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First published online April 24, 2009
doi: 10.1242/10.1242/dev.035436

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Floral stem cell termination involves the direct regulation of AGAMOUS by PERIANTHIA

Toshiro Ito^1,3, Frank Wellmer^1,4, Teva Vernoux², Annick Dedieu², Jan Traas² and Elliot M. Meyerowitz^1,*

¹ Division of Biology 156-29, California Institute of Technology, Pasadena, California 91125, USA.
² Laboratoire RDP, Ecole Normale Supérieure de Lyon, 46 allée d'Italie, 69007 Lyon, France.
³ Temasek Life Sciences Laboratory, National University of Singapore, Singapore 117604, Singapore.
⁴ Smurfit Institute of Genetics, Trinity College Dublin, College Green, Dublin 2, Ireland.

View larger version (112K): [in this window] [in a new window]   Fig. 1. pan lfy double mutant flowers are indeterminate and fail to downregulate WUS. (A-G) Phenotypes of pan and lfy single and double mutant flowers. (A) Wild-type flower bearing four sepals, four petals, six stamens and two fused carpels in whorls one to four, respectively. (B) lfy-31 flower with sepal-like organs in whorls one to three and fused sepalloid/carpelloid organs (arrow) in whorl four. (C) Flower of the well-characterized strong lfy-6 allele displaying similar phenotypes to lfy-31. (D) pan-3 flower bears extra sepals and petals but no carpel defects. (E) pan-3 lfy-31 double mutant flower with sepal-like organs in whorls one to three and unfused sepalloid/carpelloid organs (arrow) in whorl four. (F) The same pan lfy flower as in E, with several organs removed to expose the ectopic floral structures growing within (arrowhead). The approximate position of the removed fourth-whorl organs is indicated (dotted line). (G) The same lfy flower as in B, dissected to reveal a relatively normal gynoecium. (H-M) In situ localization of WUS transcript in stage 2 flowers (H-J), or in stage 7 or older flowers (K-M), of pan-3 (H,K); lfy-6 (I,L); or pan-3 lfy-31 (J,M) plants. (H-J) Early WUS expression (arrows) is similar in all three genotypes. (K-M) In older flowers, no WUS expression is observed at the base of the gynoecium (arrowheads) of pan (K) and lfy (L) single mutants, but remains strong in pan lfy flowers (M) of a similar stage. Scale bars: 50 µm.

View larger version (78K): [in this window] [in a new window]   Fig. 2. A dominant-negative PAN chimera induces floral determinacy phenotypes. (A) pAP1(1.7-kb)::alcR--alcA::ER-GFP expression in an inflorescence meristem. GFP signal (green) is observed in the central domes (arrowheads) of all visible flowers after induction with ethanol vapors. Autofluorescence from the shoot apical meristem is visible (red). (B-H) Floral phenotypes of PAN wild-type or mutant plants harboring one or more copies of PAN-RD under the control of the AP1(1.7-kb) promoter. Red and green bars indicate the number of copies of PAN-RD or wild-type PAN, respectively. Open bar indicates no copies, half-filled indicates one copy and filled indicates two copies (see key, bottom right). (B) pan-2 mutant flower. (C) pan flower harboring one copy of PAN-RD displaying amplified pan-like phenotypes, as well as additional phenotypes such as extra carpels (arrow) and severe floral indeterminacy. (D) Side view of the flower in C with some organs removed to reveal ectopic floral structures (arrow) developing interior to the fourth whorl (demarcated by a dashed line). (E) A pan-like phenotype is observed in flowers arising from a cross of genotype in C to wild type. This flower harbors one copy of PAN-RD and is heterozygous at the PAN locus. (F) Wild-type flower. (G) Wild-type flower harboring one copy of PAN-RD displaying a pan-like phenotype. (H) Flower from progeny of the plant in G harboring two copies of PAN-RD presenting strong indeterminacy defects, similar to PAN-RD/+ pan plants (C). (I-K) In situ localizations of AG transcript in stage 5-6 flowers of pan-2 (I) or PAN-RD/+ pan-2 (J,K) plants. AG localization appears unperturbed in pan flowers (I), but in PAN-RD/+ pan-2 flowers a central region within the AG domain shows diminished signal intensity. Also marked are sepals (dashed arrow) that do not express AG and early stamen primordia (arrowhead) that do. (K) Magnified view of the dashed box in J. Scale bars: 10 µm.

View larger version (13K): [in this window] [in a new window]   Fig. 3. PAN directly binds AGAMOUS regulatory sequences. (A) Structure of the 5.7 kb AG locus. Exons and introns (top) are represented in bold and dashed lines, respectively. The second intron (asterisk) contains all known AG regulatory elements. The detailed view of the 3 kb intron shows the fragments used to determine enrichment in the chromatin immunoprecipitation assays (blue bars), the 3' fragment used in co-bombardment experiments (red line), and evolutionarily conserved elements and known or predicted transcription factor binding motifs (Davies et al., 1999; Hong et al., 2003; Lohmann et al., 2001; Parcy et al., 1998). Black triangles, LFY/WUS binding sites; white triangle, predicted LFY binding site; stars, CArG boxes; circles, CCAAT boxes; diamond, AAGAAT motif; green squares, predicted core bZIP binding sites; vertical tick marks: 200 bp intervals. (B) Results of chromatin immunoprecipitation experiments from stage 5-7 flowers. Anti-PAN antiserum was used to isolate protein-DNA complexes and DNA enrichment levels were measured by quantitative real-time RT-PCR (see Materials and methods). Enrichment was calculated for antibody-bound DNA relative to total input DNA and was then normalized against an internal genomic control. Vertical bars show mean enrichment levels from duplicate experiments for amplicons distributed along the intron (shown in 3A). The scale for amplicon `H' is different and is shown in red. Results are shown only for amplicons with a coefficient of correlation (r2)>0.98. (C) Results of particle co-bombardment experiments in onion epidermal cells. Vertical bars represent mean values from duplicate experiments for numbers of cells stained for GUS enzymatic activity. The pAGi-3'::GUS reporter, which recapitulates most facets of AG expression in vivo, shows some background activity (left bar) in onion epidermal cells. When co-bombarded with p35S::PAN-VP16 (right bar), the `numbers of cells' stained for GUS activity is 4- to 5-fold greater. Error bars represent standard deviation from the mean.

View larger version (41K): [in this window] [in a new window]   Fig. 4. pan mutations do not enhance the third-whorl phenotypes of a weak ag allele. (A) Flower of the weak ag-4 allele in a mixed background of wild-type accessions (L-er/Ws). Such flowers have normal outer organs but fourth-whorl carpels are replaced with a new flower (arrow). (B) A pan-2ag-4 flower in the same genetic background has the altered floral organ numbers of the pan mutant and the new fourth-whorl flower phenotype of ag-4 (arrow).

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