The floral genome: an evolutionary history of gene duplication and shifting patterns of gene expression

Through multifaceted genome-scale research involving phylogenomics, targeted gene surveys, and gene expression analyses in diverse basal lineages of angiosperms, our studies provide insights into the most recent common ancestor of all extant flowering plants. MADS-box gene duplications have played an important role in the origin and diversification of angiosperms. Furthermore, early angiosperms possessed a diverse tool kit of floral genes and exhibited developmental 'flexibility', with broader patterns of expression of key floral organ identity genes than are found in eudicots. In particular, homologs of B-function MADS-box genes are more broadly expressed across the floral meristem in basal lineages. These results prompted formulation of the 'fading borders' model, which states that the gradual transitions in floral organ morphology observed in some basal angiosperms (e.g. Amborella) result from a gradient in the level of expression of floral organ identity genes across the developing floral meristem.     

'Living stones' reveal alternative petal identity programs within the core eudicots

Petals, defined as the showy laminar floral organs in the second floral whorl, have been shown to be under similar genetic control in distantly related core eudicot model organisms. On the basis of these findings, it is commonly assumed that the petal identity program regulated by B-class MADS-box gene homologs is invariant across the core eudicot clade. However, the core eudicots, which comprise >70% of angiosperm species, exhibit numerous instances of petal and sepal loss, transference of petal function between floral whorls, and recurrent petal evolution. In the face of these complex patterns of perianth evolution, the concept of a core eudicot petal identity program has not been tested. We therefore examined the petal identity program in the Caryophyllales, a core eudicot clade in which perianth differentiation into sepals and petals has evolved multiple times. Specifically, we analyzed the expression patterns of B- and C-class MADS-box homologs for evidence of a conserved petal identity program between sepal-derived and stamen-derived petaloid organs in the 'living stone' family Aizoaceae. We found that neither sepal-derived nor stamen-derived petaloid organs exhibit gene expression patterns consistent with the core eudicot petal identity program. B-class gene homologs are not expressed during the development of sepal-derived petals and are not implicated in petal identity in stamen-derived petals, as their transient expression coincides with early expression of the C-class homolog. We therefore provide evidence for petal development that is independent of B-class genes and suggest that different genetic control of petal identity has evolved within this lineage of core eudicots. These findings call for a more comprehensive understanding of perianth variation and its genetic causes within the core eudicots--an endeavor that will have broader implications for the interpretation of perianth evolution across angiosperms.     

A novel MADS-box gene subfamily with a sister-group relationship to class B floral homeotic genes

Class B floral homeotic genes specify the identity of petals and stamens during the development of angiosperm flowers. Recently, putative orthologs of these genes have been identified in different gymnosperms. Together, these genes constitute a clade, termed B genes. Here we report that diverse seed plants also contain members of a hitherto unknown sister clade of the B genes, termed B(sister) (B(s)) genes. We have isolated members of the B(s) clade from the gymnosperm Gnetum gnemon, the monocotyledonous angiosperm Zea mays and the eudicots Arabidopsis thaliana and Antirrhinum majus. In addition, MADS-box genes from the basal angiosperm Asarum europaeum and the eudicot Petunia hybrida were identified as B(s) genes. Comprehensive expression studies revealed that B(s) genes are mainly transcribed in female reproductive organs (ovules and carpel walls). This is in clear contrast to the B genes, which are predominantly expressed in male reproductive organs (and in angiosperm petals). Our data suggest that the B(s) genes played an important role during the evolution of the reproductive structures in seed plants. The establishment of distinct B and B(s) gene lineages after duplication of an ancestral gene may have accompanied the evolution of male microsporophylls and female megasporophylls 400-300 million years ago. During flower evolution, expression of B(s) genes diversified, but the focus of expression remained in female reproductive organs. Our findings imply that a clade of highly conserved close relatives of class B floral homeotic genes has been completely overlooked until recently and awaits further evaluation of its developmental and evolutionary importance. Electronic supplementary material to this paper can be obtained by using the Springer Link server located at http://dx.doi.org/10.1007/s00438-001-0615-8.     

The ABC model and its applicability to basal angiosperms

BACKGROUND: Although the flower is the central feature of the angiosperms, little is known of its origin and subsequent diversification. The ABC model has long been the unifying paradigm for floral developmental genetics, but it is based on phylogenetically derived eudicot models. Synergistic research involving phylogenetics, classical developmental studies, genomics and developmental genetics has afforded valuable new insights into floral evolution in general, and the early flower in particular. SCOPE AND CONCLUSIONS: Genomic studies indicate that basal angiosperms, and by inference the earliest angiosperms, had a rich tool kit of floral genes. Homologues of the ABCE floral organ identity genes are also present in basal angiosperm lineages; however, C-, E- and particularly B-function genes are more broadly expressed in basal lineages. There is no single model of floral organ identity that applies to all angiosperms; there are multiple models that apply depending on the phylogenetic position and floral structure of the group in question. The classic ABC (or ABCE) model may work well for most eudicots. However, modifications are needed for basal eudicots and, the focus of this paper, basal angiosperms. We offer 'fading borders' as a testable hypothesis for the basal-most angiosperms and, by inference, perhaps some of the earliest (now extinct) angiosperms.     

Conservation and divergence in the AGAMOUS subfamily of MADS-box genes: evidence of independent sub- and neofunctionalization events

The MADS-box gene AGAMOUS (AG) plays a key role in determining floral meristem and organ identities. We identified three AG homologs, EScaAG1, EScaAG2, and EScaAGL11 from the basal eudicot Eschscholzia californica (California poppy). Phylogenetic analyses indicate that EScaAG1 and EScaAG2 are recent paralogs within the AG clade, independent of the duplication in ancestral core eudicots that gave rise to the euAG and PLENA (PLE) orthologs. EScaAGL11 is basal to core eudicot AGL11 orthologs in a clade representing an older duplication event after the divergence of the angiosperm and gymnosperm lineages. Detailed in situ hybridization experiments show that expression of EScaAG1 and EScaAG2 is similar to AG; however, both genes appear to be expressed earlier in floral development than described in the core eudicots. A thorough examination of available expression and functional data in a phylogenetic context for members of the AG and AGL11 clades reveals that gene expression has been quite variable throughout the evolutionary history of the AG subfamily and that ovule-specific expression might have evolved more than twice. Although sub- and neofunctionalization are inferred to have occurred following gene duplication, functional divergence among orthologs is evident, as is convergence, among paralogs sampled from different species. We propose that retention of multiple AG homologs in several paralogous lineages can be explained by the conservation of ancestral protein activity combined with evolutionarily labile regulation of expression in the AG and AGL11 clades such that the collective functions of the AG subfamily in stamen and carpel development are maintained following gene duplication.     

Patterns of gene duplication and functional evolution during the diversification of the AGAMOUS subfamily of MADS box genes in angiosperms

Members of the AGAMOUS (AG) subfamily of MIKC-type MADS-box genes appear to control the development of reproductive organs in both gymnosperms and angiosperms. To understand the evolution of this subfamily in the flowering plants, we have identified 26 new AG-like genes from 15 diverse angiosperm species. Phylogenetic analyses of these genes within a large data set of AG-like sequences show that ancient gene duplications were critical in shaping the evolution of the subfamily. Before the radiation of extant angiosperms, one event produced the ovule-specific D lineage and the well-characterized C lineage, whose members typically promote stamen and carpel identity as well as floral meristem determinacy. Subsequent duplications in the C lineage resulted in independent instances of paralog subfunctionalization and maintained functional redundancy. Most notably, the functional homologs AG from Arabidopsis and PLENA (PLE) from Antirrhinum are shown to be representatives of separate paralogous lineages rather than simple genetic orthologs. The multiple subfunctionalization events that have occurred in this subfamily highlight the potential for gene duplication to lead to dissociation among genetic modules, thereby allowing an increase in morphological diversity.     

The duplicated B-class MADS-box genes display dualistic characters in orchid floral organ identity and growth

Orchidaceae are an excellent model to examine perianth development because of their sophisticated floral architecture. In this study, we identified 24 APETALA3 (AP3)-like and 13 PISTILLA (PI)-like genes from 11 species of orchids and characterized them into four AP3- and two PI-duplicated homologs. The first duplication event in AP3 homologs occurring in the early evolutionary history of the Orchidaceae gave rise to AP3A and AP3B clades. Further duplication events resulted in four subclades, namely AP3A1, AP3A2, AP3B1 and AP3B2, during the evolution of Orchidaceae. The AP3 paralogous genes were expressed throughout inflorescence and floral bud development. From the in situ hybridization results, we noticed that the transition timings from ubiquitous to constrained expression in floral organs for both clades are different. The transition point of expression of the AP3A clade (clades 3 and 4) was at the late floral organ primordia stage. In contrast, that for the AP3B clade (clades 1 and 2) was not observed until the late inflorescence and floral bud stages. In addition, the AP3 orthologous genes revealed diverse expression patterns in various species of orchids, whereas the PI homologs were uniformly expressed in all floral whorls. AP3A2 orthologs play a noticeable role in lip formation because of their exclusive expression in the lip. Further evidence comes from the ectopic expression of AP3A2 detected in the lip-like petals extending from the lip in four sets of peloric mutants. Finally, a Homeotic Orchid Tepal (HOT) model is proposed, in which dualistic characters of duplicated B-class MADS-box genes are involved in orchid perianth development and growth.     

Missing links: the genetic architecture of flower and floral diversification

To understand the genetic architecture of floral development, including the origin and subsequent diversification of the flower, data are needed not only for a few model organisms but also for gymnosperms, basal angiosperm lineages and early-diverging eudicots. We must link what is known about derived model plants such as Arabidopsis, snapdragon and maize with other angiosperms. To this end, we suggest a massive evolutionary genomics effort focused on the identification and expression patterns of floral genes and elucidation of their expression patterns in ‘missing-link’ taxa differing in the arrangement, number and organization of floral parts.     

Floral variation and floral genetics in basal angiosperms

Recent advances in phylogeny reconstruction and floral genetics set the stage for new investigations of the origin and diversification of the flower. We review the current state of angiosperm phylogeny, with an emphasis on basal lineages. With the surprising inclusion of Hydatellaceae with Nymphaeales, recent studies support the topology of Amborella sister to all other extant angiosperms, with Nymphaeales and then Austrobaileyales as subsequent sisters to all remaining angiosperms. Notable modifications from most recent analyses are the sister relationships of Chloranthaceae with the magnoliids and of Ceratophyllaceae with eudicots. We review "trends" in floral morphology and contrast historical, intuitive interpretations with explicit character-state reconstructions using molecular-based trees, focusing on (1) the size, number, and organization of floral organs; (2) the evolution of the perianth; (3) floral symmetry; and (4) floral synorganization. We provide summaries of those genes known to affect floral features that contribute to much of floral diversity. Although most floral genes have not been investigated outside of a few model systems, sufficient information is emerging to identify candidate genes for testing specific hypotheses in nonmodel plants. We conclude with a set of evo-devo case studies in which floral genetics have been linked to variation in floral morphology.     

The expression of floral organ identity genes in contrasting water lily cultivars

The floral organs of typical eudicots such as Arabidopsis thaliana are arranged in four characteristic whorls, namely the sepal, petal, stamen and carpel, and the “ABC” floral organ identity model has been based on this arrangement. However, the floral organs in most basal angiosperms are spirally arranged with a gradual transition from the inside to outside, and an alternative model referred to as “fading borders” was developed to take account of this. The flower morphology of the water lily was tested against the “fading borders” model by determining the expression profile of the six primary floral organ identity genes AP2, AGL6, AP3, PI, AG and SEP1 in two cultivars showing contrasting floral morphology. In addition, to get accurate floatation of the genes expression level from outer to inner, we divided the floral organs into eight whorls according to morphological features. All these genes were expressed throughout all whorls of the flower, but their expression level changed gradually from the outside of the flower to its inside. This pattern was consistent with the “fading borders” model.     

Duplication of floral regulatory genes in the Lamiales

Duplication of some floral regulatory genes has occurred repeatedly in angiosperms, whereas others are thought to be single-copy in most lineages. We selected three genes that interact in a pathway regulating floral development conserved among higher tricolpates (LFY/FLO, UFO/FIM, and AP3/DEF) and screened for copy number among families of Lamiales that are closely related to the model species Antirrhinum majus. We show that two of three genes have duplicated at least twice in the Lamiales. Phylogenetic analyses of paralogs suggest that an ancient whole genome duplication shared among many families of Lamiales occurred after the ancestor of these families diverged from the lineage leading to Veronicaceae (including the single-copy species A. majus). Duplication is consistent with previous patterns among angiosperm lineages for AP3/DEF, but this is the first report of functional duplicate copies of LFY/FLO outside of tetraploid species. We propose Lamiales taxa will be good models for understanding mechanisms of duplicate gene preservation and how floral regulatory genes may contribute to morphological diversity.     

Missing links: the genetic architecture of flowers [correction of flower] and floral diversification.

To understand the genetic architecture of floral development, including the origin and subsequent diversification of the flower, data are needed not only for a few model organisms but also for gymnosperms, basal angiosperm lineages and early-diverging eudicots. We must link what is known about derived model plants such as Arabidopsis, snapdragon and maize with other angiosperms. To this end, we suggest a massive evolutionary genomics effort focused on the identification and expression patterns of floral genes and elucidation of their expression patterns in 'missing-link' taxa differing in the arrangement, number and organization of floral parts.     

Evolution of the APETALA3 and PISTILLATA lineages of MADS-box-containing genes in the basal angiosperms

The B class genes, including homologs of the Arabidopsis loci APETALA3 (AP3) and PISTILLATA (PI ), appear to play a conserved role in the determination of petal and stamen identity across core eudicot angiosperms. Understanding how and when these functions evolved is a critical component of elucidating the evolution of flowers, particularly the appearance of petaloid perianth organs. Before comparisons of gene expression patterns or functions can be made, however, it is necessary to establish the orthology of AP3 and PI homologs from basal angiosperms. Here, we report the identification and analysis of 29 new representatives of the B gene lineage from basal ANITA and magnoliid dicot angiosperms. These studies indicate that gene duplications have occurred at every phylogenetic level, both before and after the duplication that produced the separate AP3 and PI lineages. Comparison of genomic structure among PI homologs indicates that a 12-nucleotide deletion that had been considered synapomorphic for the whole PI lineage actually arose within the ANITA grade, after the split of the Nymphaeales but before the separation of the Austrobaileyales. Evidence for alternative splicing of the Nymphaea AP3 homolog is also presented. The implications of these findings for angiosperm systematics, the conservation of AP3 and PI gene function, and the evolution of the ABC program are discussed.     

Characterization of candidate class A,B and E floral homeotic genes from the perianthless basal angiosperm <Emphasis Type="Italic">Chloranthus spicatus</Emphasis> (Chloranthaceae)

The classic ABC model explains the activities of each class of floral homeotic genes in specifying the identity of floral organs. Thus, changes in these genes may underlay the origin of floral diversity during evolution. In this study, three MADS-box genes were isolated from the perianthless basal angiosperm Chloranthus spicatus. Sequence and phylogenetic analyses revealed that they are AP1-like, AP3-like and SEP3-like genes, and hence these genes were termed CsAP1, CsAP3 and CsSEP3, respectively. Due to these assignments, they represent candidate class A, class B and class E genes, respectively. Expression patterns suggest that the CsAP1, CsAP3 and CsSEP3 genes function during flower development of C. spicatus. CsAP1 is expressed broadly in the flower, which may reflect the ancestral function of SQUA-like genes in the specification of inflorescence and floral meristems rather than in patterning of the flower. CsAP3 is exclusively expressed in male floral organs, providing the evidence that AP3-like genes have ancestral function in differentiation between male and female reproductive organs. CsSEP3 expression is not detectable in spike meristems, but its mRNA accumulates throughout the flower, supporting the view that SEP-like genes have conserved expression pattern and function throughout angiosperm. Studies of synonymous vs nonsynonymous nucleotide substitutions indicate that these genes have not evolved under changes in evolutionary forces. All the data above suggest that the genes may have maintained at least some ancestral functions despite the lack of perianth in the flowers of C. spicatus. Nucleotide sequences data from this article have been deposited with the EMBL/GenBank Data Libraries under accession numbers AY316311, AY397762 and AY379963.