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First published online 24 October 2007
doi: 10.1242/dev.013136
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1 Department of Molecular, Cellular and Developmental Biology, Yale University,
New Haven, CT 06520, USA.
2 Department of Ecology and Evolutionary Biology, Yale University, New Haven, CT
06520, USA.
Author for correspondence (e-mail:
vivian.irish{at}yale.edu)
Accepted 7 September 2007
 |
SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Petal identity, Homeotic genes, MADS-box genes, Poppy, Papaver somniferum
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The evolution of flowers with elaborated petals is likely
to have increased fitness through facilitating pollinator interactions
(Clegg and Durbin, 2003).
Our current understanding of the molecular mechanisms controlling
petal-identity specification rests largely on functional analyses carried out
in several core eudicot species, including Arabidopsis thaliana and
Antirrhinum majus (Weigel and
Meyerowitz, 1994). In Arabidopsis, for example, the
APETALA3 (AP3) and PISTILLATA (PI)
MADS-box genes are required to specify petal and stamen identity, and
mutations in these genes result in homeotic transformations of petals into
sepals and stamens into carpeloid structures
(Bowman et al., 1991;
Goto and Meyerowitz, 1994;
Jack et al., 1992). The AP3
and PI gene products heterodimerize, and are likely to act in vivo as part of
larger MADS-box protein complexes, in order to specify petal as well as stamen
identity (Bowman et al., 1991;
Goto and Meyerowitz, 1994;
Honma and Goto, 2001;
Jack et al., 1992;
Pelaz et al., 2001;
Riechmann et al., 1996).
Although a considerable amount is known about the molecular mechanisms
specifying petal identity in Arabidopsis and other core eudicot
species, there is little functional evidence that homologs of these genes play
similar roles in petal-identity specification outside of the core eudicots. It
is generally accepted that a bipartite perianth with distinct petals evolved
independently multiple times within the flowering plants
(Drinnan et al., 1994;
Takhtajan, 1991). However,
exactly when such events occurred is still unresolved. Phylogenetic analyses
have been used to suggest that transitions between a unipartite and bipartite
perianth have occurred multiple times within the eudicots
(Albert et al., 1998;
Soltis et al., 2005;
Zanis et al., 2003). These
analyses, though, are equivocal in determining the direction of such
evolutionary transitions. One possibility is that a bipartite perianth
represents independent evolutionary events in core eudicots as compared with
the Ranunculales (Fig. 1A).
Alternatively, a bipartite perianth might have been ancestral, and was lost in
only a few, derived, non-core eudicot lineages
(Fig. 1B). Determining whether
core eudicots and Ranunculales species possess similar or divergent
developmental genetic mechanisms to condition petal identity would be valuable
in assessing the merits of these two hypotheses.
A variety of studies have already been carried out to assess the roles of
AP3 homologs in core eudicot species. A duplication in the
AP3 gene lineage at the base of the core eudicots gave rise to the
euAP3 and TM6 lineages, which are characterized by having
distinct C-terminal sequence motifs
(Kramer et al., 1998). The
sequence motifs in the TM6 lineage genes are more similar to those of
the paleoAP3 genes, which are found in non-core eudicot angiosperms
(Kramer et al., 1998). Genetic
analyses of euAP3 genes in core eudicot species, such as of the
Arabidopsis AP3 gene, support the idea that these genes have a common
function in petal-identity specification, as well as in stamen specification
(de Martino et al., 2006;
Jack et al., 1992;
Schwarz-Sommer et al., 1992;
Vandenbussche et al., 2004).
By contrast, core eudicot TM6 genes appear to have a more restricted
role in conditioning stamen identity (de
Martino et al., 2006; Rijpkema
et al., 2006).
The roles of paleoAP3 genes in non-core eudicot angiosperms are
somewhat unclear. A variety of expression analyses have been carried out that,
in general, support the idea that paleoAP3 genes have a conserved
role in stamen identity specification, but their role in petal specification
remains ambiguous. For instance, in many basal angiosperms, paleoAP3
genes show strong and ubiquitous expression in stamens, but often
inconsistent, weak or patchy expression in petal primordia
(Kim et al., 2005;
Kramer and Irish, 1999;
Zahn et al., 2005). In
non-grass monocots, expression of paleoAP3 genes can be observed in
developing petaloid organs in some taxa [e.g. in Lilium longiflorum
(Tzeng and Yang, 2001)], but
not in others [e.g. Aparagus officinalis
(Park et al., 2003)].
Functional analyses in several monocot grasses have demonstrated that the
paleoAP3 genes in these species are required for the development of
stamens and lodicules (Ambrose et al.,
2000; Nagasawa et al.,
2003; Xiao et al.,
2003). As these grass species lack petals, such studies cannot
directly assess the roles of paleoAP3 genes in petal development.
Furthermore, a chimeric AP3 gene containing a non-core eudicot
paleoAP3 motif has been shown to be sufficient to rescue stamen, but
not petal, identity in Arabidopsis
(Lamb and Irish, 2003). By
contrast, other studies have shown that ectopic overexpression of a monocot
paleoAP3 gene can rescue both petal and stamen development,
suggesting that levels of paleoAP3 gene expression might be important
in determining developmental function
(Whipple et al., 2004). Based
on these observations, it has been suggested that the paleoAP3 genes
lack the capacity to fully specify petal identity, although they may play
subsidiary roles in petal cell-type differentiation
(Kramer and Irish, 1999;
Kramer and Irish, 2000).
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; Kim et al.,
2004) (Fig. 1) and
so are well-placed for comparative studies. P. somniferum is one of
the oldest cultivated medicinal plants, and its continuing pharmaceutical
importance as the source of a variety of morphinan alkaloids has made it one
of the best-studied Papaver species
(Chitty et al., 2006;
Kapoor, 1995;
Millgate et al., 2004;
Zulak et al., 2007).
Furthermore, functional genetic analyses can be rapidly carried out in P.
somniferum using virus-induced gene silencing
(Hileman et al., 2005). Our
results provide the first functional evidence that petal identity can be
conditioned by the action of a paleoAP3 gene, and suggest that
petal-identity specification in non-core eudicots relies on an independently
derived, but similar, developmental program as that present in core eudicots.
In addition, we show that there has been extensive functional diversification
in the role of paleoAP3 gene products in specifying petal and stamen
identity in poppies. |
MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Phylogenetic analyses
Nucleotide sequences of AP3 and PI homologs were aligned
using CLUSTALX and refined by hand using MacClade
(Maddison and Maddison, 2000).
Aligned sequences were used to generate maximum likelihood trees under the GRT
+ gamma model of molecular evolution, as implemented in GARLI
(www.bio.utexas.edu/faculty/antisense/garli/Garli.html)
(D. J. Zwickl, PhD thesis, University of Texas at Austin, 2006).
RT-PCR
Total RNA was extracted using Trizol (Invitrogen, Cleveland, OH) and
approximately 300 ng was used in 10 µl cDNA synthesis reactions using
Superscript III Reverse Transcriptase (Invitrogen). For cDNA synthesis, the
poly(T) primer used was 5'-GACTCGAGTCGACATCGA(T)17. Primers
for testing expression of PapsAP3-1, PapsAP3-2, PapsPI-1 and
PapsPI-2 were: PAP3-11, 5'-AAGAAATAAAGCCATGGAGG-3' and
PAP3-12, 5'-CTAAATACCGATTTTGGAGTC-3'; PAP3-21,
5'-AAGCTAAGGAACCACACTGA-3' and PAP3-22,
5'-CACGCATGGTTCATAGATAT-3'; PPI-11,
5'-TTGCCAAACTACAACAAGTG-3' and PPI-12,
5'-TAGCAGCTATGATCATGATC-3'; PPI-21,
5'-ACTCAAGAAAAATGGAAGAC-3' and PPI-22,
5'-GCTTTTTATAAGTTCTTTGC-3'. Actin primers were: ACT1,
5'-ATGGATCCTCCAATCCAGAC-3' and ACT2,
5'-TATTGTGTTGGACTCTGGTG-3'.
In situ hybridization
Hybridizations were carried out as previously described
(Drea et al., 2005;
Kramer and Irish, 1999) with
minor modifications. Gene specific regions derived from the C-terminal domain
and 3' UTR of PapsAP3-1, PapsAP3-2, PapsPI-1, and
PapsPI-2 sequences were used to generate digoxygenin-labeled RNA
probes.
Virus-induced gene silencing
Gene-specific regions of PapsAP3-1, PapsAP3-2, PapsPI-1 and
PapsPI-2, as well as concatenated PapsAP3-1/PapsAP3-2 and
PapsPI-1/PapsPI-2 sequences were introduced into the TRV2 vector
(Liu et al., 2002),
transformed into Agrobacterium strain GV3101 and used to infiltrate
poppy seedlings at the 3- to 5-leaf stage as previously described
(Hileman et al., 2005).
Individual resulting plants were assayed for the presence of the viral vector
using RT-PCR as well as for any visible phenotype.
Scanning electron microscopy
Plant material was fixed overnight in 3.7% formaldehyde, 5% acetic acid,
50% ethanol (FAA) and then transferred to 70% ethanol. Samples were dehydrated
in an ethanol series before critical-point drying. Samples were coated in gold
and analyzed on a Zeiss ISI-SS40 scanning electron microscope equipped with
digital image capture.
Yeast two-hybrid assays
PapsPI-1, PapsPI-2, PapsAP3-1 and PapsAP3-2 IKC domains were fused with the
GAL4 DNA-binding domain in the pGBT9 vector and with the GAL4 activation
domain in the pGAD424 vector (Clontech, Mountain View, CA). Two independent
transformations for each vector combination were performed and five colonies
per transformation were used for ß-galactosidase liquid assays, using the
protocol available at
http://www.fhcrc.org/science/labs/gottschling/yeast/Bgal.html.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Both PapsAP3-1 and PapsAP3-2 encode characteristic
paleoAP3 PI-derived motifs
(Kramer et al., 1998), but the
PapsAP3-1 predicted product lacks the consensus paleoAP3
motif (Kramer et al., 1998)
(Fig. 2A). This pronounced
difference at the C-terminus is due to insertions/deletions between the
PapsAP3 genes that engender frameshifts beginning at the start of
exon 6. As such, PapsAP3-1 lacks pleisiomorphic sequence motifs, but
has gained a novel C-terminus, whereas PapsAP3-2 retains the
ancestral character state of a paleoAP3 C-terminal motif.
The PapsPI-1 gene encodes a product containing the PI-motif as
well as a sequence extension at the C-terminus
(Fig. 2B). The
PapsPI-2 predicted product lacks the consensus PI-motif
(Kramer et al., 1998) at the
C-terminus. This appears to be due to a single nucleotide insertion in the
3' coding region followed by a 2-nucleotide deletion 22 bp downstream,
generating a stop codon and a truncated protein
(Fig. 2B). Although this domain
has been shown to be essential for protein function in the
Arabidopsis PI protein (Lamb and
Irish, 2003), the Pisum sativum PI gene also lacks this
conserved domain but has been shown to be capable of rescuing the
Arabidopsis pi mutant phenotype
(Berbel et al., 2005).
Diversification in expression of P. somniferum genes
P. somniferum flowers contain two sepals, four petals, numerous
hypogynous stamens, and generally 8 to 12 carpels, which produce the capsular
fruit (Kapoor, 1995). The
early development of floral organ primordia in P. somniferum can be
characterized by a variety of morphological landmark stages
(Fig. 3). These include stage
P3 of development when the petal, stamen and carpel primordia are first
clearly visible, and are enclosed by the surrounding sepal primordia. By stage
P5, the anther and filament of the stamens are discernable and the capsule has
become invaginated; ovules are distinguishable by stage P5. Between stages P7
and P8 the petals undergo accelerated growth and extend to fill the space
bounded by the two sepals. This rapid growth of the petals continues to force
the sepals apart when the bud opens just after anthesis. At later stages,
P. somniferum buds (stage P8 and later) become pendant prior to
anthesis, before extending upright as the flower opens owing to asymmetric
growth of the pedicel. At maturity, each floral organ type is characterized by
distinctive epidermal cell morphologies
(Fig. 4A). Sepal cells are
somewhat irregular, with numerous stomata present on the abaxial surface, and
adaxial sepal epidermal cells being more domed. Petal cells are distinctively
long and narrow, with prominent ridges on the adaxial surface that differ from
the characteristic conical and papillar cells found in petals of many species.
Stamen filaments possess elongated epidermal cells, whereas the epidermal
cells of the anther are more irregularly shaped. The gynoecium results from a
fusion of multiple carpels and possesses distinctive ridges; the epidermal
cells between the ridges are relatively flat and are interspersed with
stomata.
RT-PCR and in situ hybridization analyses showed that each of the P.
somniferum genes had a distinct pattern of expression during floral
development. PapsAP3-1 expression was initially detected in petal and
stamen primordia at stage P3, when these primordia are first apparent
(Fig. 3A). Expression of
PapsAP3-1 throughout the developing stamens and petals persisted
through later stages of flower development
(Fig. 3A,B). Weak expression of
PapsAP3-1 could be detected in sepal and carpel primordia at later
stages of development (Fig.
3B). By contrast, PapsAP3-2 expression was initially
detected at stage P4 in stamen primordia, but was excluded from petal
primordia (Fig. 3A). This
pattern persisted until the P5 stage of flower development when
PapsAP3-2 expression was also detected in petal primordia
(Fig. 3A). PapsAP3-2
expression appeared to be more ubiquitous at later stages of development, in
that low levels of expression could be detected in sepal and carpel primordia
through stage P8 (Fig. 3B). The
sequence and expression analyses indicate that PapsAP3-2 is more
representative of paleoAP3-like genes of other Ranunculales species
(Kramer et al., 2003;
Kramer and Irish, 2000;
Shan et al., 2006), suggesting
that lack of early and ubiquitous petal-specific expression is ancestral in
this clade.
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Loss-of-function phenotypes demonstrate a distinct function in floral organ identity for each P. somniferum gene
To assess the functional roles of the AP3-like and
PI-like genes in P. somniferum, we used tobacco rattle virus
(TRV)-mediated, virus-induced gene silencing (VIGS)
(Hileman et al., 2005).
Gene-specific regions of PapsAP3-1, PapsAP3-2, PapsPI-1 and
PapsPI-2, as well as concatenated PapsAP3-1/PapsAP3-2 and
PapsPI-1/PapsPI-2 sequences, were introduced into the TRV2 vector
(Liu et al., 2002) for
silencing. We screened approximately 500 infiltrated seedlings for each of the
six constructs, with 0.5-8% of resulting plants containing the transformation
vector and showing a phenotype (see Table S1 in the supplementary material).
Transcript levels of silenced genes were considerably reduced
(Fig. 6), although not
completely eliminated, consistent with our previous observations that TRV-VIGS
is an effective tool to downregulate gene expression in P. somniferum
(Hileman et al., 2005).
Silencing of an individual gene did not affect the expression of the other
AP3-like or PI-like genes
(Fig. 6), indicating that
silencing was gene-specific.
The range of phenotypes in silenced plants was distinct for each construct. Reduction in PapsAP3-1 expression resulted in homeotic transformations of petals to more sepaloid organs (Fig. 4B), whereas stamens were unaffected. This transformation was evident at the cellular level, with both adaxial and abaxial second-whorl cells showing sepaloid characteristics (Fig. 4B). Conversely, silencing of PapsAP3-2 expression resulted in stamens that showed variable transformations to carpeloid structures containing ovules, whereas the petals remained completely unaffected (Fig. 4C). The VIGS phenotypes indicate that PapsAP3-1 and PapsAP3-2 control distinct developmental pathways. When both PapsAP3-1 and PapsAP3-2 were silenced simultaneously, both petals and stamens showed homeotic transformations (Fig. 4D, Fig. 5). The range of petal phenotypes in these plants was similar to that of the PapsAP3-1-silenced lines, but the transformation of stamens to carpels was generally much more severe than in the PapsAP3-2-silenced lines alone. These results suggest that PapsAP3-1 and PapsAP3-2, although having discrete functions, are also redundant in regulating stamen development. Furthermore, one target of their transcriptional action is PapsPI-1, as its expression was downregulated in the carpeloid stamens of plants silenced for both PapsAP3-1 and PapsAP3-2 (Fig. 6).
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Conservation and diversification in P. somniferum protein-protein interactions
Various lines of evidence have suggested that homodimerization of the
paleoAP3-like gene products was ancestral
(Winter et al., 2002), and
that in core eudicots the ability of AP3 gene products to
homodimerize has been lost (Riechmann et
al., 1996; Schwarz-Sommer et
al., 1992). To determine the dimerization capabilities of the
P. somniferum gene products, we carried out yeast two-hybrid assays
on pairwise combinations of P. somniferum proteins
(Table 1). PapsPI-2
did not dimerize with any of the tested proteins, although this does not
obviate the possibility that it interacts with other MADS-box gene products to
effect its function. This is supported by the observation that silencing of
PapsPI-2 enhances the silenced phenotype of PapsPI-1
(Fig. 4F), pointing to the
possibility that both PapsPI gene products participate in common
protein complexes. PapsAP3-2 was able to homodimerize, supporting the
idea that it has retained the ancestral character state. Furthermore,
PapsAP3-1 and PapsAP3-2 were able to heterodimerize with
PapsPI-1. This heterodimerization could serve to delimit
PapsPI-1 expression to the petal and stamen whorls as a consequence
of autoregulatory feedback control, as has been shown for Arabidopsis
and Antirrhinum (Honma and Goto,
2000; Trobner et al.,
1992). Our results are consistent with this model, because
PapsAP3-1 and PapsAP3-2 are required to maintain expression
of PapsPI-1 but not PapsPI-2
(Fig. 6). This also implies
that evolutionary changes in AP3 lineage gene expression can drive
alterations in the domains of PI gene expression, without having to
invoke secondary changes in PI regulation.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Together, these data support the idea that PapsAP3-1 and
PapsAP3-2 play qualitatively different roles in floral
development.
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;
Ng and Yanofsky, 2001);
however, orthologs of Arabidopsis AP1 do not exist in Ranunculales
(Litt and Irish, 2003),
suggesting that other gene functions, potentially AP1 paralogs, play
the equivalent regulatory role in P. somniferum.
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; Force et al.,
1999; Ohno, 1970).
Why duplicated genes persist in genomes at higher frequencies than expected,
possibly allowing for subsequent changes in protein function, remains an
important question of genome evolution. The immediate retention of duplicate
genes appears to depend, in part, on the biological roles of the encoded
proteins. Genes encoding products that function as key regulatory components,
such as transcription factors, as well as those that participate in large
multiprotein complexes [e.g. MADS-domain proteins
(Honma and Goto, 2001)],
appear to be preferentially maintained owing to the requirement for a
stoichiometric balance with other components in the pathway
(Birchler and Veitia, 2007;
Evangelisti and Wagner, 2004).
The selective retention of duplicate AP3 gene lineages, both at the
base of the core eudicots (Kramer et al.,
1998) and in Ranunculales (this work), might reflect the
requirement for their functions in a developmentally and biochemically complex
regulatory system. We have provided two scenarios for the evolution of a bipartite perianth containing petals (Fig. 1A,B). Under both of these evolutionary scenarios, the ancestor of eudicots is considered to have possessed a bipartite perianth with distinct petals, and this condition was inherited in the Ranunculales. We therefore argue that the combinatorial function of PapsAP3-1 and PapsAP3-2 to specify a bipartite perianth with distinct petals was inherited from a eudicot ancestor with a single paleoAP3 homolog capable of specifying both petals and stamens. This is also consistent with our phylogenetic analyses, which indicate that the ancestral eudicot possessed a single paleoAP3 gene (see Fig. S1 in the supplementary material). Subsequent to the duplication in the paleoAP3 lineage in Ranunculales, the two resulting paralogs became subfunctionalized such that in poppy, PapsAP3-1 retained the ancestral subfunction of specifying petal identity, and PapsAP3-2 retained the ancestral subfunction of specifying stamen identity. Interestingly, whereas PapsAP3-2 does not seem to retain any residual role in petal specification, PapsAP3-1 does retain some ability to direct the development of stamens, as evidenced by the stronger transformation of stamens to carpeloid organs in the coordinate silencing of PapsAP3-1 and PapsAP3-2, as compared with silencing of PapsAP3-1 alone (Fig. 4).
The subfunctionalization of PapsAP3-1 and PapsAP3-2 is
also evident in their patterns of gene expression and co-factor interactions.
Although they have overlapping patterns of expression in both petals and
stamens, PapsAP3-2 is expressed predominantly in stamens, and only
comes to be expressed in petals at later stages of development. It is also
clear that adaptive changes in protein-protein interactions have occurred
during the evolution of PapsAP3-1 and PapsAP3-2. The
proteins encoded by PapsAP3-1 and PapsAP3-2 can
heterodimerize with PapsPI-1, but PapsAP3-2 can also
homodimerize. The ability to heterodimerize as well as homodimerize has been
observed for paleoAP3 gene products from lily, a monocot
(Tzeng et al., 2004). Thus, it
seems likely that the ancestral eudicot paleoAP3 gene possessed both
capabilities, which have subsequently been parsed in the PapsAP3-1
and PapsAP3-2 genes. This diversification in function presumably
reflects the differences in C-terminal motifs found in PapsAP3-1 as
compared with PapsAP3-2, as these motifs have been found to play
crucial roles in protein-protein interactions and function
(Lamb and Irish, 2003;
Tzeng et al., 2004).
Therefore, subfunctionalization of PapsAP3-1 and PapsAP3-2
is likely to have occurred through evolutionary shifts in expression, in
interaction with other protein partners, and through changes in their binding
affinity for downstream cis-regulatory sequences of target genes in the
respective petal and stamen developmental pathways. The differences in
PapsAP3-1 and PapsAP3-2 functions are likely to reflect an
early diversification event in the Ranunculales. A similar AP3
subfunctionalization event has been postulated to have occurred in
Aquilegia, another member of the Ranunculales, based on disparate
expression patterns of the three Aquilegia AP3-like genes
(Kramer et al., 2007).
However, this hypothesis has yet to be functionally tested.
In contrast to the roles of PapsAP3 genes during flower
development, our genetic analyses of PapsPI genes indicates that
these duplicates have not parsed their functions in a whorl-specific manner.
PapsPI-1 appears to have retained the majority of function, based on
its robust expression levels, protein interaction capabilities, and the strong
homeotic phenotype observed for PapsPI-1-silenced plants.
PapsPI-2 could potentially be on the way to becoming a pseudogene;
our VIGS studies indicate that it possesses only a redundant role in
specifying petal and stamen identity, it is expressed at low levels, and has
accumulated mutations that result in the loss of conserved C-terminal motifs.
Duplication of PI genes appears to have occurred multiple times
across the Ranunculales. Some lineages such as Clematis and
Papaveraceae possess two PI copies, whereas other Ranunculales such
as Aquilegia have only one PI gene (see Fig. S2 in the
supplementary material). Silencing of the single PI homolog present
in Aquilegia results in a phenotype similar to that we have described
for silencing of PapsPI-1. However, the homeotic transformations
observed for silencing of Aquilegia PI are difficult to ascribe to
silencing of this locus alone, as these plants show coordinate downregulation
of AP3 homologs as well (Kramer
et al., 2007). As such, it remains to be tested whether the
function we have ascribed to PapsPI-1 represents a common role for
PI-like genes across the Ranunculales.
The evolution of petal-identity pathways
Our data provide the first functional evidence that paleoAP3 genes
play critical homeotic roles in petal specification. Based on these results,
in conjunction with a number of other observations, we propose a model to
explain the evolution of the petal-identity pathway. The role of
AP3-like and PI-like genes appears to be conserved in
regulating stamen development across angiosperms, and presumably reflects the
ancestral role of such genes in specifying male reproductive identity, a
function that is likely to have been conserved in gymnosperms where such genes
are expressed in male cones (Mouradov et
al., 1999; Sundstrom et al.,
1999). During the early diversification of the angiosperms, we
propose that, concomitant with the evolution of the angiosperm bisexual axis,
the pattern of expression of paleoAP3 genes broadened. This is
consistent with the broad and variable patterns of paleoAP3 gene
expression seen in a variety of basal angiosperm species
(Kim et al., 2005). However,
because no functional data exist for any basal angiosperm species, it is
unclear to what extent this diversification in expression is responsible for
directing petaloid identity.
We suggest that the ancestral role for AP3-like and
PI-like genes in the angiosperms might have been in specifying
regional identity, as opposed to organ identity. True petals are defined as
occupying the second whorl (or inner perianth) and having a narrow base,
distinctive pigmentation and conical reflective epidermal cells
(Endress, 2006;
Martin et al., 2002;
Weberling, 1989). A number of
species possess second-whorl structures that do not display the morphological
characteristics of petals (e.g. tepals and lodicules) and, conversely, there
are species that possess organs that display a petaloid morphology outside of
the second whorl (e.g. heterotopic petaloidy). Therefore, the development of
true petals depends on two separable processes: second-whorl regional
specification and appropriate morphological differentiation.
AP3- and PI-like gene functions have been examined in
monocot grasses, which lack petals. In addition to specifying stamen identity,
these genes are also required for the specification of a grass-specific organ,
the lodicule (Ambrose et al.,
2000; Nagasawa et al.,
2003; Whipple et al.,
2007). Lodicules appear to correspond to second-whorl structures
in that they occupy a region of the flower outside of the stamens but internal
to the other non-reproductive organs of the grass flower, but otherwise are
not homologous with petals in that they do not display the morphological
characteristics of a petal, although they do have a distinctive morphology
(Clifford, 1988;
Dahlgren et al., 1985;
Whipple et al., 2007). These
data from grasses demonstrate that AP3/PI-like genes can condition
the specification of non-petaloid second-whorl structures. Furthermore,
analyses of AP3/PI gene expression in the monocot Asparagus
officinalis, which possesses a perianth composed of two whorls of
undifferentiated tepals, has shown that AP3 and PI orthologs
are expressed in inner-whorl, but not outer-whorl, organs
(Park et al., 2004;
Park et al., 2003), suggesting
a role in regional specification. Similarly, silencing of a PI
ortholog in Aquilegia suggests that it is required for the
specification of inner, but not outer whorls of petaloid organs
(Kramer et al., 2007). The
genetic requirement for AP3/PI gene functions in specifying core
eudicot petals (de Martino et al.,
2006; Jack et al.,
1992; Schwarz-Sommer et al.,
1992; Vandenbussche et al.,
2004), non-core eudicot petals (this work), and lodicules
(Ambrose et al., 2000;
Nagasawa et al., 2003;
Whipple et al., 2007),
suggests a common role for these genes in specifying a spatially limited
regional domain within the flower. As such, this might facilitate multiple
instances of re-recruitment of AP3-like genes to direct a petal
developmental program in the second whorl as petals are evolutionarily lost
and regained across angiosperms.
Consistent with this idea, our data support a model whereby different and independent gene duplication events have allowed for the recruitment of AP3-dependent gene functions for specifying petal identity. In the core eudicots, the euAP3/TM6 gene duplication event has allowed for the subfunctionalization (through shifts in both expression and dimerization capabilities) of the resulting gene copies. In basal eudicots, a distinct gene duplication event has resulted in the PapsAP3-1 and PapsAP3-2 genes that have also parsed their functions. Regardless of whether petals arose independently in the core eudicots as compared with the Ranunculales, or whether petals in the core eudicots and Ranunculales are homologous (Fig. 1), we have shown that distinct gene duplication events have allowed for the differential recruitment of gene functions to specifying petal identity in these different angiosperm lineages.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/23/4157/DC1
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