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doi: 10.1242/10.1242/dev.00295

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Separable roles of UFO during floral development revealed by conditional restoration of gene function

¹ Laboratoire de Biologie Cellulaire, INRA, Route de Saint Cyr, 78026 Versailles Cedex, France
² Department of Cell and Developmental Biology, John Innes Centre, Colney Lane, Norwich, NR4 7UH, UK

View larger version (53K): [in a new window]   Fig. 1. Ethanol-induced restoration of the ufo-2 mutant. Plants were not induced (A-D) or induced for 5 days by ethanol after 3 weeks culture under Long Days. (E-H). The apices were observed 18 days after the beginning of the treatment. No difference in floral morphology was observed between wild type (A,E) and ufo-2 (B,F) lines whether treated or not treated with ethanol. Flowers of ufo-2 35S::uidAind transgenics carrying an inducible GUS construct (C,G) were not modified by ethanol induction. (D,H) In ufo-2 35S::UFOind ethanol-induced expression of UFO restored petals and stamens and wild-type flowers (arrows) could be observed (H).

View larger version (83K): [in a new window]   Fig. 2. Floral phenotypes in wild type, ufo-2 and different phases of phenotypic restoration in ufo-2 35S::UFOind. (A) Wild-type flower, with 4 sepals (not visible), 4 petals, 6 stamens (only five visible) and a pistil with 2 carpels. (B) ufo-2 flower with no petals and stamens The four outer sepals have been removed. Two filaments (arrows), 1 out of 2 staminoid sepal (arrow), 1 carpeloid structure (asterisk), and 2 out of 4 inner sepals are visible. The pistil contains two fused carpels. (C) Type I ufo-2 35S::UFOind flower with 4 first whorl sepals, 1 petaloid sepal (arrowhead), 1 staminoid petal (arrow), 1 petal, 8 stamens and a pistil with 3 unfused carpels. (D) Type I ufo-2 35S::UFOind flower with 5 first whorl sepals, 1 sepaloid petal (arrow), 1 petal, 1 staminoid petal (black arrowhead), 8 stamens and a pistil formed by 3 unfused carpels. (E) Lateral view of a type I ufo-2 35S::UFOind flower with the 4 first whorl sepals removed to reveal the inner 2 sepals (arrowheads). (F) Type II ufo-2 35S::UFOind flower with 4 sepals, 2 petals, 1 staminoid petal (arrow), 1 petaloid stamen (arrowhead), 5 stamens (only 2 clearly visible) and a central pistil with 3 partially unfused carpels. (G) Type III ufo-2 35S::UFOind flower with 4 sepals, 4 petals, 6 stamens and a pistil with two carpels. (H) Type IV ufo-2 35S::UFOind flower with 4 first whorl sepals, 1 petal, 6 stamens and a pistil with two carpels. (I) Lateral view of a type IV ufo-2 35S::UFOind flower with 2 of the first whorl sepals removed to reveal the 4 inner stamens and a pistil with 2 carpels. Note the absence of second whorl organs. (J) SEM of a type I ufo-2 35S::UFOind flower. One sepal has been removed to show the inner organs. (K) Detail of J showing 3 stamens in an outer whorl (arrows) and 3 stamens in an inner whorl (arrowheads). (L) Type I ufo-2 35S::UFOind flower with outer organs removed showing the central pistil unfused along two sides. One of the unfused margins has a thickening of the distal part with cell types similar to the anther (compare black and white arrowheads) and cells similar to the filament of the stamen (compare black and white arrows) form the proximal region. (M) Type I ufo-2 35S::UFOind flower with outer organs removed to expose a central pistil formed by three carpels completely unfused on one side. Arrowhead points to an abnormal ovule. (N) Detail of L showing an abnormally shaped ovule. (O) Type IV ufo-2 35S::UFOind flower. No visible petals are present between the sepal whorl and the stamens. (P) Dissected type IV ufo-2 35S::UFOind flower with the sepals removed. No reduced petal is visible. The arrow points to the insertion point of a removed sepal. The arrowheads indicate 2 stamens. A-I are at the same magnification. Bars: 1mm for A-M and O; 100 µm for N and P.

View larger version (47K): [in a new window]   Fig. 3. Kinetics of UFO expression following an 8-hour pulse of ethanol induction in ufo-2 35S::UFOind apices. (A,B) The expression levels of UFO (A) and the control APT (B) were analysed by RT-PCR at the indicated times following the beginning of an 8h-vapour induction in ufo-2 35S::UFOind apices. (C) The expression level of UFO was quantified as a ratio of the level of APT and was given the arbitrary value 1 at time 0h. Bars represented SE of 6 replicates. (D) Equal amounts of RT-PCR products obtained after amplification of the UFO transcripts from apices of wild-type (WT), ufo-2, or ufo-2 35S::UFOind plants following an 8-hour ethanol induction were digested by AflIII to discriminate between wild-type and mutated transcripts.

View larger version (74K): [in a new window]   Fig. 4. UFO expression pattern during flower development. The expression pattern of UFO was examined by RNA in situ hybridisation in wild type (A-G) and in ufo-2 35S::UFOind before ethanol induction (H,I) and 24 hours after the beginning of an 8-hour ethanol pulse (J,K). (A-E) Transverse sections, (F-K) longitudinal sections. (A-E) UFO is expressed in four discrete spots corresponding to expected positions of the petals. The expression subsists weakly in the petal primordia after their initiation in a stage 6 flower (arrowheads). *, stamens. (F) In wild-type early stage 3 flowers, UFO is expressed in a cup-shaped domain. (G) A stage 4 wild-type flower with UFO expressed at the boundary of the meristem and the sepals. (H) UFO is expressed in a non-induced ufo-2 35S::UFOind early stage 3 flower in a cup-shaped domain similar to the wild-type. (I) In a non-induced ufo-2 35S::UFOind stage 4/5 flower, UFO is expressed at the axils of the sepals. (J,K) 24 hours after an 8-hour ethanol vapour treatment, UFO is uniformly expressed in a ufo-2 35S::UFOind early stage 4 flower (J) or in a stage 4/5 flower (L). A-E are at the same magnification and F-K are at the same magnification. Bars: 100 µm.

View larger version (152K): [in a new window]   Fig. 5. Floral induction of ufo-2 35S::UFOind by LD treatment. ufo-2 35S::UFOind plants were kept under short day conditions and induced for flowering by long day conditions. (A) Apex after 5 LD. No recognisable floral meristem is visible. (B) Apex after 8 LD. Arrowhead points to a floral meristem at stage 2 subtended by a leaf. Arrow indicates an early stage 3 flower in the axil of a leaf-like structure. The oldest floral meristems are at stage 3. (C) Apex after 10 LD and (D) apex after 12 LD. The oldest floral meristems have reached stage 6 or more. Arrow in C points to a flower with reduced growth. Bars: 500 µm.

View larger version (109K): [in a new window]   Fig. 6. Induction of UFO expression at different floral stages of ufo-2 35S::UFOind. ufo-2 35S::UFOind plants were ethanol-induced (A-E) after various durations of floral induction by long day treatment or were not ethanol-induced (F). Ethanol induction after 12 LD has no major effect on floral morphology of the five oldest flowers (A). Induction after 9 LD leads to a majority of type I flowers (B). After 8 LD, ethanol induction leads to a majority of type III flowers (C). Type IV and mutant-type (mt) flowers are observed following induction after 7 LD. (D). Mutant-type flowers develop on treated apices after 5 LD (E) as in non-induced plants (F). nd, flower that could not be classified as a type I-IV or a mutant flower. Bar: 100 µm.

View larger version (18K): [in a new window]   Fig. 7. Summary of floral phenotypes and model of the role of UFO during flower development. (A) Effect on the flower morphology of the timing of UFO induction (blue shading in bars represents the window of UFO expression) in the ufo-2 35S::UFOind line. In the floral diagrams, green represents sepals, white petals, yellow stamens, red carpels, blue filaments and black ovules. Bi-color represent chimeric organs. (1) In the absence of UFO induction or expression later than late stage 6, flowers with a mutant phenotype are produced. (2) Ubiquitous induction of UFO during the floral stage 4/5 or early 6 leads to stamen identity restoration and partial restoration of petal identity. An extra whorl of stamens, carpel fusion defects and abnormal ovules are observed in some flowers. (3) Ubiquitous UFO expression from the floral stage 3 onwards leads to full restoration of petal and stamen identities. The organ primordia and the floral patterning are however misaligned. An extra whorl of stamens, carpel fusion defects and abnormal ovules are observed in some flowers. (4) Ubiquitous UFO expression starting at floral stage 2 leads to complete restoration of petal and stamen development. Pistils show occasional increased carpel number or fusion defect. (5) Transient ubiquitous UFO expression from early floral stage 2 onwards or earlier leads to normal stamen development but absence of petal growth. Pistils are normal. Petaloid sepals develop occasionally. (B) Diagram of the early stages of flower development with the domain of UFO expression shown in blue. The timing of the different role of UFO is represented below. Proper patterning of the primordia in the three inner whorls requires UFO expression during the floral stage 2. The B function activity requires UFO expression during stage 2 and/or 3 but can be induced as late as stage 6 by ectopic UFO expression (dotted line). Proper petal growth requires later UFO expression.