Nearly all angiosperm flowers possess a perianth, a series of sterile
organs that surround the reproductive organs – the stamens and
carpels. In many flowering plant lineages, the perianth is
differentiated into distinct outer organs called sepals, and inner
organs called petals; this is termed a bipartite perianth. Petals are
therefore defined as occupying the spatial position outside of the
stamens but internal to the sepals, and are often large, showy and
pigmented, and contain specialized reflective papillate epidermal
cells (Endress and Matthews, 2006). 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
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
euAP3and TM6lineages, 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).
Functional analyses of genetic pathways controlling petal
specification in poppy
Sinéad Drea1, Lena C. Hileman1,*, Gemma de Martino1and Vivian F. Irish1,2,†
MADS-box genes are crucial regulators of floral development, yet how their functions have evolved to control different aspects of
floral patterning is unclear. To understand the extent to which MADS-box gene functions are conserved or have diversified in
different angiosperm lineages, we have exploited the capability for functional analyses in a new model system, Papaver
somniferum (opium poppy). P. somniferum is a member of the order Ranunculales, and so represents a clade that is evolutionarily
distant from those containing traditional model systems such as Arabidopsis, Petunia, maize or rice. We have identified and
characterized the roles of several candidate MADS-box genes in petal specification in poppy. In Arabidopsis, the APETALA3 (AP3)
MADS-box gene is required for both petal and stamen identity specification. By contrast, we show that the AP3 lineage has
undergone gene duplication and subfunctionalization in poppy, with one gene copy required for petal development and the other
responsible for stamen development. These differences in gene function are due to differences both in expression patterns and co-
factor interactions. Furthermore, the genetic hierarchy controlling petal development in poppy has diverged as compared with that
of Arabidopsis. As these are the first functional analyses of AP3 genes in this evolutionarily divergent clade, our results provide new
information on the similarities and differences in petal developmental programs across angiosperms. Based on these observations,
we discuss a model for how the petal developmental program has evolved.
KEY WORDS: Petal identity, Homeotic genes, MADS-box genes, Poppy, Papaver somniferum
Development 134, 4157-4166 (2007) doi:10.1242/dev.013136
1Department of Molecular, Cellular and Developmental Biology, Yale University, New
Haven, CT 06520, USA. 2Department of Ecology and Evolutionary Biology, Yale
University, New Haven, CT 06520, USA.
*Present address: Department of Ecology and Evolutionary Biology, University of
Kansas, Lawrence, KS 66045, USA
†Author for correspondence (e-mail: firstname.lastname@example.org)
Accepted 7 September 2007
The roles of paleoAP3genes 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 paleoAP3motif 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 paleoAP3gene 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
In order to explicitly and critically test the role of paleoAP3 and
PI homologs in the specification of petals in a non-core eudicot
lineage, we have carried out functional analyses of paleoAP3 and
PI-like genes in the non-core eudicot Papaver somniferum (opium
poppy). The Ranunculales, which includes Papaver species (the
poppies), are well-supported as the sister group to all other eudicots
(Angiosperm Phylogeny Group, 2003; 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
paleoAP3gene, 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
Isolation of Paps genes
RT-PCR reactions using the following degenerate primers were used to
isolate poppy sequences from floral poly(A) RNA: PapsAP3DF [5?-
and PapsAP3DR [5?-ATC(T/G)T(G/C)TCC(T/C/A)(T/G/A)(T/G/C)CCT -
(C/T)TGC(T/C)AT(C/T)TC-3?] for the PapsAP3 genes; PapsPIDF [5?-
(G/A)A-3?] and PapsPIDR [5?-AG(A/G)(C/T/A)G(C/T)TT (A/G)TT -
(G/A/T/C)TC(C/T)(A/G)C(C/T)TC(T/C/A)A-3?] for the PapsPI genes.
Full-length sequences were obtained using RACE (rapid amplification of
cDNA ends) according to the manufacturer’s instructions (GIBCO-BRL,
Development 134 (23)
gain of bipartite perianth (petals)
loss of bipartite perianth (petals)
inferred gene duplication
Fig. 1. Inferred evolutionary history
of petals and MADS-box genes in the
eudicots. (A,B) Alternative scenarios for
the evolution of petals. (A) The evolution
of a bipartite perianth occurred
independently in the lineage leading to
the Ranunculales as compared with the
core eudicots. (B) The evolution of a
bipartite perianth occurred prior to the
radiation of the eudicots, with losses of
this character in multiple lineages.
(C,D) Summary of phylogenetic analyses.
For comprehensive phylogenetic analyses,
see Figs S1, S2 in the supplementary
material. (C) The duplication of
paleoAP3-like genes resulting in
PapsAP3-1 and PapsAP3-2 occurred
within the Ranunculales, pre-dating the
divergence of the Ranunculaceae and
Papaveraceae. (D) The duplication of PI-
like genes occurred relatively recently in
the Papaveraceae, leading to PapsPI-1
and PapsPI-2 in P. somniferum. Clades in
bold represent those containing Paps
genes described in this study.
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