AUXIN RESPONSE FACTOR 3 integrates the functions of AGAMOUS and APETALA2 in floral meristem determinacy

Abstract

In Arabidopsis, AUXIN RESPONSE FACTOR 3 (ARF3) belongs to the auxin response factor (ARF) family that regulates the expression of auxin-responsive genes. ARF3 is known to function in leaf polarity specification and gynoecium patterning. In this study, we discovered a previously unknown role for ARF3 in floral meristem (FM) determinacy through the isolation and characterization of a mutant of ARF3 that enhanced the FM determinacy defects of agamous (ag)-10, a weak ag allele. Central players in FM determinacy include WUSCHEL (WUS), a gene critical for FM maintenance, and AG and APETALA2 (AP2), which regulate FM determinacy by repression and promotion of WUS expression, respectively. We showed that ARF3 confers FM determinacy through repression of WUS expression, and associates with the WUS locus in part in an AG-dependent manner. We demonstrated that ARF3 is a direct target of AP2 and partially mediates AP2's function in FM determinacy. ARF3 exhibits dynamic and complex expression patterns in floral organ primordia; altering the patterns spatially compromised FM determinacy. This study uncovered a role for ARF3 in FM determinacy and revealed relationships among genes in the genetic network governing FM determinacy.

Keywords: AGAMOUS; APETALA2; AUXIN RESPONSE FACTOR 3; Arabidopsis; WUSCHEL; floral determinacy.

Figure 1. ARF3 is required for FM determinacy

(a) Wild-type (Ler) siliques. (b) ag-10 siliques. The one on the right is an example of a bulged silique with internal floral organs. (c) An ag-10 arf3-29 flower with additional floral organs growing inside of the unfused sepaloid carpels. (d) An ag-10 ett-3 flower with a similar phenotype as ag-10 arf3-29. (e) Siliques from two ARF3∷ARF3-GFP transgenic lines (middle and right pairs) and ag-10 arf3-29 siliques as the control (left pair). (f) An arf3-29 flower (top view). (g) An arf3-29 flower (side view). The arrows mark the ends of the gynophore. Bars: 1mm in (a–g).

Figure 2. Genetic interactions between arf3-29 and wus-1, ag-1 and knu-1

(a) WUS expression in a stage 6 arf3-29 flower as examined by in situ hybridization. No WUS signal was detected. (b) WUS expression in ag-10 arf3-29 flowers as examined by in situ hybridization. Arrows indicate WUS signal, and numbers indicate the floral developmental stage. (c) A wus-1 flower. (d) An ag-10 arf3-29 wus-1 flower. (e) An ag-1 flower. (f) An ag-1 arf3-29 flower. (g,h) Longitudinal sections through stage 9 ag-1 (g) and ag-1 arf3-29 (h) flowers. Arrows indicate the floral meristem. (i) FM diameter of ag-1 and ag-1 arf3-29 flowers. The values indicate means ± SD (n=10). The mean values for ag-1 and ag-1 arf3-29 were significantly different according to a Student’s t-test (p<0.01). (j) Siliques of ag-10 arf3-29 (left pair) and ag-10 arf3-29 knu-1 (right pair). Bars: 50µm in (a,b) and (g,h); 1mm in (c–f,j).

Figure 3. ARF3 is an AP2 target gene

(a) ChIP-qPCR showing that AP2 binds the ARF3 locus. The tested regions of ARF3 are diagramed in the upper panel. ATG and TAG represent 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. The black lines indicate the tested regions. Anti-GFP antibody was used for the analysis, and “no antibody” served as the negative control. Inflorescences containing all unopened flowers were dissected for ChIP assay. (b) Real-time RT-PCR analysis of ARF3, SOC1 and AGL15 in 35S∷GR and 35S∷AP2m3-GR inflorescences treated with dexamethasone (DEX) for 6 hours with or without cyclohexamide (CHX). Inflorescences containing stage 8 and early flowers were used. (c) AG and ARF3 expression in Ler, ap2-2 and 35S∷AP2m3 as determined by RT-qPCR Inflorescences containing stage 8 and early flowers were used. (d) A 35S∷AP2m3 flower. Bars: 0.5mm. (e) A 35S∷AP2m3 ag-10 arf3-29 flower. Bars: 0.5mm. Error bars in (a–c) represent SD calculated from three biological replicates. Statistically significant changes are indicated by ★ (p-value < 0.05) and ★★ (p-value < 0.01).

Figure 4. ARF3 RNA and protein distribution in flowers

(a–d) in situ hybridization with an ARF3 antisense probe. ARF3 expression patterns in the IM and stage 3 (a), stage 2 (b), stage 5 (c) and stage 6 (d) FMs are shown. Numbers indicate the developmental stage. (e,f) A global view of ARF3-GFP signal distribution in the FM and floral organ promordia during early floral development as observed with a confocal microscope (e: GFP and chlorophyll fluorescence merged channels; f: GFP channel alone). Numbers indicate the developmental stages of the FMs, and the arrow indicates GFP signal in a sepal primordium. Note that the GFP signal is present in the center of the IM but is masked by the strong chlorophyll fluorescence. (g–k) ARF3-GFP signal in the IM and stage 2 (g), stage 3 (h), stage 5 (i), stage 6 (j) and stage 8 (k) FMs and flowers. Numbers indicate the floral developmental stage. The arrow in (j) indicates GFP signal in a sepal, and the arrows in (k) indicate the abaxial distribution of ARF3-GFP signals. g: gynoecium; s: stamen. Note that in (g) to (i), the regions showing strong chlorophyll fluorescence (red) also had GFP signals, which were masked by the red fluorescence. (l) Coexpression of ARF3 and WUS in the FM. ARF3∷ARF3-GFP (green) and WUS∷DsRed-N7 (red) were detected in an ARF3∷ARF3-GFP WUS∷DsRed-N7 inflorescence using a confocal microscope, and the signals were merged. Red arrows indicate coexpression regions, and the white arrow indicates chlorophyll autofluorescence. Numbers indicate the floral developmental stage. Bars: 50µm in (a,b) and (e–j); 25µm in (c,d) and (k,l).

Figure 5. ARF3’s proper spatial expression is important for its function in FM determinacy

(a) An ag-10 arf3-29 flower. (b) A WUS∷ARF3m-GFP∷WUS3’ ag-10 arf3-29 flower. (c) Fluorescence detection of ARF3m-GFP in a WUS∷ARF3m-GFP∷WUS3’ ag-10 arf3-29 flower. White arrows indicate the GFP signals. (d,e) An ARF3∷ARF3-GFP ag-10 arf3-29 plant (d) and its siliques (e). (f,g) An ARF3∷ARF3m-GFP ag-10 arf3-29 plant (f) and its siliques (g). (h,i) An ARF3∷ARF3m-PHB-GFP ag-10 arf3-29 plant (h) and its siliques (i). Bars: 1mm in (a,b); 50µm in (c); 1cm in (d,f,h); 1mm in (e,g,i).

Figure 6. AG promotes the binding of ARF3 to the WUS locus in vivo

(a) A diagram of the WUS genomic region with “+1” corresponding to the transcription start site. The gray, black and white rectangles represent the 5’ or 3’ untranslated regions, coding regions and introns or intergenic regions, respectively. The black lines indicate the regions examined in ChIP. The sequences of the putative ARF3 binding sites are also shown. (b) ARF3 occupancy at WUS and atIPT5 as determined by ChIP-qPCR. Anti-GFP antibody was used for ChIP, and “no antibody” served as the negative control. Error bars represent SD calculated from four biological replicates. Statistically significant changes are indicated by ★★ (p-value < 0.01). (c) Yeast one-hybrid analysis revealing the direct binding of ARF3 to WUSp2. Yeast strains containing WUSP2∷LacZ or WUSp4∷LacZ reporters were transformed with AD-ARF3 or AD vectors, respectively. Two independent transformants growing on selective medium (SD/-Leu) were selected for β-galactosidase assay.

Figure 7. A model of ARF3 in FM determinacy

ARF3 terminates the floral stem cells by directly or indirectly repressing WUS expression. Besides the direct repression of WUS by AG through the recruitment of PcG to WUS and the indirect repression of WUS through the activation of KNU expression (Sun et al., 2009; Liu et al., 2011), AG indirectly enhances ARF3 expression and promotes ARF3 binding to the WUS locus. As a target gene repressed by AP2, ARF3 partially mediates the FM maintenance function of AP2. Red and green arrows indicate positive and negative effects, respectively. Solid and dotted arrows indicate direct and indirect effects, respectively. ARF3 is likely to have a direct effect on WUS expression, but this has not been definitively proven in vivo, thus a dotted arrow is used.