Figure S1. The wat1-1D mutant has a single insertion of T-DNA (a) that segregates
with the tolerance to intracellular acidification by acetic acid (b).
(a) Southern analysis of the wild type (wt) and the wat1-1D mutant DNA using T-DNA
specific probes that hybridize with the BAR gene (left) or the 35S enhancers (right). (b)
Cosegregation analysis of the F2 generation. PCR using the 35S primers described in
Matherials and Methods, of 10 representative acetic acid resistant individuals. The two
bands observed in the figure correspond to the amplification of a copy (≈ 300bp) or two
copies (≈ 600 bp) of the four 35S enhancers present in tandem in the T-DNA. M: Size
marker.
Figure S2. The morphological phenotype of wat1-1D in normal conditions is
slightly different to that of the wild type.
Percentage of seedlings with green, expanded cotyledons of the wild type (wt) and the
mutant in MS medium after 3 and 5 days of sowing. (b) Representative 5 day-old
seedlings of the wild type and the mutant (left). Scale bar corresponds to 2 mm.
Average and standar error of the cotyledon area of 50 of those seedlings (right). (c)
Measurement of root length increment and fresh weight of wild type and mutant plants
after 10 days growing in MS medium. A picture of these plants is shown (right). Scale
bar corresponds to 1cm. (d) Representative rosettes (left), plants (middle) and
inflorescences (right) of wild type and mutant plants growed under greenhouse
conditions during 20 and 30 days respectively. Scale bars correspond to 2 cm. * P<0.05
by Student’s test.
days
Figure S3. Phenotypes of wat1-1D mutant are not due to over-expression of
At3g55470 gene.
(a) Localization of the T-DNA insertion of the two knock-out mutants studied for the
At3g4470 gene (470-1 and 470-2). fp: forward primer. rp: reverse primer. (b) Northern
analysis of the wild type (wt), the wat1-1D mutant, and two transgenic lines overexpressing the At3g55470 gene (T4 (1) and T4(2)) using a probe obtained by
amplification with the following primers: At3g55470F (fp) and At3g55470R (rp). (c)
Percentage of green, expanded cotyledons of the wild type, wat1-1D, knock-out mutants
and over-expression lines for the At3g55470 in the presence of acetic acid (3.5 mM).
Averages and standard error of three independent experiments are given.
Figure S4. Northern analysis of At3g55480 mRNA from wild type (wt), wat1-1D
mutant, and two transgenic lines over-expressing the truncated At3g55480 gene
(T3(1) and T3(2)).
Figure S5. The wat1-1D mutant has similar phenotypes than the pat2 mutant.
(a) Representative plates showing the phenotype of 7 day-old wild type (wt) and wat11D mutant seedlings growing with (left) or without (right) sucrose in the medium. (b)
Percentage of non-arrested seedlings of the wild type and the mutant. Average and
standar error of three experiments like the one in (a) are given. * P<0.05 by Student’s
test. (c) Shoot gravitropic response. Histogram showing the distribution of wild type
and mutant seedlings in function of the angle generated by their hypocotyl with respect
to the horizontal after the gravitropic stimulus of 90º (see Feraru et al., 2010). The angle
of 40 seedlings for each genotype was measured. (d) Representative seedlings of the
wild type and the mutant after the gravitropic stimulus.
Figure S6. Initial rate of acetic acid uptake is not affected in the wat1-1D mutant.
12 day-old seedlings were incubated for different time periods (0, 7, 15, 30 min) in
liquid MS medium supplemented with 3.5 mM 14C-acetate and the uptake of
radioactivity determined as indicated in Experimental Procedures.
Figure S7: Change in internal malate concentration during acetic acid treatment is
not affected in the wat1-1D mutant
16 day-old seedlings were transferred to liquid MS medium supplemented with 3.5 mM
acetic acid for 0, 5 and 15 minutes. After the treatments, samples were frozen with
liquid nitrogen. The mean and standard error of six experiments are given.
Figure S8. The efflux of radioactivity after loading with 14C-acetic acid
corresponds to acetic acid.
Plants (wild type and wat1-1D mutant) were loaded with 14C-acetic acid, washed and
incubated 1 h for efflux of the radioactivity as described in Experimental Procedures.
The external liquid was concentrated and injected in an HPLC system. A similar
amount of 14C-acetic acid standard was also analyzed. A single peak is observed in all
three samples, with a retention time of 34 minutes approximately. Arrows indicate the
radioactivity value of each peak in cpm (counts per minute).
GFP
Chlorophyll
Transmission
Overlay
Supplementary Figure S9. The vacuolar channels TPK1 and ALMT9 are not misslocalized in the wat1-1D mutant
Constructs with 35S promoter-TPK1-GFP (Voelker et al., 2006) and 35S promoterAMLT9-GFP (Kovermann et al., 2007) were used for transient expression in
Arabidopsis mesophyll protoplasts (Yoo et al., 2007) and images were taken with a
confocal fluorescence microscope. Scale bar corresponds to 8 μm.