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First published online 19 October 2005
doi: 10.1242/dev.02085

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Evolution of regulatory interactions controlling floral asymmetry

Department of Cell and Developmental Biology, John Innes Centre, Norwich NR4 7UH, UK

View larger version (39K): [in a new window]   Fig. 1. CYC protein binds to DNA. (A) Specificity of CYC recombinant protein binding to class I and class II TCP consensus binging sites. Oligonucleotide probes contain a shared consensus binding site for class I GGNCCCAC (red) and class II GTGGNCCC (blue) to which CYC protein binds (arrow; lane 1). Bold underlined nucleotides indicate mutations introduced that affect both class I and II binding (lanes 2 and 3) or specifically only class II (lane 4) or class I (lane 5). (B) Consensus DNA-binding sequence for CYC protein obtained by a random binding site selection method. After five rounds of selection, a total of 31 binding sites of CYC were piled up using MEME (meme.sdsc.edu) and a consensus sequence generated. Nucleotides that were not occupied by a specific residue are indicated by N.

View larger version (38K): [in a new window]   Fig. 2. Interaction of CYC recombinant protein with RAD promoter and intron. (A) RAD genomic region showing three potential CYC-binding sites in the promoter and intron. (B) PCR fragments of promoter and intron sequences with putative CYC-binding sites were amplified and used as probes in an EMSA with recombinant CYC protein. + or – indicates the presence or absence of CYC protein; asterisks indicate retardation of the mobility of CYC-DNA binding complex.

View larger version (48K): [in a new window]   Fig. 3. Northern blot of RNA from 35S::CYC:GR DEX-grown T2 plants derived from three independent transformants, probed with CYC (top) or NPTII as control (bottom). For each transformant, seedlings were grouped according to whether they had a strong (*) or a wild-type phenotype. Progeny testing showed that in all cases, plants with the strong phenotype were hemizygous, whereas plants with wild-type phenotypes were homozygous for the construct.

View larger version (31K): [in a new window]   Fig. 4. Effect of CYC in the growth of the petals of Arabidopsis. Flowers along the inflorescence of (A) wild-type and (B) 35S::CYC:GR plants grown in DEX. (C) Petal area (mm2) of consecutive flowers in the inflorescence. Flowers are numbered from the first flower with visible petals (flower 1). The inflorescence remaining after removal of flowers equal or older than stage 1 is labelled `inf'. Error bars indicate standard deviations. Number of petals measured for each stage was between five and eight. Yellow bars indicate wild-type and orange bars indicate 35S::CYC:GR plants (+CYC) grown with DEX. Scale bars: 1 mm.

View larger version (90K): [in a new window]   Fig. 5. Effect of CYC on Arabidopsis petal cell size. SEM images of the tip and middle areas of wild-type and 35S::CYC:GR petals show that the increase in petal area when CYC is active is due to an increase in cell size. In each SEM image, the same number of cells are highlighted in orange. Numbers indicate cell size (µm2±s.e.m.) in the tip and middle regions of the petals, determined using the CellFinder programme. On average between 200 and 500 cells were measured per image and five petals were used to obtain the average cell size in the different petal areas. Left panel shows wild-type and right panel shows 35S::CYC:GR plants (+CYC) grown with DEX. Scale bar: 50 µm on the SEM images; 1 cm on the whole petals.

View larger version (61K): [in a new window]   Fig. 6. Effect of overexpressing CYC in Arabidopsis. 35S::CYC:GR plants have smaller leaves and bigger petals. (A) Wild type; (B) 35S::CYC:GR plants grown on DEX; (C) higher magnification of B (6x). Scale bars: 1 cm.

View larger version (31K): [in a new window]   Fig. 7. CYC represses leaf growth in Arabidopsis. (A) Leaves from wild-type and 35S::CYC:GR plants grown on DEX (+CYC), at early (day 14; left) and late (day 36; right) developmental stages. In all cases, the fourth rosette leaf was measured. Scale bar: 1 mm. (B) SEM images of adaxial epidermal cells from leaves at the same developmental stage as in A with individual cells highlighted in diverse colours. Scale bar: 50 µm. (C) Measurements of leaf area (mm2) and (D) epidermal cell area (µm2) show that CYC reduces leaf surface area by a reducing cell proliferation and cell expansion. Three to five leaves were measured and the area of 19-87 cells in each leaf was obtained. Error bars indicate standard deviations.

View larger version (59K): [in a new window]   Fig. 8. Effect of CYC activation or inactivation on the growth of Arabidopsis leaves. (A,B) 35S::CYC:GR plants were grown in media without DEX (A) or with DEX (B) and left to grow to maturity. (C,D) Alternatively, 10 day after germination, seedlings in which leaf 3 was less than 0.5 mm wide were transferred from media without DEX to media with DEX (C) or from media containing DEX to media without DEX (B). Numbers correspond to consecutive rosette leaves in the mature plants. Scale bar: 1 cm.

View larger version (24K): [in a new window]   Fig. 9. CYC upregulates RAD expression in Arabidopsis. RT-PCR analysis of RAD transcript in 35S::CYC:GR RAD::RAD plants transferred to DEX. RNA was extracted after 6 hours after transfer. APT (adenosine phosphotransferase) was used as a constitutive control.

View larger version (25K): [in a new window]   Fig. 10. Schematic representation of evolution of gene interactions in the Antirrhinum lineage controlling flower dorsoventral asymmetry. Potential direct and indirect targets of CYC are highlighted in orange and red, with the subset of actual targets indicated in orange. Downstream targets regulating growth are in green and the rest of the regulatory network in which CYC interactions are embedded is shown in grey. Steps where divergence involving CYC interactions are lettered a-d (see text for explanation).

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