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
Author for correspondence (e-mail:
cathie.martin{at}bbsrc.ac.uk)
Accepted 16 November 2004
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SUMMARY |
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Key words: MYB transcription factor, AmMYBML1, DIVARICATA, Trichome, Conical cell, Petal
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Introduction |
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Trichomes develop in specific regions of the petals of
Antirrhinum, including the inner epidermis of the corolla tube and
the outer epidermis of the petal lobes
(Fig. 1A). Inside the mouth of
the flower, in the throat of the corolla tube, a mass of trichomes forms that
collects pollen from the undersides of pollinators. Beyond this mass,
extending down the ventral/lateral petal border to the base of the anther
filaments, are two strips of yellow trichomes that act as nectar guides for
bees (Raman, 1990;
Galego and Almeida, 2002
).
Analysis of graminifolia mutant flowers, in which the petals do not
fuse to form the corolla tube, shows that the mass of trichomes in the throat
of the tube forms largely on the ventral petal. The strips of yellow
trichomes, which serve as nectar guides, are determined in part by the ventral
petal and in part by the lateral petals (C.M., unpublished). The glandular
heads of the trichomes are one of the sites of scent (methyl benzoate)
production (Kolosova et al.,
2001
). Given that Antirrhinum flowers produce numerous
multicellular glandular trichomes, but that MIXTA function is
confined to the induction of conical cells on the inner petal epidermis, we
investigated whether other MYB transcription factors related structurally to
MIXTA might be involved in controlling trichome formation.
The AmMYBML1 (Antirrhinum majus MYB MIXTA LIKE 1) gene
encodes a protein structurally very similar to MIXTA. Here, we describe the
functional characterisation of AmMYBML1. Ectopic expression of the
gene in tobacco shows that it can induce the production of both trichomes and
conical cells on floral tissues. In Antirrhinum, the
AmMYBML1 gene is expressed only in the ventral petal. Within the
ventral petal its expression is localised to the conical epidermal cells of
the hinge, the expanded mesophyll of the hinge and the inner (adaxial)
epidermis, including trichomes, within the corolla tube. Floral expression is
determined by the promoter of AmMYBML1. Loss of function of the
DIVARICATA (DIV) gene, which specifies ventral petal
identity (Galego and Almeida,
2002), results in a large reduction in AmMYBML1
transcript levels and loss of the three specialised cell types in which
AmMYBML1 is highly expressed. Weak alleles of DEFICIENS
(DEF), which encodes one of the B-function MADS-domain transcription
factors required for petal identity, also result in reduced expression of
AmMYBML1 in petals, and cause similar losses in the three specialised
cell types as mutants of DIV. These data suggest that
AmMYBML1 is involved in the differentiation of three distinct
specialised cell forms and so contributes to petal form and function; this
role is dictated, in large part, by the specific expression pattern of
AmMYBML1, which is controlled by DIV in association with B-function
MADS-domain proteins.
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Materials and methods |
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Construct for ectopic expression of AmMYBML1 in tobacco
A sense construct for ectopic expression of AmMYBML1 in tobacco
was created by excising the whole AmMYBML1 cDNA from pCR2.1 using
SnaBI and BamHI and inserting it into the HincII
and BamHI sites of pJIT60
(Guerineau and Mullineaux,
1993). A cassette containing the cDNA driven by the double CaMV
35S promoter and with the CaMV terminator sequence was excised using
SstI and BamHI sites, and inserted into the SstI
and BglII sites of the pBin19 binary vector
(Bevan, 1984
).
AmMYBML1 Promoter::GUS reporter constructs
The genomic sequences of AmMYBML1 were identified by screening a
genomic library from A. majus in lambda EMBL4 (a gift from Hans
Sommer, MPI Koln). Primers were designed to amplify a large region of promoter
sequence (1.6 kb), and a selection of shorter promoter fragments, each with
BamHI and XbaI restriction sites on the end. These PCR
products were digested and ligated into a
BamHI/XbaI-digested pGREEN-GUS binary vector
(Hellens et al., 2000). The
AmMYBML1 promoter sequence has been submitted to EMBL/GenBank
(AY661653), along with the AmMYBML2 and AmMYBML3 cDNA
sequences (AY821655 and AY661654, respectively).
Plant transformation
Constructs were transformed into Agrobacterium tumefaciens LBA4404
using electroporation (Mattanovich et al.,
1989). Tobacco leaf discs were transformed using the method of
Horsch et al. (Horsch et al.,
1985
).
Expression analysis
RNA was extracted and transcript levels were monitored by northern
analysis, using the method of Martin et al.
(Martin et al., 1985).
Quantitative RT-PCR was undertaken using the method of Jin et al.
(Jin et al., 2000
) using
oligonucleotide primers G3540 (5'-ATTTGGTGCTGAGGTTGAGA-3') and
G3543 (5'-ACAACTGACTCCAGCAAACG-3') for the ubiquitin
cDNA, and F4266 (5'-CATTGTACGTACGTTCATCTTTAGTTAGCTTC-3') and F4267
(5'-GGGCGCGCCAGCTTCCATGACCATGTTCTT-3') for the AmMYBML1
cDNA. Twenty cycles were used to amplify the ubiquitin cDNA within
the linear range and 15 cycles for AmMYBML1.
Histochemical GUS assays
Tissue samples were immersed in GUS staining solution (40 mg/ml X-Gluc in
100 mM NaH2PO4, 10 mM Na2EDTA, 0.5 mM K
ferrocyanide, 0.5 mM K ferricyanide, 0.1% Triton X-100, pH 7) and incubated at
37°C for 3 hours. Samples were destained with 70% ethanol and examined
using a light microscope.
Protoplast transfection assays for transcriptional regulators of the AmMYBML1 promoter
Transformation of tobacco (Nicotiana tabacum cv Samsun)
protoplasts was performed as described by Negrutiu et al.
(Negrutiu et al., 1987).
Plasmid DNA (10 µg) containing the AmMYBML1 promoter region (1571
bp) fused to the GUS gene was used for each transfection. Different
combinations of 4 µg of plasmids containing cDNA sequences encoding DIV,
DEF, GLO and DEF-VP16 under the control of the double CaMV35S promoter were
used to test expression from the AmMYBML1 promoter. Varying amounts
of the plasmid pJIT60 were used to ensure that equal total amounts of DNA were
introduced for each transfection and to control for any possible effects of
the CaMV35S promoter in titrating out transcription factors. After
transfection, protoplast extracts were assayed for ß-glucuronidase (GUS)
activity according to Jefferson
(Jefferson, 1987
). All
transfections were performed in duplicate, and GUS assays were performed in
duplicate for each transfection. Similar results were obtained in at least two
independent experiments for each effector combination. GUS activity was
calculated as nmol methylumbelliferone per mg protein per minute.
Scanning electron microscopy
Plant tissue was examined under a CamScan mark IV scanning electron
microscope with a Hexland cryostage.
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Results |
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Phylogenetic analysis of AmMYBML1, AmMYBML2 and AmMYBML3
The amino acid sequences of the three MIXTA-like proteins, AmMYBML1,
AmMYBML2 and AmMYBML3, were compared with the primary sequences of other R2R3
MYB proteins using CLUSTALW. The amino acid sequence in the two MYB domains of
R2R3-MYBs is highly conserved, and phylogenetic analysis places AmMYBML1,
AmMYBML2 and AmMYBML3 in subgroup 9 of the 24 subgroups that have been
identified among the 125 Arabidopsis R2R3 MYB proteins
(Stracke et al., 2001).
Subgroup 9 contains the MIXTA-LIKE MYB genes, which are defined by
both the conservation of their DNA-binding domains and by motifs present in
the C-terminal domains (Kranz et al.,
1998
; Stracke et al.,
2001
). The functions of only two members of this group have been
characterised. PhMYB1 from Petunia hybrida plays a similar
role to MIXTA in conical petal cell development
(Avila et al., 1993
;
van Houwelingen et al.,
1998
).
AmMYBML2 is most closely related to PhMYB1 from Petunia hybrida
(Fig. 1B) (Avila et al., 1993). AmMYBML2
and AmMYBML3 are also closely related to AtMYB16 and AtMYB106. In fact,
AmMYBML2 appears to be orthologous to PhMYB1 from our alignments, and MIXTA
and AmMYBML1 appear to be products of more recently derived gene duplications
of a subgroup 9 ancestral gene. We were interested in whether the
developmental function of these related genes had radiated along with the
changes in floral morphology that adapt members of the
Scrophulariaceae to specific pollinators. Consequently, we examined
the function of AmMYBML1 in Antirrhinum in greater
detail.
Analysis of AmMYBML1 expression by RNA gel blots
Previous analysis had indicated that AmMYBML1 is expressed in
petals, early in tissue development
(Glover et al., 1998).
Investigation of AmMYBML1 expression in other plant organs showed the
AmMYBML1 transcript to be restricted to the floral tissues; no
expression was detected in leaves or roots
(Fig. 1C). Petal tissues
express AmMYBML1 only during the early stages of development, with
maximum transcript found in buds of length 0-5 mm. Analysis of expression in
young, medium and old leaves indicated that AmMYBML1 is not expressed
in leaves, irrespective of developmental stage.
In situ hybridisation to determine cell specific expression of AmMYBML1
In situ hybridisation of Antirrhinum flower buds showed that
AmMYBML1 expression is limited to the ventral petal, and is absent
from the other floral whorls and the lateral and dorsal petals
(Fig. 2A,B). Probing equivalent
sections with the cell cycle marker Cyclin D3B, showed that
AmMYBML1 is expressed in the ventral petal while its cells are still
competent for further division (Fig.
2B,C). In a double mutant of A. majus (cyc,dich)
that has radially symmetrical flowers with only ventral petals
(Luo et al., 1996)
AmMYBML1 is expressed in all the petals, confirming that its
expression is specific to the ventral petal
(Fig. 2D).
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Activity of AmMYBML1 ectopically expressed in Nicotiana tabacum
To test the biological function of AmMYBML1, and to compare it
with MIXTA, a construct containing the AmMYBML1 cDNA under
the control of the double CaMV35S promoter was used for transformation of
tobacco. Expression of AmMYBML1 in regenerated tobacco plants was
confirmed by RNA gel blot analysis. Eight independent lines with detectable
levels of AmMYBML1 expression in leaves were identified and used for
phenotypic characterisation (Fig.
3H). In the vegetative tissues, the phenotypes of the plants
expressing AmMYBML1 were identical to those of wild-type tobacco
plants. However, significant differences were observed in floral tissues.
Scanning electron microscopy showed that conical cells and trichomes developed
ectopically in the epidermis of several floral organs. The strongest phenotype
was observed in carpel epidermal tissue, but the epidermal cells of the other
floral organs, and the bracts, were also affected. Microscopic analysis of
leaf and stem epidermal samples confirmed that, unlike MIXTA, AmMYBML1 was not
able to regulate the production of conical cells or trichomes in vegetative
tissues (Fig. 3A,B). However,
bracts, sepals and stamens were found to develop conical cells in response to
AmMYBML1 expression, while the petals and carpels were found to
differentiate full multicellular trichomes, as well as ectopic conical cells
(Fig. 3C-G). These data
demonstrate that the AmMYBML1 protein can function in the same way as the
MIXTA protein to direct the differentiation of specialised epidermal cell
forms, both conical cells and trichomes. However, the activity of AmMYBML1 is
restricted to the reproductive phase of plant development, suggesting that
AmMYBML1 requires the presence of additional factors, which are restricted to
floral tissues, to induce cellular differentiation. As tobacco provides an
excellent model for testing gene function in Antirrhinum, as
evidenced by the identical effects of ectopic expression of MIXTA in
the two species (Martin et al.,
2002), these data indicate that AmMYBML1 can promote both conical
cell and trichome formation in Antirrhinum.
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Regulation of AmMYBML1 expression
The AmMYBML1 promoter, 1610 bp upstream from the initiating ATG
codon, was sequenced and analysed using databases of transcription factor
binding sites. Fig. 5A shows
the AmMYBML1 promoter with key regions highlighted. The putative TATA
box is located between 171 and 162 bp upstream of the initiating ATG codon.
Two motifs of interest were identified between 364 and 358 bp
and between 1113 and 1109 bp upstream of the ATG. These
contained putative I-box motifs, which is interesting because these sequences
have been shown to be bound by proteins similar to DIVARICATA (DIV)
(Rose et al., 1999;
Galego and Almeida, 2002
).
DIV influences specifically the growth of ventral and lateral petals
(Almeida et al., 1997
;
Galego and Almeida, 2002
). In
div mutant plants, each half of the ventral petal becomes a mirror
image of the adjacent part of the lateral petal
(Almeida et al., 1997
),
resulting in a loss or significant reduction of the hairs in the throat of the
tube that collect pollen and in the size of the strips of hairs that pass down
the corolla and mark the junction between ventral and lateral petals
(Almeida et al., 1997
). The DIV
protein is a MYB-related transcription factor which has two MYB domains, which
are separated in the primary sequence of the protein. Structurally, it is most
similar to LeMYB1 from tomato (Rose et
al., 1999
). LeMYB1 binds the I-box sequence GATAAG, while StMYB1
(a related protein from potato) binds to a similar sequence, GGATAAG, or to a
core sequence, GGATA (Baranowskij et al.,
1994
; Rose et al.,
1999
). The putative I-box at 364 in the AmMYBML1
promoter is AGATAAA. As A and G are both purines, and therefore functionally
similar, the sequence AGATAAA may be similar enough to GGATAAG to be bound by
similar proteins. Alternatively, between 1113 and 1109 bp the
sequence GGATA, the core for StMYB1 binding, was found.
|
The expression pattern determined by the 223 bp promoter fragment showed
that this sequence is all that is required for expression in trichomes.
Consequently, this region is likely to include the important cis-acting
elements regulating trichome-specific expression. Aside from the TATA box and
one potential CAAT box, there are two GAMYB (MBSII)
(Solano et al., 1995) binding
motifs within this region.
The 1260 bp promoter was enough to limit the expression of AmMYBML1 to the trichomes of the flowers but the 884 bp promoter directed GUS expression in trichomes and vascular tissue in both floral and vegetative tissues. Thus, reducing the promoter length from 1260 bp to 884 bp resulted in GUS expression being directed outside the flower. These results suggest that negative regulatory elements silencing expression in vegetative tissues are located within the promoter of AmMYBML1 between 1260 bp and 884 bp upstream of the ATG. Within this region there are at least four potential MADS domain protein-binding sites (CArG boxes; Fig. 5A). One of the two putative I boxes is also located in this region, and may be involved in specifying ventral petal-specific expression in Antirrhinum.
Unfortunately, because tobacco flowers are radially symmetrical, we were unable to use expression in tobacco to identify sequences active in determining expression in the ventral petal only.
Comparison of AmMYBML1 expression in wild type and divaricata mutant flowers
The observation that AmMYBML1 transcript is restricted to the
ventral petal in wild-type Antirrhinum flowers
(Fig. 2A) led us to investigate
its expression in the div mutant. The div mutant lacks
ventralising signals in its flowers, and all five petals are consequently
dorsalised (Galego and Almeida,
2002). The characteristic mass of trichomes in the throat of the
tube in the wild-type flower is absent from this mutant
(Fig. 6A-D). The mutant also
fails to make the folds of tissue around the hinge region of the lower petals
(Fig. 6A-D,F,G) and lacks
conical cells in the region of the corolla epidermis just below the hinge
(Fig. 6H). On the inner
epidermis of the rest of the ventral petal lobe, conical cells develop
normally (Fig. 6I,J),
suggesting that MIXTA activity is unimpaired in div mutants
(Fig. 6E). Because DIV
encodes a MYB-related transcription factor responsible for determining ventral
petal identity (Galego and Almeida,
2002
), the restriction of AmMYBML1 expression to the
ventral petal suggested that it may be a target for transcriptional control by
DIV, and that the phenotype of div mutants might reflect the loss of
AmMYBML1 expression as a consequence of the loss of activity of its
upstream regulator. Gel blots of RNA from petals of wild-type and div
mutant flowers showed that AmMYBML1 was expressed in both lines, but
the expression in wild-type petals was at least 10-fold higher than in the
div mutant petals (Fig.
6E). MIXTA expression was unaffected by mutation of
DIV (Fig. 6E).
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Discussion |
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The AmMYBML1 gene encodes a protein with very strong structural
similarity to MIXTA (Fig. 1B),
such that both proteins belong to R2R3 MYB subgroup 9
(Stracke et al., 2001). On the
basis of this structural similarity, the AmMYBML1 protein was predicted to
play a role in conical cell and trichome formation. This was confirmed by
ectopic expression of AmMYBML1 in tobacco. AmMYBML1 induced the
formation of both ectopic conical cells and trichomes on floral organs of
tobacco. No phenotypic consequences of AmMYBML1 expression were
observed in vegetative tissues, in contrast to the conical cells and trichomes
induced on leaves in response to ectopic MIXTA expression
(Glover et al., 1998
).
Therefore, AmMYBML1 can function only in floral tissues. This restriction of
AmMYBML1 function is most probably attributable to the interaction of AmMYBML1
with other transcription factors with activity localised to floral
tissues.
Petal and carpel tissue were most responsive to AmMYBML1 expression. Tobacco petals are already covered with conical cells, but in the transgenic lines, ectopic trichomes were formed among the conical cells (Fig. 3D). The wild-type carpel epidermal surface was smooth, but in the transgenic lines ectopic conical cells and trichomes were produced (Fig. 3E-G). The excess trichomes on both organs were multicellular and competent to form glandular heads. These data confirmed that the AmMYBML1 protein, as predicted from its structural similarity to MIXTA, is able to induce the formation of both conical cells and trichomes but, unlike MIXTA, only in floral organs.
AmMYBML1 expression is restricted to the ventral petal of the corolla
The endogenous role of a gene is dependent not only on the function of its
encoded protein but also on its expression pattern. To investigate the role of
AmMYBML1 in Antirrhinum, its expression pattern was
determined using northern analysis and in situ hybridisation. Northern
analysis indicated that expression of AmMYBML1 is restricted to
floral tissues (Fig. 1C). From
this evidence it can also be concluded that any role of AmMYBML1 in the
differentiation of cells is restricted to the flower.
In situ hybridisation demonstrated that AmMYBML1 expression was restricted to the second whorl of the flower and, within the corolla, to the ventral of the five petals (Fig. 2A,B). Confirming this, in floral symmetry mutants where all five petals adopt ventral identity (dich, cyc) all five petals expressed AmMYBML1 (Fig. 2D). Several specialised cell types form on the ventral petal, including trichomes in the throat of the corolla tube, conical cells on the adaxial epidermis of the petal lobes and hinge region, and expanded adaxial mesophyll cells in the hinge region.
Analysis of the timing of AmMYBML1 expression relative to a
predictive marker of cell division, Cyclin D3b
(Forbert et al., 1996),
indicated that AmMYBML1 is expressed in the ventral petal while cells
are still dividing (Fig. 2A-C).
Previous studies have shown that that the ability of MIXTA to direct the
formation of conical cells or trichomes is linked to the competence of cells
for further division when the gene is expressed
(Glover et al., 1998
). The
observation that AmMYBML1 is expressed while cells are still
competent for further division indicates that it may play a role in the
formation of either trichomes or conical cells, or both.
AmMYBML1 expression is required for the development of specialised trichomes within the corolla tube
In situ hybridisation studies identified AmMYBML1 expression in
the trichomes of the ventral petal, especially those that form in a mass in
the throat of the corolla tube and that collect pollen from pollinating bees
(Fig. 2A,
Fig. 6A,C). Expression in
trichomes was confirmed by promoter::GUS analysis, which identified GUS
staining in all floral trichomes (Fig.
5B). Deletions of the promoter expanded GUS expression to the
trichomes of vegetative organs, suggesting that flower-specific expression is
the result of negative regulation of AmMYBML1 expression
(Fig. 5E). The activity of the
AmMYBML1 promoter in trichomes was consistent with the observed
ability of AmMYBML1 to induce trichome formation on the petals and
carpels of tobacco, when ectopically expressed
(Fig. 3D,G). Taken together
with the early expression of AmMYBML1 relative to the progression of
cell division (Fig. 2B,C) and
the expression of AmMYBML1 in the trichomes of the ventral petal
(Fig. 2E), these data suggest
strongly that AmMYBML1 directs the formation of specialised trichomes in the
ventral petal.
AmMYBML1 initiates the development of some conical cells in the ventral petal epidermis
In situ hybridisation experiments also identified AmMYBML1
transcript in cells of the inner epidermis of the hinge of the ventral petal
(Fig. 2E,F). This epidermis
develops conical cells, similar to those of the main expanse of the petal
lobes. Observation of transcript in this region, in combination with the
ability of AmMYBML1 to direct conical cell formation when ectopically
expressed, led us to investigate further the role of this gene in the
differentiation of these specialised cells. Previous studies had indicated the
importance of MIXTA in the differentiation of petal conical cells in
Antirrhinum and suggested that in mixta mutants, no conical
cells are produced on the adaxial epidermis. Re-investigation of
mixta mutant plants, which have flattened epidermal cells on the main
expanse of their petal lobes, did reveal a role for AmMYBML1. The
epidermis of the ventral petal in the region of the hinge still produced some
conical cells in the mixta mutants
(Fig. 4A,B), indicating that a
gene other than MIXTA is sufficient for the development of conical
cells in this restricted region of the corolla. In combination with the
ability of AmMYBML1 to direct the formation of conical cells, the
localisation of its transcript to this region and the fact that promoter
analysis confirmed the expression of AmMYBML1 in conical cells of
tobacco petals (Fig. 5C)
strongly support the idea that AmMYBML1 is responsible for the differentiation
of conical cells on the epidermis of the ventral petal hinge.
AmMYBML1 induces differential expansion of the mesophyll of the ventral petal, which reinforces the hinged landing platform for pollinating bees
In situ hybridisation also identified AmMYBML1 expression in the
adaxial mesophyll cells of the ventral petal hinge
(Fig. 2E,F and
Fig. 4C). Expression in
mesophyll cells was not observed for MIXTA
(Fig. 2G) and suggests a novel
role for AmMYBML1. Scanning electron microscopy revealed that the
mesophyll cells in this hinge region were expanded and tightly packed
together, creating folds of tissue (Fig.
4D). Differential expansion of the mesophyll on the adaxial side
may contribute to the overall form of the ventral petal which facilitates
pollination. The folded region reinforces the corolla, which is important
because the ventral petal functions as a landing platform for large
pollinating bees. When AmMYBML1 was expressed in tobacco under the
control of the CaMV35S promoter, petal lobes were considerably thicker than
controls, and thickening was correlated with increased expansion of mesophyll
cells (Fig. 4G,H). Therefore
the combination of expression of AmMYBML1 in the hinge mesophyll and
ability of AmMYBML1 to enhance mesophyll cell expansion in transgenic tobacco
flowers leads us to conclude that AmMYBML1 is involved in expansion of the
adaxial mesophyll cells of the ventral petal, helping to create the folds of
the reinforced hinged landing platform. Interestingly, MIXTA was not
as effective as AmMYBML1 at promoting mesophyll thickening in
transgenic tobacco, suggesting a further functional distinction between the
two proteins.
DIV controls AmMYBML1 expression and defines AmMYBML1 function in cell specialisation during corolla development
We have been unable to identify a mutant of AmMYBML1, despite
extensive reverse genetic screens. However, examination of the div
mutant of A. majus supported our interpretation of the function of
AmMYBML1. DIV promotes AmMYBML1 expression significantly,
although it does not affect expression of MIXTA. div mutants lack
trichomes in the throat of their corolla tubes. This supports the idea that
AmMYBML1 promotes trichome formation in the ventral petal tube directly.
div mutants also lack conical cells in the region of the ventral
petal epidermis around the hinge (Fig.
6H). As MIXTA expression is not affected by DIV, although
DIV is expressed throughout the corolla in the early stages of development
(Galego and Almeida, 2002),
this observation supports a role for AmMYBML1 in promoting conical
cell formation in this restricted region. Finally, div mutants lack
the folds of tissue that normally form at the hinge, supporting the view that
AmMYBML1 expression in the adaxial mesophyll in this region promotes
the formation of the folds of petal tissue that reinforce the hinge and
provide a robust landing platform for pollinating bees.
DIV determines ventral petal identity in A. majus
(Almeida et al., 1997;
Galego and Almeida, 2002
) and
promotes AmMYBML1 expression in the ventral petal. Our data suggest
that DIV is an activator of AmMYBML1. Certainly, a significant number
of the specific events in ventral petal development appear to be realised
through DIV activation of AmMYBML1 expression.
The B-function floral homeotic genes, DEF and GLO, also
contribute to the control of AmMYBML1 expression, as evidenced by the
reduction in transcript levels of the gene in flowers from plants homozygous
for weak def alleles (Fig.
7E), and by the reduction of those cellular features promoted by
AmMYBML1 in the ventral petal of flowers of def101
(Fig. 7A-D). Traditionally, DEF
and GLO are defined as functioning in the determination of organ identity, and
consequently it might be that their regulation of AmMYBML1 expression
is indirect. However the inhibition of AmMYBML1 promoter activity by
the combination of DEF and GLO in protoplast transfection assays (see Fig. S2
in the supplementary material) suggests that they may interact directly with
the promoter. The absence of transcriptional activation of the
AmMYBML1 promoter by DIV/DEF/GLO in any combination suggests that
there are additional components in the transcriptional activation complex.
Potential candidates are the SEPALLATA-like genes and
SQUAMOSA encoding MADS box transcription factors known to form
ternary complexes with DEF and GLO, and to be essential for their
transcriptional regulatory activity
(Egea-Cortines et al., 1999;
Gutierrez-Cortines and Davies,
2000
).
Evolution of AmMYBML1 function
Phylogenetic analysis suggests AmMYBML1 to be the product of a
relatively recent duplication of an ancestral subgroup 9 gene. The specialised
function of AmMYBML1 may have evolved at the same time as the
zygomorphic floral structure of Antirrhinum and other
Scrophulariacae. Its activity contributes to the adaptations of the
Antirrhinum flower to specialised pollination by bees. Our data
suggest that the divergence of developmental function between MIXTA
and AmMYBML1 is largely the result of differences in the expression
patterns of the two genes although there are also subtle differences in the
biochemical functions of the proteins they encode. Gene duplication and
specialisation of the AmMYBML1 expression pattern may have been the
primary changes underpinning the evolution of the specialised cell types to
provide some of the features of Antirrhinum flowers that adapt them
to bee pollination. The AmMYBML1 protein directs the formation of three
different specialised cell types in the ventral petal of Antirrhinum.
The conical epidermal cells on the hinge epidermis may play a role in
directing pollinators towards their nectar reward. The hinge structure created
by the thickening and folding of the mesophyll acts as both a landing platform
for bees and a deterrent to potential nectar robbers, as it requires an animal
with the weight of a bumblebee to open the corolla at the hinge. The trichomes
in the throat of the corolla tube, on the ventral petal, collect pollen from
the surface of pollinating bees and redistribute it to the stigmatic surface
for fertilisation. The coordination of development of these three different
specialised cell forms results in the morphogenesis of a structurally complex
petal, which is the function of this one transcription factor, AmMYBML1. The
divergence of AmMYBML1 function from that of MIXTA and other
R2R3 MYB genes of subgroup 9 in A. majus illustrates one of
the ways specialised floral forms have been achieved in plants.
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ACKNOWLEDGMENTS |
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Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/132/2/359/DC1
* Present address: Departamento de Biología Bolecular y
Bioquímica, Facultad de Ciencias, Universidad de Málaga, Campus
de Teatinos, 29071-Málaga, Spain
Present address: CSIRO Plant Industry, Glen Osmond, SA 5064, Australia
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