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First published online 20 February 2008
doi: 10.1242/dev.015842

Development 135, 1235-1245 (2008)
Published by The Company of Biologists 2008
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An Arabidopsis F-box protein acts as a transcriptional co-factor to regulate floral development

Eunyoung Chae1, Queenie K.-G. Tan1, Theresa A. Hill1,* and Vivian F. Irish1,2,{dagger}

1 Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520, USA.
2 Department of Ecology and Evolutionary Biology, Yale University, New Haven, CT 06520, USA.


Figure 1
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  		Fig. 1. The stoichiometry of LFY and UFO is important in flower development. (A) Inflorescences of 35S::UFO-Myc, 35S::UFO-Myc; lfy-26/+ and 35S::UFO-Myc; lfy-26 plants (from the left). Most flowers of 35S::UFO-Myc; lfy-26/+ produce siliques, whereas 35S::UFO-Myc flowers are completely infertile. Arrows indicate extra co-florescences on 35S::UFO-Myc; lfy-26 plants. Representative plants were siblings obtained from a segregating F4 population. (B) 35S::UFO-Myc flower with extra petals and stamens. Carpels are transformed to stamenoid organs. (C) A representative 35S::UFO-Myc;lfy-26/+ flower; most such flowers are phenotypically normal, except that they produce curved siliques. (D) A representative 35S::UFO-Myc; lfy-26 flower that is indistinguishable from lfy-26 mutant flowers. (E) Comparison of stamenoid fourth whorl organs from 35S::UFO-Myc (left) and two siliques from 35S::UFO-Myc; lfy-26/+plants (right). Scale bar: 2 mm. (F) Comparison of a rosette leaf from 35S::UFO-Myc (left), 35S::UFO-Myc; lfy-26/+ (middle) and 35S::UFO-Myc; lfy-26 plants (right).

 

Figure 2
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  		Fig. 2. LFY and UFO physically interact. (A) UFO induces a supershift of a LFY-DNA complex. Electrophoretic mobility shift assay (EMSA) using an AP3 promoter sequence. BMV, non-specific brome mosaic virus control; LFY, in vitro transcribed and translated LFY protein; UFO, in vitro transcribed and translated UFO protein; LFY+UFO lane shows a supershift of the LFY-DNA complex, while UFO alone does not bind to the AP3 promoter sequence, indicating that UFO binds to the LFY-DNA complex. (B) Upper panel: GST pull-down assay showing interaction between a bacterially produced GST-LFY fusion protein and 35S-labeled UFO protein; GST alone does not show an interaction with UFO. Input lane represents 10% of total protein. Lower panel shows same blot probed with {alpha}GST antibody, demonstrating equivalent loading of GST and GST-LFY lanes. (C) Interaction of LFY and UFO in yeast two-hybrid assays. The AD-LFY/BD-UFO interaction shows a significant increase in β-gal activity over background controls. This interaction is enhanced 8.6-fold when the F-box is deleted in the AD-LFY/BD-{Delta}FUFO combination compared with that of AD-LFY/BD-UFO. AD-{Delta}FUFO and BD-LFY also shows a significant interaction, while AD-UFO and BD-LFY shows a background level of β-gal activity. AD, Gal4 activation domain; BD, Gal4 DNA-binding domain. Bars represent mean±s.e.m. for five replicates. (D) Co-immunoprecipitation of UFO-Myc with LFY-FLAG in planta. Protein from wild-type, 35S::LFY-FLAG, 35S::UFO-Myc; ufo-2 (LF; UM) or 35S::UFO-Myc (UM) floral buds were used to precipitate the LFY immune complex using {alpha}-FLAG. For control immunoprecipitations, normal mouse IgG serum conjugated to agarose beads was used. Myc-tagged UFO specifically co-precipitated with LFY-FLAG from 35S::LFY-FLAG, 35S::UFO-Myc tissue only.

 

Figure 3
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  		Fig. 3. Mapping of the LFY interaction domain. (A) Diagram of the mutated and truncated forms of LFY. Gray boxes indicate the conserved domains at the N terminus and C terminus. Either D139 or S140 were mutated to alanine and used for yeast two-hybrid assays. Four different truncated versions of LFY, LFYN1 (amino acids 1-141), LFYC1 (amino acids 142-420), LFYN2 (amino acids 1-375) and LFYC2 (amino acids 376-420) are shown. (B) Yeast two-hybrid assays testing the interaction between various LFY truncations/mutants and {Delta}FUFO. Quantitative measurements of β-GAL activities (average of five independent assays) are indicated. LFYN2 fails to interact with {Delta}FUFO, while LFYC1 retains the interaction. Neither of the mutant forms D139 nor S140 affected the interaction with {Delta}FUFO. Bars represent mean±s.e.m. for the five replicates. (C) GST pull-down assays performed with bacterially expressed GST fusion proteins and inflorescence protein extracts from 35S::UFO-Myc plants. Top panel, western blot probed with anti-Myc antibody. Bottom panel, Coomassie Blue stained gel showing GST fusion proteins used in the assays. LFYN2 does not pull down UFO as efficiently as does full-length LFY from plant extracts.

 

Figure 4
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  		Fig. 4. Recruitment of LFY and UFO to the AP3 promoter. (A) The AP3 genomic region. Three different regions of the AP3 promoter, DEE (distal early element), PEE (proximal early element) and INT (inter-region between DEE and PEE) (Hill et al., 1998Go) are illustrated. (B) (Left) Chromatin immunoprecipitation (ChIP) was performed with anti-FLAG antibody or mouse normal IgG serum from inflorescence tissue of plants of the indicated genotypes. Promoter regions from AP1 and AP3 were amplified using PCR as indicated. A region of the Mu transposon was used as a positive control for amplification. LFY specifically associates with both the DEE and PEE elements of the AP3 promoter, as well as with the AP1 promoter fragment. The ufo-2 mutation does not compromise the ability of LFY to bind to target sequences. (Right) DEE and PEE levels were normalized to Mu and the fold change of experimental IP over IgG control IP is indicated. The values are mean±s.e.m. from three PCR experiments. (C) (Left) ChIP was carried out using inflorescence tissues obtained from genotypes as indicated. Chromatin immunoprecipitations carried out using anti-Myc antibody or normal mouse IgG serum. UFO specifically associates with both AP3 promoter regions, DEE and PEE; however, the presence of the lfy-26 mutation abolished these interactions, indicating that LFY is required for UFO to associate with AP3 promoter sequences. (Right) Quantitation as in B.

 

Figure 5
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  		Fig. 5. UFO-SRDX represses endogenous LFY activity. (A,H) WT (L. er) plants. (B-G,I) 35S::UFO-SRDX plants. (A) A wild-type flower. (B) The first arising flower-like structure in a transgenic 35S::UFO-SRDX line. The structure is subtended by a bract and consists of leafy organs, sepal-like organs and carpelloid sepals arranged in a spiral. (C) The fourth structure produced on the primary inflorescence. Organs are arranged in a spiral pattern. Occasionally, such flowers produce petals with reduced number and size. The flowers are infertile. (D) A later-arising flower with floral organs arranged in a whorl and subtended by bracts (arrow). The bracts are spirally arranged and show internode elongation. (E) A transgenic primary inflorescence in which the apex terminates with fused carpelloid sepal-like organs. Even apical flowers are subtended by bracts. (F) A representative 35S::UFO-SRDX flower from the line that resembles ufo-2. Like ufo-2 flowers, petals and stamens are absent or reduced in number and size or are filamentous. Carpelloid sepals and mosaic organs are often also observed. (G) A lateral structure from one of the most severe transgenic lines. No floral organs are produced; rather, only leaf-like organs arise in a spiral phyllotaxy with internodes elongated resembling ap1 lfy double mutants. (H) A wild-type inflorescence subtended by cauline leaves (arrow). (I) An inflorescence from a 35S::UFO-SRDX plant. Flowers are subtended by prominent bracts (arrow) or stipules (arrowhead). Only inflorescences but not flowers are subtended by cauline leaves in wild type (H).

 

Figure 6
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  		Fig. 6. Proteasome activity is required for the ectopic induction of AP3 expression in seedlings expressing 35S::UFO and 35S::LFY-GR. (A) The hlr mutation abrogates DEX-dependent induction of AP3 expression. Levels of AP3 expression monitored by qRT-PCR and normalized to EF-1{alpha}. (B) Epoxomicin treatment reduces DEX induced AP3 expression in seedlings that express 35S::UFO-Myc and 35S::LFY-GR. Gray and white bars represent two different biological replicates. Levels of AP3 expression monitored by qRT-PCR and normalized to GAPDH. Ten plants assayed for each condition. Values represent mean±s.e.m. for the three technical replicates. (C) A subpopulation of LFY-FLAG protein is post-translationally modified and stabilized in the presence of epoxomicin. SDS-PAGE of LFY-FLAG protein as detected by {alpha}FLAG antibodies shows a prominent band of LFY-FLAG protein at approximately 55 kDa (lane 1), as well as a smear of higher molecular weight proteins. The levels of these higher molecular weight proteins are reduced in a ufo-2 mutant background (lane 2). Conversely, an increase in the levels of these higher molecular weight proteins is seen in epoxomicin-treated 35S::LFY-FLAG inflorescences (lane 4) when compared with mock (DMSO)-treated tissue (lane 3). No crossreacting proteins are detected in wild-type samples (lane 5). Blot was reprobed with {alpha}RPN6 as a loading control. (D) LFY-FLAG protein is ubiquitylated. (Top panel) Immunoprecipitated LFY-FLAG protein or control IgG probed with anti-ubiquitin antibodies; (bottom panel) probed with anti-FLAG antibodies. Protein extracts from wild-type (WT), 35S::LFY-FLAG (LF) or 35S::LFY-FLAG; ufo-2 (LF; ufo2) inflorescences. LFY-FLAG protein migrates at ~55 kDa (arrowhead), while polyubiquitylated species can be detected in the 150-220 kDa range (bracket). A ~115 kDa UFO-dependent band can be detected (asterisk) in the LF lane, suggesting LFY-FLAG is post-translationally modified in a UFO-dependent manner.

 

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© The Company of Biologists Ltd 2008