Supporting Information Legends
Figure S1. Identification of an ea7 suppressor mutant. (a) A 15-day-old Landsberg
erecta (Ler) seedling. (b) A 15-day-old ae7 (Ler) single mutant plant. (c) A
15-day-old ae7 suppressor mutant, designated Line 89/ae7. Images from (a-c) are in
the same magnification. Bar in (a), 1 cm. (d) Quantitative analysis of cell number of
the first rosette leaves, showing that the decreased cell number in ae7 leaves was
rescued in Line 89/ae7. The value of cell numbers from wild-type Ler was arbitrarily
fixed at 1.0, and the data are shown as the averages with s.d., n = 10. **, P < 0.01.
Figure S2. Molecular identification of the CCR1 gene. (a) Fine structure mapping to
localize the ae7 suppressor locus on chromosome 1 (upper panel), and sequencing of
putative genes in the mapped region revealed the mutation corresponding to the CCR1
gene (lower panel). Black and white boxes in the lower panel indicate the
protein-coding regions and the untranslational regions of CCR1, respectively, lines
show introns, and red arrows mark the position of primers used in genotyping the
CCR1pro:CCR1-GUS/ccr1-4 transgenic plants. (b-e) An allelism test between ccr1-4
and ccr1-g. (b) A 12-day-old wild-type Col-0 plant. (c) A 12-day-old ccr1-4 plant. (d)
A 12-day-old ccr1-g plant. (e) A 12-day-old F1 plant which was from a cross between
ccr1-4 and ccr1-g. (f) A diagram showing the construct used in the ccr1-4
complementation test. (g) A 28-day-old wild-type Col-0 plant. (h) A 28-day-old
ccr1-4 plant. (i) A 28-day-old CCR1pro:CCR1-GUS/ccr1-4 plant. The lower panels of
(g-i) show results from genotyping the CCR1pro:CCR1-GUS/ccr1-4 plant according to
the method described by Bui and Liu (2009). (j) Sequences of primers used in
genotyping. Blue and black letters represent genomic and primer sequences,
respectively, while red letters indicate the nucleotides which do not match those of the
corresponding genomic sequences. For genotyping the ccr1-4 plants were transformed
with the CCR1pro:CCR1-GUS construct through an Agrobacterium-mediated
transformation method. Seeds of T1 plants were grown on MS medium containing
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hygromycin. A total of 17 independent hygromycin-resistant plants were obtained,
which all showed normal phenotype. A wild-type, a ccr1-4, and a T2 transgenic line
were genotyped with primers 1 and 3, or 2 and 3. (k) Cell numbers in the first leaves
on day 25 of a T2 CCR1pro:CCR1-GUS/ccr1-4 plants were close to those of the
same-age wild-type plants. The value of cell numbers from wild-type Col-0 was
arbitrarily fixed at 1.0. Bars show the average with s.d., n = 10. **, p < 0.01. Bars in
(b-e, g-i), 1 cm.
Figure S3. Cell size and cell number per leaf of the ccr1-g mutant. (a, b) Palisade
cells of the first rosette leaves on day 12 from wild-type (a) and ccr1-g (b) seedlings.
Leaves were analyzed by a DICM and images were taken at the position as shown in
Figure 4i. Bars in (a, b), 50 m. (c) Analysis of palisade cell numbers of the ccr1-g
plants. The first ccr1-g leaves on day 25 were used in analyses. The value of cell
numbers from wild-type Col-0 was arbitrarily fixed at 1.0, and the data are shown as
the average with s.d., n = 10. **, P < 0.01.
Figure S4. Expression patterns of CCR1 in the root. (a-d) GUS staining to determine
CCR1 expression patterns in the root of CCR1pro:CCR1-GUS/Col-0 transgenic plants.
Seedlings on day 7 were used in the experiment, and roots stained with 10 min (a), 30
min (b), 1 h (c), 2 h (d), and 16 h (e) are shown. CCR1 is expressed everywhere
except the root cap. The vascular cylinder showed the strongest GUS staining,
whereas staining in epidermis and cortex appeared relatively weak. Bar in (a-d),
100m. (f) Quantitative analysis of FeA content in the root of seedlings on day 7.
FeA was not detected in wild-type Col-0 roots, whereas ccr1-4 roots accumulated
high levels of FeA. For comparison, the FeA level in Col-0 leaves on day 7 was
served as a control, which was arbitrarily fixed at 1. Data are means + s.e. of three
biological replicates.
Figure S5. Effects of FeA and H2O2 on cell numbers of cortex in the root
meristematic zone. (a-c) Root meristematic zones of seedlings on day 7. (a) Col-0. (b)
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Col-0 treated with 50 M FeA. (c) ccr1-4. (d) The number of root cortex cells was
altered by treatments with FeA or H2O2. The value of cell numbers from wild-type
Col-0 was arbitrarily fixed at 1.0. Shown are the averages with s.d., n = 10. **, p <
0.01. Bars in (a-c), 50 m.
References
Bui, M. and Liu Z. (2009). Simple allele-discriminating PCR for cost-effective and
rapid genotyping and mapping. Plant Methods 5:1, doi:10.1186/
1746-4811-5-1.
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