Download Weber et al_rev Legends of supplementary figures and tables

Weber et al_rev Legends of supplementary figures and tables
Figure S1. Isolation of ozs mutants.
(a) Step one: seeds were grown vertically on agar plates with 60µM ZnSO 4, and screened for growth
reduction stronger than Col-0. Red arrows mark seedlings showing this phenotype. (b) Step two:
seedlings with reduced root growth were transferred to plates with control medium. Per plate two plants
with normal root growth were co-transferred as positive control. Plates were turned by 90° and cultivated
for additional 5 days. Seedlings that showed recovery of root growth were selected as candidates (marked
by asterisks) and chosen for further analysis, bar=1cm.
2+
Figure S2. Dose-dependent Zn -hypersensitivity of ozs1.
Seedlings of wild type (Col-0) and two independent ozs1 backcross lines (BC) were grown vertically in the
presence of different ZnSO4 concentrations. Length of primary roots was determined after ca. 12d of
cultivation (when Col-0 seedlings on control plates had reached the bottom of the plate). Data in (a)
represent mean values ±SD of 3 independent experiments (n=66-81). Significant differences from WT
were determined by two way ANOVA and Tukey test, *P<0.05, ***P<0.001. BC=backcross line. (b)
Representative pictures documenting growth of Col-0 and ozs1 seedlings after 12d, bar=1 cm.
Figure S3. The ozs1 phenotype is caused by a mutation in AtMTP1.
(a) The Col-0 wild-type and the mutant ozs1 version of AtMTP1 were expressed as C-terminally HAtagged proteins in the Saccharomyces cerevisiae strain zrc1cot1. Wild-type AtMTP1 was able to rescue
2+
growth in the presence of Zn -excess while cells expressing the ozs1 version did not grow better than
empty vector controls (pYES2). S. cerevisiae cells were grown at 30°C in yeast nitrogen base
supplemented with the appropriate amino acids and carbohydrates. Shown in (b) is detection of MTP1
protein expression using a monoclonal anti-HA antibody (Covance) (upper panel) and the Coomassiestained loading control (lower panel). (c) Wild type (Col-0), ozs1, and two homozygous ozs1 lines
transformed with a genomic fragment of AtMTP1 (primers gf-MTP1-1-1 and gf-MTP1-5-1, Tab. S2) were
grown on control medium or in the presence of 40µM ZnSO 4. Length of the primary root was determined
after ca. 12d of cultivation (when Col-0 seedlings on control plates had reached the bottom of the plate).
Data represent means ±SD of 3 independent experiments (n=67-81). Significant differences from WT
were determined by two-way ANOVA and Tukey test, ***P<0.001.
Figure S4. Hypersensitivity of ozs2 towards Zn
2+
is highly specific.
Seedlings of Col-0 and two independent ozs2 backcross lines were grown on medium containing growthinhibiting concentrations of different metal cations, NaCl, paraquat (=Pa) or mannitol (=Man). Length of the
primary root was determined after ca. 12d of cultivation (when Col-0 seedlings on control plates had
reached the bottom of the plate). Data represent means ±SD of 3 independent experiments (n=43-102).
Significant differences from WT were determined by two-way ANOVA and Tukey test, *P<0.05.
Figure S5. Zn accumulation of wild type Col-0 and ozs2 mutant seedlings.
Col-0 and two independent ozs2 backcross lines (BC) were grown vertically on agar plates in the
presence of different ZnSO4 concentrations. Roots (a) and shoots (b) were harvested separately, washed
with Millipore-water and CaCl2, and analysed by ICP-OES following microwave digestion. Data represent
mean ±SD of 3-7 independent experiments. Significant differences from wild type were determined by
Student´s t-test, *P<0.05.
2+
Figure S6. Semi-dominance of the ozs2 Zn -hypersensitivity phenotype.
Seedlings of Col-0, ozs2 and the F1 generation of a cross between both lines were grown vertically on
control medium or in the presence of 80µM ZnSO 4. (a) Length of the primary root was determined after
ca.12d of cultivation (when seedlings on control plates had reached the bottom of the plate). Shown in (b)
is relative root growth in the presence of Zn
2+
excess. Data represent means ±SD of 3 independent
experiments (n=54-178).
Figure S7. Characterization of the atpme3 T-DNA insertion line 002A10 (GABI-Kat).
(a) Genomic DNA was extracted from leaf material of Col-0 and atpme3 plants. Homozygosity for insertion
into the AtPME3 gene was determined using both T-DNA and AtPME3 specific primer combinations. (b,c)
Total RNA was extracted from root material of Col-0 and atpme3 plants grown hydroponically for 4 weeks.
Following cDNA synthesis AtPME3 transcript levels were determined by conventional RT-PCR (b) and by
quantitative real-time PCR (c). In (c) transcript level is expressed relative to EF1. CT value was
calculated by subtracting the cycle threshold (CT) value of the reference gene EF1 from the CT value of
the target gene PME3. Relative transcript level (RTL) was calculated as: RTL = 1000x2
represent means of 3-4 independent experiments.
-CT
. Data
2+
Figure S8. AtPME3 overexpressing lines are specifically Zn -hypersensitive.
In Col-0 background transgenic lines were generated overexpressing either wild-type PME3 (=PME3-ColOX), ozs2 mutant version (=PME3-mut-OX) or an enzymatically inactive version of AtPME3-mut (=PME3mut-ia-OX) (these lines were also used for the experiments presented in Figure 6a,b). (a) PME3 transcript
levels, expressed relative to EF1, were determined by quantitative real-time PCR for seedlings grown in
liquid culture to comparable size (7-9d). Data represent means of three independent experiments. (b)
Seedlings of Col-0 as well as homozygous transgenic PME3-Col-OX and PME3-mut-OX lines were grown
vertically under control conditions and in the presence of growth-inhibiting metal cation concentrations.
Length of the primary root was determined after ca.12d of cultivation (when seedlings on control plates
had reached the bottom of the plate). Data represent means ±SD (n=18-31), Significant differences from
wild type were determined by two-way ANOVA and Tukey test, ***P<0.001.
Figure S9. Total pectin methylesterase activity in AtPME3 overexpressors and T-DNA insertion
lines.
Col-0, atpme3 and lines overexpressing either the wild type AtPME3 (=PME3-Col-OX) or the ozs2 mutant
version (=PME3-mut-OX) in atpme3 background were grown in hydroponic culture (modified Hoagland
medium). After 4-5 weeks under short-day conditions root material was harvested and frozen in liquid
nitrogen. Total soluble protein was extracted according to Guénin et al. (2011). PME activity was
measured according to Reca et al. (2012).
Figure S10. Ca
2+
2+
suppresses the Zn -hypersensitivity phenotype caused by overexpression of
AtPME3.
2+
Zn
tolerance of atpme3 and a line overexpressing the WT version of AtPME3 in atpme3 was assayed
2+
either in the absence or the presence of 0.5mM CaCl2. Seedlings were exposed to 60µM Zn . Length of
primary roots was determined after ca. 12d of cultivation (when Col-0 seedlings on control plates had
reached the bottom of the plate). Data represent means ±SD of 2 independent experiments (n=30-32).
Significant differences to atpme3 were determined by a two-way ANOVA analysis, ***P<0.001.
Figure S11. Pectin staining of Col-0 and ozs2 roots with monoclonal antibodies JIM7 and CCRC
M38.
2+
Roots from 4d old seedlings, grown in the presence of 60µM Zn , were embedded in Technovit 8100 and
5µm transversal sections were incubated with antibody JIM7 (binding to highly methylated pectin) or
antibody CCRC M38 (binding to stretches of demethylesterified pectin). A Cy3-labeled secondary antibody
was used to visualize the different pectin epitopes in a Leica TCS SP5 confocal microscope. Shown in (a)
are four representative sections each for Col-0 (WT) and ozs2, bar=20µm. (b) The fluorescence pattern
between individual sections from roots differs during development. Therefore, the fluorescence intensities
were quantified with ImageJ software separately for the cortex and for the stele part of the root. All
pictures had intensities between 0 to 255 gray values. Intensities below 10 were considered as
background and subtracted. The graph shows the ratio of (absolute) integrated fluorescence intensity of
cortex vs. stele. Error bars represent standard deviation of n=10 quantified sections. The differences in
fluorescence intensities between wild type and osz2 are not statistically significant.
Figure S12: Root ultrastructure of ozs2 displays no difference to Col-0 wild type. Ultrastructural
characterizations of the stele of the ozs2 mutant (a-eozs2) in comparison to Col-0 wild type (a-eCol-0) were
performed. (a) An overview of root cross sections with corresponding cell types is shown. Cc: companion
cell, ed: endodermis, pa: parenchyma cell, pc: pericycle, se: sieve element, x: xylem. (b) The endodermis
(ed) is the innermost layer of the cortex, which surrounds the stele. The pericycle (pc) builds the outermost
layer of the stele. (c) The stele consists of the phloem with its sieve elements (se), companion cells (cc)
and (d) parenchyma cells, which are orthogonally arranged to (e) the xylem. Two arrowheads facing each
other visualize the distance between the middle lamella and inner membrane of the cell wall. The cell wall
of the endodermis and the lignified secondary cell wall of the xylem have a parallel texture, in contrast to
the scattered texture of the cell wall of the pericycle and phloem cells. Upper scale bar in aozs2 applies to
(a) with 5µm. Lower scale bar in eozs2 applies to (b)-(e) with 0.5µm.
Table S1. Cell wall thickness of the different Col-0 and ozs2 root
cell types
Cell wall thickness (in µm) was determined in ultrathin sections (~6070nm) of Col-0 and ozs2 seedlings. Data represent mean values ±SD
of about 200 cells analyzed per cell type. Significant differences
between the genotypes were determined by one-way ANOVA and
Tukey test. They are indicated by different letters, P<0.05.
Table S2. Primers used