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First published online 11 April 2007
doi: 10.1242/dev.003103

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Specification of Arabidopsis floral meristem identity by repression of flowering time genes

Chang Liu^1,*, Jing Zhou^1,*, Keren Bracha-Drori², Shaul Yalovsky², Toshiro Ito¹ and Hao Yu^1,

¹ Department of Biological Sciences and Temasek Life Sciences Laboratory, National University of Singapore, Singapore 117543, Singapore.
² Department of Plant Sciences, Tel Aviv University, Tel Aviv 69978, Israel.

View larger version (102K): [in this window] [in a new window]   Fig. 1. Phenotypes of constitutive expression of AGL24, SVP and SOC1. (A) The 35S:AGL24 Arabidopsis flower had leaf-like sepals and a secondary flower (arrow) without petals. (B) The 35S:SOC1 flower had light green sepaloid petals. (C) The 35S:SVP flower was converted into a shoot-like structure. Note the formation of stamens (arrows). (D) Internode elongation in a 35S:SVP flower. (E) The elongated 35S:SVP flower terminated with a chimeric structure of leaves, carpelloid leaves (arrowheads) and stamens (arrows). (F) The 35S:AGL24 35S:SVP flower developed like an inflorescence meristem. Note the formation of secondary flowers (arrows). (G) A floral structure arising from an individual floral meristem at a basal position in the main inflorescence of ap1-1. (H) The 35S:AGL24 35S:SOC1 had an increased production of secondary flowers in floral meristems at basal positions in the main inflorescence. The main inflorescence of 35S:AGL24 35S:SOC1 terminated soon after the generation of several floral structures. (I) A floral structure arising from an individual floral meristem at a median position in the main inflorescence of ap1-1.

View larger version (90K): [in this window] [in a new window]   Fig. 2. Rescue of ap1-1 by loss-of-function of AGL24, SVP and SOC1. (A,B) Phenotypes of Arabidopsis ap1-1. (C,D) Phenotypes of ap1-1 agl24-1. (E,F) Phenotypes of ap1-1 svp-41. (G,H) Phenotypes of ap1-1 soc1-2. Top view of a developing inflorescence is shown in A,C,E,G, while side view of a floral structure arising from an individual floral meristem at a basal position in the main inflorescence is shown in B,D,F,H.

View larger version (123K): [in this window] [in a new window]   Fig. 3. In situ localization of SVP in wild-type and ap1-1 Arabidopsis plants. (A) A longitudinal section of a wild-type inflorescence apex hybridized with AP1 antisense probe. (B,C) Two successive longitudinal sections of a wild-type inflorescence apex hybridized with SVP antisense probe. (D,E) Longitudinal sections of wild-type floral meristems at stage 3 (D) and stage 7 (E) hybridized with SVP antisense probe. (F) A longitudinal section of an ap1-1 inflorescence apex hybridized with SVP sense probe. (G-I) Serial longitudinal sections of an ap1-1 inflorescence apex hybridized with SVP antisense probe. An arrow in I indicates another floral meristem appearing at the edge of the inflorescence meristem. (J) Longitudinal section through ap1-1 floral meristems at stage 3 or later stages hybridized with SVP antisense probe. Arrows indicate the ectopic expression of SVP. (K,L) Two successive longitudinal sections of an ap1-1 stage-4 floral meristem hybridized with SVP antisense probe. Note the ectopic expression of SVP in the adaxial surface of the first whorl organs. Numbers in A-L indicate floral stages (Smyth et al., 1990). Scale bars: 100 µm for all panels.

View larger version (86K): [in this window] [in a new window]   Fig. 4. In situ localization of SOC1 in wild-type and ap1-1 Arabidopsis plants. (A-D) Serial longitudinal sections of a wild-type inflorescence apex hybridized with SOC1 antisense probe. An arrow in D indicates another floral meristem appearing at the edge of the inflorescence meristem. (E,F) Longitudinal sections of wild-type floral meristems at stage 4 (E) and stage 7 (F) hybridized with SOC1 antisense probe. (G) A longitudinal section of an ap1-1 inflorescence apex hybridized with SOC1 sense probe. (H) A longitudinal section of an ap1-1 stage-7 floral meristem hybridized with SOC1 sense probe. (I-N) Serial longitudinal sections of an ap1-1 inflorescence apex hybridized with SOC1 antisense probe. Note the ectopic expression of SOC1 in floral meristems at stage 1 to early stage 3. (O,P) Longitudinal sections of ap1-1 floral meristems at stage 3 (O) and stage 7 (P) hybridized with SOC1 antisense probe. Note the SOC1 expression throughout the stage 3 and stage 7 floral meristems. Numbers in A-P indicate floral stages (Smyth et al., 1990). Scale bars: 100 µm for all panels.

View larger version (16K): [in this window] [in a new window]   Fig. 5. Induced AP1 activity can transcriptionally repress SVP and SOC1. Transcript levels were determined by quantitative real-time PCR analyses of three independently collected replicates. Results were normalized against the expression of TUB2, then against the value of the first set of samples. Error bars indicate SD. (A,B) Timecourse expression of SVP (A) and SOC1 (B) in inflorescence apices of ap1-1 35S:AP1-GR plants mock-treated (Mock) or treated with 10 µM dexamethasone (Dex). (C) Expression of SVP and SOC1 in inflorescence apices of ap1-1 35S:AP1-GR, which were mock-treated (M), treated with 10 µM dexamethasone (D), with 10 µM cycloheximide (C) and with 10 µM cycloheximide plus dexamethasone (CD). Expression analyses were performed after 4 hours of treatment. (D) Expression of AGL24, SVP and SOC1 in inflorescence apices of wild-type and ap1-1 plants. Gene expression in wild-type Arabidopsis plants was set to one.

View larger version (29K): [in this window] [in a new window]   Fig. 6. AP1 directly binds to the regulatory regions of AGL24, SVP and SOC1. (A) Schematic of the genomic regions of Arabidopsis AGL24, SVP and SOC1. Bent arrows denote translational start sites and stop codons. Exons and introns are shown by black and white boxes, respectively. The arrowheads indicate the sites containing either single mismatch or perfect match from the consensus binding sequence (CArG box) for MADS-domain proteins. The hatched boxes represent the DNA fragments amplified in the ChIP assay. (B) Western analysis of nuclear extracts from inflorescences (i) of ap1-1, and inflorescences (i) and leaves (l) of wild-type plants probed with the purified AP1 antibody. AP1 protein was only detectable in wild-type inflorescences. (C) Western analysis of the specificity of anti-AP1 serum in the ChIP procedure. After sonication, the supernatant containing solubilized chromatin from wild-type inflorescence served as an input for immunoprecipitation either with IgG(-) or with anti-AP1 serum (+). Anti-AP1 serum could specifically precipitate AP1 protein. (D) Western analysis of the specificity of anti-AP1 serum to precipitate AP1-GR fusion protein in the ChIP procedure. After sonication, the supernatant containing solubilized chromatin from inflorescences of wild-type and ap1-1 35S:AP1-GR (Dex- or Mock-treated) plants served as an input for immunoprecipitation either with IgG(-) or with anti-AP1 serum (+). Anti-AP1 serum could specifically precipitate AP1-GR protein. (E-G) ChIP analysis of AP1 binding to regulatory sequences of AGL24 (E), SVP (F) and SOC1 (G). Real-time PCR assay of immunoprecipitated DNAs was conducted in triplicate. Relative enrichment of each target DNA fragment was calculated first by normalizing the amount of a target DNA fragment against a TUB2 genomic fragment, and then by normalizing the value for anti-AP1 serum against the value for IgG. The enrichment of an ACTIN 2/7 gene fragment was used as a negative control. Error bars indicate the standard error of the mean.

View larger version (85K): [in this window] [in a new window]   Fig. 7. Mutagenesis of AP1-binding site causes ectopic expression of AGL24 in young floral meristems. (A) Schematic of the ProAGL24:GUS construct where the 4.7 kb Arabidopsis AGL24 genomic sequence was translationally fused with the GUS gene. The native CArG box near the AGL24-4 fragment identified in Fig. 6E was mutated. (B,C) GUS staining in inflorescence apices of the transformants containing ProAGL24:GUS (B) and its derived construct with the mutated CArG box (C). At least 12 independent lines for each construct were analyzed and representative images are shown. Scale bars: 100 µm for B,C.