FIGURE LEGENDS
Figure 1. Mitochondrial localization of AtMTM1 and AtMTM2, and their interactions with AtMSD1. (A) Arabidopsis protoplasts were transfected with AtMTM1-YFP and AtMTM2-YFP to observe mitochondrial localization. (B) The YFPN was fused to AtMTM1 and AtMTM2, and the YFPC was fused to AtMSD1. Constructs were co-transfected in protoplasts for BiFC assay as indicated. The reconstituted YFP signals were observed by confocal microscopy. MitoTracker staining and chlorophyll autofluorescence were used to identify mitochondria and chloroplasts, respectively. Bars = 20 μm.
Figure 2. AtMTM1 and AtMTM2 recovered yeast MnSOD activities inymtm1Δ cells. The ymtm1Δ cells were transfected withAtMTM1 and AtMTM2 , and in-gel SOD activities were analyzed. Yeast CuZnSOD (ySOD1) and MnSOD (ySOD2) activities are indicated by arrowhead and bracket (top) , respectively, and immunoblotting used α-alcohol dehydrogenase1 (ADH1) antibody(bottom) . ADH1 was an input control.
Figure 3. AtMTM1 enhanced AtMSD1 activity and AtMSD1 restored the WT phenotype in ysod2Δ cells in response to MV stress. (A)Co-expression of AtMTM1 enhanced AtMSD1 activity in ysod2Δcells. AtMSD1 activity (top) and protein (bottom) were analyzed. Yeast without (-) and with (+) 100 μM MnSO4supplementation are indicated. Immunoblotting used α-AtMSD1 antibody and mitochondrial porin was an input control. (B) AtMSD1complemented ysod2Δ cells to the WT phenotype with MV stress. The amount of 5 μL diluted culture from A 600 = 1 to 10-4 was grown on YPD medium containing 3 to 5 mM MV.
Figure 4. Expressions of AtMTM1, AtMTM2, andAtMSD1 in various organs. Two to five-week-old organs were used for qPCR analysis. Expression level was normalized relative to theAtMTM1 in root. Data are mean ± SE of three biological replicates. *, significant at P < 0.05 compared with the root value. AtPP2A was an internal control.
Figure 5. Post-transcriptional regulation of MnSOD and gene expressions of AtMTM1, AtMTM2, and AtMSD1 under MV stress. (A) Two-week-old seedlings were treated without or with 0.1 to 10 μM MV for 24 h. An amount of 30 μg protein was used for in-gel SOD activity assay (top) and immunoblotting used α-AtMSD1 antibody (bottom) . Actin was an input control. AtMSD1 activity and protein were normalized relative to the control without MV treatment. (B) Two-week-old seedlings were treated without (-, control) or with 5 μM MV for 2 to 12 h. Expression level was normalized relative to the control of AtMTM1 or AtAOX1A . The oxidation-responsive gene of mitochondrial AtAOX1A was used as a reference. Data are mean ± SE of three biological replicates. *, significant at P < 0.05 compared with the control.AtPP2A was an internal control.
Figure 6. Expressions of AtMTM1, AtMTM2, andAtMSD1 in response to different metal treatments. Two-week-old seedlings were treated without (-; control) or with 100 μM metal ions of MnCl2, Fe citrate, CuSO4, ZnSO4, MgCl2, and CaCl2for 24 h. Expression level was normalized relative to the control. Data are mean ± SE of three biological replicates. *, significant at P< 0.05 compared with the control. AtPP2A was an internal control.
Figure 7. Expressions of AtMTM2 in mtm1-i andAtMTM1 in mtm2 mutants in adapting to a shock of MV stress. Two-week-old seedlings were treated without (control) or with 1 to 10 nM MV for 12 h. (A and B) AtMTM2expression in mtm1-i mutant and AtMTM1 expression inmtm2 mutant were analyzed by qPCR, respectively. Expression level was normalized relative to the control of WT. Data are mean ± SE of three biological replicates. *, significant at P < 0.05 compared with the WT. AtPP2A was an internal control.
Figure 8. Root lengths of mtm1-i and mtm2 mutants in adaption to long-term MV stress. (A and B) Root lengths ofmtm1-i seedlings grown on 1 to 50 nM MV for 8 d and mtm2on 0.5 to 10 nM MV for 10 d were measured, respectively. Data are mean ± SE of three biological replicates. *, significant at P< 0.05 compared with the WT.
Figure 9. Characterization of mtm1-i mtm2-double mutant. (A) Expression level was normalized relative to the WT. AtPP2Awas an internal control. (B) Mn and FeSOD activities in six siblings of mtm1-i mtm2-#1 line (top) . Coomassie blue staining gel showed the ribulose bisphosphate carboxylase large subunit (RbcL) that was used as an input control (bottom) . (C)Mn and FeSOD activity of three independent plants was normalized relative to the WT, respectively. Data are mean ± SE of three biological replicates. *, significant at P < 0.05 compared with the WT. (D) Early-flowering phenotype of one-month-oldmtm1-i mtm2-#1 plants.
Figure 10. Root lengths of mtm1-i, mtm2, andmtm1-i mtm2-double mutants under Mn supplementation. WT and three mutants grown on 1/2 MS basal medium supplemented without (-) or with additional 10, 50, and 500 μM MnCl2 for 6 d and root lengths were measured. Data are mean ± SE of three biological repeats. *, significant at P < 0.05 compared with the WT.
Figure 11. Subcellular localization and SOD activity of modified AtMSD1. (A) Localizations of mitochondrial AtMSD1, cytosol-destined Δ-TP-AtMSD1, and chloroplast-destined Chl-TP-AtMSD1 were analyzed by confocal microscopy. YFP was fused to the C-terminal end of each tester. Bar = 20 μm. (B) Transient expressions of AtMSD1-Tag ,Δ-TP-AtMSD1-Tag , and Chl-TP-AtMSD1-Tag in WT protoplasts. Transfections without (-) or with (+) 30 μg plasmid DNA ofAtMSD1-Tag in 106 protoplasts are indicated. In-gel SOD activity assay (top) and immunoblotting with α-AtMSD1 and α-Actin antibodies (bottom) were conducted. Actin was an input control.
Figure 12. Exogenous expression of AtMSD1-3xFLAG in WT and mtm1-i mtm2 mutant protoplasts. Transfections with 5 and 10 μg plasmid DNA in 106 protoplasts are indicated. In-gel SOD activity assay (top) and immunoblotting with α-FLAG and α-Actin antibodies (bottom) were conducted. Actin was an input control.
Figure 13. Mn and Fe contents in mtm1-i, mtm2, andmtm1-i mtm2 mutants under MnCl2 treatment.Two-week-old seedlings were incubated without (control) or with 100 μM MnCl2 for 24 h. (A and B) Mn and Fe contents in root and shoot were measured by ICP-OES, respectively. The index of metal retention ability represents the ratio of ion content in the Mn treatment to the control. Data are mean ± SE of three biological repeats. *, significant at P < 0.05 compared with the WT.