QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 5 January 2005
doi: 10.1242/dev.01600

This Article Summary Full Text Full Text (PDF) Supplementary Material Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Gómez-Mena, C. Articles by Sablowski, R. Search for Related Content PubMed PubMed Citation Articles by Gómez-Mena, C. Articles by Sablowski, R. Social Bookmarking                         What's this?

Transcriptional program controlled by the floral homeotic gene AGAMOUS during early organogenesis

¹ Department of Cell and Developmental Biology, John Innes Centre, Norwich NR4 7UH, UK
² Business Unit Bioscience, Plant Research International, PO Box 16, 6700 AA Wageningen, The Netherlands

View larger version (51K): [in a new window]   Fig. 5. AG binds to candidate target genes. (A) Binding sites identified by sequence analysis. `m' is a mutated version of the binding site in AP3, used as a negative control. Next to each numbered binding site, the corresponding gene and sequence are shown, with mismatches to the consensus AG binding site TT(A/T/G)CC(A/T)6GG(A/T/C)AA (Shiraishi et al., 1993) marked in boldface. The white boxes represent sequences upstream of the start codon (except for AG, where the reference point is the 5' splice site of the second intron), with vertical lines indicating the position of the AG binding sites. The horizontal bars above some of the binding sites indicate the fragment amplified in the ChIP experiment (C). (B) Binding to AG in vitro, shown by electrophoretic mobility shift assays (EMSA). The probes contained the binding sites numbered in A. Each probe was incubated with extract from bacteria induced to express AG (+) or an empty expression vector (-). In all experiments, the same amount of labelled probe was used and a lane with probe 2 (not shown) was included to adjust the exposure to comparable levels. (C) Binding to AG in vivo, shown by ChIP. Numbers correspond to the binding sites shown in A; in each panel, PCR amplification (35 cycles) of sequences containing the binding site (black bars in A) is compared in immunoprecipitates obtained with antiserum against AG (+) or CLV3 (-). In the last panel on the left, the fourth exon of EIF4A1 was used as a negative control lacking AG binding sequences: with 35 cycles (not shown), no band was seen; with 40 cycles (panel), similar levels of contaminating template were amplified. The results shown were replicated in two fully independent ChIP experiments.

View larger version (102K): [in a new window]   Fig. 1. Steroid-inducible stamen and carpel development. (A,B) ag-3, AGGR inflorescences, mock treated (A) or treated with dexamethasone (DEX; B). (C,D) Close-up view of flowers from the plants shown in A,B; while the mock-treated plant shows an indeterminate number of sepals and petals (C), DEX treatment has induced stamen (st) and carpel (ca) development (D). (E,F) cal-1, ap1-1, AGGR inflorescences, two weeks after mock treatment (E) or treatment with DEX (F). Note the mass of meristems in E (similar to those shown at high magnification in G) compared with the mature stamens and carpels in F. (G-I) Scanning electron micrographs of cal-1, ap1-1, AGGR inflorescences 1 day (G), 3 days (H) and 7 days (I) after DEX treatment. The arrows in H indicate meristems that are beginning to produce organ primordia; in I, developing stamens (st) and carpels (ca) are morphologically identifiable. Scale bars: 5 mm in A,B; 1 mm in C-F; 100 µm in G-I.

View larger version (48K): [in a new window]   Fig. 2. Summary of changes in gene expression after AGGR activation. (A,B) Venn diagrams showing the number of genes significantly activated (A) or repressed (B), 1, 3 or 7 days after DEX treatment. The grey area contains the 12 genes chosen for more detailed analysis because their activation was sustained during the time course. (C) Predicted functions of the proteins encoded by the genes shown in A. The ratio between the number of genes and the area of the coloured sections is the same across the diagrams for 1, 3 and 7 days after AGGR activation.

View larger version (40K): [in a new window]   Fig. 3. Expression levels of selected genes after AGGR activation. (A) Expression detected on the oligonucleotide array. M1 to M7 and D1 to D7 indicate 1, 3 and 7 days after mock treatment and DEX treatment, respectively. The coloured rectangles show normalised mean expression according to the colour scale on the left; the levels are shown only when the difference between mock and DEX treatment was statistically significant. The 12 genes in the grey box showed sustained activation and correspond to the grey area in Fig. 2A. Additional genes with previously characterised roles in stamen or carpel development and with significant activation at day 7 only are also shown. (B) Activation of the 12 genes in the grey box in Fig. 2A, confirmed by RT-PCR (in the case of AG, the primers used did not amplify AGGR). M1-7 and D1-7 are as described in A; APT1 (adenosine phosphotransferase) was used as a constitutive control.

View larger version (120K): [in a new window]   Fig. 4. RNA in situ hybridisation of selected genes during wild-type floral development. (A,B) At4g21590. The arrows show expression in the centre of a stage 3 bud (A), and later in developing stamens (B). (C) At1g13400. Arrows indicate expression in emerging stamen primordia (st), and in the placental region in early carpels (ca). (D,E) At3g17010. Expression in emerging stamen primordia (st) is indicated in D; arrows in E indicate expression in the sporogenous tissue of stamens (st) and in the placental region of carpels (ca). (F) Expression of GA4 in stamen filaments (arrow). (G,H) Expression of ATH1 in developing stamens (st). For better contrast, the sections in A, B, G and H were photographed in aqueous medium, before the tissues were permanently mounted.

View larger version (133K): [in a new window]   Fig. 6. Maintenance of AP3 expression requires either AG or AP1. (A,C,E) Top view of inflorescence in ag-3 (A), ap1-1 (C), and in the ag-3, ap1-1 double mutant (E). (B,D,F) RNA in situ hybridisation with the AP3 probe hybridised to longitudinal sections through the inflorescence apex of ag-3 (B), ap1-1 (D), and the ag-3, ap1-1 double mutant (F). Arrows point at stage 3 buds, where AP3 expression is initiated, and arrowheads indicate later expression during petal (B) and stamen (D) development. The sections of the three genotypes were hybridised in parallel on the same slides, to allow comparison of the expression levels. Scale bars: 1 mm in A,C,E.

View larger version (17K): [in a new window]   Fig. 7. Model for the co-ordinated regulation of floral organ identity regulators. (A) In stamen development, AG, AP3, PI and SEP3 are initially activated independently (grey arrows) (Ferrario et al., 2004; Zik and Irish, 2003a). LFY is responsible for the initial activation of AG, whereas early activation of AP3/PI occurs in areas of the meristem that express both LFY and UFO (Parcy et al., 1998). The AG, AP3, PI and SEP3 proteins (circles) function together in a complex to promote stamen development (Honma and Goto, 2001), and to amplify and maintain their own expression. Solid black arrows indicate direct interactions supported by ChIP; feedback activation of PI may be indirect (dashed arrow) (Honma and Goto, 2000). (B) The protein complex proposed to control carpel development contains AG and SEP3 (Honma and Goto, 2001). As in A, the initially independent expression of AG and SEP3 is maintained by a positive-feedback loop. In addition, CRC expression is reinforced, although CRC expression can promote carpel development independently of AG (indicated by the parallel grey arrow) (Alvarez and Smyth, 1999). As in A, interactions supported by ChIP are indicated by solid black arrows. The possibility that CRC might also promote AG activity is indicated by the dashed arrow with a question mark.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter