Gene networks controlling the initiation of flower development

The onset of flower formation is a key regulatory event during the life cycle of angiosperm plants, which marks the beginning of the reproductive phase of development. It has been shown that floral initiation is under tight genetic control, and deciphering the underlying molecular mechanisms has been a main area of interest in plant biology for the past two decades. Here, we provide an overview of the developmental and genetic processes that occur during floral initiation. We further review recent studies that have led to the genome-wide identification of target genes of key floral regulators and discuss how they have contributed to an in-depth understanding of the gene regulatory networks controlling early flower development. We focus especially on a master regulator of floral initiation in Arabidopsis thaliana APETALA1 (AP1), but also outline what is known about the AP1 network in other plant species and the evolutionary implications.     

Patterns of molecular evolution among paralogous floral homeotic genes.

The plant MADS-box regulatory gene family includes several loci that control different aspects of inflorescence and floral development. Orthologs to the Arabidopsis thaliana MADS-box floral meristem genes APETALA1 and CAULIFLOWER and the floral organ identity genes APETALA3 and PISTILLATA were isolated from the congeneric species Arabidopsis lyrata. Analysis of these loci between these two Arabidopsis species, as well as three other more distantly related taxa, reveal contrasting dynamics of molecular evolution between these paralogous floral regulatory genes. Among the four loci, the CAL locus evolves at a significantly faster rate, which may be associated with the evolution of genetic redundancy between CAL and AP1. Moreover, there are significant differences in the distribution of replacement and synonymous substitutions between the functional gene domains of different floral homeotic loci. These results indicate that divergence in developmental function among paralogous members of regulatory gene families is accompanied by changes in rate and pattern of sequence evolution among loci.     

Evolutionary dynamics of genes controlling floral development

Advances in the understanding of floral developmental genetics in model species such as Arabidopsis continue to provide an important foundation for comparative studies in other flowering plants. In particular, floral organ identity genes are the focus of many projects that are addressing both ancient and recent evolutionary questions. Expanded analyses of the evolution of these gene lineages have highlighted the dynamic nature of the gene birth-and-death process, and may have significant implications for the evolution of genetic pathways. Crucial functional studies of floral organ identity genes in diverse taxa are allowing the first real insight into the conservation of gene function, while findings on the genetic control of organ elaboration offer to open up new avenues for investigation. Taken together, these trends show that the field of floral developmental evolution continues to make significant progress towards elucidating the processes that have shaped the evolution of flower development and morphology.     

The making of a flower: control of floral meristem identity in IT>Arabidopsis/IT>

During the reproductive phase of a plant, shoot meristems follow one of two developmental programs to produce either flowers or vegetative shoots. The decision as to which meristems give rise to flowers, and when they do so, determines the general morphology of an inflorescence. Molecular and genetic research in Arabidopsis and other model species has identified several genes that control the identity that a meristem will adopt. These meristem identity genes are activated in response to developmental and environmental cues, and can be assigned to three basic categories: those required either to initiate or maintain the floral program in some meristems and those required to maintain the vegetative program in others.     

Quantitative trait loci for inflorescence development in Arabidopsis thaliana

Variation in inflorescence development patterns is a central factor in the evolutionary ecology of plants. The genetic architectures of 13 traits associated with inflorescence developmental timing, architecture, rosette morphology, and fitness were investigated in Arabidopsis thaliana, a model plant system. There is substantial naturally occurring genetic variation for inflorescence development traits, with broad sense heritabilities computed from 21 Arabidopsis ecotypes ranging from 0.134 to 0.772. Genetic correlations are significant for most (64/78) pairs of traits, suggesting either pleiotropy or tight linkage among loci. Quantitative trait locus (QTL) mapping indicates 47 and 63 QTL for inflorescence developmental traits in Ler x Col and Cvi x Ler recombinant inbred mapping populations, respectively. Several QTL associated with different developmental traits map to the same Arabidopsis chromosomal regions, in agreement with the strong genetic correlations observed. Epistasis among QTL was observed only in the Cvi x Ler population, and only between regions on chromosomes 1 and 5. Examination of the completed Arabidopsis genome sequence in three QTL regions revealed between 375 and 783 genes per region. Previously identified flowering time, inflorescence architecture, floral meristem identity, and hormone signaling genes represent some of the many candidate genes in these regions.     

Genetic Interactions That Regulate Inflorescence Development in Arabidopsis

In Arabidopsis, floral meristems arise in continuous succession directly on the flanks of the inflorescence meristem. Thus, the pathways that regulate inflorescence and floral meristem identity must operate both simultaneously and in close spatial proximity. The TERMINAL FLOWER 1 (TFL1) gene of Arabidopsis is required for normal inflorescence meristem function, and the LEAFY (LFY), APETALA 1 (AP1), and APETALA 2 (AP2) genes are required for normal floral meristem function. We present evidence that inflorescence meristem identity is promoted by TFL1 and that floral meristem identity is promoted by parallel developmental pathways, one defined by LFY and the other defined by AP1/AP2. Our analysis suggests that the acquisition of meristem identity during inflorescence development is mediated by antagonistic interactions between TFL1 and LFY and between TFL1 and AP1/AP2. Based on this study, we propose a simple model for the genetic regulation of inflorescence development in Arabidopsis. This model is discussed in relation to the proposed interactions between the inflorescence and the floral meristem identity genes and in regard to other genes that are likely to be part of the genetic hierarchy regulating the establishment and maintenance of inflorescence and floral meristems.     

Diversification and co-option of RAD-like genes in the evolution of floral asymmetry

To understand how changes in gene regulatory networks lead to novel morphologies, we have analysed the evolution of a key target gene, RAD, controlling floral asymmetry. In Antirrhinum, flower asymmetry depends on activation of RAD in dorsal regions of the floral meristem by the upstream regulators CYC and DICH. We show that Arabidopsis, a species with radially symmetric flowers, contains six RAD-like genes, reflecting at least three duplications since the divergence of Antirrhinum and Arabidopsis. Unlike the situation in Antirrhinum, none of the Arabidopsis RAD-like genes are activated in dorsal regions of the flower meristem. Rather, the RAD-like genes are expressed in distinctive domains along radial or ab-adaxial axes, consistent with a range of developmental roles. Introduction of a RAD genomic clone from Antirrhinum into Arabidopsis leads to a novel expression pattern that is distinct from the expression pattern of RAD in Antirrhinum and from the endogenous RAD-like genes of Arabidopsis. Nevertheless, RAD is able to influence developmental targets in Arabidopsis, as ectopic expression of RAD has developmental effects in this species. Taken together, our results suggest that duplication and divergence of RAD-like genes has involved a range of cis- and trans-regulatory changes. It is possible that such changes led to the coupling of RAD to CYC regulation in the Antirrhinum lineage and hence the co-option of RAD had a role in the generation of flower dorsoventral asymmetry.     

Integration of floral inductive signals by flowering locus T and suppressor of overexpression of Constans 1

Genetic analyses have identified four major genetic pathways which control the timing of floral transitions in Arabidopsis . The floral promotion or inhibitory signals from these distinct pathways ultimately converge into a subset of genes, commonly known as the floral integrators, such that the plants are compelled either to switch into a flowering phase or to remain in the vegetative stage. In this review, we have discussed the recent findings regarding cross-talk between floral promotion pathways and flowering locus T and suppressor of overexpression of Constans 1 , the major floral integrators within Arabidopsis . The spatial regulation of these integrators has also been discussed, as these spatial patterns may help us to understand the manner in which floral inductive signals are transmitted. We have also discussed conservation and divergence with regard to the regulation of floral integrators between Arabidopsis and rice. Finally, we have provided a series of insights into the complex signaling network which coordinates plant development.     

The identification of candidate genes for a reverse genetic analysis of development and function in the Arabidopsis gynoecium

The screening for mutants and their subsequent molecular analysis has permitted the identification of a number of genes of Arabidopsis involved in the development and functions of the gynoecium. However, these processes remain far from completely understood. It is clear that in many cases, genetic redundancy and other factors can limit the efficiency of classical mutant screening. We have taken the alternative approach of a reverse genetic analysis of gene function in the Arabidopsis gynoecium. A high-throughput fluorescent differential display screen performed between two Arabidopsis floral homeotic mutants has permitted the identification of a number of genes that are specifically or preferentially expressed in the gynoecium. Here, we present the results of this screen and a detailed characterization of the expression profiles of the genes identified. Our expression analysis makes novel use of several Arabidopsis floral homeotic mutants to provide floral organ-specific gene expression profiles. The results of these studies permit the efficient targeting of effort into a functional analysis of gynoecium-expressed genes.     

Similar genetic mechanisms underlie the parallel evolution of floral phenotypes

The repeated origin of similar phenotypes is invaluable for studying the underlying genetics of adaptive traits; molecular evidence, however, is lacking for most examples of such similarity. The floral morphology of neotropical Malpighiaceae is distinctive and highly conserved, especially with regard to symmetry, and is thought to result from specialization on oil-bee pollinators. We recently demonstrated that CYCLOIDEA2-like genes (CYC2A and CYC2B) are associated with the development of the stereotypical floral zygomorphy that is critical to this plant-pollinator mutualism. Here, we build on this developmental framework to characterize floral symmetry in three clades of Malpighiaceae that have independently lost their oil bee association and experienced parallel shifts in their floral morphology, especially in regard to symmetry. We show that in each case these species exhibit a loss of CYC2B function, and a strikingly similar shift in the expression of CYC2A that is coincident with their shift in floral symmetry. These results indicate that similar floral phenotypes in this large angiosperm clade have evolved via parallel genetic changes from an otherwise highly conserved developmental program.     

Beyond Arabidopsis. Translational biology meets evolutionary developmental biology

Developmental processes shape plant morphologies, which constitute important adaptive traits selected for during evolution. Identifying the genes that act in developmental pathways and determining how they are modified during evolution is the focus of the field of evolutionary developmental biology, or evo-devo. Knowledge of genetic pathways in the plant model Arabidopsis serves as the starting point for investigating how the toolkit of developmental pathways has been used and reused to form different plant body plans. One productive approach is to identify genes in other species that are orthologous to genes known to control developmental pathways in Arabidopsis and then determine what changes have occurred in the protein coding sequence or in the gene's expression to produce an altered morphology. A second approach relies on natural variation among wild populations or crop plants. Natural variation can be exploited to identify quantitative trait loci that underlie important developmental traits and, thus, define those genes that are responsible for adaptive changes. The possibility of applying comparative genomics approaches to Arabidopsis and related species promises profound new insights into the interplay of evolution and development.     

Functional analyses of genetic pathways controlling petal specification in poppy

MADS-box genes are crucial regulators of floral development, yet how their functions have evolved to control different aspects of floral patterning is unclear. To understand the extent to which MADS-box gene functions are conserved or have diversified in different angiosperm lineages, we have exploited the capability for functional analyses in a new model system, Papaver somniferum (opium poppy). P. somniferum is a member of the order Ranunculales, and so represents a clade that is evolutionarily distant from those containing traditional model systems such as Arabidopsis, Petunia, maize or rice. We have identified and characterized the roles of several candidate MADS-box genes in petal specification in poppy. In Arabidopsis, the APETALA3 (AP3) MADS-box gene is required for both petal and stamen identity specification. By contrast, we show that the AP3 lineage has undergone gene duplication and subfunctionalization in poppy, with one gene copy required for petal development and the other responsible for stamen development. These differences in gene function are due to differences both in expression patterns and co-factor interactions. Furthermore, the genetic hierarchy controlling petal development in poppy has diverged as compared with that of Arabidopsis. As these are the first functional analyses of AP3 genes in this evolutionarily divergent clade, our results provide new information on the similarities and differences in petal developmental programs across angiosperms. Based on these observations, we discuss a model for how the petal developmental program has evolved.     

Analysis of PEAM4, the pea AP1 functional homologue, supports a model for AP1-like genes controlling both floral meristem and floral organ identity in different plant species

APETALA1 (AP1) and its homologue SQUAMOSA (SQUA) are key regulatory genes specifying floral meristem identity in the model plants Arabidopsis and Antirrhinum. Despite many similarities in their sequence, expression and functions, only AP1 appears to have the additional role of specifying sepal and petal identity. No true AP1/SQUA-functional homologues from any other plant species have been functionally studied in detail, therefore the question of how the different functions of AP1-like genes are conserved between species has not been addressed. We have isolated and characterized PEAM4, the AP1/SQUA-functional homologue from pea, a plant with a different floral morphology and inflorescence architecture to that of Arabidopsis or Antirrhinum. PEAM4 encodes for a polypeptide 76% identical to AP1, but lacks the C-terminal prenylation motif, common to AP1 and SQUA, that has been suggested to control the activity of AP1. Nevertheless, constitutive expression of PEAM4 caused early flowering in tobacco and Arabidopsis. In Arabidopsis, PEAM4 also caused inflorescence-to-flower transformations similar to constitutive AP1 expression, and was able to rescue the floral organ defects of the strong ap1-1 mutant. Our results suggest that the control of both floral meristem and floral organ identity by AP1 is not restricted to Arabidopsis, but is extended to species with diverse floral morphologies, such as pea.     

Production and characterization of diverse developmental mutants of Medicago truncatula

The diploid annual legume Medicago truncatula has been developed as a tractable genetic system for studying biological questions that are unique to, or well suited for study in legume species. An efficient mutagenesis protocol using ethyl-methyl sulfonate and a polymorphic ecotype with properties appropriate for use as a mapping parent are described. Isolation and characterization of three developmental mutants are described. The mtapetala mutation results in homeotic conversions of floral organ whorls 2 and 3 into sepals and carpelloid structures, respectively, similar to mutations in the apetala3/pistillata genes of Arabidopsis. The palmyra mutation primarily affects seedling shoot meristem initiation, and thus phenocopies meristem function mutations identified in Arabidopsis such as the zwille locus. The phenotype of the palmyra and mtapetala double mutant is additive, with seedling shoot meristems and floral organs indistinguishable from those of the single palmyra and mtapetala mutants, respectively. These results are consistent with a lack of genetic interaction between these loci. A third mutant, speckle, is characterized by spontaneous necrotic lesion formation on leaves, root, and stems, similar to necrosis mutants identified in other plant species. In addition to documenting the efficient mutagenesis of M. truncatula, the availability of developmental mutants that phenocopy characterized Arabidopsis mutants will provide a basis for establishing orthologous gene function between M. truncatula and Arabidopsis, once the genes responsible are cloned. Moreover, the male-sterile, female-fertile nature of the mtapetala mutant provides a convenient tool for genetic analyses in M. truncatula.     

Separation of genetic functions controlling organ identity in flowers

Comparative studies on the ABC model of floral development have revealed extensive conservation of B and C class genes, but have failed to identify similar conservation for A class genes. Using a reverse genetic approach, we show that the previous inability to obtain Antirrhinum mutants corresponding to the A class gene AP2 of Arabidopsis reflects greater genetic redundancy in Antirrhinum . Antirrhinum has two genes corresponding to AP2, termed LIP1 and LIP2, both of which need to be inactivated to give a mutant phenotype. Analysis of interactions between LIP and class B/C genes shows that unlike AP2 in Arabidopsis, LIP genes are not required for repression of C in outer whorls of the flower. However, like AP2, LIP genes play a role in sepal, petal and ovule development, although some of their detailed effects are different, reflecting the diverse morphologies of Antirrhinum and Arabidopsis flowers. The dual functions for which AP2 is required in Arabidopsis are therefore separate in Antirrhinum, showing that the genetic basis of some aspects of organ identity have undergone major evolutionary change.     

Response: Missing links: the genetic architecture of flower and floral diversification

The genomic approach to understanding how evolution has generated the extraordinary diversity of flowers is to assemble a floral EST database for several missing-link taxa and then use gene phylogenies and expression data to identify genes that are important in flower evolution. However, such a genomic approach is likely to miss important genes that are not members of gene families that control flower development in Arabidopsis, and can overlook genes that are not expressed, or are weakly expressed, at their site of action. Therefore we propose complementary genetic approaches in which a few phylogenetically well distributed species are developed as model systems and floral differentiation among closely related species is studied using functional approaches.     

Evolution of regulatory interactions controlling floral asymmetry

A key challenge in evolutionary biology is to understand how new morphologies can arise through changes in gene regulatory networks. For example, floral asymmetry is thought to have evolved many times independently from a radially symmetrical ancestral condition, yet the molecular changes underlying this innovation are unknown. Here, we address this problem by investigating the action of a key regulator of floral asymmetry, CYCLOIDEA (CYC), in species with asymmetric and symmetric flowers. We show that CYC encodes a DNA-binding protein that recognises sites in a downstream target gene RADIALIS (RAD) in Antirrhinum. The interaction between CYC and RAD can be reconstituted in Arabidopsis, which has radially symmetrical flowers. Overexpression of CYC in Arabidopsis modifies petal and leaf development, through changes in cell proliferation and expansion at various stages of development. This indicates that developmental target processes are influenced by CYC in Arabidopsis, similar to the situation in Antirrhinum. However, endogenous RAD-like genes are not activated by CYC in Arabidopsis, suggesting that co-option of RAD may have occurred specifically in the Antirrhinum lineage. Taken together, our results indicate that floral asymmetry may have arisen through evolutionary tinkering with the strengths and pattern of connections at several points in a gene regulatory network.     

The blooming of grass flower development

The past half decade has provided a wealth of information concerning the molecular and genetic control of floral organ and meristem identity in dicotyledonous plants. Comparatively little is understood about these processes in grass species in spite of the importance that these species play in human agriculture. The isolation of grass genes that are homologous to dicot floral homeotic genes in combination with recent advances in reverse genetic technology and improvements in cereal transformation opens the door for understanding molecular mechanisms of grass flower development. Such information will also focus attention on the evolutionary relationships between grass and dicot flowers and the degree to which the developmental pathways leading to reproductive organ development in divergent angiosperms have utilized conserved mechanisms.     

Genetics of flower initiation and development in annual and perennial plants

Flowering is an integral developmental process in angiosperms, crucial to reproductive success and continuity of the species through time. Some angiosperms complete their life cycle within a year (annual plants), and others have a longer reproductive life, which is characterized by the generation of new flowering and vegetative shoots every year (perennial plants). Despite the differences in their lifespan, the underlying genetics of flower induction and floral organ formation appears to be similar among these plants. Hence, the knowledge gained from the study of flowering mechanism in Arabidopsis thaliana can be used to better understand similar processes in other plant species, especially the perennials, which usually have a long generation time and are not amenable to genetic analysis. Using Arabidopsis as a model, we briefly discuss the current understanding of the transition from vegetative to reproductive growth and the subsequent formation of individual floral organs, and how this knowledge has been successfully applied to the identification of homologous genes from perennial crops. Although annuals appear to share many similarities with perennials in terms of gene function, they differ in their commitment to flowering. Once an annual reaches the reproductive phase, all meristems are typically converted into either floral or inflorescence meristems. In contrast, each year, each meristem of a mature perennial has the choice to produce either a vegetative or a reproductive shoot. The physiology and genetics of flowering in Citrus are used to highlight the complexity of reproductive development in perennials, and to discus possible future research directions.