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

High sulphur phenotype of <i>msa1-1</i>.

<p>(A) Five-week-old WT (right row) and <i>msa1-1</i> mutant (left row) plants grown in soil. The picture was taken before harvesting samples for ICP-MS. (B) Total sulphur in the leaf of five-week-old WT and <i>msa1-1</i> mutant plants grown in soil. (C) Total sulphur in the shoot of WT and <i>msa1-1</i> mutant plants grown on agar solidified MGRL media. (D) Sulphate content in the shoot of WT and <i>msa1-1</i> mutant plants grown on agar solidified MGRL media. (E) Sulphite content in the shoot of WT and <i>msa1-1</i> mutant plants grown on agar solidified MGRL media. (F) Total Se content in the leaf of five-week old plants grown in soil. (G) Percentage difference of 20 elements of <i>msa1-1</i> mutant compared with the WT, visualized as the radar chart. Data in (B) to (F) are presented as means ± SD with <i>n</i> = 12 in (B) and (F), <i>n</i> = 6 in (C), <i>n</i> = 3 in (D) and (E). *, <i>P</i> ≤ 0.05; **, <i>P</i> ≤ 0.01, Student’s <i>t</i> test. DW, dry weight; FW, fresh weight.</p

Expression pattern and subcellular localization of <i>MSA1</i>.

<p>(A-G) Histochemical GUS staining of <i>MSA1</i> promoter-GUS transgenic plants. One-week-old seedlings grown on agar solidified MGRL media with 1500 μM sulphate (A) or without sulphate (B). (C) A leaf from a two-week-old plant grown on agar solidified MGRL media with 1500μM sulphate; (D) the inflorescence of a plant grown in soil; (E) a flower; (F) developing siliques; (G) a mature silique. (H) Expression of <i>MSA1</i> was strongly induced by S-deficiency. Plants were grown in S sufficient conditions (S1500) or S deficient conditions (S0) for two weeks. Expression level of <i>MSA1</i> was normalized to the internal control gene <i>UBQ10</i>, and presented as 2^(-deltaCt) with means ± SD (<i>n =</i> 3). Columns with different letters indicate significant differences (<i>P</i> ≤ 0.01, LSD test). (I) Subcellular localization of MSA1. Constructs encoding GFP alone and GFP fused of wild type MSA1 (<i>GFP-MSA1w</i>) and mutated MSA1 (<i>GFP-MSA1m</i>) were transformed into <i>Arabidopsis</i> under the control of the CaMV 35S promoter. The GFP-MSA1 fusion protein was specifically expressed in the nucleus as stained by DAPI. Scale bar, 10μm.</p

Identification the causal gene for <i>msa1-1</i>.

<p>(A) Bulk segregant analysis (BSA) of the high leaf S phenotype in an <i>msa1-1</i> × Ler-0 F2 population. Blue lines represent allele frequency differences between the pools of F2 plants with high and low leaf S (<i>n</i> = 40) at the polymorphic SNPs between Col-0 and L<i>er</i>-0. (B) Identification of genes with mutations in the BSA confidence interval identified by SOLiD sequencing. Blue bars, grey lines and white bars represent exons, introns and untranslated region, respectively. (C, E) Genetic complementation of T-DNA insertion alleles by crossing with WT Col-0 or <i>msa1-1</i>. The S (C) and Se (E) content in leaves of F1 plants were determined. Data are presented as means ± SD (<i>n =</i> 6 to 12). (D, F) Transgenic complementation of the high S phenotype of <i>msa1-1</i>. The S (D) and Se (F) content in the leaves of six independent transgenic complementation lines were determined. Data are presented as means ± SD (<i>n =</i> 3 to 12). Columns with different letters indicate significant differences (<i>P</i> ≤ 0.01, least significant difference test). DW, dry weight. ICP-MS data is accessible using the digital object identifier (DOI) <a href="http://dx.doi.org/10.4231/T95Q4T1C" target="_blank">10.4231/T95Q4T1C</a> (see <a href="http://dx.doi.org/" target="_blank">http://dx.doi.org/</a>).</p

Effects of <i>MSA1</i> mutation on genome-wide DNA methylation.

<p>(A) Normalized DNA methylation level on CG, CHG and CHH contexts in the shoot and root of WT and <i>msa1-1</i> on chromosome 1. DNA methylation level was calculated as the density of methylated C in each 100 kb window, and the highest density window in each contexts was designated as 100%. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006298#pgen.1006298.s007" target="_blank">S7 Fig</a> for other chromosomes. (B) Numbers of overlapping DMRs between the shoot and root of WT and <i>msa1-1</i>. (C-E) DNA methylation profile of <i>SULTR1;1</i> (C), <i>SULTR1;2</i> (D) and <i>APR3</i> (E) in the shoot and root of WT and <i>msa1-1</i>. DNA methylation levels are indicated by the height of vertical lines. The positive and negative values represent the methylation level in sense and antisense strand, respectively. The blue and red lines at the bottom indicated the location of the shoot and root DMRs, respectively. The vertical magenta line in (C) shows the location of the SURE element and arrows indicate the location of primers used for the chop-PCR in (F). (F) Determination of DNA methylation level in the promoter of <i>SULTR1;1</i> by chop-PCR in shoots and roots of WT and <i>msa1-1</i> plants grown on agar solidified MGRL media under S sufficient (1500 μM sulphate; +S) or S deficient (no added sulphate; -S) conditions. Genomic DNA was digested without or with McrBC, an endonuclease which only cleaves DNA containing methylcytosine residues, followed by PCR using the primers shown in (C).</p