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. 2016 Jul 28;113(33):9375–9380. doi: 10.1073/pnas.1602960113

Fig. S2.

Fig. S2.

Molecular and genetic analysis of WUS and AG and FHY3 in FM determinacy. (A–D) In situ hybridization examining the WUS expression pattern (marked by a red arrow) in Ler (A), ag-10 (B), fhy3-68 (C), and fhy3-68 ag-10 (D). Floral development stage was marked. (Scale bars, 100 µm.) (E and F) Flower phenotypes of wus-1 (E) and fhy3-68 ag-10 wus-1 (F). (Scale bars, 500 µm.) (G) The WUS transcript abundance in FHY3:FHY3-GR fhy3-4 inflorescences measured by real-time PCR. Inflorescences containing stage 8 and younger flowers were treated with DEX or DMSO and then harvested at the indicated time point. UBQ5 served as the internal control. Three biological replicates were performed. (H) ChIP with anti-FLAG antibody to examine FHY3 binding at WUS in 35S:3FLAG-FHY3-3HA fhy3-4 inflorescences. The regions examined are diagrammed in the upper row, with “+1” indicating the TSS. The gray, black, and white rectangles represent the 5′ or 3′ UTR, coding regions, and introns or intergenic regions, respectively. (Scale bar, 500 bp). ELF4, a FHY3 target gene, served as a positive control, and eIF4A1 (EU.K.ARYOTIC TRANSLATION INITIATION FACTOR 4A1) served as a negative control. Error bars represent SD from three biological repeats. No significant FHY3 occupancy was detected at WUS. **P < 0.01. (I and J) Real-time RT-PCR to measure AG (I) and FHY3 (J) transcript levels in the indicated plants. UBQ5 served as the internal control. Three biological replicates were performed. Error bars represent SD from three biological repeats. (K) Real-time RT-PCR to measure AG transcript levels in FHY3:FHY3-GR fhy3-4 inflorescences. Inflorescences containing stage 8 and younger flowers were treated with DEX or DMSO (control) then harvested at the indicated time point. UBQ5 served as the internal control. Three biological replicates were performed. Error bars represent SD from three biological repeats. No significant change in AG transcript levels in the DEX-treated sample compared with the DMSO control indicated that FHY3 does not directly regulate AG expression.