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First published online 21 March 2007
doi: 10.1242/dev.02836
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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.
* Author for correspondence (e-mail: cathie.martin{at}bbsrc.ac.uk)
Accepted 14 February 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Petal, Cell shape, Petunia, Antirrhinum, MYB transcription factor
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Ashman et al., 2000;
Moller, 1995;
Spaethe et al., 2001;
Stanton and Preston, 1988;
Totland, 2004;
Young and Stanton, 1990).
Indeed, in some plants the size of the visual signal is effectively increased
by the clustering of flowers on inflorescences
(Ohara and Higashi, 1994). The
colour signal provided (usually by petals) influences the distance at which
flowers of a particular size can be recognised
(Menzel et al., 1997;
Spaethe et al., 2001). The
colour signal is determined not only by the composition of different pigments
produced in the petals, but also by the patterns of pigmentation, colour
intensity, colour saturation and colour brightness
(Lunau, 2000). In flowers that
are coloured by anthocyanin pigments (blue, purple and red pigments), colour
hue, intensity and brightness are determined not only by the chemical nature
of the pigments that accumulate in the vacuoles of petal epidermal cells, but
also by their environment (pH, presence of metal ions, presence of flavonoid
co-pigments) and by the morphology of the epidermal cells themselves. An
estimated 80% of angiosperms have specialised petal epidermal cells that are
conical or papillate in shape, particularly on the epidermal surfaces that
face prospective pollinators (Kay,
1988; Kay et al.,
1981). Conical cells enhance the colour intensity and brightness
of petal surfaces by reflecting a higher proportion of incident light into the
epidermal cells, where more is absorbed by the vacuolar pigments, relative to
flat petal epidermal cells (which appear pale and dull in comparison)
(Kay, 1988;
Kay et al., 1981;
Noda et al., 1994). Flowers of
Antirrhinum majus with conical epidermal cells are more attractive to
pollinating bees than those with flat petal epidermal cells
(Comba et al., 2000;
Glover and Martin, 1998).
The activity of the MIXTA gene in petal epidermal cells of the
snapdragon, A. majus, controls the development of conical cell shape
(Noda et al., 1994). The
activity of the MYB-related transcription factor encoded by MIXTA
appears to be both necessary and sufficient to drive the formation of conical
epidermal cells from the default flat epidermal cells
(Glover et al., 1998;
Martin et al., 2002).
MIXTA-like genes, encoding MYB-related transcription factors that are
structurally closely related to MIXTA, have been identified in a number of
other plant species and include the PhMYB1 gene from Petunia
hybrida (van Houwelingen et al.,
1998) and the AtMYB16 gene (also referred to as
AtMIXTA) of Arabidopsis thaliana
(Romero et al., 1998).
However, three genes that encode R2R3 MYB transcription factors that are very
closely related to MIXTA (AmMYBML1, AmMYBML2 and AmMYBML3)
(Perez-Rodriguez et al., 2005)
are also expressed in petals of A. majus, showing that there are
multiple genes encoding proteins belonging to this subclass of transcription
factors expressed in the same tissues of one species. These genes are not
functionally redundant, as evident by the clear phenotype of the
mixta mutant. Indeed, discrete functions have already been
established for AmMYBML1 as compared with MIXTA, the former
controlling trichome, conical cell and mesophyll cell morphogenesis in the
ventral petal of Antirrhinum flowers
(Perez-Rodriguez et al.,
2005).
Emerging data from the study of transcription factors belonging to large
families of structurally related proteins suggest that very similar members of
phylogenetically-clustered subgroups usually share closely related functions,
even though functions may have diverged over the entire family. Structural
similarity has led to claims of orthology and functional equivalence between
MIXTA, PhMYB1 and AtMYB16
(van Houwelingen et al., 1998;
Romero et al., 1998). However,
to achieve a general understanding of the control of morphogenesis of petal
epidermal cells, the function of new genes needs to be assayed and compared
with that of the prototype, MIXTA. In addition, the relevance of
these genes to cell shaping and, specifically, to their roles in adapting
petals for pollinator attraction, needs to be established in different
angiosperm species.
We have examined the function of three of the genes encoding proteins very closely related to MIXTA; PhMYB1 from Petunia hybrida, AmMYBML2 from A. majus and AtMYB16 from Arabidopsis thaliana. Structurally, these proteins are most closely related to each other and their genes are therefore orthologous. All three proteins promote directional cell expansion in a bioassay in tobacco. The similarities between the phenotypes induced by these proteins in this bioassay (their biochemical functions) and the similarities in their expression patterns in the three different species, suggest that these proteins have equivalent effects on cellular morphology (their developmental functions). More detailed analysis of the function of PhMYB1 in Petunia, using a transposon-induced unstable mutant, showed that PhMYB1 does indeed have a primary function in determining the degree of extension growth of the epidermal cells of the petal and its activity contributes to the final shape of these cells. Thus, MIXTA and PhMYB1 have overlapping developmental functions despite not being encoded by orthologous genes. The loss of PhMYB1 activity affects petal placement as well as petal cell shape. As a result of loss of PhMYB1 activity, petals are much more reflexed than in the wild type. Re-examination of the mixta mutant phenotype revealed it to have a similar effect on the angle of presentation of the dorsal petal lobes in A. majus. It would appear that cell shaping affects not only petal appearance, but also overall petal design. Both aspects of the activity of these MIXTA-like genes in angiosperm flowers might contribute positively to pollinator attraction.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) plus 60 amino acids downstream, including the nuclear
localisation signal and a conserved QWSAR motif
(Kranz et al., 1998), were
used to create the alignment (see Fig. S1 in the supplementary material).
Phylogenetic analysis was performed with PAUP*4.0b10
(Swofford, 2001). An optimal
tree according to the distance criterion (minimum evolution; mean character
difference) was obtained with a heuristic search (TBR). One thousand
bootstrapped data sets were used to estimate the confidence of each tree
clade. The following sequence accessions were used:
AmMYBMIXTA: X79108.1 GI:485866 from Antirrhinum majus (snapdragon)
AmMYBML1: CAB43399.1 GI:4886264 from A. majus
AmMYBML2: AAV70655.1 GI:56069813 from A. majus
AmMYBML3: AAU13905.1 GI:51895758 from A. majus
OsMYB75: NT_107239.1 GI:50953764 from Orysa sativa (rice)
OsMYB44: NT_079863.2 OsJNBa0072F16.11 from O. sativa
AtMYB16: X99809.1 GI:1514441 from Arabidopsis thaliana
AtMYB17: AF062866.1 GI:3941423 from Arabidopsis thaliana
AtMYB106: NP_186763.2 GI:79386566 from Arabidopsis thaliana
PhMYB1: CAA78386.1 GI:20563 from Petunia hybrida
PpMYB1: X67051.1 GI:22639 from Physcomitrella patens (moss)
For the phylogeny of all R2R3 subgroup-9 members (at the time of going to
press) shown in Fig. S2 (see Fig. S2 in the supplementary material), protein
sequences were aligned using the ClustalW (version 1.83) program
(Thompson et al., 1994).
Phylogenetic analysis was performed with Phylip programs (version 3.63) using
only the MYB domain and the adjacent MYB subgroup-9 motif (see Fig. S2 in the
supplementary material) (Kranz et al.,
1998). A distance matrix method employing the
Jones-Taylor-Thornton model (Jones et al.,
1992) was used to compare the sequences and a tree was built using
the Neighbour-joining clustering method
(Saitou and Nei, 1987). One
thousand bootstrapped data sets were used to indicate the confidence of each
tree clade.
Constructs for ectopic expression of PhMYB1, AmMYBML2 and AtMYB16
The full-length cDNA clone of PhMYB1 was a generous gift from
Javier Paz-Ares (Centro Nacional de Biotecnologia, Madrid, Spain). The
isolation of the AmMYBML2 cDNA has been described previously
(Perez-Rodriguez et al.,
2005). AtMYB16 (At5g15310) was amplified from
first-strand cDNA prepared from RNA from seedlings of Arabidopsis
thaliana ecotype Colombia. The primers used for amplification were 16ats
(5'-GACCTCTCAAAACAATGGGTAGATCAC-3') and 16atas
(5'-GAACATCGGTGAATCCGACGGTGAAG-3'. All three cDNAs were cloned in
sense orientation into pJIT60 (Guerineau
and Mullineaux, 1993) for expression driven by the double CaMV 35S
promoter and terminated by the CaMV 35S terminator sequence. The expression
cassettes were excised with KpnI and XhoI and cloned into
the KpnI and SalI sites of pBin19.
Plant transformation and growth conditions
The constructs in pBin19 were transferred into Agrobacterium
tumefaciens strain LBA4404, and used for transformation of tobacco
(Nicotiana tabacum var. Samsun) by the leaf disc method
(Mattanovich et al., 1989;
Horsch et al., 1985). Tobacco
and Petunia plants were grown in a glasshouse at 22°C with 16
hours light. The binary vector expressing AtMYB16 under the control
of the CaMV 35S promoter was also used for transformation of
Antirrhinum (Colombia) as described by Jin et al.
(Jin et al., 2000).
Petunia hybrida lines
The progeny of two plants of the unstable phmyb1 mutant line from
Petunia hybrida (van Houwelingen
et al., 1998), one showing a mutant phenotype with clear revertant
sectors (line KB1/7) and the other showing a wild-type revertant phenotype
(line KB1/11), were analysed. The offspring of the first plant (KB1/7) showed
sectors like its parent. The wild-type revertant (KB1/11) was heterozygous for
the dTph1 insertion, and progeny segregated - one mutant to three wild-type
individuals, indicating that the unstable phmyb1 allele is recessive
to the wild-type, revertant allele. The absence of the dTph1 transposon
insertion was screened in individual wild-type plants from seed of KB1/11 by
PCR to identify homozygous, wild-type revertants. These plants were used
subsequently as references for phenotypic comparison with the mutant
allele.
Antirrhinum accessions
A stock laboratory line of A. majus (JI:7) was used for species
comparisons. Wild-type revertant (Mixta+) and
mixta mutant lines have been described previously
(Perez-Rodriguez et al., 2005;
Noda et al., 1994). Accessions
of A. barrelieri and A. australe were obtained as vouchers
from the Herbarium at Harvard University.
Localisation of dTph1 insertion in the PhMYB1 gene
Genomic DNA was extracted from two young leaves or from one pair of
prophylls as described by Souer et al.
(Souer et al., 1995). The
entire coding region of the PhMYB1 gene was amplified using
gene-specific primers Ph1-A (5'-GTTGCATTTTTCTCCAATGGG-3') and
Ph1-B (5'-AACTCAACACTCGATCACTAG-3'), subcloned into pGEM-T-easy
(Promega) and sequenced.
RNA extraction and expression analysis
Total RNA was extracted from petals of Petunia (wild-type
revertant line KB1/11) and RNA gels were run and blotted as described by
Martin et al. (Martin et al.,
1985). Equivalent loading of RNA (20 µg per lane) was confirmed
by staining the membrane with a Methylene Blue solution [0.02% (w/v) Methylene
Blue, 0.3 M Na acetate pH 5.5]. A gene-specific probe for PhMYB1 was
used which consisted of a 782 bp DNA fragment from the 3' end of the
gene. This was obtained by PCR amplification using the PhMYB1 cDNA
from plasmid pJAM1354, primers Phmyb1-Probe5
(5'-CGAAGCCGAAGCTCGACTAG-3') and Phmyb1-Probe3
(5'-GAATCTGAGGGTGAAGAATTCAC-3'), and labelled by random priming
with [32P]dCTP.
|
).
Poly(A+) RNA was prepared using poly(A+) columns
(Promega). Poly(A+) RNA (5 µg per lane) was separated on
denaturing gels and blotted onto nitrocellulose
(Martin et al., 1985). The
levels of RNA loaded were checked by probing the membrane with a
UBIQUITIN gene fragment. Probes for MIXTA and
AmMYBML2 were prepared from full-length cDNA clones as described by
Perez-Rodriguez et al. (Perez-Rodriguez et
al., 2005).
RNA in situ hybridisation
In situ hybridisation with digoxigenin-labelled antisense RNA was performed
on 7 µm sections of Petunia hybrida and A. majus flowers
as described previously (Perez-Rodrigues et al., 2005). RNA probes were
generated using T7 polymerase. To produce PhMYB1 sense and antisense
RNA probes, the 3' end of the gene was amplified using primers
Phmyb1-Probe5 and Phmyb1-Probe3 and subcloned into pGEM-T vector in both
orientations. The probe for AmMYBML2 was the full-length cDNA
(Perez-Rodriguez et al.,
2005).
Scanning electron microscopy
Plant tissue was frozen in nitrogen slush at -190°C. Ice was sublimed
at -95°C, and the specimens were then sputter-coated with platinum and
examined in a Philips XL 30 FEG scanning electron microscope (SEM) fitted with
a cold stage. For freeze-fractures, frozen samples were warmed to -100°C
prior to fracture. Cell counts and cell size measurements were made from SEM
micrographs at 400x magnification.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Stracke et al., 2001;
Jiang et al., 2004). Among the
126 members of the R2R3 MYB family in Arabidopsis thaliana, there are
24 subgroups, characterised by their highly conserved DNA-binding domain as
well as conserved amino acid motifs in their C-terminal domains
(Kranz et al., 1998;
Stracke et al., 2001). The
PhMYB1 protein from Petunia hybrida falls into subgroup 9, which also
contains the MIXTA and MIXTA-LIKE MYB proteins AmMYBML1, AmMYBML2 and AmMYBML3
from A. majus, and AtMYB16 (At5g15310) and AtMYB106 (At3g01140) from
Arabidopsis thaliana (Fig.
1) (Perez-Rodriguez et al.,
2005).
PhMYB1 is thought to encode the structural and likely functional
counterpart of MIXTA in Petunia
(Avila et al., 1993;
van Houwelingen et al., 1998).
However, the isolation of AmMYBML1, AmMYBML2 and AmMYBML3
cDNAs from A. majus suggests that PhMYB1 is, in fact,
orthologous to AmMYBML2 rather than to MIXTA
(Fig. 1; see Figs S1 and S2 in
the supplementary material)
(Perez-Rodriguez et al.,
2005). This raised the question of whether these structurally
related proteins share homologous functions, and whether the functions of
MIXTA and AmMYBML1 have diverged from those of AmMYBML2 and PhMYB1.
PhMYB1 is the only MIXTA-like gene in Petunia hybrida
We first undertook experiments to identify R2R3 MYB genes belonging to
subgroup 9 in Petunia hybrida. First-strand cDNA, made from RNA from
developing flower buds, was amplified by 3' RACE and by RT-PCR, using
oligonucleotides from regions of the MIXTA sequence that encode the
amino acid sequences conserved in subgroup-9 MYB proteins
(Kranz et al., 1998;
Stracke et al., 2001). No
transcripts were amplified, even at low annealing temperatures. In addition,
genomic DNA from Petunia was digested with different restriction
enzymes, blotted onto nitrocellulose and probed with labeled DNA fragments
encoding the different parts of the MIXTA protein. Blots were washed at low
stringency. No hybridizing bands were detected, despite DNA fragments
corresponding to PhMYB1 being readily detectable on the same blots
(see Fig. S3 in the supplementary material). We concluded that the
Petunia genome does not contain genes equivalent to the
MIXTA gene of A. majus. PhMYB1 was the only
MIXTA-like gene we could detect in Petunia.
DNA gel blots of Petunia genomic DNA, probed with fragments of the PhMYB1 gene encoding the C-terminal domain of the protein, gave a single DNA fragment when washed at either high or low stringency (see Fig. S2 in the supplementary material). This suggested that PhMYB1 is a single-copy gene and that the Petunia genome does not contain additional genes highly homologous to PhMYB1.
The Antirrhinum genome contains genes encoding three members of
R2R3 MYB subgroup 9: AtMYB16, AtMYB17 and AtMYB106
(Kranz et al., 1998). AtMYB16
and AtMYB106 are structurally very similar to PhMYB1, AmMYBML2 and AmMYBML3
(Fig. 1). No orthologue of
MIXTA is encoded by the Arabidopsis genome.
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), neither
AmMYBML2, PhMYB1 nor AtMYB16 induced the formation of
outgrowths of epidermal cells on organs other than flowers and inflorescence
leaves (see Fig. S4 in the supplementary material). The ectopic outgrowths
induced by 35S:PhMYB1, 35S:AtMYB16 and 35S:AmMYBML2 were
clearest on the carpel surface. The outgrowths never developed into
multicellular trichomes, unlike the response to high ectopic expression of
MIXTA or AmMYBML1 (see Fig. S5 in the supplementary
material) (Glover et al., 1998;
Martin et al., 2002;
Perez-Rodriguez et al., 2005).
Freeze-fractured leaves and petals also showed that ectopic expression of
these genes had no effect on cell layers other than the epidermis (not shown).
These data suggested that AmMYBML2, PhMYB1 and AtMYB16 are
functionally homologous and are involved in the control of cell shape, but
function in a manner distinct from that of MIXTA and
AmMYBML1. We also ectopically expressed AtMYB16 under the control of the double 35S promoter in Arabidopsis. Five independent transgenic lines were analysed. The inner epidermal cells of the petals grew to a greater length and changed from cone-shaped to bullet-shaped (Fig. 2G,H). No other changes in cell morphology in flowers were observed in these lines.
These data from high-level expression in tobacco demonstrated that PhMYB1,
AmMYBML2 and AtMYB16 have equivalent biochemical functions. They can, in
certain tissues, promote the formation of outgrowths, but their primary
function seems to be to promote unidirectional cell expansion once an
outgrowth has been initiated. However, developmental function, particularly
that of transcription factors, is also dependent on the cellular context in
which proteins are active (Lee and
Schiefelbein, 2001).
Expression patterns of PhMYB1, AmMYBML2 and AtMYB16
The expression of the PhMYB1 gene in different organs of
Petunia has been reported by Avila et al.
(Avila et al., 1993). The gene
is highly expressed in flowers, but is also expressed in sepals and at a low
level in leaves. No expression was detected in roots. We analysed the timing
of expression of the PhMYB1 gene, relative to different stages of
flower development, by RNA gel blots. PhMYB1 transcript levels peaked
at stage 3 (Fig. 3A). In situ
hybridisation showed PhMYB1 to be expressed in both the inner and
outer epidermal cells of the corolla limb
(Fig. 3B).
For comparison, we analysed the expression of AmMYBML2 in Antirrhinum. AmMYBML2 was expressed relatively late in petal development, being maximally expressed in the corollas of flowers which had just opened. The peak of AmMYBML2 expression was significantly later during petal development than the expression of MIXTA. AmMYBML2 was expressed in leaves, particularly leaves undergoing expansion growth and mature leaves. Expression was also detected in roots (Fig. 3C). In situ hybridisation showed that AmMYBML2 was expressed at an apparently low level in both epidermal layers of the petals of A. majus (Fig. 3D).
|
).
Both AtMYB16 and AtMYB106 are most highly expressed in
flowers, particularly petals, but expression of both genes was also detectable
in leaves, particularly younger rosette leaves. These data confirmed previous
findings reported by Kranz et al. (Kranz
et al., 1998) and Stracke et al.
(Stracke et al., 2001).
Together with the bioassay analyses these data showed that PhMYB1, AmMYBML2
and AtMYB16 have the same biochemical functions and are expressed in the same
tissues, implying that they probably play equivalent developmental roles.
These proteins are functionally distinct from MIXTA in terms of their
biochemical functions and expression patterns. This separation of function is
also clear in Antirrhinum, where AmMYBML2 is expressed in
the petals of mixta mutants but cannot rescue the conical cell
phenotype that is lost with loss of MIXTA activity (C.M. and M.P.-R.,
unpublished).
|
). To define in more detail the developmental role of
PhMYB1 in Petunia hybrida and to relate its activity to that
of MIXTA, AmMYBML1 and AmMYBML2 in A. majus, we
analysed the changes in cell shape caused by mutation of the PhMYB1
gene.
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), in the PhMYB1 gene. dTph1 is able to undergo
excision, both somatic and germinal, to generate two types of derivative
allele; stable mutant alleles (when the transposon leaves a footprint upon
excision) and stable, wild-type revertant alleles (where the excision event
restores the wild-type sequence) (Gerats et
al., 1990; Kroon et al.,
1994). When derivative alleles are created in germinal cells,
these events give rise to stable derivative alleles in the progeny.
|
;
van Houwelingen et al., 1998).
No obvious phenotypes were seen in any tissues of phmyb1 mutants
other than in the petal epidermis.
dTph1 is inserted into the coding region of PhMYB1
The exact position of the transposon insertion in the PhMYB1 gene
was determined by PCR of the mutant allele using genomic DNA from the
phenotypically unstable line. Two PCR products were obtained, differing in
size by approximately 300 bp (Fig.
5A). Sequencing of the larger DNA fragment established that dTph1
was inserted in the third exon of PhMYB1, a region encoding part of
the C-terminal domain that is conserved among members of R2R3 MYB subgroup 9
(Kranz et al., 1998). In
subgroup-9 MYB proteins, the C-terminal domain comprises two conserved motifs
that are 10 and 22 amino acids long; dTph1 was inserted in the region encoding
the second one, where it introduced a stop codon into the coding sequence
(Fig. 5B). The smaller PCR band
from genomic DNA corresponded to the products of somatic reversion, as
confirmed by sequencing.
Changes in cell shape resulting from the phmyb1 mutation
Differences in cell shape induced by the mutation in the PhMYB1
locus were observed only in the flower petal epidermis. SEM micrographs showed
that cells of the inner epidermis of wild-type Petunia plants had a
conical-papillate shape, with a pentagonal or hexagonal base, appearing very
similar to those found in Antirrhinum
(Fig. 6A,B). However, unlike
Antirrhinum, the cells of the outer epidermis of the petals were
conical, although they grew out less than those of the inner epidermis and
were irregularly shaped at their base (Fig.
6A,C).
The somatically unstable phmyb1 line of Petunia allowed
us to compare the cellular morphology of revertant wild-type sectors with the
background mutant cells, at exactly the same stage of development and in a
uniform genetic background. Fig.
7 shows SEM micrographs of the inner petal epidermis of the
unstable line. phmyb1 mutant cells still developed as small cones,
although they were not as conical as wild-type revertant cells
(Fig. 7A-C). This was in
contrast to the effect of MIXTA in Antirrhinum petals, because
mixta mutant cells of the inner epidermis are flat (see Fig. S6 in
the supplementary material) (Noda et al.,
1994). In the outer epidermis of Petunia, phmyb1 mutant
cells were completely flat and had an irregular shape, like the pavement
epidermal cells of leaves (Fig.
7D), whereas the revertant cells were conical but still had their
lobed base shape. In the inner epidermis, the developmental function of PhMYB1
is distinct from, although related to, that of MIXTA in A. majus. The
developmental function of PhMYB1 in the inner epidermis is probably more
closely aligned to that of AmMYBML2. By contrast, in the outer epidermis, the
role of PhMYB1 appears to be equivalent to that of MIXTA. The phmyb1
mutation had no effect on the development of the multicellular trichomes that
formed in the outer epidermis of flower petals
(Fig. 7D).
|
To understand the basis for this PhMYB1-dependent difference in petal presentation, the number of epidermal cells in a given revertant or mutant area was counted (Fig. 8B). In the inner epidermis, there were more wild-type cells (14% increase on average) than mutant cells (Fig. 8B). These cell counts showed that, on average, phmyb1 mutant cells occupied a greater surface area than equivalently positioned wild-type cells in the inner epidermis. However, in the outer epidermis, there was no significant difference in the number of cells per unit area between mutant and wild-type revertant sectors.
Effect of mutation of the MIXTA gene on petal presentation in A. majus
The effect of the phmyb1 mutation on petal presentation was an
unexpected phenotypic consequence of its effects on cell shape. To determine
whether cell shaping affected the presentation of petals in other species, we
re-examined mixta mutants of A. majus and compared them with
wild-type lines. Differences in petal presentation were observed only for the
dorsal petals of the corolla. In freshly opened flowers of wild-type lines
with conical inner epidermal cells, the dorsal petal lobes were relatively
straight and presented at an angle to the direction of approach of prospective
pollinators (Fig. 9). In a
somatically stable mixta mutant line caused by the insertion of the
Tam4 transposon (Noda et al.,
1994), and in an independent EMS-generated
mixta- allele
(Perez-Rodriguez et al.,
2005), the dorsal petals were reflexed, as compared with wild-type
lines, in a manner analogous to the phmyb1 mutant phenotype
(Fig. 9).
Cell shape and petal presentation in species of Antirrhinum
In some of the wild species of the genus Antirrhinum there are
marked differences in the presentation of the dorsal petals. In A.
barrelieri, for example, the dorsal petals are erect with no reflexing
(Fig. 10H), whereas in A.
australe the petals are highly reflexed
(Fig. 10I) so that the area
visible (looking at the flowers straight on) is only about 50% of the total
area of the dorsal lobes. We examined the petal epidermal cells in A.
barrelieri and A. australe by SEM and found that although the
cells in A. barrelieri were significantly smaller than those in
A. majus and A. australe, they were steeply conical
(Fig. 10B,E). By contrast, the
cells of A. australe were very shallow cones
(Fig. 10C,F). RNA gel blot
analysis showed MIXTA to be more highly expressed in A.
barrelieri than in A. majus and much more highly expressed than
in A. australe. AmMYBML2 was expressed equally in A.
barrelieri and A. australe, and at higher levels than in
equivalent flowers of A. majus
(Fig. 10J). This suggested
that differences in the degree of petal reflexing between Antirrhinum
species are probably a function of the differences in extension growth of the
inner epidermal cells of the dorsal petals which, in turn, are a function of
the relative activity of the MIXTA-like genes in the different
species.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The
AmMYBML1 protein from A. majus can also induce the formation of
ectopic conical cells and trichomes when overexpressed in tobacco
(Perez-Rodriguez et al.,
2005). The similarity of the tobacco phenotypes induced in the
same bioassay by the activity of each of the three genes, AmMYBML2,
AtMYB16 and PhMYB1, suggests that these genes are functionally
equivalent. It also suggests they are involved in controlling cell shape, but
that their activity, although similar, is distinct from that of MIXTA
and AmMYBML1 (see Fig. S5 in the supplementary material). In fact,
the phylogenetic analysis of the subgroup-9 proteins, and our failure to find
a gene orthologous to MIXTA in Petunia hybrida, suggest that
PhMYB1, AmMYBML2 and AtMYB16 are the more ancestral genes in
dicotyledonous flowers and that MIXTA and AmMYBML1 might
have resulted from more recent gene duplications
(Fig. 1 and see Fig. S2 in the
supplementary material). Sub-functionalisation following gene duplication
might have given rise to MIXTA, which has very strong activity in
inducing cellular outgrowths, and to AmMYBML1, which promotes the
formation of trichomes and conical cells and tissue folding in the ventral
petal of the corolla, a feature specific to the Scrophulariaceae
(Perez-Rodriguez et al.,
2005).
|
) and
Antirrhinum (Martin et al., 2001). Such co-factors might be present
in epidermal tissues in a gradient, the maximum level being in flowers. In
some cases, R2R3 MYB factors are known to interact with other proteins. For
example, the C1 and Pl proteins of maize interact with bHLH proteins to induce
the synthesis of anthocyanin (Goff et al.,
1992; Lloyd et al.,
1992), and the R2R3 MYB transcription factor GL1 interacts with
the bHLH proteins GL3 and EGL3 and the WD40-repeat-containing protein TTG1 to
promote the formation of trichomes in Antirrhinum leaves
(Larkin et al., 1994;
Payne et al., 2000;
Schiefelbein, 2003;
Zhang et al., 2003;
Zimmermann et al., 2004a).
However, the R2R3 MYB subgroup-9 proteins lack the consensus signature motif
for interaction with bHLH partners
(Grotewold et al., 2000;
Zimmermann et al., 2004a;
Serna and Martin, 2006), so
interacting proteins that contribute to the functional specificity of this
group of proteins are unlikely to be group IIIf bHLH co-factors
(Heim et al., 2003;
Zimmermann et al., 2004a). An
alternative explanation for the phenotypic differences between
2x35S::MIXTA and 2x35S::PhMYB1 is that, in the cells lacking
a visible phenotype, other factors may negatively regulate responses to the
specific MIXTA-like genes.
PhMYB1 and MIXTA play related but distinct roles in petal conical cell development
The phenotype of unstable phmyb1 mutant petals resembles that of
unstable mixta mutant lines: darker revertant sectors are visible on
a pale background of mutant cells. However, whereas the mutation in the
MIXTA gene results in flat inner epidermal cells (see Fig. S6 in the
supplementary material), the inner epidermal cells in the phmyb1
mutant are still able to develop into cones
(Fig. 7B). The conical shape of
phmyb1 mutant cells is shallower than that of wild-type cells in
Petunia. This result reinforces the view that MIXTA and
PhMYB1 are not functionally identical, and suggests that the
formation of fully developed conical cells in the inner epidermis of petals
might require two distinct activities: one, conferred by MIXTA in
Antirrhinum, might initiate the change in growth direction and direct
the cells to grow in a polar manner, mainly along one axis; whereas the second
activity, conferred by PhMYB1 in Petunia and
AmMYBML2 in Antirrhinum, might be responsible for a second
phase of elongation that leads to the formation of a complete cone. In
Antirrhinum, where the first activity is conferred by MIXTA,
and the second by AmMYBML2, the relative activity of these two genes
might determine the final shape of the petal epidermal cells. In
Petunia, the identity of the first activity is unknown; it could be
another R2R3 MYB member of subgroup 9, very similar to PhMYB1 (although we
could find no molecular evidence for the existence of additional genes
encoding subgroup-9 proteins in Petunia), or it might not be a MYB
protein at all, but rather the result of a mechanism for preparing cells for
shape changes (A. Gouveia, PhD thesis, University of East Anglia, 2005).
Unlike the inner epidermal cells, mutant cells of the petal outer epidermis of the phmyb1 mutant of Petunia are flat. This suggests that the formation of cones in the outer epidermis of Petunia petals is not controlled in exactly the same way as in the inner epidermal cell layer, as the formation of cones in the outer epidermis is completely dependent upon the activity of PhMYB1. Moreover, this shows that although PhMYB1 is not orthologous to MIXTA, its function is closely related. Interestingly, the orthologous gene to PhMYB1, AmMYBML2, is unable to induce conical cell formation in the outer epidermis of the petals of Antirrhinum, even though it is expressed in these cells. Perhaps it is not expressed at high enough levels to induce outgrowths of the outer epidermal cells.
|
).
Therefore, it is likely that the effects of MIXTA on petal
presentation result from similar effects on periclinal expansion of the inner
epidermal cells relative to the expansion of the outer epidermal cells, as
suggested for PhMYB1 in Petunia. Three species of Antirrhinum show different degrees of reflexing of their dorsal petal lobes and the shapes of their cells in the inner epidermis differ significantly. A. barrelieri has the most upright dorsal petals and the most conical epidermal cells. A. majus has more-reflexed dorsal petals and less-steep cones on its inner epidermis. The highly reflexed petals of A. australe have the flattest conical cells of the three species. The degree of steepness of the cones in the three species is inversely correlated with the degree of petal reflexing, but positively correlated with MIXTA transcript levels, emphasising the importance of epidermal cell shape in determining petal form.
The effect of loss-of-function of R2R3 MYB subgroup-9 proteins on petal
curvature is likely to have appreciable effects on the attractiveness of the
flowers to prospective pollinators. The petal reflexing, observed in both the
phmyb1 and the mixta mutants, causes an effective reduction
in the diameter of the corolla, the parameter that is the principal visual
signal identified by pollinators at a distance
(Menzel et al., 1997). We
estimate that the effect of the phmyb1 mutation is to reduce the
apparent diameter of the corolla by at least 20%. This would affect the
distance at which the floral signal could be recognised. Extrapolation of
empirical data for bees suggests the recognition distance might be reduced by
as much as 12 cm (Menzel et al.,
1997). Reflexing of the petals also affects the degree of colour
saturation across the corolla (Eckert and
Carter, 2000). Varying domains of colour saturation might provide
pollinators with targeting information over shorter distances. Consequently,
the effects of these mutations might extend to modifying these short-range
signals as well as affecting recognition at distance through changing
perceived corolla size. The shape of the petal epidermal cells also affects
the perceived intensity of the colour signal from the petals and its
brightness as a result of differences in the reflection and absorption of
light by these differently shaped cells
(Gorton and Vogelmann, 1996;
Kay, 1988;
Kay et al., 1981;
Noda et al., 1994). Field
trials have shown that flowers of A. majus with conical cells
(Mixta+) are more attractive to bees than flowers with
flat epidermal cells (mixta-), particularly under
conditions of low pollinator density (Comba
et al., 2000; Glover and
Martin, 1998). This might be due partly to the differences in the
perceived colour intensity of the flowers, but pollinators showed preferences
for flowers with conical epidermal cells even where no anthocyanin pigments
were produced and the flowers were white. The explanation for this additional
dimension to the positive signal provided by conical petal cells might be the
effect of epidermal cell shaping on corolla reflexing and perceived corolla
size. These aspects of petal design represent additional parameters under the
control of members of the MIXTA-like family of MYB transcription factors that
direct the morphogenesis of petal epidermal cells for their specialised
functions in pollinator attraction.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/9/1691/DC1
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ACKNOWLEDGMENTS |
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