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First published online 19 July 2006
doi: 10.1242/dev.02498

Development 133, 3159-3166 (2006)
Published by The Company of Biologists 2006
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APETALA1 and SEPALLATA3 interact with SEUSS to mediate transcription repression during flower development

Vaniyambadi V. Sridhar*, Anandkumar Surendrarao{dagger} and Zhongchi Liu{ddagger}

Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD 20742, USA.


Figure 1
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  		Fig. 1. Interactions between SEU and AP1/SEP3 in yeast and in vitro. (A) A yeast two-hybrid interaction assay showing positive interactions between the SEU-BD bait and the AP1-AD or SEP3-AD prey. A truncated SEU without its Q2 and C-terminal domains was used in constructing SEU-BD, which no longer self-activates in yeast (Sridhar et al., 2004Go). Full-length AP1, SEP3, AP3 and PI were fused to GAL4-AD. Activation of HIS3 and lacZ is indicated by growth on -HIS media and by the blue color, respectively. Red colonies indicate a lack of ADE2 reporter activation (James et al., 1996Go). The relative level of lacZ (ß-galactosidase) activity is shown to the right. (B) A similar yeast two-hybrid interaction assay showing positive interactions between SEU-BD and the C-terminal domain of AP1 and SEP3 (AP1-C and SEP3-C). (C) An in vitro pull-down assay showing 35S-labeled AP1 and SEP3 proteins retained by GST-SEU (right). Equal amounts of in vitro translated products were loaded onto the NuPAGE gel (INPUT lanes). GST alone failed to retain any of the 35S proteins (data not shown). (D) A three-protein pull-down assay with SEU-GST serving as a bridging protein. The ability of MBP-LUFS/amylose beads to retain 35S-labeled AP1, SEP3 or AP3 was tested in the presence (+) or absence (-) of SEUGST. MBP was used as a negative control.

 

Figure 2
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  		Fig. 2. Synergistic genetic interactions between seu and ap1. (A) A seu-1 flower. (B) An inflorescence of seu-1. (C) An ap1-3 flower. (D) Comparing the height of ap1-3 plants heterozygous or homozygous for seu-1. (E) An ap1-3 seu-1 double mutant flower. Note the complete absence of petals and the carpelloid whorl 1 organs with horn-like projections (arrows). Secondary flowers are absent. (F) An ap1-3 flower heterozygous for seu-1. Note the complete loss of petals and the formation of carpelloid whorl 1 organs (arrows) in the primary and the secondary flowers. (G) An ap1-1 flower. (H) An inflorescence of ap1-1. (I) An ap1-1 seu-1 double mutant flower that produced secondary and higher order flowers. Many of the flowers exhibit carpelloid sepals (arrows). (J) The inflorescence of an ap1-1 seu-1 double mutant plant. The inflorescence resembles cauliflowers, with many more higher order floral meristems.

 

Figure 3
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  		Fig. 3. AP1 and SEP3 mediate the repressor activity of SEU/LUG. (A) AP1-BD and SEP3-BD, with GAL4 BD at the N terminus of the fusion proteins, mediate the repressor activity of SEU/LUG in yeast, as indicated by the ß-galactosidase activity from the GAL7-lacZ reporter. AP1-BD (lane 3) and SEP3-BD (lane 9), but not BD (pGBT9 vector; lanes 1-2), activate lacZ expression. This activity is reduced in the presence of SEU (lanes 4, 10). Full-length LUG (lanes 5, 6, 11, 12) and LUG without its LUFS domain (LUGdeltaLUFS; lanes 7, 8, 13, 14) were tested in the presence or absence of SEU. Error bars show the standard deviation of means of triplicate assays. (B) Diagram of the pAG3'I::LUC reporter. An ~900 bp fragment from the 3' AG enhancer is inserted upstream of the TATA box of the 35S promoter that drives firefly luciferase (LUC). The two LFY/WUS-binding sites (black circles) and the two CArG boxes (diamonds) are indicated. (C) AP1 and SEP3 mediate the repression of the pAG3'I::LUC reporter in onion cells. The ratio of pAG3'I::LUC and 35S::Renilla LUC expression is shown. Onion epidermal cells were transiently transformed with 35S::AP1 or 35S::SEP3, together with 35S::SEU or 35S::LUG, or 35S::SEU plus 35S::LUG. The pART7 vector was a negative control. Error bars show standard deviation of means of triplicate assays.

 

Figure 4
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  		Fig. 4. In vivo association of SEU protein with AG 3' enhancer. (A) Diagram of the AG second intron, which coincides with an ~3 kb HindIII fragment (Bao et al., 2004Go; Busch et al., 1999Go; Deyholos and Sieburth, 2000Go). Numbers indicate the nucleotide sequence, with the 5' HindIII site designated as 1. The location of LFY/WUS-binding sites (back circles), CArG boxes (diamonds), and BLR-binding sites (triangles) is indicated. The two `redundant' enhancers defined by KB14 (5' enhancer) and KB18 (3' enhancer) reporter lines (Busch et al., 1999Go), as well as the position of the AG-5 and AG-3 PCR products, are shown. The ~900 bp AG fragment in the pAG3'I::LUC reporter is indicated. Drawing is not to scale. (B) Association of SEU with the AG 3' enhancer revealed by ChIP with an anti-SEU antibody. AG-5 and AG-3 are PCR products detecting immunoprecipitated wild-type and seu-3 chromatin, respectively. The control E1F4P primer amplifies a non-regulatory target of LUG/SEU. `No Ab' and `{alpha}SEU Ab' correspond to chromatin treated without or with anti-SEU antibodies, respectively.

 

Figure 5
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  		Fig. 5. A proposed model of how the inner whorl-specific activation of AG is achieved. In whorls 1 and 2, multiple negative regulatory activities impinge upon the AG cis-regulatory region (such as the AG intron II) to prevent AG transcription. These negative regulatory factors include AP2, BLR and SEU/LUG/AP1 or SEU/LUG/SEP3. In whorls 3 and 4, multiple positive regulatory factors antagonize the negative effect of SEU/LUG/SEP3 to promote AG transcription. These positive regulatory factors include the combined activities of LFY and WUS, as well as positive autoregulation by AG/SEP3. Additionally, the AG/SEP3 complex inhibits AP1 transcription and an interaction between AG and SEP3 may preclude the SEU/LUG co-repressors from interacting with SEP3. Arrows leading from AP1 or SEP3 to respective LUG/SEU/AP1, LUG/SEU/SEP3 or AG/SEP3 complexes indicate the incorporation of these MADS box proteins into the respective protein complexes.

 

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© The Company of Biologists Ltd 2006