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Figure S1. OsVIT1 and OsVIT2 expression in response to external zinc status. 14 days-old rice seedlings grown in hydroponics were exposed to 1 mM ZnSO4 or zinc deficiency treatments at indicated time intervals. Expression levels of OsVIT1 and OsVIT2 under 1 mM ZnSO4 (a), zinc deficiency (b) in rice roots, or 1 mM ZnSO4 (c), zinc deficiency (d) in rice shoots were determined. Actin 1 was used as an intern control, data are mean ± SD, n = 3.

Figure S2. OsVIT1 and OsVIT2 fail to rescue Cd2+ sensitivity in ∆ycf1. Yeast mutant ∆ycf1 and its wild type (WT) DTY7 cells containing pYES2 (vector), pYES2-OsVIT1 (OsVIT1) or pYES2-OsVIT2 (OsVIT2) grown on SD-Ura plates without (left panel) or with 60 μM CdCl2 (right panel) for 72 h and photographed.

Figure S3. Spatial iron distribution in mature embryos of rice mutants. (a) Schematic directions of cross-section in rice embryos. (b) Representative Perls’ images at direction ①, indicating iron distribution in seeds of Zhonghua11 (1–a), Dongjin (1–b), osvit1-1 (1–c), osvit2-1 (1–d), OsVIT1/osvit1-1 (1–e) and OsVIT2/osvit2-1 (1–f). (c) Representative Perls’ images at direction ②,indicating iron distribution in seeds of Zhonghua11 (2–a), Dongjin (b), osvit1-1 (c), osvit2-1 (d), OsVIT1/osvit1-1 (e) and OsVIT2/osvit2-1 (f).

Figure S4. Metal levels in rice seedlings and xylem sap. Roots, shoots or xylem sap were sampled from rice seedlings grown for 14 days in hydroponics and subjected to ICP-MS determination of metal contents. (a–b) Fe, Mn, Zn and Cu levels in roots (a) and shoots (b). (c) Fe and Mn concentrations in xylem sap. (d) Zn and Cu concentrations in xylem sap. Data are mean ± SD, n = 8.

Figure S5. Expression levels of Fe uptake- and transport-related genes. Quantitative RT-PCR determination of gene expression levels in rice seedlings grown for 14 days in hydroponics. (a) OsIRT1 and OsNAAT1 expression in roots. (b) OsYSL15 expression in roots and shoots. (c) OsNAS3 expression level in roots and shoots. Data are mean ± SD, normalized to that of Actin 1, n = 8.

Figure S6. Seedlings grown under Fe and Zn deficiency. Rice seedlings grown in hydroponics for 12 days without (Ctl) or with deficiency of Zn (−Zn), Fe (−Fe) or both (−Fe−Zn) in growth chamber, and subjected to visual inspection of whole seedling (a), leaf blade (b) and determination of root length (c) and fresh weight of seedlings (d). Bars represent 5 cm, data are mean ± SD, n = 8.

Figure S7. Seedlings grown under excess Fe and Zn. Rice seedlings grown in hydroponics for 20 days without (Ctl) or with 1 mM ZnSO4 (+Zn), 1 mM FeSO4 (+Fe) or both (+Fe+Zn) in growth chamber, and subjected to visual inspection of whole seedling (a), leaf blade (b) and determination of root length (c) and fresh weight of seedlings (d). Bars represent 5 cm, data are mean ± SD, n = 8.

Figure S8. Increased Cd2+ accumulation in mutant rice grains. Rice were grown in polluted field containing 0.55 ppm Cd2+ and seeds were sampled. Cd2+ contents (a), Fe, Zn, Mn and Cu contents (b) in brown rice were determined by ICP-MS. Data are mean ± SD, n = 8. Significant differences from their relative wild type were determined by students’ t test. **P < 0.01.

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