jipb13187-sup-0001-Supplement_data.docx10.9 MB Figure S1. MTP gene structure and genetic characterization of mtp mutants. (A) The structure of the MTP genes and the position of T-DNA insertion sites of the mutant lines. Locations of the T-DNA insertions are indicated by the solid triangle. Exons are shown as black boxers and the introns as black lines, grey boxers represent untranslated regions. (B) Genomic PCR of mtp mutants. The wild type genomic DNA was used as a positive control and H2O was used as negative control. (C) Predicated four-transmembrane domains of MTP10. Figure S2. Phenotype of mtp mutants under high Mg2+ conditions. (A) The growth phenotype of Col-0 and mtp mutants under high Mg2+ conditions. Seeds were planted on 1/6 MS medium containing 0.25 mM or 10 mM of MgCl2. Seedlings were cultured for 14 days. Bars= 1 cm. (B, C) Quantification of seedling fresh weight (B) and primary root length (C) of the seedlings in panel (A). The biomass and root length of untreated wild type were set to 1. Data represent means ± SD of six replicate experiments. Asterisks indicate significant difference between the wild type and mtp10 mutant (Student's t-test, *P < 0.05,**P<0.01). Figure S3. Phenotype of mtp mutants under low Mg2+ conditions. (A) The growth phenotype of Col-0 and mtp mutants under low Mg2+ conditions. Seeds were planted on 1/6 MS medium containing 0 mM or 0.05 mM of MgCl2. Seedlings were cultured for 14 days. Bars=1 cm. (B, C) Quantification of fresh weight (B) and root length (C) in panel (A). The biomass and root length of untreated wild type were set to 1. Data represent means ± SD of six replicate experiments. Asterisks indicate significant difference between the wild type and mtp10 mutant (Student's t-test, *P < 0.05,**P<0.01). Figure S4. Quantification of lateral root numbers in Figure 1. Asterisks indicate significant difference between the wild type and mtp10 mutant (Student's t-test, *P < 0.05,***P<0.01). Figure S5. Development of shoot of mtp10 is dramatically affcted by Mg2+ toxicity. (A) 4-day-old seedlings grown on 1/2 MS medium were transferred to 1/6 MS liquid medium or 1/6 MS liquid medium containing different levels of MgCl2. Plants were photographed after grown for 10 days. Bars=2 mm. (B) After grown for 5 weeks in 1/6 MS liquid medium, plants were transferred to 1/6 MS liquid medium containing different concentrations of MgCl2. Phenotype of flowers were photographed. Bars=1 cm. Figure S6. The mtp10 mutant was hypersensitive to excess MgSO4. (A) Wild type and mtp10 seedlings were transferred to1/6 MS medium or 1/6 MS containing 2, 4, 6, 8, or 10 mM MgSO4. Plants were photograghed after grown on the medium for 14 days. Bars=1 cm. (B, C) Quantification of primary fresh weight (B) and root length (C). Data represent means ± SD of six replicate experiments. The biomass and root length of untreated wild type were set to 1. Asterisks indicate significant difference between the wild type and mtp10 mutant (Student's t-test, *P < 0.05, **P<0.01). Figure S7. The mtp10 mutants are sensitive to high-Mg stress specifically. (A) After germination on 1/2 strength MS medium for 3 days, wild type and mtp10 young seedlings were transferred onto 1/6 strength MS with extra high concentrations of different cation ions as showed in the figure. Plants were photographed after growing for 14 days. Bars=1 cm. (B, C) Quantification of fresh weight (B) and root length (C) of the seedlings at the end of treatment. The biomass and root length of untreated wild type were set to 1. Data represent means ± SD of six replicate experiments. Asterisks indicate significant difference between the wild type and mtp10 mutant (Student's t-test, *P < 0.05,**P<0.01). Figure S8. Complementation of MM281 mutant by MTP family proteins. The MM281 strains and MM281 strains transformed with the pTrc99A vector, MGT5, MGT10, MTP1, 9, 10, or 11 cDNA in pTrc99A vector. Growth of different bacterial cells on the N-minimal medium containing 10 mM, 5 mM, 1 mM, or 100 μM MgSO4. From left to right is a 10-fold dilution series of bacterial cultures. Figure S9. Localization of MTP10-GFP in plasma membrane. (A-D) Confocal microscopy of GFP signals in the epidermal cells from a cotyledon of a 5-d-old transgenic wild type seedling expressing 35Spro:MTP10-GFP. The Columns, from left to right, show the GFP signals (green), the plasma membrane fluorescence stained with FM4-64 (red), an overlay (of green and red) from the same sample and bright-field differential interference contrast (DIC). Bars = 20 μm. Figure S10. Expression pattern of MTP10pro::GUS different tissues of Arabidopsis. Histochemical GUS staining was carried out in different parts of Arabidopsis tissues. Images from left to right in the first line are seedlings grown for 1, 2, 3, 5, 7, and 14 days. Images in the last two lines are different tissues including shoot, root, later root, leaf, stem, flower, anther and siliques. Bars = 0.5 mm. Figure S11. Quantitative real-time PCR analysis of the mRNA levels of MTP10 in shoots of Arabidopsis. 7-day-old seedlings were planted on 1/6 MS medium and then transferred to 1/6 MS medium containing 10 mM MgCl2 and grew for 1, 7 days respectively. Expression level of MTP10 was detected at the end of the treatment. Data are means ± SD. n = 4. *P < 0.05. Significant differences are indicated as *P < 0.05 (Student's t-test). Asterisks indicate significant difference between the wild type and mutant lines. Three independent experiments were performed. Figure S12. Metal ion content in wild type and mtp10 mutant. 7-day old seedlings grown on 1/2 MS medium were transferred to 1/6 MS or 1/6 MS containing 6 mM MgCl2 liquid medium. Samples were collected after being treated for another 10 days. The metal ions (Mg, Ca, Mn and Zn) contents were measured by ICP-MS (A-D). Data are means ± SD. n = 4. *P < 0.05. Significant differences are indicated as *P < 0.05 (Student's t-test). Asterisks indicate significant difference between the wild type and mtp10 mutant. Three independent experiments were performed. Figure S13. Genetic characterization of mtp10, mgt6 and mtp10 mgt6 double mutants. RT-PCR (A) and genomic PCR (B) analysis of MTP10 and MGT6 in wild-type, mtp10, mgt6 and mtp10 mgt6 double mutant seedlings. Figure S14. Phenotype of wild type, mtp10, mgt6 and mtp10 mgt6 double mutants under high Mg2+ conditions. (A, B) Seedlings were grown for 3 days on half strength MS medium and then transferred to 1/6 strength MS medium (A) or 1/6 MS medium containing 10 mM MgCl2 (B) for another 10 days. Bars= 1 cm. (C, D) Quantification of fresh weight (C) and root length (D) in panel (A) and (B). The biomass and root length of untreated wild type were set to 1. Data are mean ± SD. n = 4. Significant differences are indicated as *P < 0.05 (Student's t-test). Asterisks indicate significant difference between the wild type and mutant lines. Three independent experiments were performed. Table S1. List of PCR Primers.