SUPPLEMENTARY LEGENDS
Figure S1. Screening strategy used in this study. (a) Schematic model of light signaling
during Arabidopsis seedling photomorphogenesis under white light from fluorescent
tubes. (b) In the conventional screening for phyB signaling mutants, the seedlings are
grown on agar plates under cR, and the hypocotyl orientation is randomized by the
action of phytochromes (left panel), which makes it difficult to detect small differences
in hypocotyl length. However, in the screening of this present study, seedlings were
grown on soil under cW, and the unidirectional hypocotyl elongation by phototropism
facilitated the high-throughput detection of subtle differences in hypocotyl length (right
panel). The complete loss of cryptochrome function enabled us to monitor only the
effect of phytochrome signaling during seedling photomorphogenesis, even under cW,
while inducing phototropism by activating phototropins. Yellow arrow indicates
long-hypocotyl individual.
Figure S2. rrc1 mutants are defective in all of the phyB-mediated light responses that
were examined. (a) Cotyledon area of 7-day-old seedlings grown under cR (left panel,
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8.0 μmol m-2 sec-1; right panel, 8.6 μmol m-2 sec-1). Data represent the mean ± SE (left
panel, n = 45; right panel, n = 35). (b) Chlorophyll content of 7-day-old seedlings
grown under cR (left panel, 9.0 μmol m-2 sec-1) or cW (right panel, 40 μmol m-2 sec-1).
Data represent the mean ± SE (left panel, two biological replicates; right panel, three
biological replicates). (c) Petiole length of plants grown for 4 weeks under cW (40 μmol
m-2 sec-1). Data represent the mean ± SE (left panel, n = 19; right panel, n = 22). (d)
Flowering time expressed as days to 1-cm bolting under long-day (LD, 16-h light/ 8-h
dark) or short-day (SD, 8-h light/ 16-h dark) conditions. Data represent the mean ± SE
(n = 25). N.A., not analyzed.
Figure S3. Identification of the gene responsible for rrc1-1. (a) T-DNA insertion site in
the rrc1-1 mutant. The genes that are located within 10 kb of the CaMV35S enhancer of
the T-DNA and their exon (shown in black)/intron (shown in white) structure are shown.
(b) RT-PCR analysis in rrc1-1 and the parental line cry1 cry2. UBQ10 was used as an
internal control. Primer positions are indicated by arrowheads in (a). (c) RT-PCR
analysis of RRC1 in the two independent RRC1-overexpressing lines in the Col-0
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background (OX31-1 and OX36-3). Primer positions are indicated by arrowheads in (a).
(d) Hypocotyl lengths of 5-day-old seedlings grown under cR (8.9 μmol m-2 sec-1) or
cFR (30 μmol m-2 sec-1). Data represent the mean ± SE (n = 25).
Figure S4. Genomic sequence of RRC1 in rrc1-1, rrc1-2 and rrc1-3 mutants. The
genomic sequence of the 3’ end of RRC1 (exons, uppercase; introns and 3’UTR,
lowercase). The amino acid sequences of the RRC1 proteins that are encoded by Col-0
or the rrc1 alleles are presented in single letter code underlying the genomic nucleotide
sequence. The positions of the T-DNA insertions in the rrc1 alleles are indicated by
arrowheads. In rrc1-2 and rrc1-3, the T-DNAs are inserted within the same intron
between exon15 and exon16, and the predicted stop codon (shaded) is present in this
intron. In rrc1-1, the T-DNA is inserted within the final exon. Although the genomic
sequence of RRC1 in rrc1-1 was normal except for the region around the T-DNA
insertion site, the mutant mRNA sequence showed an altered alternative splicing event.
The aberrantly spliced-out intron is underlined. The T-DNA sequence contains an
in-frame stop codon, and the rrc1-1 transcript encodes a predicted 899-amino acid
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protein that is in-frame.
Figure S5. Alignment and phylogenetic relationship between RRC1 and various SR140
homologs. (a) Alignment of the amino acid sequences of the SR140 homologs from
Arabidopsis (RRC1 and At5g10800), rice (Q6ETK6) and human (O15042). The
alignment was generated using CLUSTALX 2.1 (http://www.clustal.org/clustal2/) with
the default parameters. The conserved domain structures are shown in boxes. (b)
Phylogenetic tree of SR140 homologs. The published and predicted SR140 homolog
sequences in the UniProtKB (http://www.uniprot.org/help/uniprotkb) database were
aligned using CLUSTALX 2.1 as in (a), and the BOOTSTRAP N-J TREE algorithm
was adopted to calculate a tree. The tree was then drawn using Njplot 2.3
(http://pbil.univ-lyon1.fr/software/njplot.html). The accession numbers of the sequences
that were used are as follows: human (Homo sapiens), O15042; mouse (Mus musculus),
Q6NV83; dog (Canis familiaris), E2R1V3; Xenopus tropicalis, B5DEC9; zebrafish
(Danio rerio), B0S5J3; ant (Camponotus floridanus), E2B1D1; fruitfly (Drosophila
melanogaster), Q9W2N6; rice (Oryza sativa subsp. japonica), Q6ETK6; Coprinopsis
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cinerea, A8N661; Cryptosporidium muris, B6AAY3; Physcomitrella patens subsp.
patens, A9TAZ0 [a] and A9SYF0 [b]; grape (Vitis vinifera), D7SV56; Selaginella
moellendorffii, D8SG98 [a] and D8R633 [b]; Chlamydomonas reinhardtii, A8J7H0; and
Arabidopsis thaliana, At1g10800 (F4KIA8) and RRC1(Q9C5J3). The percentage
bootstrap values of 1000 replications are shown on the internal nodes. Branch lengths
are proportional to phylogenetic distances. Scale bar represents 0.05 amino acid
substitutions per site.
Figure S6. Expression analysis of RRC1. (a) RRC1 and the paralogous gene At5g10800
are almost ubiquitously expressed in Arabidopsis. Total RNA was extracted from
various organs of light-grown Col-0 mature plants. RL, rosette leaves; CL, cauline
leaves; IS, inflorescence stem; RT, roots; FB, flower buds; OF, open flowers; and SI,
siliques. UBQ10 was used as an internal control. (b) Various wavelengths of light did
not affect the expression of RRC1, as determined by RT-PCR analysis. Total RNA was
extracted from Col-0 and phyB-9 seedlings that had been grown for 4 days in dark, cR
(8.6 μmol m-2 sec-1), cFR (27 μmol m-2 sec-1), cB (8.7 μmol m-2 sec-1) or cW (55 μmol
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m-2 sec-1) conditions. UBQ10 was used as an internal control. (c) Various wavelengths
of light did not affect the accumulation of RRC1. Four-day-old etiolated Col-0 seedlings
were kept in the dark for an additional 1 day (Dark) or exposed to cR (9.2 μmol m -2
sec-1) (R), cFR (26 μmol m-2 sec-1) (FR), cB (9.2 μmol m-2 sec-1) (B), or cW (37 μmol
m-2 sec-1) (W) for 1 day, and total protein was extracted. Each lane contained 50 µg of
total protein. The CBB-stained gel image is shown as a loading control. Similar results
were obtained in two independent experiments.
Figure S7. T-DNA insertional mutations in At5g10800 do not affect photomorphogenic
phenotypes. (a) Schematic illustration of the At5g10800 gene structure and the positions
of the T-DNA insertion in two independent mutant alleles of At5g10800. (b) RT-PCR
analysis of At5g10800. Total RNA was extracted from mature plants that had been
grown under cW. The primer sets that were used in the analysis are shown on the left
and correspond to the primers shown in (a). UBQ10 was used as an internal control. (c)
Hypocotyl lengths of 5-day-old seedlings grown under cR (8.5 μmol m-2 sec-1). Relative
hypocotyl lengths are expressed as a ratio to the respective dark value. Data represent
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the mean ± SE (n = 25).
Figure S8. RT-PCR analysis of PIF genes in the rrc1 mutants. The same cDNA
templates as in Figure 4 were used. For PIF1, PIF3 and PIF6, the number of PCR
cycles was 26, 28 and 36, respectively. The mass of each splice variant was quantified
using an Agilent 2100 Bioanalyzer, and the percentage of the minor variant to the total
amount of the transcript was shown as the mean value of two biological replicates ± SD.
Each band was cloned and sequenced, and the schematic diagrams of sequenced splice
variants are shown on the right. Gray and white boxes represent translated and
untranslated regions, respectively. Lines represent introns. The positions of the primers
and the predicted stop codons are indicated by arrowheads and asterisks, respectively.
TUB2 was used as an internal control.
Figure S9. Linearity in the quantification of splice variants of RS31 and SR34b. (a)
RT-PCR analysis of RS31 and SR34b. cDNA from 4-day-old dark-grown Col-0
seedlings was used as a template. The number of PCR cycles in each lane is indicated
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above. Details are as in Figure S8. (b) Quantification of the ratio of splicing variants
amplified with different numbers of PCR cycles. The mass of each splice variant in (a)
was quantified using an Agilent 2100 Bioanalyzer, and the ratio of splice variants was
calculated. The mean values ± SD among the four different cycles are shown.
Figure S10. Representative gel images of the RT-PCR analysis of RS31 and SR34b.
Four-day-old etiolated seedlings of Col-0, phyB-9 and rrc1-3 were exposed to cR (9.0
μmol m-2 sec-1) for the indicated times (DR), and total RNA was extracted for the
RT-PCR analysis. Total RNA from the seedlings kept in darkness for the same period of
time was used as a control (DD). Representative gel images of the RT-PCR analysis of
RS31 and SR34b are shown in (a) and (b), respectively. For RS31 and SR34b, the
number of PCR cycles used was 24 and 27, respectively.
Figure S11. Alternative splicing of RRC1 was not affected by red light. (a) Schematic
illustration of RRC1 structure and the positions of the T-DNA insertion in the three rrc1
mutant alleles. Black boxes, exons. White boxes, untranslated regions. Black lines,
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introns. (b) RT-PCR analysis of RRC1 in the rrc1 mutants. Two different sets of primers
were used. The same cDNA templates as in Figure 4 were used. (c) RT-PCR analysis of
RRC1 during the transition from dark to cR. The same cDNA templates as in Figure 5
were used. Primer positions are indicated by arrowheads in (a). TUB2 was used as an
internal control.
Table S1. Primer sequences used in this study.
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