Supporting Information Figure Legends
Figure S1. Molecular characterization of arf3-29 and the phenotype of ett-3.
(a) A diagram of the ARF3 gene showing the affected sites in the arf3-29 and ett-3 mutants. ATG
and TAG correspond to the start and stop codons, respectively. The gray, black and white
rectangles represent the 5’ or 3’ untranslated regions, coding regions, and introns or intergenic
regions, respectively. arf3-29 harbors a G-to-A point mutation at the junction of the eighth exon
and eighth intron of ARF3. ett-3 is a nonsense mutation in the eighth exon (Sessions et al., 1997).
The primers used for the characterization of arf3-29 are marked as F and R. The sizes of the 8th
intron, 9th exon and 9th intron are labeled.
(b) Characterization of the arf3-29 mutant by RT-PCR. Inflorescences of Ler and arf3-29 were
collected and RNA was extracted followed by RT-PCR with the primers marked above. Genomic
DNA from Ler served as a control. The sizes of PCR products are as indicated to the right of the
gel image.
(c) A side view of an ett-3 flower. The extended gynophore is delimited by arrows and maybe
indicative of a weak FM determinacy defect. Bar: 0.5mm.
Figure S2. WUS, AG and ARF3 expression in various genotypes.
(a,b) In situ hybridization for WUS expression in stage 6 Ler (a) and ag-10 (b) flowers. No WUS
signals were detected. Bars: 50μm.
(c) AG expression in the indicated genotypes as examined by RT-qPCR. Inflorescences containing
unopened flowers of the indicated genotypes were dissected and RNA was extracted followed by
RT-qPCR. Error bars represent SD calculated from three biological replicates. The Student’s t test
was performed to determine the significance of difference when one genotype was compared with
its corresponding control, for example, arf3-29 was compared to Ler; ag-10 arf3-29 was
compared to ag-10. No statistically significant changes were found.
(d) ARF3 expression in the indicated genotypes as examined by RT-qPCR. Inflorescences
containing stage 8 and early flowers of Ler and ag-10 were dissected and RNA was extracted
followed by RT-qPCR. Error bars represent SD calculated from three biological replicates.
Statistically significant changes are indicated by ★ (p-value < 0.05).
(e) ARF3 expression in 35S::AG-GR ag-1 after DEX treatment for 0 day (0d), 2 days (2d) and 4
days (4d). UBQ5 served as an internal reference gene. ARF3 expression is shown as the ratio of
DEX-treated vs. DMSO-treated (DEX/DMSO) samples. Inflorescences containing stage 8 and
early flowers of 35S::AG-GR ag-1 were dissected after chemical treatment and RNA was
extracted and subjected to RT-qPCR. Error bars represent SD calculated from three biological
replicates. Statistically significant changes are indicated by ★★ (p-value < 0.01).
Figure S3. ARF3 protein distribution in 35S::AP2m3 floral meristems
(a-c) ARF3-GFP signal in a stage 4 flower of ARF3::ARF3-GFP (a) and stages 4 (b) and 8 (c)
flowers of ARF3::ARF3-GFP 35S::AP2m3. GFP signals are weaker in the sepals (white arrows)
and the meristem rib zone (red arrows) in ARF3::ARF3-GFP 35S::AP2m3 than in
ARF3::ARF3-GFP (compare (b) with (a) and (c) with Figure 4i). Bars: 50μm
Figure S4. ARF3 transcript and protein distribution in FMs and floral organs.
(a) In situ hybridization with an ARF3 sense probe. No specific signal was detected.
(b) In situ hybridization with an ARF3 antisense probe. Abaxial distribution of ARF3 transcripts
in the gynoecium of a stage 8 Ler flower was detected.
(c) Microscopic
three-dimensional
image
reconstruction
of
early-stage
flowers
of
ARF3::ARF3-GFP. Flowers beyond stage 6 were removed.
(d) ARF3-GFP signal in the stage 7 flower. GFP signal was evenly distributed throughout the
newly generated gynoecium.
(e) ARF3-GFP distribution in floral organs. An inflorescence from an ARF3::ARF3-GFP
pWUS::DsRed-N7 plant was observed under a confocal microscope in the GFP channel. The GFP
signal (green color) corresponds to the distribution of ARF3 protein.
(f) WUS reporter expression (red) in inflorescence tissue. The same inflorescence in (c) was
scanned in the red channel for DsRed. The red and white arrows indicate DsRed signal and
chlorophyll autofluorescence, respectively.
Bars: 50μm in (a-f).
Figure S5. ARF3 expression in various transgenic plants.
(a-c) In situ hybridization for ARF3 expression in ARF3::ARF3-GFP (a), ARF3::ARF3m-GFP (b)
and ARF3::ARF3m-PHB-GFP (c) flowers. The arrow in (c) indicates the presence of ARF3 RNA
in the adaxial domain of floral organ primordia..
Bars: 50μm in (a-c).