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First published online 16 December 2004
doi: 10.1242/dev.01584

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Development of three different cell types is associated with the activity of a specific MYB transcription factor in the ventral petal of Antirrhinum majus flowers

Maria Perez-Rodriguez^1,*, Felix W. Jaffe^2,, Eugenio Butelli¹, Beverley J. Glover² and Cathie Martin^1,

¹ Department of Cell and Developmental Biology, John Innes Centre, Norwich, NR4 7UH, UK
² Department of Plant Sciences, University of Cambridge, Downing Street, Cambridge, CB2 3EA, UK

View larger version (41K): [in a new window]   Fig. 1. Cell types in Antirrhinum petals and AmMYBML1 structure. (A) Trichomes are present on both epidermal surfaces of the tube and on the outer epidermis of the petal lobes. The inner epidermis of the petal lobes comprises conical cells. The sheet of conical epidermal cells is highly folded in the region of the hinge to give an undulating surface. T. tube; DP, dorsal petal; VP, ventral petal; LP, lateral petal. (B) Phylogenetic relationships between the protein sequences encoded by R2R3 MYB proteins of subgroup 9. AtMYB16, 106 and 17 are from Arabidopsis thaliana (Kranz et al., 1998; Strake et al., 2001), PhMYB1 is from Petunia hybrida (Avila et al., 1993), AmMYBMIXTA, AmMYBML1, AmMYBML2 and AmMYBML3 are from Antirrhinum majus (Noda et al., 1994; Glover et al., 1998) (this paper). The sequences of AtMYBWER and AtMYBGL1 from Arabidopsis (which are R2R3 MYB proteins belonging to subgroup 15 of the R2R3 MYB gene family) are included as outliers (Kranz et al., 1998; Stracke et al., 2001). (C) RNA gel blot showing expression of AmMYBML1 in corollas of flower buds of different ages (between 0-5 mm and 25-30 mm long), leaves (young, yo; medium, med; old) and roots.

View larger version (117K): [in a new window]   Fig. 2. In situ hybridisation of AmMYBML1 in Antirrhinum flower buds. (A) Entire bud probed with AmMYBML1. Expression was observed only in the ventral petal. Se, sepal; DP, dorsal petal; St, staminal filament; C, carpel; VP, ventral petal; B, bract; arrow indicates expression. (B) Higher magnification view to show AmMYBML1 expression on the adaxial side of the ventral petal. (C) Flower bud of equivalent age (to B) probed with Cyclin D3b. There is strong labelling in ovules, anthers and in the ventral petal of the corolla (arrowed). (D) Flower bud from cyc dich double mutant showing AmMYBMl1 expression in the developing petals. All the petals in this line have ventral identity, and AmMYBML1 is expressed (arrowed) in the petals that lie in the dorsal position (DP) as well as in the ventral position (VP). Scale bars: 100 µm. (E) In situ hybridisation of bud (15-25 mm) showing expression of AmMYBML1 in the ventral petal. Labelling was observed in the adaxial epidermis of the tube, in the trichomes of the tube, and in the hinge but not in the main part of the petal lobe epidermis. (F) Expression of AmMYBML1 in the ventral petal. In the hinge region the gene is expressed in the adaxial (inner) epidermis and in mesophyll cells on the adaxial side. (G) In contrast to B, MIXTA is expressed only in epidermal cells, even in the hinge region. Scale bars: 100 µm.

View larger version (156K): [in a new window]   Fig. 3. Morphological consequences of strong constitutive expression of AmMYBML1 in transgenic tobacco. (A) Scanning electron micrograph (SEM) of control (N. tabaccum var. Samsun) leaf, upper epidermis. (B) Tobacco leaf of plant expressing AmMYBML1 under the control of the CaMV 35S promoter (35S-AmMYBML1 plant), upper epidermis. (C) Petal inner epidermis of control. (D) Inner petal epidermis of 35S-AmMYBML1 plant; note glandular trichomes. (E) Carpel of control. (F) Carpel of line expressing AmMYBML1. (G) Higher magnification SEM of carpel epidermis of 35S-AmMYBML1 line; note ectopic production of both conical cells and trichomes. (H) RNA gel blot of transgenic tobacco lines carrying 35S-AmMYBML1 or a control line (Samsun) probed with the complete AmMYBML1 cDNA sequence.

View larger version (150K): [in a new window]   Fig. 4. Further exploration of the function of AmMYBML1. (A) Freeze fracture SEM through the corolla hinge of A. majus. This flower was taken from a mixta mutant, which has flat epidermal cells on the inner epidermis of the corolla lobes. However, a few conical cells are present in the hinge region (indicated by arrows), presumably as a result of AmMYBML1 activity, which is expressed, in lobes, just in the hinge region. (B) Conical cells (indicated by arrows) in the hinge region of an EMS-induced mixta mutant line are shown in more detail. (C) In situ hybridisation showing the expression of AmMYBML1 in the mesophyll tissue of the folds of the ventral petal (arrowed) on the adaxial side of the hinge. ct indicates the corolla tube tissues; cl the corolla lobe tissues. (D) The mesophyll of the folds of the petal hinge consists of compact and tightly packed cells, especially towards the adaxial side. (E) The mesophyll cells of the petal lobes are more loosely packed (compare with D). (F) The cells of the petal mesophyll have projections which contact other cells. (G) Transverse section through wild-type tobacco petal lobe for comparison with H. (H) Section through petal lobe of a tobacco line ectopically expressing AmMYBML1. Expression of AmMYBML1 thickens the petal through increased expansion of the mesophyll cells (compare with G). Scale bars: 200 µm.

View larger version (91K): [in a new window]   Fig. 5. Analysis of the AmMYBML1 promoter. (A) The AmMYBML1 promoter, showing the locations of the TATA box, putative I-boxes, CArG boxes and the MYB-binding site II (MBSII), recognised by proteins such as GAMYB (Solano et al., 1995). The different lengths of the promoter fragments tested in GUS reporter assays in tobacco are indicated in different shades of grey; 1571 bp fragment in white, 1260 bp fragment in very pale grey, 884 bp promoter in mid grey, 223 bp promoter in darker grey. (B) Expression of GUS driven by the full-length AmMYBML1 (1571 bp) promoter in tobacco petal trichomes. (C) Expression of GUS driven by the full-length AmMYBML1 promoter in tobacco petal conical cells. (D) Expression of GUS driven by the 884 bp AmMYBML1 promoter in tobacco leaf vascular tissue. (E) Expression of GUS driven by the 884 bp AmMYBML1 promoter in the trichomes of tobacco leaves.

View larger version (113K): [in a new window]   Fig. 6. Effects of mutation of the DIV gene on development of the ventral petal of A. majus and expression of AmMYBML1. (A) Light micrograph of the corolla tube of the ventral petal of a wild-type flower of A. majus, showing the mass of hairs in the throat of the tube that collect pollen, the strips of yellow hairs (nectar guides) and the folds of the ventral petal that reinforce the hinge. (B) Light micrograph of the corolla tube of the ventral region of a div mutant flower for comparison with A. The div mutant petal lacks the hairs in the throat of the tube and the folds around the hinge. (C) SEM of the corolla tube of the ventral petal of a wild-type flower. Scale bar: 1mm. (D) SEM of the corolla tube of the ventral region of the corolla of a div mutant flower. Scale bar: 1 mm. (E) RNA gel blots showing the effects of the div mutation on expression of AmMYBML1 and MIXTA. Div and div represent RNA from wild-type corollas and div mutant corollas, respectively. (F) SEM of folds of ventral petal in the region of the hinge of a wild-type flower. Scale bar: 100 µm. (G) SEM of region equivalent to F around the hinge in div mutant flower. Only very slight undulations of tissue are observed compared with F. Scale bar: 100 µm. (H) SEM of surface of ventral petal lobe of a div mutant flower. The hinge is on the left. F indicates flat cells; C indicates conical cells. Scale bar: 1 mm. (I) SEM of petal epidermal cells of ventral petal lobe in a region more distal to the hinge (than in G) in a wild-type flower. Scale bar: 100 µm. (J) SEM of petal epidermal cells of petal lobe in the ventral position in a region more distal to the hinge (than in H) in a div mutant flower. Scale bar: 100 µm.

View larger version (60K): [in a new window]   Fig. 7. Effects of weak alleles of the DEF gene on development of the ventral petal of A. majus and expression of AmMYBML1. (A) Light micrograph of the corolla tube of the ventral region of a def101 mutant flower for comparison with wild type shown in Fig. 6A. The def101 mutant petal has many fewer hairs in the throat of the corolla tube and less folds around the hinge. (B) SEM of the corolla tube of the ventral petal of a def101 mutant flower. There are far fewer hairs in the throat of the tube and fewer folds around the hinge. Scale bar: 1 mm. (C) SEM of folds of ventral petal in the region of the hinge of a def101 mutant ventral petal for comparison to the structure of this region in the wild-type corolla shown in Fig. 6F. (D) SEM of surface of the ventral petal lobe of a def101 mutant flower in the region of the hinge. The hinge is on the left. All the epidermal cells are flat. This should be compared with the wild type shown in Fig. 6F. (E) Quantitative RT-PCR of AmMYBML1 transcript levels in corollas of wild type (Def), def101 mutant, def chlor mutant and lines heterozygous for def101 and def chlor. Transcript levels of AmMYBML1 should be compared with the levels of control gene, encoding ubiquitin (UBI) in the same tissues.