Exogenous Cytokinin Treatment Enhances the FM Determinacy Defect of ag-10.
(A) and (B) Longitudinal sections of Ler siliques after DMSO (A) and 6-BA (B) treatment. No obvious FM determinacy defect was detected in 6-BA-treated siliques.
(C) and (D) Longitudinal sections of ag-10 siliques after DMSO (C) and 6-BA (D) treatment. Severe FM indeterminacy was observed in 6-BA-treated ag-10 siliques, as characterized by additional tissues in primary carpels (marked by a white arrow).
(E) and (F) Longitudinal sections of ag-10 arf3-29 siliques after DMSO (E) and 6-BA (F) treatment. Slightly enhanced FM indeterminacy was detected in the siliques of 6-BA-treated plants compared with DMSO-treated plants. Prolonged FMs and additional tissues are marked by yellow and white arrows, respectively.
Bars = 50 µm.

ARF3 Represses Cytokinin Activity and Levels.
(A) to (D) Expression of ProTCSn:GFP (green) in FMs. Images show transverse sections of early flower buds of Ler (A), ag-10 (B), arf3-29 (C), and ag-10 arf3-29 (D). Yellow arrows indicate GFP in the OC regions of FMs. Bars = 50 µm.
(E) and (F) Transcript levels of GFP (E) and genes involved in cytokinin biosynthesis, signaling, and metabolism (F) in the indicated plants. RNA was extracted from inflorescences containing stage 6 and younger flowers for RT-qPCR. Error bars indicate the sd from three biological replicates with independently prepared inflorescence materials. *P < 0.05 and **P < 0.01 (Student’s t test).
(G) and (H) Mass spectrometric measurements of tZRMP (G) and tZR and tZ (H) in inflorescences of the indicated plants. Mean values of three replicates with independently prepared inflorescence materials containing unopened flowers are shown. Error bars indicate the sd of three biological experiments. **P < 0.01 (Student’s t test) between the inflorescences of wild type and the indicated mutants.

ARF3 Directly Inhibits the Expression of IPT3, IPT5, and IPT7 to Regulate FM Determinacy.
(A) to (E) Representative silique phenotypes of ag-10 (A), ag-10 arf3-29 (B), ag-10 arf3-29 ipt3 ipt7 (C), ag-10 arf3-29 ipt3 ipt5 (D), and ag-10 arf3-29 ipt5 ipt7 (E). In (B) to (E), the leftmost silique has one carpel removed from the primary gynoecium to show the structures inside. ipt3 ipt7, ipt3 ipt5, and ipt5 ipt7 partially rescued the FM determinacy defect of ag-10 arf3-29, as characterized by a fertile and elongated ovary with fewer excess tissues inside.
(F) to (I) Longitudinal sections of representative siliques of ag-10 (F), ProARF3:IPT3-GFP ag-10 (G), ProARF3:IPT5-GFP ag-10 (H), and ProARF3:IPT7-GFP ag-10 (I). Increased IPT gene expression in ARF3 expression regions resulted in FM determinacy defects, such as extra tissues (marked by red arrows) growing within the primary ovaries of transgenic plants.
(J) Transcript levels of the indicated genes in ProARF3:ARF3-GR arf3-29 inflorescences after 8 h treatment with CHX or CHX+DEX. Inflorescences containing stage 6 and younger flowers were collected for RT-qPCR. Error bars indicate the sd from three biological replicates with independently prepared inflorescence materials. *P < 0.05 and **P < 0.01 (Student’s t test).
(K) Diagrams of the IPT3 and IPT7 genomic regions with an arrow indicating the transcription start site (+1). Black, coding sequences; dark gray, untranslated regions. Black bold lines with P1 and P2 indicate fragments examined by ChIP-qPCR. “TGTCTC” is the typical ARF binding motif. Bar = 500 bp.
(L) ChIP assay with anti-GFP antibody to examine ARF3 binding at IPT3 and IPT7 in Ler and ProARF3:ARF3-GFP inflorescences. The regions from IPT3 and IPT7 marked in were examined. IPT5, an ARF3 target gene, served as a positive control, and eIF4A (EUKARYOTIC TRANSLATION INITIATION FACTOR4A) served as a negative control. Error bars represent the sd from three biological repeats. **P < 0.01 (Student’s t test) between Ler and ProARF3:ARF3-GFP inflorescences.
Bars = 1 mm in (A) to (E) and 50 µm in (F) to (I).

ARF3 Represses LOG Gene Expression.
(A) and (B) Mass spectrometric measurements of tZRMP (A) and tZR and tZ (B) in inflorescences of Ler and ProARF3:ARF3-GR arf3-29 under DMSO and DEX treatment. Mean values of three replicates with independently prepared inflorescence materials containing unopened flowers are shown. Error bars indicate the sd of three biological experiments. **P < 0.01 (Student’s t test).
(C) Expression of LOG family genes in the inflorescences of the indicated plants. Inflorescences containing stage 6 and younger flowers were collected for RT-qPCR. Error bars indicate the sd from three biological replicates with independently prepared inflorescence materials. **P < 0.01 (Student’s t test) between Ler and mutant inflorescences.
(D) and (E) Expression of LOG2, LOG3, LOG4, and LOG7 in ProARF3:ARF3-GR arf3-29 inflorescences after 8 h treatment with the indicated chemical. Inflorescences containing stage 6 and younger flowers were collected for RT-qPCR. The DEX treatment data indicate that ARF3 represses the expression of some LOG genes, and the CHX+DEX treatment data indicate that the repression by ARF3 is indirect. Error bars indicate the sd from three biological replicates with independently prepared inflorescence materials. *P < 0.05 and **P < 0.01 (Student’s t test).

ARF3 Directly Represses AHK4 to Regulate FM Determinacy.
(A) and (B) Expression of ProAHK4:GUS in Ler (A) and arf3-29 (B) inflorescences under the same staining conditions. The stronger GUS staining in arf3-29 compared with Ler indicates that AHK4 expression was derepressed in arf3-29.
(C) Quantitative GUS activity in inflorescences of the indicated plants. Mean values of three biological replicates with independently prepared inflorescence materials containing unopened flowers are shown. **P < 0.01 (Student’s t test).
(D) to (F) Representative silique phenotypes of ag-10 (D), ag-10 arf3-29 (E), and ag-10 arf3-29 ahk4 (F). In (E) and (F), the leftmost silique has one carpel of the primary gynoecium removed to show the structures contained therein. The ahk4 mutation partially rescued the severe FM determinacy defect of ag-10 arf3-29.
(G) to (I) Longitudinal sections of representative siliques of the following: ag-10 (G) and ProARF3:AHK4-3FLAG ag-10 with severe (H) and intermediate (I) FM indeterminacy. Increased AHK4 gene expression in ARF3 expression regions resulted in severe FM determinacy defects in 10 out of 32 plants, as characterized by a sustained FM (red arrow in [H]), and intermediate FM indeterminacy in 12 out of 32 plants, as indicated by excess tissues growing within the primary ovary (yellow arrow in [I]).
(J) Diagram of the AHK4 genomic region, with the arrow indicating the transcription start site (+1). Dark-gray rectangles, untranslated regions; black rectangles, exons; white rectangles, introns. Black bold lines with P1, P2, and P3 indicate fragments examined by ChIP-qPCR. “TGTCTC” is the typical ARF binding motif. Bar = 500 bp.
(K) ChIP assay with anti-GFP antibody to examine ARF3 binding at AHK4 in Ler and ProARF3:ARF3-GFP arf3-29 inflorescences. The regions marked in were examined. ARF3 could bind the P1 and P2 regions containing the “TGTCTC” motif but not the P3 region. eIF4A served as a negative control. Error bars represent the sd from three biological repeats with independently prepared inflorescence materials containing unopened flowers. **P < 0.01 (Student’s t test) between Ler and ProARF3:ARF3-GFP arf3-29 inflorescences.
(L) EMSA confirmation of ARF3-GST binding to the AHK4 P2 region. Upper panel: native and mutated AHK4P2 sequence used for the EMSA. The mutated nucleotides are shown in red. The arrowhead indicates band shifts (complexes of ARF3-GST protein and probe DNA), and the arrow indicates free probe. Nonlabeled oligonucleotides were used as a competitor.
Bars = 100 µm in (A) and (B), 1 mm in (D) to (F), and 50 µm in (G) to (I).

Expression Pattern of WUS in the Indicated Flowers.
In situ hybridization with a WUS antisense probe. The arrows indicate WUS signal. WUS expression was not detected in a stage 8 ag-10 flower (A), but it was detected in stage 8 or 9 flowers of ProARF3:IPT3-GFP ag-10 (B), ProARF3:IPT5-GFP ag-10 (C), ProARF3:IPT7-GFP ag-10 (D), and ProARF3:AHK4-3FLAG ag-10 (E). Red numbers indicate the floral developmental stage. Bars = 50 µm.

AG Regulates Cytokinin Activity.
(A) to (D) Expression of ProTCSn:GFP (green) in the inflorescences of Pro35S:AG-GR ag-1 under DMSO (A) and DEX (B) treatment and Pro35S:AG-GR ag-1 arf3-29 under DMSO (C) and DEX (D) treatment for 4 d.
(E) GFP transcript levels in inflorescences of the indicated plants. Inflorescences containing stage 6 and younger flowers were collected for RT-qPCR.
(F) to (H) Representative silique phenotypes of ag-11 (F), ag-11 ipt3 ipt5 ipt7 (G), and ag-11 ahk4 (H). The leftmost silique has one carpel of the primary gynoecium removed to show the structures contained therein. ag-11 produces short and bulged siliques with additional tissues growing inside. ipt3 ipt5 ipt7 and ahk4 largely rescued the severe FM determinacy defect of ag-11.
(I) and (J) Longitudinal sections of representative ag-1 (I) and ag-1 ipt3 ipt5 ipt7 (J) flowers at similar developmental stages. The FM size (marked by red lines) of ag-1 ipt3 ipt5 ipt7 is smaller than that of ag-1.
(K) Quantification of FM size (µm) in ag-1 (n = 15) and ag-1 ipt3 ipt5 ipt7 (n = 12). **P < 0.01 (Student’s t test).
(L) Time-course expression analysis of the indicated genes in Ler and Pro35S:AG-GR ag-1 inflorescences under DEX treatment. Ler inflorescences containing stage 6 and younger flowers and Pro35S:AG-GR ag-1 inflorescences containing unopened flowers were collected for RT-qPCR.
(M) IPT5 expression in inflorescences of the indicated plants. RNA was extracted from Ler and arf3-29 inflorescences containing stage 6 and younger flowers for RT-qPCR. Pro35S:AG-GR ag-1 and Pro35S:AG-GR ag-1 arf3-29 were treated with DEX or DMSO for 4 d, and inflorescences containing unopened flowers were collected for RT-qPCR.
Error bars in (E), (L), and (M) indicate the sd from three biological replicates with independently prepared inflorescence materials. *P < 0.05 and **P < 0.01 (Student’s t test). Bars = 50 µm in (A) to (D), (I), and (J) and 1 mm in (F) to (H).

ARF3 Mediates Auxin-Mediated Repression of Cytokinin.
(A) to (D) Longitudinal sections of representative flowers of Ler (A) and ag-11 after 5 d of DMSO (B), 6-BA (C), and IAA (D) treatment. White arrows indicate prolonged FMs. Bars = 50 µm.
(E) to (H) Expression of ProTCSn:GFP (green) in Ler and arf3-29 inflorescences treated with DMSO ([E] and [G]) and IAA ([F] and [H]).
(I) GFP transcript levels in inflorescences of the indicated plants. RNA was extracted from inflorescences containing stage 6 and younger flowers for RT-qPCR. Error bars indicate the sd from three biological replicates with independently prepared inflorescence materials. **P < 0.01 (Student’s t test).
(J) Time-course expression analysis of IPT3, IPT5, and IPT7 in IAA-treated Ler and arf3-29 inflorescences. Inflorescences containing stage 6 and younger flowers were collected for RNA extraction and RT-qPCR. Error bars indicate the sd from three biological replicates with independently prepared inflorescence materials. *P < 0.05 and **P < 0.01 (Student’s t test).

Repressive Role of ARF3 in Multiple Developmental Processes.
(A) Overrepresented GO categories in the downregulated genes in ProARF3:ARF3-GR arf3-29 inflorescence under the 12 h DEX treatment. The GO terms related to polarity specification, cell proliferation, flower development, and epigenetic regulation were enriched in ARF3 repressed genes (Supplemental Data Set 1).
(B) Expression of representative genes related to polar auxin transport and pattern specification as well as cell cycle progression after induction by ARF3. Red indicates increased expression, and green indicates reduced expression compared with the mean of all samples. The values from three biological replicates (1, 2, and 3) are listed for DMSO and DEX treatment, respectively.

ARF3 Controls Cell Cycle Gene Expression and Cell Division in the FM.
(A) Expression of cell cycle genes in DEX-treated ProARF3:ARF3-GR arf3-29 inflorescences. ProARF3:ARF3-GR arf3-29 inflorescences were treated with DEX for the indicated amounts of time and collected under a microscope for RNA extraction and RT-qPCR. The transcript level at 0 h was used as a reference and was designated as 1.0. Error bars indicate the sd from three biological replicates with independently prepared inflorescence materials containing stage 6 and younger flowers. *P < 0.05 and **P < 0.01 (Student’s t test).
(B) Expression of cell cycle genes in inflorescences of the indicated plants. Inflorescences containing stage 6 and younger flowers were collected for RNA extraction and RT-qPCR. The transcript level in ag-10 was used as a reference and was designated as 1.0. Error bars indicate the sd from three biological replicates with independently prepared inflorescence materials. *P < 0.05 and **P < 0.01 (Student’s t test).
(C) to (J) Longitudinal sections of the inflorescence meristems of the indicated plants. Inflorescences containing stage 6 and younger flowers were stained with FM4-64 (red), and flowers at later stage 3 were selected for observation under a confocal microscope. The L1 layer cells are outlined in each panel, with cells undergoing division marked by a white arrowhead in (D) to (G) and (J). ProARF3:ARF3-GR arf3-29 inflorescences were treated with DMSO (G) and DEX (H) for 10 d prior to observation. Bars = 20 µm.
(K) Number of cells in the FM L1 layer. The numbers of FM L1 layer cells in the indicated plants were counted, and the mean values from multiple flowers (n = 12) are shown. *P < 0.05 and **P < 0.01 (Student’s t test).

A Model of How ARF3 Integrates the Repression of Cytokinin Biosynthesis by Auxin and AG to Regulate FM Determinacy.
(A) At stage 3-4 of floral development, AG indirectly represses ARF3 expression through GIK () and other factors and consequently enhances IPT and cell cycle gene expression to promote cell division and to maintain the meristem cell population, thereby favoring FM maintenance.
(B) At stage 5-6 of floral development, auxin and AG promote ARF3 expression, which in turn directly represses IPT3, IPT5, IPT7, and AHK4 and indirectly represses LOG gene expression. The resulting inhibition of cytokinin activity is required for the proper temporal termination of WUS expression during FM determinacy.