic variance-heterogeneity was detected for the leaf molybdenum concentrations. Several significant SNPs are detected and these define an extended vGWA associated region (vBLOCK), where the minor alleles at these significant loci define an LD-block associated with a higher phenotypic variance (<i>vBLOCK^hv</i>). (C) Illustration of the high LD across vBLOCK. The accessions that are homozygous for the minor/major allele are colored green/grey and then sorted according to the genotype of the SNP with the strongest genetic variance-heterogeneity (red dashed line, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005648#pgen.1005648.t001" target="_blank">Table 1</a>).</p

T-DNA insertion lines with significant associations to the mean leaf molybdenum concentrations.

<p>T-DNA insertion lines with significant associations to the mean leaf molybdenum concentrations.</p

Metabolites quantification and supplementation.

<p>(A) Schematic representation of sulphur assimilation in <i>A</i>. <i>thaliana</i>. Colour squares above the metabolites represent the log2 value of the <i>msa1-1</i>/WT Col-0 ratio of the concentration of each metabolite. APR: APS reductase; APS: adenosine 5’-phosphosulfate; ATPS: ATP sulfurylase; CBL: cystathionine β-lyase; CGS: cystathionine γ-synthase; Cyst: cystathionine; γ-ECS: γ-glutamylcysteine synthetase; γ-GluCys: γ-glutamylcysteine; GSHS: glutathione synthetase; Hcy: homocysteine; MS: methionine synthase; OAS: O-acetylserine; OAS-TL: OAS(thiol)lyase; SAT: serine acetyltransferase; SAMS, S-adenosylmethionine synthetase; SiR: sulphite reductase; SHM: serine hydroxymethyltransferase. (B-G) Measurement of sulphur-related metabolites. Plants were grown on agar solidified MGRL media under S sufficient (S1500) or S deficient (S0) conditions. Metabolites were extracted from shoots and roots and quantified by HPLC. Data are presented as means ± SD (<i>n</i> = 3). *, <i>P</i> ≤ 0.05; **, <i>P</i> ≤ 0.01, Student’s <i>t</i> test. (H-I) The concentrations of SAM and MTA in the shoots and roots of WT Col-0 and <i>msa1-1</i> grown under S sufficient condition. (J) Total S in the shoots of WT Col-0 and <i>msa1-1</i> grown under S sufficient condition without (CK) or with SAM added to the growth medium. Data in (B-J) are presented as means ± SD (<i>n</i> = 3 in (B-G), <i>n</i> = 5 in (H-I), and <i>n</i> = 6 in (J)). * and ** in (B-J) indicate values significantly different between WT Col-0 and <i>msa1-1</i> mutant at <i>P</i> ≤ 0.05 and <i>P</i> ≤ 0.01, respectively (Student’s <i>t</i> test). DW, dry weight. CK, control.</p

Cytosine methylation levels at CG, CHG, and CHH and total cytosine sites in WT and <i>msa1-1</i>.

<p>Cytosine methylation levels at CG, CHG, and CHH and total cytosine sites in WT and <i>msa1-1</i>.</p

High leaf S phenotype of <i>msa1-1</i> is dependent on two high-affinity sulphate transporters SULTR1;1 and SULTR1;2.

<p>(A, B) Expression of <i>SULTR1;1</i> and <i>SULTR1;2</i> in the <i>msa1-1</i> mutant. Quantitative RT-PCR analysis of <i>SULTR1;1</i> and <i>SULTR1;2</i> in the shoot and root of WT Col-0 and <i>msa1-1</i>. Plants were grown on agar solidified MGRL media for two weeks with sufficient sulphate (1500 μM; S1500) (A) or without added sulphate (S0) (B). Expression level was normalized to the internal control gene <i>UBQ10</i>, and presented as 2^(-deltaCt) with means ± SD (<i>n =</i> 3). * and ** represent significant differences between the WT and mutant at <i>P</i> ≤ 0.05 and <i>P</i> ≤ 0.01, respectively (Student’s <i>t</i> test). (C) Phenotype of five-week-old <i>msa1-1 sultr1;1 sultr1;2</i> triple mutant and control lines. Pictures were taken before harvesting for ICP-MS analysis. Scale bars in all images represent 1 cm. (D) Total S in the leaves of five-week-old <i>msa1-1 sultr1;1 sultr1;2</i> triple mutant and control lines. Data are presented as means ± SD (<i>n =</i> 11 or 12). Bars with different letters indicate significant differences (<i>P</i> ≤ 0.01, least significant difference test). DW, dry weight.</p