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BBMB 607 6 September 2006 Presented by: Tracie Hennen-Bierwagen

ALG24, SHORT VEGETATIVE PHASE, and APETALA1 Redundantly Control AGAMOUS during Early Stages of Flower Development in Arabidopsis Veronica Gregis, Alice Sessa, Lucia Colombo, and Martin M. Kater The Plant Cell, Vol. 18, 1373-1382 June 2006. BBMB 607 6 September 2006

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BBMB 607 6 September 2006 Presented by: Tracie Hennen-Bierwagen

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  1. ALG24, SHORT VEGETATIVE PHASE, and APETALA1 Redundantly Control AGAMOUS during Early Stages of Flower Development in ArabidopsisVeronica Gregis, Alice Sessa, Lucia Colombo, and Martin M. KaterThe Plant Cell, Vol. 18, 1373-1382 June 2006 BBMB 607 6 September 2006 Presented by: Tracie Hennen-Bierwagen

  2. Background • Switch from vegetative to reproductive development is under the control of floral meristem identity genes • These genes regulate the floral homeotic genes of the A-, B-, and C-classes • Homeotic genes in turn specify floral organ identity within each spatial domain • LFY regulates AG; LFY and AP1 activate AP3; LUG and SEU combine to repress AG

  3. Mutants investigated in this paper

  4. Genetic evidence of interactions • AP1 interaction with SEU • AP1 interacts with AGL24 and SVP • SEU interacts with LUG to corepress AG • Does AGL24, SVP, and AP1 recruit SEU/LUG to AG?

  5. Specific Aims • In-depth characterization of the roles of SVP, AGL24, LUG, and SEU, their specific targets and functions in floral organ identity and spatial placement, and patterns of expression • Investigate functional redundancy between homologous MADS-box transcription factors involved in floral development • Define MADS box proteins molecular roles within the ABC model (multiple functions throughout development)

  6. Floral transition timing comparisons • The similar MADS box TFs have opposing functions regulating flowering time • AGL24 is a promoter of flowering • SVP is a repressor • SVP is epistatic to AGL24 • Both expressed later - what role(s) do they continue to play in development?

  7. Floral Defects of svp alg24 double and svpagl24 ap1 triple mutants Defects appear similar to those caused by precocious and spatial misexpression of B and C class genes (like AG) due to lack of repression by LUG/SEU

  8. SEM of Early Floral Development in agl24 svp and agl24 svp ap1 Mutants • Homeotic effects - reduction of, and misplacement of organs • SEM shows mutant effects occur very early in development in organ primordia • B and C class genes targets of AGL24 and SVP after floral transition - second role?

  9. AG and AP3 Gene Expression Patterns in WT or agl24 svp Mutant Flowers • Homeotic B and C class genes deregulated? • AP3 petals/stamens; AG stamens/carpels • AP3 and AG expression explored by in situ hybridization • AG is expressed very early in and throughout development • AG and AP3 are misexpressed spatially - found in all whorls AG expression AP3 expression

  10. LUG/SEU Expression in agl24 svp Mutant • Floral phenotype of agl24 svp double mutant is similar to lug and seu mutants • Is the phenotype of the agl24 svp mutant due to misexpression or reduced expression of LUG and/or SEU

  11. Overview of Yeast Two-Hybrid • BD construct binds reporter DNA • If AD protein interacts with BD protein then transcription of reporter gene takes place • Assay for presence of reporter protein Stratagene catalog 2006

  12. Limitations of Yeast Hybrid Assays • YFP must fold correctly • Full length AP1 didn’t show an interaction • Posttranslational modifications necessary in vivo don’t exist in heterologous systems • Disulfide bridges, glycosylation, phosphorylation • Autoactivation • In multicomponent complexes, native yeast TFs could interact as “bridges” • Still an in vitro method - evidence of interaction could still be artificial and wouldn’t occur in vivo

  13. In vivo protein-protein interaction assays

  14. Results of Yeast Two-, Three-, and Four-Hybrid Assays

  15. Conclusions • SVP and AGL24 regulate flowering time in opposition • SVP and AGL24 are also involved in proper floral organ placement in later development through protein-protein interactions with AP1 by recruitingthe LUG/SEU corepressor complex to regulate AG • MADS-box transcription factors can play multiple roles via different protein-protein interactions throughout floral development • Functional redundancy exists but in this case only at permissive temperatures

  16. Future Experiments • Pull down assays • Affinity chromatography • Co-immunoprecipitation • FRET or BRET (fluorescence or bioluminescence resonance energy transfer) • BRET uses luciferase and GFP • FRET donor to excite acceptor also but has more limitations • In vivo TAP-tagging

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