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First published online 24 October 2007
doi: 10.1242/dev.013136

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Functional analyses of genetic pathways controlling petal specification in poppy

Sinéad Drea¹, Lena C. Hileman^1,*, Gemma de Martino¹ and Vivian F. Irish^1,2,

¹ Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520, USA.
² Department of Ecology and Evolutionary Biology, Yale University, New Haven, CT 06520, USA.

View larger version (26K): [in this window] [in a new window]   Fig. 1. Inferred evolutionary history of petals and MADS-box genes in the eudicots. (A,B) Alternative scenarios for the evolution of petals. (A) The evolution of a bipartite perianth occurred independently in the lineage leading to the Ranunculales as compared with the core eudicots. (B) The evolution of a bipartite perianth occurred prior to the radiation of the eudicots, with losses of this character in multiple lineages. (C,D) Summary of phylogenetic analyses. For comprehensive phylogenetic analyses, see Figs S1, S2 in the supplementary material. (C) The duplication of paleoAP3-like genes resulting in PapsAP3-1 and PapsAP3-2 occurred within the Ranunculales, pre-dating the divergence of the Ranunculaceae and Papaveraceae. (D) The duplication of PI-like genes occurred relatively recently in the Papaveraceae, leading to PapsPI-1 and PapsPI-2 in P. somniferum. Clades in bold represent those containing Paps genes described in this study.

View larger version (97K): [in this window] [in a new window]   Fig. 2. Sequence alignment of P. somniferum AP3- and PI-like sequences. Alignment of C-terminal domains of predicted AP3 (A) and PI (B) proteins from core eudicots, monocots and basal eudicots. Conserved domains are boxed. Identical residues are highlighted in black and similar residues in gray. The arrow in B indicates the position of the insertion in PapsPI-2 that mediates a frameshift.

View larger version (56K): [in this window] [in a new window]   Fig. 3. Expression analyses of PapsAP3 and PapsPI genes. (A) mRNA in situ hybridization analyses for PapsAP3-1, PapsAP3-2, PapsPI-1 and PapsPI-2 during P3 to P5 stages of poppy flower development. Light micrographs of representative stages are shown. Black arrow, petal primordia; red arrow, carpel primordia. PapsAP3-1 is expressed in petal and stamen primordia commencing at stage P3, and is maintained in these organs through later stages. This is in contrast to PapsAP3-2 expression, which is initially restricted to the stamen primordia; later, PapsAP3-2 expression expands to include developing petals at stage P5. PapsPI-1 expression commences in petal and stamen primordia at stage P3, and is maintained in this pattern through later stages. PapsPI-2 expression is weak but detectable in petal, stamen and carpel primordia at stage P3; by stage P5, expression is predominantly in stamens and on the adaxial side of the carpels. Scale bar: 200 µm, for all panels. (B) RT-PCR analyses of PapsAP3 and PapsPI expression during stages P6 to P8 of poppy flower development (as shown above). At each stage, sepals (se), petals (pe), stamens (st) and carpels (ca) were removed separately for analysis. The P. somniferum actin gene (PapsACT1) was used as an amplification control.

View larger version (115K): [in this window] [in a new window]   Fig. 4. Functional analyses of PapsAP3 and PapsPI genes using virus-induced gene silencing. (A) Wild-type poppy flower. Scanning electron micrographs (SEMs) of abaxial (a) and adaxial (b) sepals; abaxial (c) and adaxial (d) petals; anther (e) and filament (f) of stamens; carpel wall (g) and ovules (h). (B) vigsAP3-1 (silenced for PapsAP3-1) flower showing homeotic transformation of petals into sepaloid organs. SEM of abaxial (a) and adaxial (b) sepaloid petals. (C) vigsAP3-2 flower showing variable transformations of stamens to carpeloid structures. Range of carpeloidy in stamens (a). SEM of carpeloid stamen with stigmatic ray overlying anther tissue (b) and showing the presence of ectopic ovules (c). (D) vigsAP3-D (vigsAP3-1/AP3-2) flower showing a strong homeotic transformation of petals and stamens. SEM of abaxial (a) and adaxial (b) sepaloid petals; abaxial surface (c) and ectopic ovules of carpeloid stamens (d). (E) vigsPI-1 flower displaying homeotic transformations in petals and stamens. SEM of abaxial (a) and adaxial (b) sepaloid petals; emerging ovule at junction of anther and filament of carpeloid stamen (c) and examples of stigmatic tissue overlying the anther of carpeloid stamens (d). (F) vigsPI-D (vigsPI-1/PI-2) flower showing strong homeotic transformations of petals and stamens. SEM of abaxial (a) and adaxial (b) sepaloid petals; abaxial surface (c) and ovules of carpeloid stamens (d). Scale bars: 30 µm in Aa-f, Ba,b, Da,b, Ea,b, Fa,b; 10 µm in Ag, Dc, Fc; 100 µm in Ah, Cc, Dd, Ec,d, Fd; 300 µm in Cb.

View larger version (78K): [in this window] [in a new window]   Fig. 5. Functional analysis of PapsAP3 genes. Wild-type (right) and vigsAP3-D (left) mature flower buds. A dramatic homeotic conversion of petals and stamens is seen in the vigsAP3-D bud.

View larger version (70K): [in this window] [in a new window]   Fig. 6. Expression analyses of VIGS lines. Petals (pe), sepaloid petals (sp), stamens (st), carpeloid stamens (cs) and pooled stamens and carpels (s/c) were dissected out and used for RT-PCR analyses. Expression of PapsACT1, PapsAP3-1, PapsAP3-2, PapsPI-1 and PapsPI-2 in each of the six VIGS backgrounds and in wild-type flowers is shown. RT-PCR was also used to demonstrate the presence of the TRV2 vector insert in each transformation. PapsACT1 served as an amplification control.