INVITED REVIEW

Molecular Mechanisms Underlying Origin and Diversication

of the Angiosperm Flower
GUENTER THEI SSEN* and RAI NER MELZER
Friedrich-Schiller-Universitaet Jena, Lehrstuhl fuer Genetik, Philosophenweg 12, D-07743 Jena, Germany
Received: 2 March 2007 Returned for revision: 25 April 2007 Accepted: 31 May 2007 Published electronically: 31 July 2007
Background Understanding the mode and mechanisms of the evolution of the angiosperm ower is a long-standing
and central problem of evolutionary biology and botany. It has essentially remained unsolved, however. In contrast,
considerable progress has recently been made in our understanding of the genetic basis of ower development in
some extant model species. The knowledge that accumulated this way has been pulled together in two major hypo-
theses, termed the ABC model and the oral quartet model. These models explain how the identity of the different
types of oral organs is specied during ower development by homeotic selector genes encoding transcription factors.
Scope We intend to explain how the ABC model and the oral quartet model are now guiding investigations
that help to understand the origin and diversication of the angiosperm ower.
Conclusions Investigation of orthologues of class B and class C oral homeotic genes in gymnosperms suggest that
bisexuality was one of the rst innovations during the origin of the ower. The transition from dimer to tetramer
formation of oral homeotic proteins after establishment of class E proteins may have increased cooperativity of
DNA binding of the transcription factors controlling reproductive growth. That way, we hypothesize, better devel-
opmental switches originated that facilitated the early evolution of the ower. Expression studies of ABC genes in
basally diverging angiosperm lineages, monocots and basal eudicots suggest that the classical ABC system known
from core eudicots originated from a more fuzzy system with fading borders of gene expression and gradual tran-
sitions in organ identity, by sharpening of ABC gene expression domains and organ borders. Shifting boundaries of
ABC gene expression may have contributed to the diversication of the angiosperm ower many times indepen-
dently, as may have changes in interactions between ABC genes and their target genes.
Key words: ABC model, angiosperm, evo-devo, fading borders, oral quartet model, gymnosperm, shifting boundaries.
INTRODUCTION
Evolution of the angiosperm ower: news and views
from Darwin until now
Imagine roses, tulips, lilies and orchids we all feel quite
familiar with owers, dont we? Many will be quite sur-
prised, therefore, to learn that the modes and mechanisms
of the origin and early diversication of the owering
plants (angiosperms) is still one of the most hotly debated
matters of evolutionary biology.
The angiosperms provide us, directly or indirectly, with
the vast majority of human food (such as vegetables and
fruits) as well as with a lot of other economically important
products (e.g. use in the preparation of clothes, furniture
and drugs), and they also have great aesthetic value as orna-
mental plants. In addition, the contributions of angiosperms
to biodiversity in terrestrial ecosystems can hardly be over-
estimated. No wonder, therefore, that botanists and evolu-
tionary biologists made considerable efforts to clarify the
origin and diversication of the owering plants. The appar-
ently sudden origin and rapid early morphological radiation
of the angiosperms, as revealed by the fossil record, already
intrigued Charles Darwin, who considered it a perplexing
phenomenon and even an abominable mystery (Crepet,
1998, 2000).
Like most other real mysteries, explaining the early
evolution of angiosperms proved difcult, despite
considerable efforts during the last one and a half centuries
(Frohlich, 2003, 2006). Major reasons for this are a quite
uninformative (and probably extremely incomplete) fossil
record and the great morphological gap between the
owers of angiosperms and the reproductive structures of
gymnosperms, angiosperms closest relatives; gymnos-
perms comprise extant conifers (including well-known taxa
such as spruce and pine species), gnetophytes, cycads,
Ginkgo, and diverse extinct groups including Bennettitales,
Cordaitales, Corystosperms and Glossopterids (Doyle,
1998). The morphological gap between gymnosperms and
angiosperms leads to problems with homology assignments
between reproductive organs from owering plants and
their putative ancestors and thus hampers an understanding
of the transition from gymnosperm reproductive cones to
angiosperm owers (Frohlich, 2003).
Flowers are characteristic features of angiosperms, and
their origin is a major aspect of the abominable
mystery. Given the importance of owers one wonders
that botanists have difculties to dene both precisely and
comprehensively what actually a ower is. Bateman et al.
(2006) have listed about a dozen of historical circumscrip-
tions, and these authors as well as Baum and Hileman
(2006) came up with updated denitions of a ower. As a
consensus of these contemporary attempts, one may dene
a ower as a determinate, compressed, bisexual reproductive
axis composed of megasporangia (carpels), microsporangia
(stamens) and a sterile perianth composed of at least one
sterile laminar organ. This is obviously not a comprehensive
* For correspondence. E-mail guenter.theissen@uni-jena.de
# The Author 2007. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved.
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denition, since there are numerous types of owers that
lack one or more of these organ types, probably due to
secondary simplication (e.g. unisexual owers with only
stamens or carpels).
This leads to the question as to which of these features
represent the key characteristics of angiosperm owers
(for a detailed discussion of the synapomorphies of the
angiosperms, see Donoghue and Doyle, 1989). The pre-
sence of carpels enclosing the ovules is generally con-
sidered an essential character that distinguishes typical
angiosperm owers from gymnosperms reproductive
cones (Crane et al., 1995; Endress, 2001; Stuessy, 2004).
Another key feature of angiosperm owers is a trait termed
hermaphroditism, or bisexuality, describing the fact that
male and female reproductive organs are united in one struc-
ture (or secondarily separated, as in the unisexual owers of
monoecious and dioecious angiosperms), while they might
be primarily separated in different structures in gymnos-
perms. Moreover, owers have a compressed reproductive
axis, while gymnosperms have elongate ones (Baum and
Hileman, 2006). The presence of a perianth surrounding
the reproductive organs, often including attractive organs
of petaloid appearance ( petals or tepals) is considered yet
another typical feature distinguishing angiosperm owers
from gymnosperm cones. Often double fertilization yielding
a nutritive endosperm is also considered an important
angiosperm feature (Stuessy, 2004; Kramer and Jaramillo,
2005). Obviously, a ower is not a simple characteristic,
but a complex of innovations (Baum and Hileman, 2006).
Almost all terrestrial environments are currently domi-
nated by owering plants. To what extent the evolution of
the ower contributed to the evolutionary success of the
angiosperms is unknown, but there is good reason to
assume that it was of quite some importance. In addition
to allowing efcient pollination by animals, thereby permit-
ting population densities lower than in gymnosperms
without inbreeding depression and extinction (Regal,
1977), owers also catalyse seed dispersal via the for-
mation of fruits, also often employing animal vectors.
Flowers also increase the potential for reproductive iso-
lation, e.g. by changes in pollinator specicity or owering
time (Grant, 1971).
The tempo and mode of morphological changes by
which the angiosperm ower originated has remained enig-
matic. Based on circumstantial evidence, several authors
have argued that bisexuality may have been one of the
rst steps during ower origin (see below, and Theissen
et al., 2002; Theien and Becker, 2004; Baum and
Hileman, 2006). Due to a lack of informative fossils, the
other steps are even more speculative. Stuessy (2004)
hypothesized that angiosperms may have evolved slowly
from seed ferns in the Jurassic, beginning rst with the
carpel, followed later by double fertilization, and lastly by
the appearance of owers. This series of events may well
have taken more than 100 million years to complete,
before an explosive evolutionary diversication may
have set in when the nal combination of the essential
angiosperm features was achieved (Stuessy, 2004).
Ignoring the question of carpel origin, Baum and Hileman
(2006) proposed that evolution of a bisexual axis via a
gynomonoecious intermediate was the rst step during
ower origin, followed by evolution of oral axis com-
pression and determinacy as step two, evolution of a peta-
loid perianth by sterilization of the outer stamens in step
three, and origin of the dimorphic perianth of core eudicots
in step four. What makes this scenario especially intriguing
is the fact that for each of these steps an underlying
developmental genetic mechanism is suggested by Baum
and Hileman (2006). Thus the scenario outlined by these
authors can in principle be experimentally tested by com-
parative developmental genetic studies in gymnosperms
and angiosperms. This, however, is much easier said than
done, given the poor performance of extant gymnosperms
as developmental genetic model systems; they are all
woody species requiring lots of growth space and many
years of vegetative development until they reach the repro-
ductive stage.
Various aspects of the evolutionary origin of the angios-
perm ower have been reviewed by several authors during
recent years (Crepet, 1998, 2000; Frohlich, 1999, 2003,
2006; Frohlich and Parker, 2000; Ma and dePamphilis,
2000; Albert et al., 2002; Theissen et al., 2002; Stuessy,
2004; Theien and Becker, 2004; Theissen, 2005a;
Theien and Kaufmann, 2006; Baum and Hileman, 2006;
Bateman et al., 2006; Doyle, 2006). With the accumulating
knowledge about the molecular genetic basis of ower
development in some model plants the proximate causes
and molecular mechanisms of oral evolution have found
increasing interest. Here we focus on an especially well-
understood aspect of owers from a developmental
genetic point of view: organ identity. We outline how two
leading models explaining the specication of oral organ
identity during individual plant development are helping
to better understand ower origin and diversication.
Evolution of seed plants and the ancestral ower
of crown group angiosperms
Understanding the phylogeny of seed plants (spermato-
phytes), a clade comprising angiosperms gymnosperms,
is an important prerequisite for understanding the evolution
of the reproductive structures of these taxa, including
owers. Much has been learned about the phylogeny of
seed plants in recent years by the use of molecular
markers, even though some critical aspects have remained
unresolved and controversial (Bateman et al., 2006;
Frohlich, 2006).
Extant gymnosperms are morphologically very diverse
and hence have usually been considered being paraphyletic,
with gnetophytes (Gnetum, Ephedra and Welwitschia) often
regarded as the sister group of the angiosperms (Doyle,
1998). It thus came as a surprise that the vast majority
of studies using molecular markers found moderate to
strong support for extant gymnosperms being monophyletic
(see, for example, Chaw et al., 1997, 2000; Soltis et al.
1999a; Winter et al., 1999; Bowe et al., 2000; Frohlich
and Parker, 2000; reviewed by Frohlich, 2006). Extant
gymnosperms and angiosperms may thus be sister
groups that, as also suggested by molecular evidence (see
e.g. Goremykin et al., 1997), separated roughly about
Theissen and Melzer Molecular Mechanisms of Floral Evolution 604
300 million years ago. These hypotheses, however, are not
easy to reconcile with the fact that the most ancient reliable
angiosperm fossils, i.e. those of Archaefructus, are just
about 130 million years old (Sun et al., 2002; Friis et al.,
2003), because this means there would have been about
170 million years of evolution during which the lineage
that led to extant angiosperms left no recognizable traces
in the fossil record (Bateman et al., 2006). Thus especially
some (but not all) paleobotanists do not accept the
molecular-derived hypotheses about seed plant phylogeny,
and consider the issue as unresolved (for an overview, see
Bateman et al., 2006; Frohlich, 2006).
In any case, the inability to unambiguously identify an
(extinct or extant) group of gymnosperms as the closest
relatives of the angiosperms implies that the gap between
the two kinds of seed plants could not be bridged yet.
The deep phylogeny of extant angiosperms itself is also
still quite controversial [compare, for example, Soltis and
Soltis (2004) and Soltis et al. (2004) with Goremykin
et al. (2003, 2004)], but it seems that we are much closer
to a reasonable consensus than in case of deep seed plant
phylogeny (Goremykin and Hellwig, 2006; Bateman
et al., 2006).
There is evidence that either Amborella trichopoda, or a
clade of Amborella together with Nymphaeales (water
lilies) represent the most basal angiosperms (see, for
example, Qiu et al., 1999; Soltis et al., 1999b; Barkman
et al., 2000; Graham and Olmstead, 2000; for a review
see Kuzoff and Gasser, 2000; Soltis and Soltis, 2003;
Soltis et al. 2005). As the next branch, this clade or grade
is then followed by Austrobaileyales, a monophyletic group
uniting Schisandraceae (now also including Illiciaceae;
Frohlich, 2006), Trimeniaceae and Austrobaileyaceae. [The
grade of probably most basal angiosperms was originally
termed ANITA (Qiu et al., 1999), and has recently been
named ANA (Amborella, Nymphaeales, Austrobaileyales)
(Frohlich, 2006).]
Assuming that Amborella, Nymphaeales and
Austrobaileyales indeed represent the most basal angios-
perms, the owers at the base of crown group angiosperms
can be reconstructed by consideration of oral structures
within this grade. These ancestral owers (Frohlich,
2006) were probably already hermaphroditic and had an
undifferentiated perianth, in which organs were arranged
in more than two cycles or a spiral. Given their complete-
ness and complexity these owers at the base of crown
group angiosperms, hence probably represent already a
quite late, rather than initial stage during the origin of the
ower as we know it. However, owers with differentiated
sepals and petals probably evolved even later during the
evolution of angiosperms (Albert et al., 1998; Kuzoff and
Gasser, 2000; Ronse De Craene et al., 2003; Soltis et al.,
2005).
In the framework of our current view on angiosperm phy-
logeny, the clade comprising all owering plants above the
basally diverging lineages is called the euangiosperms,
including the magnoliids, monocots, Chloranthaceae and
eudicots. The phylogenetic relationships of these groups
are unresolved. The eudicots comprise a grade of successive
branches, with Ranunculales, including Papaveraceae, as
sister to all other eudicots, and a large clade of core eudi-
cots containing the majority of all angiosperm species,
including Caryophyllales (to which Polygonaceae belong),
Rosids (to which Brassicaceae belong), Asterids, and
several additional lineages (Fig. 1). The major eudicot
model plant thale cress (Arabidopsis thaliana; henceforth
termed Arabidopsis) belongs to the plant family
Brassicaceae within the eurosids (APG II, 2003; Soltis
and Soltis, 2003).
From the ABC model to the quartet model
of oral organ identity
Eudicots represent the largest group of owering plants,
comprising about 75 % of angiosperm species (Buzgo
et al., 2005). Eudicots have relatively standardized owers
that typically consist of four different classes of organs
arranged in four (or more) whorls at the tip of a oral
shoot. The rst, outermost whorl usually consists of green
sepals resembling vegetative leaves. The second whorl is
composed of often relatively large, coloured and showy
petals. The third whorl contains the stamens, i.e. the male
reproductive organs which produce the pollen. Finally, the
fourth, innermost whorl contains the carpels, i.e. the
female reproductive organs, which are often fused and
inside of which, after fertilization, the ovules develop into
seeds.
The structures of mature sepals, petals, stamens and
carpels differ dramatically. Nevertheless, each oral organ
starts its development as a little bulge generated by anti-
clinal and periclinal divisions of undifferentiated cells of
the oral meristem. Thus each cell in the developing
oral organ primordium somehow recognizes its position
within the ower, and differentiates accordingly into a cell
type that is appropriate for the specic organ.
Based on the study of homeotic mutants in which the
identity of oral organs is changed, genetic models were
developed that explain how the different oral organs
acquire their specic identities during development
(reviewed by Theissen, 2001). In Arabidopsis, homeotic
mutants come in three classes, A, B and C. Ideal class A
mutants have carpels rather than sepals in the rst whorl,
and stamens instead of petals in the second whorl. Class B
mutants have sepals rather than petals in the second
whorl, and carpels replace stamens in the third whorl.
Class C mutants develop petals rather than stamens in the
third whorl, and sepals instead of carpels in the fourth
whorl. In addition, the owers of class C mutants grow
indeterminately, i.e. there is continued production of
mutant oral organs inside the 4th whorl; this is in contrast
to wild-type owers, whose development ceases after carpel
formation.
The phenotypes of class A, B and C mutants proved
extremely informative; they suggested that ower develop-
ment is sculpted by homeotic selector genes (also termed
oral organ identity genes). These genes act as major
developmental switches that control the entire genetic pro-
gramme required for the development of a particular organ.
The activities and interactions of oral homeotic genes
have been described in several quite similar models
Theissen and Melzer Molecular Mechanisms of Floral Evolution 605
(Haughn and Somerville, 1988; Schwarz-Sommer et al.,
1990), of which one, the ABC model (Coen and
Meyerowitz, 1991) became widely known (Fig. 1). This
classical ABC model maintains that organ identity in
each oral whorl is determined by a unique combination
of three organ identity gene activities, called A, B and C.
Expression of class A genes alone species the formation
of sepals. The combination AB species the development
of petals, and the combination BC species stamen for-
mation. Expression of C alone determines the development
of carpels. In order to explain the three classes of oral
homeotic mutants, the ABC model proposes that the class
A and class C genes negatively regulate each other, so
that the class C gene activity becomes expressed throughout
the ower when the class A gene function is compromised,
and vice versa (for reviews of the ABC model, see Weigel
and Meyerowitz, 1994; Theien, 2001).
In Arabidopsis, class A genes are represented by
APETALA1 (AP1) and APETALA2 (AP2), class B genes
by APETALA3 (AP3) and PISTILLATA (PI), and class C
genes by AGAMOUS (AG). Molecular cloning of these
genes revealed that, except AP2, they all represent MIKC-
type MADS-box genes encoding transcription factors
(MADS-domain proteins) (for a review see Theien,
2001). Thus the products of the ABC genes probably
control the transcription of other genes (target genes)
FI G. 1. On the evolution of the ABC system of oral organ specication. On the right, different versions of the ABC system are shown. The ABC models
differ either by the expression domains of the ABC genes, or by the organs specied by the different gene combinations. For every type of ABC system
only one example is shown, even though it may occur in different taxa and may have originated independently several times during evolution. For sim-
plicity, conserved class A genes are presented here even though in most cases class A gene functions might not be separable from functions as oral
meristem identity genes. Photographs of cones or owers of species sculpted by the corresponding ABC system are shown in the middle. The relationships
(according to APGII, 2003) between the plant families to which the different species shown belong are indicated by the phylogenetic tree on the left. Note
that in some cases (Macleaya, Rumex) the oral structures shown may represent exceptions rather than the rule in the respective plant families.
Abbreviations: mo, male organs; fo, female organs; sl, sepal-like tepals; pl, petal-like tepals; sd, staminodes; st, stamens; ca, carpels; te, ( petaloid)
tepals; pe, petals; pa, palea/lemma; lo, lodicules; se, sepals; sp, sepaloid petals
Theissen and Melzer Molecular Mechanisms of Floral Evolution 606
whose products are directly or indirectly involved in the for-
mation or function of oral organs.
MIKC-type proteins have a characteristic domain struc-
ture, including, from N- to C-terminus, a MADS (M-),
intervening (I-), keratin-like (K-) and C-terminal (C-)
domain (Munster et al., 1997). In all kinds of MADS-
domain proteins, the MADS-domain is by far the most
highly conserved region; it is the major determinant of
DNA binding, but it also performs functions in dimeri-
zation of MADS-domain proteins and in the binding
of accessory factors. MADS-domain proteins bind to
DNA sites with similarity to the consensus sequence
5
0
-CC(A/T)
6
GG-3
0
, termed a CArG-box (for CC-A
rich-GG). The I-domain, located directly downstream of
the MADS-domain, is relatively variable in length and not
very strongly conserved. At least in some MIKC-type pro-
teins, it constitutes an important determinant for the selective
formation of DNA-binding dimers. The K-domain is charac-
terized by a conserved, quite regular spacing of hydrophobic
residues, which is proposed to allow for the formation of
amphipathic helices. These are assumed to interact with
amphipathic helices of other K-domain-containing proteins
via the formation of coiled-coils to promote protein dimeri-
zation or multimeric complex formation (Kaufmann et al.,
2005). The most variable region is the C-domain at the
C-terminal end of MIKC-type proteins. In some MIKC-
type proteins it is involved in transcriptional activation, or
in the formation of multimeric complexes (structural and
phylogenetic aspects of MIKC-type proteins have been
reviewed in detail by Kaufmann et al., 2005).
Despite its heuristic value, the ABC model has two major
shortcomings: mutant and transgenic studies revealed that
the ABC genes are required, but not sufcient for the
specication of oral organ identity. Moreover, the ABC
model does not provide a molecular mechanism for the
specication of organ identity by the interaction of oral
homeotic genes. Development of the ABCDE and the
oral quartet models allowed both of these shortcomings
to be overcome. Based on the analysis of multiple mutants
generated via reverse genetics, the ABC model was rst
extended to an ABCD model by addition of class D
genes that are closely related to class C genes and specify
ovule identity (Angenent and Colombo, 1996). Further con-
sideration of class D genes is not required in the framework
of this review.
Knowing about another class of closely related
MIKC-type MADS-box genes originally known as
AGL2-like genes (Theien et al., 1996), but later renamed
into SEPALLATA (SEP)-like genes (Pelaz et al., 2000), is
essential here, however. Arabidopsis has four different
SEP-like genes, termed SEP1SEP4. While all single
and the double mutants of SEP revealed only very weak,
if any, deviations from wild-type phenotype, in sep1 sep2
sep3 triple mutants the organs in all whorls of the ower
develop into sepals, and ower development becomes inde-
terminate (Pelaz et al., 2000); in sep1 sep2 sep3 sep4 quad-
ruple mutants vegetative leaves rather than sepals develop
in all whorls of indeterminate owers (Ditta et al., 2004).
The SEP genes are still expressed in loss-of-function
mutants of class B and class C genes, and the initial
expression patterns of B and C class genes are not altered
in the sep1 sep2 sep3 triple mutant (Pelaz et al., 2000).
Therefore, the SEP genes are considered as yet another
class of oral organ identity genes, termed class E genes
(Theien, 2001), required for the development of all cate-
gories of oral organs.
According to the ABCDE model class A E genes are
required to specify sepals, A B E petals, B C E
stamens, C E carpels and D E ovules (Theien,
2001; Ditta et al., 2004). But by which molecular mechan-
ism do the different oral homeotic genes interact?
All MADS-domain proteins tested bind to DNA in a
sequence-specic way as dimers. Explaining the interaction
of oral homeotic genes by dimerization of their gene pro-
ducts soon failed, however, because the expected dimers
were not observed in respective studies (Riechmann et al.,
1996; Fan et al., 1997). For example, the class B proteins
AP3 and PI bind to DNA only as obligate AP3PI
heterodimers, but DNA-binding dimers between AP3 or
PI and AP1 (class A) or AG (class C) proteins were
not observed (Riechmann et al., 1996). An obvious way
to explain the combinatorial interactions would be that
class A and class B proteins (for specifying petals) or
class B and class C proteins (for specifying stamens) bind
separately to different cis-regulatory elements in the pro-
moters of the same target genes that are activated or
repressed during petal or stamen formation, respectively.
Alternatively, class A and class B (class B and class C) pro-
teins could have distinct sets of target genes involved in
petal (stamen) formation, so that the combinatorial inter-
action of the homeotic genes would be mechanistically rea-
lized only at the level of target genes, or even further
downstream in the gene cascades involved.
Things turned out to be more interesting. An important
clue was provided when Egea-Cortines et al. (1999)
reported that DEFICIENS (DEF), GLOBOSA (GLO) and
SQUAMOSA (SQUA) from snapdragon (Antirrhinum
majus) putative orthologues of AP3, PI and AP1,
respectively form multimeric complexes in electro-
phoretic mobility shift assays and yeast three-hybrid ana-
lyses. The authors hypothesized that the protein complex
is actually a protein tetramer, composed of a DEFGLO
heterodimer and a SQUASQUA homodimer, in which
the DEFGLO and SQUASQUA dimers recognize differ-
ent CArG-boxes (Egea-Cortines et al., 1999).
When Pelaz et al. (2000) reported that not only the ABC
but also the SEP genes are required for the formation
of petals, stamens and carpels, the available data about
the mechanisms of oral organ specication were pulled
together in the oral quartet model (Theien, 2001). It
suggests that complexes of four oral homeotic proteins
including SEP proteins control oral organ identity.
According to the original model there is at least one unique
quaternary complex for each type of oral organ (Theien,
2001). These quaternary protein complexes might function
as transcription factors by binding to CArG-boxes in the
promoters of target genes, which they either activate or
repress as appropriate for the development of the identities
of the different oral organs (Theien, 2001). There is evi-
dence that in these complexes, the AP1 or SEP proteins
Theissen and Melzer Molecular Mechanisms of Floral Evolution 607
provide the transcription-activation domain, while the other
proteins might be important for organ-specicity of gene
regulation (Honma and Goto, 2001). The oral quartet
model suggests that two protein dimers of each tetramer
recognize two different CArG-boxes, which might be
brought into close vicinity by bending the DNA between
the CArG-boxes (Egea-Cortines et al., 1999; Theien,
2001; Theien and Saedler, 2001), so the quartet (or tetra-
mer) is actually a dimer of dimers.
The oral quartet model was soon corroborated by
Honma and Goto (2001) who demonstrated the formation
of the complexes postulated for stamens and petals,
namely AP3/PI/AG/SEP and AP3/PI/AP1 (or SEP),
respectively, in yeast three-hybrid and four-hybrid assays.
Moreover, it was shown that ectopic co-expression of
class A/E B genes AP1 AP3 PI or SEP3 AP3
PI in transgenic Arabidopsis plants leads to a reprogram-
ming of supposed-to-be leaf primordia in a way that they
develop into petaloid organs. AP1 and SEP3 can substitute
each others which probably reects their close evolutionary
relationship (Becker and Theien, 2003) and hence partial
redundancy (Honma and Goto, 2001; Pelaz et al., 2001).
The co-expression of the class B C E genes AP3
PI AG SEP3 converts vegetative leaves into stamenoid
organs (Honma and Goto, 2001). These data add not only
support for the oral quartet model but also demonstrate
that just a few proteins are both necessary and sufcient
to superimpose petal and stamen identity upon a vegetative
developmental programme.
Clearly, the empirical basis of the oral quartet model is
still quite weak, especially since there is no direct evidence
so far that multimeric complexes of oral homeotic proteins
really exist, loop DNA, and regulate target gene expression
in the nuclei of plant cells. Moreover, a number of import-
ant questions remain unanswered by the oral quartet
model, for example, how target gene specicity is achieved
by oral homeotic proteins (Melzer et al., 2006).
Limitations and possible experimental tests of the oral
quartet model are discussed by Theien and Melzer (2006).
Nevertheless, since the oral quartet model, in contrast to
the ABCDE model, demonstrates how the combinatorial
interaction of the oral homeotic genes works mechanisti-
cally, it has been widely accepted and became the standard
model for work on oral homeotic genes (see, for example,
Jack, 2001; Winter et al., 2002a; Ferrario et al., 2003; Lee
et al., 2003; Ferrario et al., 2004; Shchennikova et al.,
2004; Krizek and Fletcher, 2005; Robles and Pelaz,
2005). The oral quartet model may thus facilitate research
on ower development and evolution in a similar way as the
ABC model did 10 years before.
In the following, we outline the impact of the oral
quartet model and the ABC model on understanding
ower origin and diversication, respectively.
FLORAL QUARTETS AND FLOWER ORI GI N
Molecular aspects of ower origin: a primer
In the eudicot and monocot model plants, but by inference
probably also in all other angiosperms, the identity of oral
organs is totally dependent on the uncompromised activity
of oral homeotic genes. Clarifying the origin of these
genes and of the interactions in which they and their
protein products are involved in may thus provide us
with valuable insights into the origin of the ower
(Theissen and Saedler, 1995; Theissen et al., 2000, 2002).
Characterization of the MADS-box gene families in
mosses and ferns suggested that, despite the presence of
numerous MIKC-type genes, orthologues of oral homeotic
genes are absent in non-seed plants (for a review, see
Theissen et al., 2000). However, putative orthologues of
class B and class C oral homeotic genes were isolated
from different conifers and the gnetophyte Gnetum
gnemon; orthologues of class C oral homeotic genes
were also identied in Cycas and Ginkgo (Tandre et al.,
1995, 1998; Rutledge et al., 1998; Mouradov et al., 1999;
Sundstrom et al., 1999; Winter et al., 1999; Theissen
et al., 2000; Fukui et al., 2001; Jager et al., 2003;
Theissen and Becker, 2004; Zhang et al., 2004). (More pre-
cisely, gymnosperms contain putative precursors of both
class C and D genes, which are here considered as class
C genes to simplify things, even though class C/D genes
would be a more precise term.) This suggests that class B
and class C genes were established by gene duplications
and subsequent divergence in the lineage that led to
extant spermatophytes, after the lineage that led to extant
ferns had already branched off, i.e. 300400 million
years ago; note, however, that some molecular clock esti-
mates suggested a much earlier origin of these genes
(Nam et al., 2003).
The expression patterns of gymnosperm class B and C
gene orthologues resemble those of class B and class C
genes in angiosperms, with class C genes being generally
expressed in reproductive organs (both male and female
ones), while expression of class B genes is focused on
male reproductive organs (for a review, see Theissen
et al., 2000; Theien and Becker, 2004). Class B and
class C genes from diverse gymnosperms could partially
or fully substitute for their angiosperm orthologues in
different kinds of complementation and ectopic expression
experiments, thus revealing conservation of gene function
during more than 300 million years of seed plant evolution
(Sundstrom and Engstrom, 2002; Winter et al., 2002a;
Zhang et al., 2004). Together with the expression data
in gymnosperms these ndings suggest that the ABC
system specifying oral organ identity in owering plants
evolved from a BC precursor system that was already estab-
lished in the most recent common ancestor of extant seed
plants about 300 million years ago (Winter et al., 1999).
We hypothesize that the BC system specied female
reproductive organs by the expression of class C genes,
and male reproductive organs by the expression of both
class C and class B genes (Fig. 1) (Winter et al., 1999;
Theissen et al., 2002; Theien and Becker, 2004). Thus
differential expression of class B genes specifying male
organ identity may represent the primary sex-determination
mechanism of all seed plants (Winter et al., 1999). If true,
switching from male to female organ identity, or vice versa,
could, in principle, simply result from changes in the spatial
or temporal expression of one ore more class B genes,
Theissen and Melzer Molecular Mechanisms of Floral Evolution 608
assuming that these genes encode transcription factors that
control, directly or indirectly, all the target genes required
to bring about male rather than female organ identity
during development.
Based on such a simple switch mechanism, novel
hypotheses on the origin of the bisexuality of owers
were developed; these hypotheses start with unisexual
axes as existing in most extant gymnosperms (Theissen
et al., 2002). The out-of-male scenario maintains that
bisexual owers originated from a male gymnosperm
cone, and that reduction of class B gene expression in the
upper region of the male cone led to the development of
female instead of male reproductive units in this upper
region (Theissen et al., 2002). The out-of-female scenario
assumes that owers originated from a female cone. In this
scenario ectopic expression of class B genes in the basal
region of the female cone led to the development of male
rather than female reproductive units in this basal region
(Theissen et al., 2002). Under both hypotheses a perianth-
less ower-like structure with male reproductive units in
the basal region (outer whorls) and female reproductive
units in the apical region (inner whorls) would have been
established. Mutant analysis indicates that the structural
transition predicted in these hypotheses is developmentally
possible (Theien and Becker, 2004). Among the molecular
changes which have been suggested to have caused the
modications in B gene expression are changes in genes
that control B gene expression (encoding trans-acting
factors), or changes in the cis-regulatory elements of the
B genes themselves (for a more detailed discussion, see
Theissen and Becker, 2004).
Under the hypotheses discussed here the perianth origi-
nated later than oral bisexuality. A major reason for this
assumption is that the identity of all the organs of perianth-
less owers could be specied by organ identity genes
(class B and class C genes) that were probably established
already in the most recent common ancestor of extant
seed plants.
Two alternatives to the out-of-male and out-of-female
hypotheses have been inspired by phylogeny reconstruc-
tions of the oral meristem identity gene FLORICAULA/
LEAFY and its orthologues. The mostly male theory
also maintains that ower organization derives more from
the male structure of ancestral gymnosperms than from
the female structure; in contrast to the out-of-male hypoth-
esis, however, it assumes that owers originated when
female organs (ovules) started to develop ectopically in
male cones (Frohlich and Parker, 2000; Frohlich, 2003,
2006). Albert et al. (2002) proposed that sexual conden-
sation (i.e. bisexuality) in angiosperm owers might have
originated when previously distinct spatial regulation of
copies of LFY-like genes in separate male and female
apices was amalgamated into singular LEAF (the proposed
orthologue of LEAFY in gymnosperms) control in all
reproductive meristems following loss of the NEEDLE
gene, a LFY paralogue only present in gymnosperms.
Albert et al. (2002) hypothesized that positive selection
on the partly redundant LEAF paralogue trapped reproduc-
tively unisexual angiosperm-ancestors into a condensed,
bisexual state.
Why oral quartets?
All of the hypotheses on ower origin mentioned attri-
bute a major role to class B and class C proteins or to reg-
ulators of oral homeotic proteins (LEAFY-like proteins).
Even though these transcription factors may well have
been of fundamental importance during the origin of the
angiosperm ower, we would like to bring to your attention
yet another player among the oral homeotic proteins,
namely class E proteins. Why? In contrast to the other
classes of oral homeotic genes, class E genes are absol-
utely required for the identity of all oral organs to
develop (Ditta et al., 2004). Moreover, in contrast to class
B and class C genes (for that matter comprising class D
genes), class E genes are unique to owering plants and
appear to be absent in gymnosperms. Attributing class E
genes a special role during ower origin thus appears not
too far fetched (Zahn et al., 2005).
Hence the question arises as to what makes class E genes
so special from a functional, or mechanistic, point of view.
A clue may be provided by the oral quartet model; it accu-
rately reects the developmental importance of the proteins
encoded by the class E genes, because class E proteins are
constituents of all the predicted multimeric transcription
factor complexes (Honma and Goto, 2001; Theien,
2001; Theien and Saedler, 2001), and perhaps of others
as well (Theien and Melzer, 2006; Melzer et al., 2006).
But why should tetrameric rather then dimeric protein com-
plexes specify organ identity? One could easily imagine a
scenario in which all oral organs are determined by only
three classes (ABC) of proteins that form specic dimers
which then act in a combinatorial way to activate organ-
specic target gene expression (see above) (Riechmann
et al., 1996).
A null hypothesis could postulate that the establishment
of tetramer formation represents just a neutral drift in
proteinprotein interaction patterns without any conse-
quences for gene regulation or the tness of the affected
organisms. We consider this as unlikely, however, and
suggest that the transition from a dimeric to a tetrameric
mode of protein interaction improved gene regulatory
mechanisms by increasing the cooperativity by which
MIKC-type MADS-domain proteins bind to DNA.
Cooperative binding describes the phenomenon that
binding afnity increases with the amount of molecules
already bound. In the case of MADS-domain proteins it
means that a dimer bound to a CArG-box increases the like-
lihood that a nearby CArG-box is also occupied by a
MADS-domain protein dimer. Cooperative binding is a
common theme among prokaryotic as well as eukaryotic
DNA-binding proteins (for examples, see Tsai et al.,
1989; Ptashne, 2005; Licht and Brantl, 2006; Svingen and
Tonissen, 2006). It offers the (unique) advantage to increase
the ratio of specic to non-specic binding while still
keeping the system exible enough for fast and robust regu-
lation (Ptashne, 2004).
In addition, in case of transcription factors binding coop-
eratively, a genetic switch can be created, in that small
changes in protein concentration within a certain, relatively
small critical range, can cause a dramatic response on target
Theissen and Melzer Molecular Mechanisms of Floral Evolution 609
genes being regulated. Thus by a small change in the con-
centrations of transcription factors developmental pro-
grammes can be changed dramatically (for discussions on
the switch function of cooperative binding proteins, see
Ackers et al., 1982; Ptashne, 2004, 2005). Moreover, tran-
scription factors often control their own expression via
feedback loops, with this reinforcing a possible switch
function.
During developmental transitions as well as organ for-
mation, livings cells need regulatory processes of great
sensitivity and high specicity, otherwise the transition
to reproductive growth may occur at the wrong time or
the specication of organ identity may fail. Therefore,
signal-to-noise ratios must be high when relevant infor-
mation is received and transmitted between the cell
surface, the cytoplasm and the nucleus (Blundell and
Fernandez-Recio, 2006). Hence, living cells have complex
signalling pathways that are moderated by feedback mech-
anisms. The transcription factors encoded by oral homeo-
tic genes can be considered as late elements in signal
transduction pathways specifying organ identity; there is
considerable evidence that they are subject to feedback
mechanisms including autoregulatory control (Schwarz-
Sommer et al., 1992; Gomez-Mena et al., 2005). Combi-
natorial tetramer formation by oral quartet formation
could well represent an important aspect of the switch
and feedback mechanisms that distinguishes the develop-
ment of one oral organ identity from the others. This
may explain the fundamental developmental importance
of the function of class E genes. All our admittedly
limited knowledge about the interaction behaviour of
oral homeotic proteins points towards a function of class
E (and, to some extent, class A) proteins as mediators of
higher order complex formation, i.e. they might constitute
the bridge proteins that make the oral quartets possible.
We assume that without such bridge proteins no higher
order complexes will form and, consequently, the system
will loose a considerable component of its cooperative
behaviour and maybe also (at least partly) its ability to
function as a genetic switch. Note, however, that yet
another function of SEP-like and AP1-like proteins might
be to provide transcriptional activation domains to the cor-
responding transcription factor complexes (Honma and
Goto, 2001).
Baum and Hileman (2006) proposed a model about
the role of oral quartets during ower origin that nicely
ts to our views. Previously, Theien and Becker (2004)
suggested that during ower origin, changes in cis-
regulatory elements, typically located in the promoter
region of a respective gene, in enhancers or silencers up-
or downstream of the coding region, or even in introns,
might have rendered class B gene expression more suscep-
tible to a hypothetical apical basal gradient already present
within the reproductive cone. The authors suggested that
such a gradient could be based on either a low molecular
weight compound (e.g. a phytohormone) or on a protein
(e.g. a transcription factor). Baum and Hileman (2006)
suggested that the apical basal gradient being employed
is made up of an orthologue of the oral meristem identity
protein LFY, indeed being a transcription factor.
In Arabidopsis, LFY is an important signalling integrator
for the transition from vegetative to reproductive develop-
ment. LFY expression appears to respond positively to a
developmental clock and to signalling by the phytohormone
giberellin. Baum and Hileman (2006) suggest that repro-
ductive shoots in the most recent common ancestor of ow-
ering plants may have gradually accumulated higher and
higher levels of LFY protein during development. Under
the hypothesis of Baum and Hileman (2006), both class B
and class C genes are activated by LFY-like proteins,
but class C genes are more responsive to LFY than class
B genes, so that the maximal level of class C gene
expression is nally much higher than that of class B
gene expression. Therefore, while class C gene expression
continues to increase during development due to increased
LFY expression, class B gene expression reaches a plateau
and then declines because complexes of class C and class
E proteins outcompete B/C/E complexes, and hence the
autoregulatory circuit of B gene expression is interrupted.
Consequently, at the base of the reproductive cone, where
both B and C genes are expressed, male organs develop,
while at the top, where only C genes are expressed,
female organs develop (Baum and Hileman, 2006).
The model of Baum and Hileman (2006) already
includes class E proteins and the formation of higher
order complexes. To emphasize the evolutionary impor-
tance of the class E proteins, we consider the hypothesis
of Baum and Hileman (2006) in the light of a hypothetical
scenario in which just protein dimers rather than tetramers
control oral organ formation (Fig. 2A). We assume that
either dimers of class B proteins, together with dimers of
class C proteins, and/or B/C heterodimers are responsible
for development of male reproductive organs, whereas
female organs are specied by class C protein homodimers.
Under this scenario the system would lack a considerable
component of its cooperative behaviour and might conse-
quently also be a less efcient developmental switch
distinguishing male from female development. The critical
threshold level of protein concentration needed for organ
formation would need to be much higher, because with
the loss of some cooperativity DNA-binding afnity
would also decrease. Moreover, as class C protein concen-
tration increases along the cone axis, a quite broad zone in
which neither unambiguously male nor female organs
develop may appear, because the critical threshold concen-
tration for developing female organs is not yet reached,
while class C protein level is so high that male organs
also do not develop properly (Fig. 2A). According to such
a scenario an elongated axis with male reproductive organs
on the bottom, followed by a broad zone of unclear organ
development, and nally followed by female organs might
develop.
In contrast, if cooperative binding exists (Fig. 2B) DNA-
binding afnity would increase. Consequently, protein
levels needed for specication of reproductive organs
would be much lower (as the DNA-binding afnity is
higher, fewer molecules are needed to occupy all the
relevant target gene promoters). Also, the concentration
of protein complexes bound to target gene promoters
along the cone axis would increase with a much steeper
Theissen and Melzer Molecular Mechanisms of Floral Evolution 610
slope and by this constituting the developmental switch.
Finally, as a consequence of this cooperative binding, a
compressed axis with female organs on the top and male
organs on the bottom will develop (Fig. 2B), and the
zone of unclear organ formation might be considerably
smaller. The resulting structure thus resembles an angios-
perm ower without a perianth, as predicted in some
models of ower origin (see above). We speculate that it
is cooperativity, brought about by the oral quartets, that
facilitated the development of male and female organs so
closely together on a compressed axis. Furthermore, we
propose that the many different kinds of organs within a
typical angiosperm ower (sepals, petals, stamens, carpels
with ovules) can only develop in such close vicinity to
each other because each of them is dened by its own
genetic switch, i.e. by its own oral quartet.
One should note that our model considers cooperativity
of DNA binding, but not positive autoregulation of class
C genes by corresponding proteins, which may further
help to dene the expression domains of class B and C
genes and thus to reduce or eventually even eliminate
the zone of unclear organ formation (Fig. 2).
It is further noteworthy that the increase of cooperative
DNA binding might also have increased the spectrum of
target genes that can be regulated as the afnity to the
respective promotors is increased. This broadening of the
target gene spectrum could be another important aspect in
the evolution of the angiosperm ower.
Importantly, the scenario presented here is not in contra-
diction to any of the proposed hypotheses on ower origin
outlined above. Rather, it can be understood as an addition
to all these theories. The invention of oral quartets might
have been one of the few crucial molecular steps for the
origin of the angiosperm ower. To test this hypothesis,
one needs to trace back the origin of the oral quartets,
i.e. the origin of the proteins that confer tetramerization.
Good candidates would be ( phylogenetically dened)
SEP- and AP1-like genes, which gave rise to (functionally
dened) class E and class A genes, respectively.
How did oral quartets originate?
The quartet (or tetramer) of oral homeotic proteins is
actually a dimer of dimers, which provides a rst clue as
FI G. 2. Two models for the specication of male and female organ identity in reproductive cones as a function of oral homeotic protein concentrations.
Both models assume that oral homeotic protein expression responds positively to a gradient of the oral meristem identity protein LEAFY, which forms
a concentration gradient along the cone axis from bottom (low) to top (high). The graphs represent the concentration of DNA-bound protein complexes
determining male (in green) and female (in blue) organ identity, respectively. When reproductive organs are specied by dimers of MADS-domain pro-
teins (we assume that B-protein homodimers and C-protein homodimers and/or B/C-heterodimers specify male organs whereas female organs are speci-
ed by C-protein homodimers), the slope with which protein concentration increases and hence target gene activation occurs would be less steep
compared with a system where cooperativity due to tetramerization (i.e. male organs are specied by a B/C/E-protein tetramer and female organs by
a C/E/C/E-protein tetramer) is taken into account (compare A and B). As a consequences, the zone of unclear organ formation (symbolized by a question
mark) is narrower when organ identity is specied by tetramers rather then by dimers. Also, although not depicted here for simplicity, the total protein
concentration (in contrast to the DNA-bound protein complexes) necessary to occupy a critical threshold of target gene promoters would be higher when
dimers specify organ identity. Note, however, that also a system composed of protein dimers can already posses a certain level of cooperativity, and that
other factors like feedback regulation that are not entirely taken into account here might also inuence the system.
Theissen and Melzer Molecular Mechanisms of Floral Evolution 611
to how it originated during evolution (Kaufmann et al.,
2005). An extreme hypothesis about the origin of oral
quartet formation suggests that tetramerization could be
an intrinsic capacity of all MIKC-type proteins, based on
the unique domain structure of these proteins; if so,
quaternary complexes of MIKC-type proteins should exist
not only in seed plants, but also in ferns, mosses and
even some green algae. Alternatively, quartet formation
might be a phenomenon that is restricted to some derived
groups of angiosperms, such as core eudicots (and maybe
also monocots) (Kaufmann et al., 2005). Pulling together
what has been said before, however, we consider another
hypothesis more likely that the capacity of quartet for-
mation was provided by the origin of SEP-like or SEP/
AP1-like genes either before or close to the origin of
extant angiosperms. Quartet formation may have improved
the developmental switch mechanism underlying the speci-
cation of organ identity, as required, e.g. for the sharp
transition from female to male (or vice versa) organ identity
within one and the same, condensed reproductive structure
(ower). So what is known about the origin of the clades
of SEP-like and AP1-like genes? (This clade terminology is
used for simplicity here, although the correct clade names
would be AGL2- and SQUAMOSA-like genes, respectively;
see Theien et al., 1996; Becker and Theien, 2003.)
As already mentioned above, class B and class C oral
homeotic proteins probably originated 300400 million
years ago in the lineage that led to the extant seed plants
(Theissen et al., 2000). However, the origin of the gene
subfamilies comprising class A and class E genes
AP1-like genes and SEP-like genes, respectively (Theien
et al., 1996; Pelaz et al., 2000) is unresolved. Despite
extensive efforts neither AP1-like nor SEP-like genes
have been isolated from conifers, gnetophytes, cycads or
Ginkgo, strongly suggesting that these gene types are
absent in extant gymnosperms (Becker and Theien, 2003;
Zahn et al., 2005). It is less clear, however, whether AP1-
and SEP-like genes originated in the lineage that led to
extant angiosperms after the gymnosperm lineage had
branched-off, or whether they originated earlier and had
been established in the most recent common ancestor of
extant seed plants about 300 million years ago already, and
then got lost in the lineage that led to extant gymnosperms.
Unfortunately, the phylogenetic relationship among the
major clades of MIKC-type genes is poorly resolved,
undermining a clear distinction between these two scen-
arios. AP1- and SEP-like genes consistently form a super-
clade together with AGL6-like genes (a subfamily of
MADS box genes whose function is unknown), but the
relationship among these gene clades and to other
MIKC-type genes is largely unclear. In quite a number of
gene trees AGL6-like genes appear as the sister group to
SEP-like genes, to the exclusion of AP1-like genes at the
base of the superclade (Fig. 3A; Theissen et al., 2000;
Becker and Theien, 2003; Nam et al. 2003). AGL6-like
genes, however, have been isolated from gymnosperms as
well as from angiosperms. Thus, if these trees represent
the true phylogenetic relationships among the gene clades
in question, both AP1- and SEP-like genes must have
been lost independently in the lineage that led to extant
gymnosperms (Fig. 3A). According to other phylogenies,
SEP- and AP1-like genes are sister groups, with
AGL6-like genes, in turn, forming the sister group to
both of the former (Fig. 3B) (Carlsbecker et al., 2003;
FI G. 3. Three different scenarios of how SEP-, AP1- and AGL6-like pro-
teins might be phylogenetically related. The phylogenies shown in (A) and
(B) are supported by various publications [compare Theissen et al. (2000);
Becker and Theien (2003) and Nam et al. (2003) with Carlsbecker et al.
(2003) and Kim et al. (2005)]. The phylogeny shown in (C) is discussed by
Becker and Theien (2003). Dotted lines represent lineages that probably
have been lost in the extant gymnosperms. Dimeric and tetrameric com-
plexes bound to DNA are symbolized as in Fig. 2. Asterisks mark the
hypothetical origin of higher order complexes. We assume that higher
order complex formation was established in the most recent common
ancestor of SEP-like and AP1-like proteins.
Theissen and Melzer Molecular Mechanisms of Floral Evolution 612
Kim et al., 2005). If this reects the true phylogeny, then
the gene lineage that later diverged into SEP-like and
AP1-like genes in angiosperms must have been lost in
the common ancestor of extant gymnosperms (Fig. 3B).
A third hypothesis, emphasized by Becker and Theien
(2003), would be that gymnosperm AGL6-like genes are
sister to all angiosperm genes, with angiosperm AGL6-
like genes being sister to SEP-like and AP1-like genes
(Fig. 3C). This would imply that angiosperm AGL6-like
genes originated from AGL6-like genes in the common
ancestor of extant gymnosperms and angiosperms, and
gave rise to AP1-like and SEP-like genes by two consecu-
tive gene duplications followed by sequence divergence.
Unfortunately, most phylogeny reconstructions do not corro-
borate such a scenario so far. However, an even more challen-
ging task is to gure out where and when the developmental
functions conferred by these genes have their origin. While
the function of class A genes (A-function) is not well con-
served even within eudicots it might just be an offspring of
a more widespread function in the specication of meristem
identity (Theissen et al., 2000; Litt, 2007) the opposite is
true for the function of class E genes (E-function). Class
E gene mutants have been described in eudicots as well as
monocots (Pelaz et al., 2000; Ferrario et al., 2003; Agrawal
et al. 2005), and the expression patterns of SEP-like genes
suggests that there is an E-function already in basally diver-
ging angiosperm lineages (Kim et al., 2005). Due to its fun-
damental importance in ower development, clarifying the
origin of the E-function may well be a major step in under-
standing the origin of the ower. However, although the
A-function, as originally dened speciying sepals and
petals is poorly conserved, the respective proteins show
characteristics that might not only be well conserved, but
that are also shared with E-function proteins. Indeed, it is
hard to clearly distinguish the function of class A and class
E genes, even in eudicot model plants. For example, if AP1
(an A-function gene) is overexpressed together with AP3
and PI (B-function genes), leaves are transformed into
petals. However, the same phenotype can be achieved by
overexpressing SEP3 (an E-function gene) instead of AP1
(Honma and Goto, 2001). Moreover, recent reports indicate
that both, SEP3 and AP1, play a similar role in oral induc-
tion (Sridhar et al., 2006). Also, class A and class E proteins
appear to be the only oral homeotic proteins that show
considerable transcription activation ability (Honma and
Goto, 2001). Finally, and maybe most importantly, E- and
A-function-related proteins are to date the only ones that
have been shown to serve as bridge proteins in higher order
complex formation (see Kaufmann et al., 2005, and refer-
ences cited therein).
The properties shared by A- and E-function proteins (the
transcription activation ability, the ability to mediate higher
order complex formation) are the ones that are evolutionary
most important. One might therefore speak of an E-function
that is also shared by A-function-related proteins at the base
of extant angiosperms. This is supported by the broad
expression pattern of A-genes in basally diverging angios-
perm lineages, which more closely resembles the
expression of E-genes than that of A-genes in eudicots
(Kim et al., 2005).
To trace back the origin of higher order complex for-
mation, both the function and the phylogenetic position of
AGL6-like genes, the third player in the superclade,
needs to be determined. If AGL6-like proteins are nested
within SEP- and AP1-like proteins, the most parsimonious
assumption would be that higher order complex formation
originated once at the base of this superclade and hence
also gymnosperm AGL6-like proteins should posses this
ability (Fig. 3A). If, on the other hand, AGL6-like genes
are sister to both SEP-like and AP1-like genes, one could
imagine that the ability to form higher order complexes ori-
ginated near the base of the SEP/AP1-lineage and is there-
fore specic for extant angiosperms (Fig. 3B). A similar
scenario can be envisaged if the phylogenetic scenario
depicted in Fig. 3C is true.
Unfortunately, there are almost no genetic or biochemical
data available for AGL6-like proteins that could strongly
support either of the views. It therefore remains one of
the main goals of future research to examine whether
AGL6-like proteins from angiosperms and maybe more
importantly from gymnosperms have the ability to
mediate tetramerization of oral homeotic proteins. This
will probably tell us when the oral quartets originated
and with this bring us closer to the molecular heart of
how the angiosperm ower originated.
FADI NG BORDERS AND SHIFTING
BOUNDARIES: THE ABCS OF
FLORAL DI VERSITY
Homeosis and oral diversication
The owers of the more than 250 000 extant angiosperm
species vary in many different ways, including the
number, arrangement, identity and shape of oral organs,
and the symmetry of the ower as a whole. Arguably the
best understood aspect from a developmental genetic
point of view is organ identity.
As previously discussed, changes in the expression pat-
terns of oral homeotic genes in transgenic or mutant
plants can bring about homeotic transformations of oral
organs, so that at some places the wrong organs develop.
For example, the expression of class C genes in the
whorls of the perianth leads to the development of carpel-
loid organs in the rst oral whorl, where actually sepals
should develop; in the second whorl, where petals are
usually formed, staminoid organs or even true stamens
develop (Bradley et al., 1993). The ectopic expression of
class B genes throughout the ower of Arabidopsis results
in the development of petaloid organs rather than sepals
in the rst oral whorl and of staminoid organs rather
than carpels in the fourth oral whorl (Krizek and
Meyerowitz, 1996). Thus it is tempting to speculate that
such changes in organ identity represent suitable models
for evolutionary processes that generated some aspects of
oral diversity. Homeotic transitions and other very rapid
(saltational) morphological changes have long been neg-
lected by mainstream evolutionary biology that considered
gradualistic changes the only credible mode of evolution;
in the framework of evolutionary developmental biology,
Theissen and Melzer Molecular Mechanisms of Floral Evolution 613
however, homeotic transitions have been reconsidered as a
reasonable mode of evolution, and ample evidence has
been gathered that they indeed played an important role
during the evolution of land plants (Theissen et al., 2000,
2002; Bateman and DiMichele, 2002; Kramer et al.,
2003; Ronse De Craene, 2003, 2007; Theissen, 2005b,
2006; Hintz et al., 2006; Nutt et al., 2006).
To determine whether changes in the spatial or temporal
expression of oral homeotic genes have contributed to the
structural diversication of the ower during angiosperm
evolution, comparative expression studies in owers with
different architectures are required. On the following
we discuss a few molecular case studies that corroborate
the view that homeotic transitions played a role during
the evolution of owers.
Fading borders by fuzzy expression domains
The classical ABC model was developed based on
studies in core eudicots, so it could well represent a
derived situation within angiosperms.; indeed, studies in
other groups of angiosperms support that view. The basal
lineages of angiosperms represent only a few per cent of
the owering plant species, but it is here where the greatest
diversity in oral structure and form is found. The owers
of basal angiosperms vary considerably in size, the
number of oral parts and the arrangement of oral
organs in spirals or whorls.
The mRNA expression patterns of putative oral
homeotic genes (orthologues of the oral organ identity
genes from core eudicots) were determined in quite a
number of basally diverging angiosperms lineages, including
Amborella, Nuphar (Fig. 1), Illicium, Magnolia and Asimia
(Kim et al., 2005). Generally, orthologues of class B genes
were found to be expressed in stamens and perianth organs,
orthologues of class C genes in stamens and carpels, and
class E gene orthologues in all oral organs (Kim et al.,
2005). For example, water lilies (Nymphaeaceae) such as
Nuphar (Fig. 1) have outer oral organs sometimes referred
to as sepals because of their greenish color, even though they
exhibit expression of class B gene orthologues, as do the
petals, staminodes and stamens (Kim et al., 2005). The peri-
anth of Amborella, another basal angiosperm, shows a
gradual transition from outer to inner oral organs (lateral
receptacular bracts to tepals, from outer tepals to inner
tepals, and from tepals to reproductive organs) (Soltis
et al., 2006); all these organs express class B gene ortho-
logues (Kim et al., 2005).
Class A and class C gene orthologues have broader
expression domains in the oral apices of basal angios-
perms than in core eudicots. The expression patterns
obtained for ABC genes from basal angiosperms are gene-
rally consistent with the morphology of the owers as pre-
dicted by the ABC model. For example, oral organs in
Amborella, Nuphar and Illicium expressing class B gene
orthologues are always more or less petaloid (or stamens).
These ndings gave rise to the fading borders model,
suggesting that gradual transitions in organ morphology
across the oral meristem result from gradients in level
of expression of ABC genes across the oral meristem
(Fig. 1) (Buzgo et al. 2004; Soltis et al., 2006).
Relatively weak expression at the edge of each genes
expression domain overlaps with expression of another
oral homeotic gene, while strong expression of each
oral organ identity gene occurs distant from the adjacent
genes centre of strong expression. According to this
hypothesis, in Nuphar, for example, staminodes develop
in the area where relatively weak C-gene expression over-
laps with strong B-gene expression. As C-gene expression
increases, stamens instead of staminodes develop in the
succeeding inner whorls (see ABC model for Nuphar in
Fig. 1). This fuzzy pattern of ABC gene expression would
impose some features of adjacent organs onto each other
and thus produce morphologically intergrading rather than
distinct oral organs. Such views about ambiguous organ
identity may rst appear unusual; they have been anticipated,
however, by a discussion ongoing for decades about
even more radical concepts such as fuzzy morphology
and continuum morphology to explain peculiarities in
the body plans of plants; good cases in point are bladderworts
(Utricularia) and river-weeds (Podostemaceae) (Rutishauser
and Isler, 2001; Rutishauser and Moline, 2005; and refer-
ences cited therein).
Sharpening the borders by autoregulatory control
If having broad and somewhat fuzzy expression domains is
the ancestral condition of oral homeotic genes in angios-
perm owers, the question arises as to how these expression
domains became constrained and sharpened during evolu-
tion. We suggest that the establishment of positive autoregu-
latory control contributed signicantly to this, because it led
to the amplication of small differences in expression levels
so that eventually only domains of no expression or full
expression remain, with sharp borders between both
expression domains (and hence organs). Autoregulatory
control may well have originated when CArG-boxes origi-
nated by chance (e.g. point mutations) in the regulatory
regions (such as promoters) of oral homeotic genes.
However, also changes in proteinprotein interaction
patterns and more sophisticated changes in gene regulatory
control may have been involved. Class B genes might serve
as a well-studied example here. The class B proteins from
the eudicot model plants Arabidopsis and Antirrhinum are
stable and functional in the cell only as heterodimers of a
DEF/AP3-like and a GLO/PI-like protein. These obligate
heterodimers regulate their own transcription by binding
to CArG-boxes in the promoters of their own genes (or
by binding to promoters of positive regulators) and are,
therefore, co-expressed in petals and stamens throughout
ower development. Heterodimerization is also absolutely
required for movement of B proteins into the nucleus and
DNA binding in vitro (reviewed by Theien and Becker,
2004).
In contrast to the obligatory heterodimerization in core
eudicots, a diversity of interaction patterns of class B
proteins, including obligate and facultative heterodimeriza-
tion as well as homodimerization, has been found
in monocots, such as tulip (Tulipa gesneriana) and lily
(Lilium regale) (Kanno et al., 2003; Winter et al., 2002b).
Theissen and Melzer Molecular Mechanisms of Floral Evolution 614
All class B gene orthologues from gymnosperms tested so
far revealed the ability to homodimerize (Sundstrom and
Engstrom, 2002; Winter et al., 2002b).
These data suggest that the interaction of class B proteins
evolved from homodimerization in gymnosperms to facul-
tative and obligate heterodimerization in monocots on the
one hand, and to exclusively obligate heterodimerization
in core eudicots on the other hand (Winter et al., 2002b).
Some types of obligate heterodimerization may have origi-
nated in several grasses such as maize (Zea mays) a second
time independently from its origin in core eudicots
(Whipple and Schmidt, 2006). This trend towards obligate
heterodimerization raises the question as to whether it is
more than a neutral drift in protein interaction patterns.
One hypothesis maintains that it was exactly the origin of
obligate heterodimerization that helped to restrict functional
class B gene expression to sharp domains (whorls) within
the ower, and thus may have contributed to the evolution
of morphologically distinct and unambiguous rather than
intergrading oral organs (Winter et al., 2002b).
Getting diversity by sliding boundaries
Once sharp borders of ABC gene expression and hence
unambiguous organ identities originated, diversication of
the ower in terms of oral organ identity was everything
but over. One obvious way, already mentioned above, to
further diversify oral structures is to change the expression
domains of oral homeotic genes and hence to change
organ identity in the affected whorls. It seems that nature
made rich use of this possibility during evolution. Two dif-
ferent scenarios come to mind: either the expression
domain of class B genes changes, or the expression
domain of both class A and class C genes changes simul-
taneously (due to the mutual antagonism between class A
and class C genes, at least as revealed in Arabidopsis).
Changes in class B gene expression appear of greater
evolutionary importance than changes in class A and
class C gene expression. Using the classical ABC model
as the starting point, two different cases can be distin-
guished: outward and inward shifts of class B gene
expression. Tulip (e.g. Tulipa gesneriana), lily (e.g.
Lilium regale), and most of their relatives in the lily
family (Liliaceae), for example, have owers displaying
organ identities quite similar to the ones of higher eudicots.
However, rst whorl organs are typically petaloid like
second whorl organs rather than sepaloid, i.e. the perianth
is a perigon composed of two whorls of petaloid tepals
(Fig. 1). This suggests a modied ABC model that is
characterized by an outward shift of class B gene expres-
sion, so that it is not only found in the organs of whorls
two and three, but also in the organs of the rst whorl
(Theissen et al., 2000, and literature cited therein). When
putative class B genes, i.e. DEF-like and GLO-like genes,
were investigated in lily and tulip, they were found to be
expressed in the organs of the rst three whorls of the
ower, as predicted by the modied ABC model (Kanno
et al., 2003). A similar situation is realized in orchids
(Orchidaceae). Orchid owers have a unique structure
including three types of perianth organs: three outer
tepals (T1T3) in the rst oral whorl, and two lateral
inner tepals (t1, t2) as well as a median inner tepal (t3)
called the lip (or labellum) in the second oral whorl. All
these tepals are usually of petaloid appearance and
express putative class B oral homeotic genes (DEF-like
and GLO-like genes) (Tsai et al., 2004; Xu et al., 2006).
Outside of the monocots, a similar situation is found, for
example, in some basal eudicots. Many owers of the
Ranunculaceae have distinctly different petaloid organs in
the rst two whorls (even though the rst whorl organs
are usually called sepals). Kramer et al. (2003) identied
many duplication events of putative class B genes at differ-
ent phylogenetic levels, with DEF/AP3-like genes display-
ing early duplications near the base of eudicots and GLO/
PI-like genes more recent duplications. Again, expression
studies suggested that petaloidy of organs in the rst
oral whorl is due to a shift of class B gene expression
towards the rst oral whorl (as in lily and tulip), but
also that differential expression of a particular lineage of
DEF/AP3-like genes has contributed to the distinction of
the petaloid organs in the rst and second oral whorl
(Kramer et al., 2003).
A somehow complementary case, i.e. an inward shift
in class B gene expression, is given in sorrel (Rumex
acetosa), where only the stamens, but none of the perianth
organs express DEF/AP3-like or GLO/PI-like (class B)
oral homeotic genes; these ndings are in line with the
sepaloid appearance of rst and second whorl organs of
the wind-pollinated owers of sorrel (Fig. 1) (Ainsworth
et al., 1995).
These cases strongly suggest that oral diversity can be
achieved by outward or inward shifts of class B oral
organ identity gene expression (Fig. 1). Respective mech-
anisms have been suggested by several authors and are
now known as sliding boundary or shifting boundary
models (Bowman, 1997; Albert et al., 1998; Theissen
et al., 2000; Kramer et al., 2003). Shifts in class A and
class C gene expression have been less frequently con-
sidered. A plausible possibility would be the outward
shift of class C gene expression towards the second
oral whorl, thus transforming petals into stamens. Good
candidates for owers specied that way are found in
some basal eudicots, such as Macleaya (Papaveraceae)
(Fig. 1) (Ronse De Craene, 2003) and in some naturally
occurring oral homeotic varieties of Capsella bursa-
pastoris (Brassicaceae), a close relative of Arabidopsis
(Hintz et al., 2006; Nutt et al., 2006).
Unfortunately, all the cases outlined above await corro-
boration by mutant analysis.
Right on target: different organs by changes
in downstream genes
In some cases very differently appearing organs are
specied by very similar ABC systems. An intriguing
example is revealed by the comparison between the
owers of grasses (Poaceae, such as rice, Oryza sativa),
and the owers of most core eudicots, including
Arabidopsis (Fig. 1). While class A genes alone specify
sepals in Arabidopsis, they may specify palea and lemma
Theissen and Melzer Molecular Mechanisms of Floral Evolution 615
in grasses. Even more remarkably, while the combination of
class A and class B genes species (usually) showy petals,
the same combination of oral homeotic genes species
lodicules in grasses. Lodicules are grass-specic, small,
glandular-like organs that swell at anthesis to spread the
lemma and palea apart so that the wind can disperse the
pollen produced by the stamens. They are very different
from petals indeed. According to their position in the
ower, however, lodicules might be petal homologues.
Recent comparative expression studies of class B genes in
a basal grass and close relatives support that view
(Whipple et al., 2007). The development of tremendously
different, yet homologous oral organs under the control
of orthologous homeotic genes is most plausibly explained
by changes in target genes of oral homeotic genes. Future
determination and characterization of larger numbers of
target genes in both grass and eudicot species than has
been possible so far will be required to work out the
details of this process.
OUTLOOK
There are quite a number of studies about the importance
of gene duplications, changes in proteinprotein inter-
actions, or morphological innovations during evolution.
Only few investigations, however, combine these elds of
inquiry, even though the processes they study often may
be intimately linked. For example, according to one of
our hypotheses, the origin of the ower may have been
facilitated by a transition from dimerization to tetrameriza-
tion of oral homeotic proteins, which again could have
depended on the establishment of SEP-like or AP1-like
genes by gene or genome duplication and sequence diver-
gence. To test such hypotheses, evolutionary biology
has to become an even more interdisciplinary endeavour,
ranging from hard-core physico-chemistry (e.g. of protein
protein and proteinDNA interactions) via molecular
evolution, comparative genomics and bioinformatics (e.g.
modelling the consequences of changes in proteinDNA
and proteinprotein interactions), to eld ecology (e.g.
investigating reproductive tness in natural environments),
all focused on one research topic (such as oral origin
or diversication) rather than being scattered. Stay tuned!
ACKNOWLEDGEMENTS
G.T. thanks Mike Jackson and Thomas L. Rost for their
invitation to write a review on the molecular origins of
the angiosperm ower, and for their willingness to
include it in this EvoDevo Highlight issue of the
Annals of Botany. Special thanks to Cristina Ferrandiz
and Charlie Scutt for their organizational efforts concerning
the Evo-Devo session at the XV FESPB Congress Plants,
People, Ecosystems and Applications, July 2006, Palais
des Congre`s, Lyon, France. R.M. thanks the
Studienstiftung des deutschen Volkes for his fellowship.
Both authors thank Hannelore Simon and Guilan Yang
for providing pictures of tulip and rice, respectively.
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