SUPPORTING INFORMATION
Figure S1. Rootward auxin transport is compromised in the exo70A1-1 mutant, but not
in the GFP:EXO70A1-complemented exo70A1-2 mutant.
Rootward auxin transport was measured in the roots of 7-d-old seedlings after application of
3
H-IAA to the root-shoot junction. After 12 h, the amount of 3H-IAA was determined in roots,
and revealed significant reduction in auxin transport in exo70A1 mutants, but not in
GFP:EXO70A1 complemented plants. Variance of samples was tested by F-test, two sample
t-test for equal variance was subsequently used to evaluate difference between WT and
exo70A1-1 mutant (t-test p-value = 0.0006). Two sample t-test adjusted for unequal variance
was used to evaluate difference between WT and exo70A1-2/GFP-EXO70A1 plants (t-test pvalue = 0.1454). More efficient auxin transport in the case of complemented plants may
reflect overexpression or a slight difference in the genetic background (despite all plant were
Col-0). DPM = disintegrations per minute; ±SD are indicated; n = 20 for each column.
Figure S2. Quantitative analysis of auxin-response distribution in exo70A1-2 roots after
a local IAA application.
The quantification is related to the experiment in Figure 1e (i.e. local application of IAA to
the root-shoot junction). Pink bars demonstrate lines of the GUS signal measurement in each
root evaluated. Intensity of the GUS signal is plotted in graphs including standard deviation
bars. Middle part of each graph corresponds to stele, while left and right rims to epidermis.
Measurement in the upper 3 h after IAA application illustrates a massive auxin canalization
defect in exo70A1 mutants. The reduced GUS signal in the lower site, in the case of exo70A1
seedlings, reflects compromised efficiency in rootward auxin transport 3 hours after IAA
application. At least 5 seedlings were evaluated for each line in graphs.
Figure S3. Root epidermal cells of exo70A1-1 retain PIN2:GFP in abnormally-expanded
endomembrane compartments and exhibit recycling defect of PIN2:GFP.
WT and exo70A1-1 seedlings expressing PIN2:GFP were imaged before and after 2h
treatment with brefeldin A (BFA), 30 min and 90 min after the BFA washout, and after
prolonged 12h BFA treatment. (a) Root cells of untreated exo70A1-1 mutants exhibit
abnormally-expanded endomembrane compartments (marked by arrowheads) similar to
exo70A1-2 mutant cells (Figure 3b). (b) Homozygous exo70A1-1 mutants show identical
defect in re-established of the PIN2 plasma membrane localization after both transient and
chronic BFA treatment was documented as in the case of exo70A1-2 mutants. Bars = 10 µm.
Figure S4. Impaired recycling of PIN1 and PIN2 from brefeldin A compartments to the
plasma membrane visualized by immunolabelling in exo70A1-2 mutants.
PIN1 (a) and PIN2 (b) proteins were visualized by specific antibodies in WT and exo70A1-2
seedlings after 2h brefeldin A (BFA) treatment, 30 min and 90 min after BFA washout, after
prolonged 12h BFA treatment, and in untreated controls. Identical defect in re-established of
the PIN1 and PIN2 plasma membrane localization after both transient and chronic BFA
treatment was documented as in experiments using GFP-tagged PIN1 and PIN2 proteins
(Figure 3). Bars = 10 µm.
Figure S5. Impaired recycling of PIN1 and PIN2 from brefeldin A compartments to the
plasma membrane as visualized by the FM4-64 dye staining in exo70A1-2 mutants.
Seedlings were stained with FM4-64 for 15 min and imaged before and after 2h treatment
with brefeldin A (BFA), 30 min and 90 min after BFA washout, and after prolonged 12h BFA
treatment. Bars = 10 µm. BFA compartments were disaggregated within 30 min after BFA
washout in WT. A defect in such disaggregation was observed in exo70A1 mutants even 90
min after the BFA washout. Similar defect was documented also after chronic 12h BFA
treatment.
Figure S6. The GFP:EXO70A1 expression complements the EXO70A1 mutation.
Homozygous mutants exo70A1-2 exhibit WT phenotypes after transformation with the
Pro35S:GFP:EXO70A1 construct.
Table S1. Number of analysed plants and cells and percentage of affected cells in brefelin A
experiments.