Figure legends: Supplemental data
Figure S1
Mn2+ accumulation by yeast transformed with AtMTP11 or empty vector. MnCl2 (5
mM) was added to cultures at an initial density of 2.5 x 106 cells per mL (OD600nm
~0.20) and subsamples collected for Mn2+ analysis at the times indicated. Growth of
both strains was similar to cells grown with basal Mn2+. Data show means ± SE with
n = 3.
Figure S2
Transgenic plants that express an RNAi construct targeting AtMTP11 expression
phenocopy the mtp11 mutant when grown on agar medium. (A) AtMTP11 expression
in wild type and two independent RNAi lines grown with a basal Mn2+ supply, (B)
shoot dry weight of seedlings grown with 1 mM Mn2+ supply expressed as a percent
of the control treatment (basal Mn) and (C) Mn concentrations in shoots of seedlings
grown with a basal Mn2+ supply. Seedlings for shoot dry weights and elemental
analysis were harvested after 20 d growth. Error bars denote the means ± SE (n = 3)
and the LSD (P < 0.05) is shown.
Figure S3
The effects of Mn2+ deficiency on dry weight and Mn2+ concentrations in wild type
(WT) and mtp11 mutant plants. Seedlings were grown on agar medium that was
supplemented with either all mineral nutrients including Mn2+ at 4.5 μM (basal) or all
mineral nutrients except that the Mn2+ was omitted (Minus Mn). Seedlings were
harvested after 20 d for (A) shoot dry weights and (B) Mn2+ concentrations in shoots
(means ± SE with n = 3). The bar denotes the LSD (P < 0.05).
Figure S4
The effects of Cu2+ and Zn2+ toxicity on dry weights of wild type (WT) and mtp11
mutant plants. Seedlings were grown on agar medium that was supplemented with all
mineral nutrients (control) or all mineral nutrients and additional Cu2+ at 30 M
(Cu30) or Zn2+ at 0.4 mM (Zn0.4). Data show means ± SE with n = 4. The bar
denotes the LSD (P < 0.05).
Figure S5
Effect of Mn2+ supply on root growth and Mn2+ accumulation of wild-type, mtp11
mutant and mtp11 mutants expressing AtMTP11-GFP. (A) Effect of 1.0 mM Mn2+
supply on root dry weights and (B) Mn concentration in roots of seedlings for plants
grown with 1 mM Mn2+ supply (Mn concentration at basal supply is shown in Figure
7D). Seedlings were grown for 20 d and data show means ± SE with n = 5. The
various genotypes are wild type (WT), mutant (mtp11) and independent transgenic
lines expressing the transgene in mtp11 (L9 and L15). The bars denote the LSD (P <
0.05) where the data show statistically significant differences.
Figure S6
Silencing of the AtMTP11-GFP transgene in the T2 generation is associated with (A
and B) loss of Mn2+ tolerance and (C and D) increased Mn concentrations in shoots at
basal Mn2+ (4.5 μM) supply. Seedlings were grown for 20 days on agar medium
supplemented with all mineral nutrients (basal) or the same medium supplemented
with an additional 1 mM MnCl2. The various genotypes are wild type (WT), mutant
(mtp11) and a transgenic line transformed with AtMTP11:GFP in the mtp11 genetic
background (L10). Data show means ± SE with n = 4 (A and C) or n = 5 (B and D).
The bars denote the LSD (P < 0.05). Seedlings in the T1 generation for L10 were
segregating for the transgene at a ratio of 3:1 and were homozygous for the transgene
in the T2 generation. Expression of AtMTP11 relative to the cyclophilin reference
gene was as follows: L10 in the T1 generation (selected on kanamycin so all were
transgenic), 0.310 ± 0.034; L10 in the T2 generation, 0.007 ± 0.00; wild type, 0.460 ±
0.002; and mtp11, 0.008 ± 0.001 (means ± SE).
Figure S7
Stable expression of GFP-AtMTP11 in mtp11 Arabidopsis seedlings.