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First published online 21 March 2007
doi: 10.1242/dev.02836

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Control of cell and petal morphogenesis by R2R3 MYB transcription factors

¹ Department of Cell and Developmental Biology, John Innes Centre, Norwich NR4 7UH, UK.
² Departamento de Biología Molecular y Bioquímica, Universidad de Málaga, 29071 Málaga, Spain.
³ University of California, Riverside, Department of Plant Pathology, Center for Plant Cell Biology, 3447 Boyce Hall, Riverside, CA 92521, USA.
⁴ Department of Developmental Genetics, Institute for Molecular Biological Sciences, Vrije Universiteit, de Boelelaan 1087, 1081 HV Amsterdam, The Netherlands.

View larger version (7K): [in this window] [in a new window]   Fig. 1. Phylogram of R2R3 MYB proteins belonging to subgroup 9. Bootstrap values are indicated at the nodes of the branches (values inferior to 50% have been omitted). PpMYB1 was used as an outgroup to root the tree. For the sources of the sequences, see Materials and methods.

View larger version (150K): [in this window] [in a new window]   Fig. 2. Effect of ectopic expression of AmMYBML2, AtMYB16 and PhMYB1 on epidermal cells. (A-D) SEM micrographs of conical inner epidermal cells of wild-type tobacco petal limb (A), and of tobacco petals transformed with 2x35S::AmMYBML2 (B), 2x35S::PhMYB1 (C), or 2x35S::AtMYB16 (D). The activity of each of the three genes causes the inner epidermal petal cells to grow to a greater length than the wild-type cells. (E,F) Micrographs of carpel epidermis of wild-type tobacco (E) and of cellular outgrowths on tobacco carpels transformed with 2x35S::PhMYB1 (F); tobacco carpels overexpressing AmMYBML2 or AtMYB16 have the same phenotype (not shown). (G,H) Micrographs of conical inner epidermal cells of wild-type Arabidopsis thaliana petals (G), and of petals transformed with 2x35S::AtMYB16 (H), where the cells grew to a greater length than in wild-type. Scale bars: 20 µm.

View larger version (48K): [in this window] [in a new window]   Fig. 3. Comparison of expression patterns of PhMYB1 and AmMYBML2. (A) Expression of PhMYB1 during petal development of Petunia hybrida. RNA gel blot analysis of total RNA from petals of increasing size (1, 0-1 cm; 2, 1-2 cm; 3, 2-3 cm; 4, 3-4 cm; 5, open flower). The RNA was probed with a fragment corresponding to the 3' end of the PhMYB1 gene. (B) In situ hybridisation of PhMYB1 (antisense probe) in petals of Petunia between stages 2 and 3. The control shows hybridisation to the sense probe. Expression of PhMYB1 was detected in both the inner and outer epidermis of the corolla lobe. (C) Expression of AmMYBML2 in A. majus. RNA gel blot analysis of poly(A+) RNA from petals of increasing size (1, 0-0.5 cm; 2, 0.5-1 cm; 3, 1-1.5 cm; 4, 1.5-2 cm; 5, 2-2.5 cm; 6, 2.5-3 cm), leaves (y, young; me, medium; ma, mature) and roots, probed with the full-length AmMYBML2 cDNA. Petal RNA was also probed with MIXTA to show that expression of AmMYBML2 peaks later than that of MIXTA. (D) In situ hybridisation of AmMYBML2 in petals of A. majus at stage 4, showing weak expression in both the inner and outer epidermal cell layers. The control shows hybridisation to the sense probe. Scale bars: 100 µm.

View larger version (89K): [in this window] [in a new window]   Fig. 4. Phenotype of the phmyb1 mutant from Petunia hybrida. (A) Petals show darker revertant sectors (white arrow) distinguishable from a paler background of mutant cells. (B) Micrographs of the inner epidermis of an unstable phmyb1 line showing a revertant sector (the boxed region in A). Mutant cells and wild-type revertant cells have different shapes. Scale bar: 100 µm.

View larger version (21K): [in this window] [in a new window]   Fig. 5. The dTph1 transposon is inserted in the coding region of the PhMYB1 gene. (A) Agarose gel stained with ethidium bromide showing the bands obtained by PCR amplification of the PhMYB1 sequence from genomic DNA of six unstable phmyb1 lines. The difference in size between the lower and the upper bands corresponds to the size of dTph1. (B) Diagram of the phmyb1 allele showing the position of the transposon insertion. Hatched boxes represent introns. The blue boxes correspond to the R2R3 MYB domain and the stippled box corresponds to the conserved region in the C-terminal domain that identifies subgroup 9 within the R2R3 MYB gene family. This region comprises two conserved stretches of 10 and 22 amino acids; dTph1 (purple box) is inserted in the second one, where it introduces a stop codon into the phmyb1 open reading frame.

View larger version (90K): [in this window] [in a new window]   Fig. 6. Cell shapes in wild-type Petunia hybrida flower petals as seen by SEM. (A) A freeze-fracture across a wild-type petal showing the presence of conical cells in both the inner/adaxial and outer/abaxial epidermis. ie, inner epidermis; m, mesophyll; oe, outer epidermis. (B) Inner epidermal cells of wild-type petals. (C) Cells of the outer epidermis. The base shapes of one cell in the inner and outer epidermal layers are outlined in pink. t, trichome. Scale bars: 50 µm.

View larger version (134K): [in this window] [in a new window]   Fig. 7. Differences in the shape of wild-type and phmyb1 mutant cells in petals of the unstable phymb1 mutant. (A) SEM micrograph of the inner epidermis in which a revertant sector can be seen on a background of mutant cells that retain a flatter conical shape. (B,C) Freeze-fracture across one sector of the inner epidermis showing in detail the shape of revertant wild-type cells (C), which grow to a greater length than mutant cells (B). (D) The outer epidermis of unstable phmyb1 lines, showing sectors of revertant conical cells and flat mutant cells, both with lobed base shapes. Trichomes (t) also develop in the outer epidermis of Petunia hybrida petals but are unaffected by the inactivation of the PhMYB1 gene. Scale bars: 100 µm in A,D; 20 µm in B,C.

View larger version (48K): [in this window] [in a new window]   Fig. 8. Effect of the phmyb1 mutation on the shape of Petunia flowers. (A) Wild-type flowers seen from the top (a) and side (d). Top view (b) and side view (e) of an unstable phmyb1 flower. A revertant sector occupies two-thirds of the corolla lobe (left). The mutation causes the corolla (right third) to recurve downwards, whereas the wild-type sector stays in a straight position. (c) The effect of the mutation can also be clearly observed in flowers where most of the corolla is recurved downwards, except for the area (top left) corresponding to a PhMYB1+ revertant sector. (B) PhMYB1 affects the lateral expansion of cells. Cells were counted in a given area of mutant and wild-type sectors in petals of phmyb1 unstable lines, in the inner and outer epidermal cell layers. (Left) Example areas of the inner epidermis chosen for cell counts in a revertant sector (black box), and in an equivalently sized region of an adjacent mutant area (white box). The bar chart shows that in the inner epidermis, the number of cells per unit area was lower in phmyb1 mutant areas as compared with wild-type revertant sectors, whereas in the outer epidermis there was no significant difference. The values shown are the means from ten values±s.e.

View larger version (32K): [in this window] [in a new window]   Fig. 9. Effects of mutation of MIXTA on the reflexing of the dorsal petal lobes of A. majus. (A) Wild-type Mixta+ line, side and top view. (B) Isogenic stable mixta- line (mixta::Tam4) (Noda et al., 1994), side and top view. (C) Wild-type Mixta+ line, side and top view. (D) EMS-induced mixta- line (Perez-Rodriguez et al., 2005), side and top view.

View larger version (36K): [in this window] [in a new window]   Fig. 10. Comparison of petal reflexing, epidermal cell shape and the levels of MIXTA and AmMYBML2 transcripts in petals of recently opened buds of A. majus, A. barrelieri and A. australe. (A-C) Freeze-fracture SEM micrographs of sections across the mid-point of the left dorsal petal of A. majus (A), A. barrelieri (B) and A. australe (C). The cells in A. barrelieri are more conical than in A. majus. The cells in A. australe are the flattest. (D-F) This is supported by surface SEM views of these cells in A. majus (D), A. barrelieri (E) and A. australe (F). Scale bars: 50 µm. (G-I) The degree of reflexing of the dorsal petals is small in A. majus (G), minimal in A. barrelieri (H) and very strong in A. australe (I). (J) RNA gel blots of poly(A+) RNA from buds just prior to opening of A. majus (A.m), A. australe (A.a) and A. barrelieri (A.b), showing the relative expression of MIXTA and AmMYBML2 in the different species of Antirrhinum. A cDNA fragment encoding ubiquitin was used to probe the RNA gel blots as a loading control. The ubiquitin transcript was polymorphic in A. majus (two bands). Only the larger transcript was detected in A. australe, whereas in A. barrelieri only the smaller transcript was detected.

View larger version (4K): [in this window] [in a new window]   Fig. 11. Model suggesting how competing directional growth of the inner epidermal cells of the petal might influence the periclinal expansion of the inner epidermis and influence the overall presentation of petals.