APETALA2 negatively regulates multiple floral organ identity genes in Arabidopsis by recruiting the co-repressor TOPLESS and the histone deacetylase HDA19

Abstract

The development and coordination of complex tissues in eukaryotes requires precise spatial control of fate-specifying genes. Although investigations of such control have traditionally focused on mechanisms of transcriptional activation, transcriptional repression has emerged as being equally important in the establishment of gene expression territories. In the angiosperm flower, specification of lateral organ fate relies on the spatial regulation of the ABC floral organ identity genes. Our understanding of how the boundaries of these expression domains are controlled is not complete. Here, we report that the A-class organ identity gene APETALA2 (AP2), which is known to repress the C-class gene AGAMOUS, also regulates the expression borders of the B-class genes APETALA3 and PISTILLATA, and the E-class gene SEPALLATA3. We show that AP2 represses its target genes by physically recruiting the co-repressor TOPLESS and the histone deacetylase HDA19. These results demonstrate that AP2 plays a broad role in flower development by controlling the expression domains of numerous floral organ identity genes.

Fig. 1.

Physical association of AP2, TPL and HDA19. (A) Yeast two-hybrid (Y2H) assays. Protein domains of TPL and AP2 are depicted. TPL and AP2 interact by Y2H (bottom right) as indicated by blue coloration. Deletion of the CTLH domain or introduction of the N176H missense mutation into the TOP domain of TPL disrupts interaction with AP2. Mutation of the conserved leucines within the EAR motif of AP2 prevents binding to TPL. (B) In a semi-in vivo pull-down assay, GST N-TPL binds AP2-GFP from floral bud lysates but not AP2 mEAR-GFP. GST N-TPL N176H does not bind AP2-GFP. Both GST N-TPL and GST N-TPL N176H interact with HDA19-HA. Ponceau Red and Coomassie Blue staining shows equal protein loading and efficient GST protein expression, respectively. (C) Bimolecular fluorescence complementation (BiFC) assays. Nuclear fluorescence (arrowheads) shows physical association between HDA19 and both TPL and TPL N176H in transiently transfected tobacco cells. Homodimerization of TPL serves as a positive control; the right column depicts negative control combinations.

Fig. 2.

AP2, TPL and HDA19 cooperatively repress the C-class gene AG in outer whorl floral organs. (A) Wild-type Arabidopsis flower composed of four sepals (se), four petals (p), six stamens (st) and two fused carpels (c). (B-D) Mutant flowers exhibiting carpel-like whorl 1 organs (arrowheads) with stigmatic tissue at their margins. Arrow in B designates stamen-like identity. (E-H) AG in situ hybridizations on stage 7-8 flowers. (E) Wild-type flower with expression restricted to stamen and carpel primordia. (F-H) Mutants expressing AG throughout the flower (arrowheads). (I) Anti-HA ChIP showing specific binding of AP2, TPL and HDA19 to the second AG intron. Control ChIPs were performed on non-transgenic wild-type tissue (Ler) or without antibody (no Ab). Data were normalized relative to input and ACT2 abundance. Data are represented as mean ± s.e.m. of at least two biological replicates. Student's t-test was used to determine the significance of target enrichment relative to Ler IP (*P≤0.05, **P≤0.005) and the significance of decreased binding in ap2-2 (a, P≤0.08; b, P≤0.05). (J) Transgenes used in K,L. Domains are described in Fig. 1A. The TPL promoter is depicted as a black oval. (K) TPLp::TPL-AP2DBs rescues ap2-2 floral defects. Compare with A,B. (L) TPLp::AP2DBs is insufficient to rescue ap2-2. Scale bars: 1 mm in A-D; 50 μm in E-H.

Fig. 3.

AP2, TPL and HDA19 repress the B-class gene AP3 in whorl 1 organs. (A-D) Mutant Arabidopsis flowers with sepal-to-petal homeotic conversions (arrowheads). Compare with wild type in Fig. 2A. (E-H) Scanning electron micrographs of abaxial surfaces of floral organs. (E) Elongated epidermal cells of wild-type sepals are variable in size. (F) Slightly conical cells of wild-type petals are relatively uniform in size. (G) tpl-1 and (H) hda19-1 mosaic whorl 1 organs with a sharp boundary between sepal and petal identity. (I-N) AP3 in situ hybridizations on stage 4 (I,J) and stage 7-8 (K-N) flowers. (I) AP3 is not expressed in young wild-type sepal primordia. (J) Ectopic AP3 expression in a young tpl-1 whorl 1 organ (arrowhead). (K) AP3 is detectable in later-stage wild-type sepals, but only at the organ base (arrows). (L-N) AP3 expression domains are expanded in mutant whorl 1 organs (arrowheads). (O) Anti-HA ChIP showing AP2, TPL and HDA19 specifically bind the AP3 promoter. Control ChIPs were performed on non-transgenic wild-type tissue (Ler) or without antibody (no Ab). Data were normalized relative to input and ACT2 abundance. Data are represented as mean ± s.e.m. of at least two biological replicates. Student's t-test was used to determine the significance of target enrichment relative to Ler IP (*P≤0.05, **P≤0.005) and the significance of decreased binding in ap2-2 (b, P≤0.05). Scale bars: 1 mm in A-D; 100 μm in E-H; 50 μm in I-N.

Fig. 4.

The B-class gene PI is misexpressed in the outer whorl of tpl-1 and hda19-1 mutant backgrounds. (A-F) PI in situ hybridizations. (A,B) Stage 4 and (C-F) stage 7-8 Arabidopsis flowers. (A,C) PI is not expressed in whorl 1 organs in either stage of floral development. (B,D-F) PI is ectopically expressed in mutant whorl 1 organs (arrowheads). Scale bars: 50 μm.

Fig. 5.

The E-class gene SEP3 is repressed in whorl 1 organ primordia by AP2, TPL and HDA19. (A-F) SEP3 in situ hybridizations on stage 3 (A,C,E,F) and stage 4 (B,D) Arabidopsis flowers. (A) SEP3 is not expressed in newly initiated outer whorl organ primordia in wild type. (B) By stage 4, SEP3 expression is detectable in wild-type sepals, but only at the distal tips of the organs (arrows). (C,E,F) SEP3 is ectopically expressed in mutant outer whorl organ primordia (arrowheads). (D) Sepal of a stage 4 tpl-1 flower showing an expanded SEP3 expression domain (arrowhead). (G) sep3-2 suppresses whorl 1 homeotic conversions of tpl-1 at 21°C and 29°C. (H-J) 2x35Sp::SEP3 flowers with sepals exhibiting petal identity (arrowheads) on one margin (H), both margins (I) or throughout the entire organ (J). (K) Scanning electron micrograph of a mosaic 2x35Sp::SEP3 sepal with ectopic petal epidermal identity (upper left; compare with Fig. 3E,F). (L) Anti-HA ChIP showing binding of AP2, TPL and HDA19 to two regions of the SEP3 promoter. Control ChIPs were performed on non-transgenic wild-type tissue (Ler) or without antibody (no Ab). Data were normalized relative to input and ACT2 abundance. Data are represented as mean ± s.e.m. of at least two biological replicates. Student's t-test was used to determine the significance of target enrichment relative to Ler IP (*P≤0.05, **P≤0.005) and the significance of decreased binding in ap2-2 (a, P≤0.08; b, P≤0.05). Scale bars: 20 μm in A-F; 500 μm in H-J; 50 μm in K.

Fig. 6.

The effects of hda19-1 and ap2-2 mutation on histone acetylation levels. (A) Histone acetylation modifications H4K16Ac, H4K5Ac and H3K9Ac are increased in hda19-1 Arabidopsis flowers compared with wild type (left). H4K16Ac levels in ap2-2 flowers are comparable to that of wild type (right). Band intensities were normalized relative to total histone H3 loading controls. (B) Anti-H4K16Ac ChIP showing enrichment at the second intron of AG and at two promoter regions of SEP3 in ap2-2 flowers. A slight enrichment is seen at the promoter of AP3. Data are represented as mean ± s.e.m. of three biological replicates and are presented as the ratio of enrichment in ap2-2 over that of wild type. Data were normalized relative to INPUT and ACT2 abundance. The constitutively expressed PP2A gene serves as a negative control. Student's t-test was used to determine the significance of target enrichment relative to PP2A (*P≤0.08; **P≤0.05).

Fig. 7.

Model of AP2 function during flower development. AP2 controls the outer expression boundaries of B-, C- and E-class genes by recruiting TPL and HDA19. This AP2 repression complex directly represses the C-class gene AG in whorls 1 (sepal) and 2 (petal) and the B-class gene AP3 and E-class gene SEP3 in whorl 1. AP2-mediated repression of the B-class gene PI in whorl 1 may occur through negative regulation of upstream activators, such as SEP3. MADS-domain products of the floral homeotic genes (colored circles) are predicted to interact with one another and with SEP proteins to form quartet complexes that regulate gene expression required for organ identity specification (Honma and Goto, 2001). Intermediary proteins might facilitate TPL and HDA19 association.