Download Figure S1. Interactions between AS1 and CO proteins that contains

Figure S1. Interactions between AS1 and CO proteins that contains B-box mutations.
The full-length of co mutant cDNAs (co-2, co-3, co-4, and co-6) were amplified using mRNA
extracted from each mutant allele and introduced into the pDEST15 GST-fusion vector. All co
mutant proteins were purified by glutathione affinity chromatography and used for in vitro
pull down assays. Since the amount of the degradation products of the GST-CO variants
varied, we used a similar amount of full length of GST-CO variants (indicated by the
arrowhead) when we performed the GST pull-down assay for 6xHis-AS1.
Figure S2. AS1 is mainly expressed in the vascular tissues. 14-day-old plants were
incubated in a solution containing 50 mM sodium phosphate buffer pH 7.2, 10 mM EDTA,
0.1% Triton X-100, 0.002% (w/v) chloramphenicol, 1.25 mM potassium ferricyanide, 1.25
mM potassium ferrocyanide, 0.1% (w/v) 5-bromo-4-chloro-3-indolyl β-D-glucuronide (XGluc) at 37°C for 1-4 hours. After the incubation, plant samples were treated with 70%
ethanol to remove chlorophyll.
(a) AS1-GUS activity was detected mainly in the vascular tissues of a cotyledon, a rosette leaf
(left upper panels from left to right), and the whole plant (left lower panel) grown in LD
conditions. Scale bars are 1cm.
(b) AS1-GUS expression in 12L/12D and SD conditions was same as that in LD conditions.
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Scale bar: 1cm.
Figure S3. Location of the T-DNA insertion in the as1-21 mutant.
(a) The position of T-DNA insertion in AS1 gene. We determined the precise position of TDNA insertion in as1-21. T-DNA was inserted in the 1st intron of AS1 gene (131 bases
downstream from the transcription start site of AS1). +1 represents the transcriptional start site.
(b) Altered AS1 transcript in as1-21. We found that AS1 mRNA is expressed in the as1-21, but
the AS1 transcript possesses different 5’-UTR sequences than the AS1 transcript expressed in
the wild type. Nucleotide sequences of the AS1 mRNA expressed in the as1-21 mutant are
compared with those of AS1 mRNA expressed in WT. The difference in 5’-UTR sequences in
as1-21 is shown in gray. This data suggests that the insertion of T-DNA in the 1st intron
changed the splicing pattern of AS1 in as1-21. The AS1 mRNA in as1-21 includes left border
(LB) sequences of the T-DNA.
(c) Leaf morphological phenotypes of as1-21 mutant and WT plants are shown. The as1-21
showed a similar leaf morphological phenotype to other as1 alleles.
(d) Comparison of phenotypes in flowering time and shape of whole plants among as1 mutant
alleles grown in 12L/12D conditions.
(e, f) Complementation of the as1 mutant using the AS1 genomic fragment. This result
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confirmed that the flowering phenotype of as1-21 is due to the loss of AS1 activity.
Comparison of phenotypes in whole plants (e) and flowering time (f) of wild-type, as1-21,
and gAS1:HA/as1-21 grown in LD (f), 12L/12D (e, f), and SD (f) conditions. The phenotypes
of as1-21 mutant were complemented by the insertion of genomic AS1 fragment. The means
are ± SD (n = 14-18).
(g) Flowering phenotype of as1 mutant under different light/dark conditions. as1 mutant
showed late flowering when light is less than 14 hours. The means are ± SD (n = 16-18).
Figure S4. Expression levels of KNAT1 and AtGA20ox1 in as1 mutants.
Quantification of KNAT1 (a) and AtGA20ox1 (b) mRNA abundances in as1 alleles and ga5-3.
Twenty four-day-old plants grown on soil in short days were harvested at ZT 4. ACT2 was
used as an internal control. The means and S.D. were based on three independent experiments.
Figure S5. Flowering time of SUC2:CO as1 plants under various photoperiods.
Plants were grown in LD, 12L/12D, and SD conditions, and total leaf number was counted. In
LD and 12L/12D conditions, flowering time of the SUC2:HA-CO as1-21 lines were similar to
that of the SUC2:HA-CO plants, assuming that higher expression levels of FT in SUC2:CO
as1-21 compared to those in WT (Figure 5e,f) are sufficient to induce early flowering. In SD
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conditions, the SUC2:CO as1-21 line flowered later than the SUC2:CO line. This might be
due to the effect of the as1 mutation on the down-regulation of AtGA20ox1 expression in the
GA-dependent flowering pathway.
Figure S6. Effects of AS1 overexpression on flowering time and leaf shape regulation.
(a) SUC2:AS1-HA and 35S:AS1-HA plants were grown under indicated conditions and
flowered slightly earlier than the wild-type plants under 12L/12D and 10L/14D conditions.
The levels of AtGA20ox1 transcripts were increased in the transgenic plants (Fig. S4) but FT
transcripts were not changed, indicating that AS1 alone may not be sufficient for FT induction,
and that slight early flowering of the transgenic plants may be due to the elevated AtGA20ox1
activity. The means are ± SD (n = 12).
(b) A leaf phenotype in AS1 overexpression plants. Leaf width in AS1-ox plants is narrower
than that in WT (Theodoris et al., 2003).
Figure S7. AS1 specifically binds to FT promoter sequences in vitro.
E. coli crude extract containing 6xHis-AS1 protein was incubated with radio-isotope labeled
FT fragments and separated on 5% non-denaturing polyacrylamide gels. The DNA-AS1
complex represents the binding of AS1 to the FT fragment. The presence or absence of AS1
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protein are shown as plus or minus symbols. Water was added instead of a protein extract for
“No protein” sample. Empty vector and 6xHis-AS1 indicate 1.25 μg of total extracts
containing 6xHis protein or 6xHis-AS1 fusion protein, respectively. (a) The DNA-AS1
protein complex was only detected in the 32P-labeled amplicon 11 (-482/-189) fragment. (b)
The effect of CO on the binding of AS1 to FT promoter. CO by itself does not bind to the
amplicon 11 DNA fragment. CO seemed not to affect AS1 binding ability to the amplicon 11
fragment. There are three faint non-specific bands in the empty vector control lane and the
middle band migrated at a similar position to the AS1-specific band. As shown in Figure S6c,
we confirmed that the presence of the AS1-specfic band by adding non-specific single
stranded DNA into the reaction. (c) Confirmation of specific association of AS1 protein with
the FT promoter sequences. We found that adding a different amount of non-specific single
stranded DNA (S•S DNA) changed the patterns of non-specific bands without changing the
AS1-specific band. This data demonstrated that what we indicated by the arrowhead is the
AS1-specific band, and under our EMSA condition there is a faint non-specific band that
migrates at the same position as the AS1-specific band.
Figure S8. Anti-AS1 and anti-CO antibodies can detect AS1 and CO expressed in plants.
Specificities of anti-AS1 (a, b) and anti-CO (c, d) antibodies were analyzed. (a) 100 μg of WT
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and as1-21 nuclear proteins and 5 μg of 35S:AS1-HA nuclear proteins were used to test
whether anti-AS1 antibody can detect AS1 protein. Anti-HA antibody was also used to detect
the AS1-HA protein. Although we could not detect endogenous AS1 protein, we could detect
HA-tagged AS1 protein using our anti-AS1 antibody. Anti-histone H3 antibody (Abcam) was
used to control protein loading. (b) 10 μg of nuclear proteins derived from the as1 mutant and
three epitope-tagged AS1 expressing lines were used to test whether anti-AS1 antibody can
detect other epitope-tagged AS1 proteins. (c, d) 100 μg of whole protein extracts were used
for detecting CO protein. Anti-CO antibody (c) and anti-GFP (d) antibodies were used to
check whether anti-CO antibody specifically recognizes CO-GFP protein in Arabidopsis.
Coomassie staining of membranes used for western blots indicated that similar amounts of
proteins were loaded. For 35S:CO-GFP, the pMDC83-CO (Figure 1f) construct was
introduced into Arabidopsis wide-type. Although we could not detect endogenous levels of
CO, our anti-CO antibody detected CO-GFP proteins.
Table S1. Primer sequences used in this study.
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