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.