Loss of deeply conserved C-class floral homeotic gene function and C- and E-class protein interaction in a double-flowered ranunculid mutant.
ABSTRACT: In the model plant Arabidopsis thaliana, a core eudicot, the floral homeotic C-class gene AGAMOUS (AG) has a dual role specifying reproductive organ identity and floral meristem determinacy. We conduct a functional analysis of the putative AG ortholog ThtAG1 from the ranunculid Thalictrum thalictroides, a representative of the sister lineage to all other eudicots. Down-regulation of ThtAG1 by virus-induced gene silencing resulted in homeotic conversion of stamens and carpels into sepaloid organs and loss of flower determinacy. Moreover, flowers exhibiting strong silencing of ThtAG1 phenocopied the double-flower ornamental cultivar T. thalictroides 'Double White.' Molecular analysis of 'Double White' ThtAG1 alleles revealed the insertion of a retrotransposon causing either nonsense-mediated decay of transcripts or alternative splicing that results in mutant proteins with K-domain deletions. Biochemical analysis demonstrated that the mutation abolishes protein-protein interactions with the putative E-class protein ThtSEP3. C- and E-class protein heterodimerization is predicted by the floral quartet model, but evidence for the functional importance of this interaction is scarce outside the core eudicots. Our findings therefore corroborate the importance and conservation of the interactions between C- and E-class proteins. This study provides a functional description of a full C-class mutant in a noncore ("basal") eudicot, an ornamental double flower, affecting both organ identity and meristem determinacy. Using complementary forward and reverse genetic approaches, this study demonstrates deep conservation of the dual C-class gene function and of the interactions between C- and E-class proteins predicted by the floral quartet model.
Project description:The floral meristem (FM) is self-maintaining at the early stages of flower development, but it is terminated when a fixed number of floral organs are produced. The FLORAL ORGAN NUMBER4 (FON4; also known as FON2) gene, an ortholog of Arabidopsis CLAVATA3 (CLV3), is required for regulating FM size and determinacy in rice. However, its interactions with floral homeotic genes remain unknown. Here, we report the genetic interactions between FON4 and floral homeotic genes OsMADS15 (an A-class gene), OsMADS16 (also called SUPERWOMAN1, SPW1, a B-class gene), OsMADS3 and OsMADS58 (C-class genes), OsMADS13 (a D-class gene), and OsMADS1 (an E-class gene) during flower development. We observed an additive phenotype in the fon4 double mutant with the OsMADS15 mutant allele dep (degenerative palea). The effect on the organ number of whorl 2 was enhanced in fon4 spw1. Double mutant combinations of fon4 with osmads3, osmads58, osmads13, and osmads1 displayed enhanced defects in FM determinacy and identity, respectively, indicating that FON4 and these genes synergistically control FM activity. In addition, the expression patterns of all the genes besides OsMADS13 had no obvious change in the fon4 mutant. This work reveals how the meristem maintenance gene FON4 genetically interacts with C, D, and E floral homeotic genes in specifying FM activity in monocot rice.
Project description:The identification of mutants in model plant species has led to the isolation of the floral homeotic function genes that play crucial roles in flower organ specification. However, floral homeotic C-function genes are rarely studied in basal eudicots. Here, we report the isolation and characterization of the AGAMOUS (AG) orthologous gene (PaAG) from a basal eudicot London plane tree (Platanus acerifolia Willd). Phylogenetic analysis showed that PaAG belongs to the C- clade AG group of genes. PaAG was found to be expressed predominantly in the later developmental stages of male and female inflorescences. Ectopic expression of PaAG-1 in tobacco (Nicotiana tabacum) resulted in morphological alterations of the outer two flower whorls, as well as some defects in vegetative growth. Scanning electron micrographs (SEMs) confirmed homeotic sepal-to-carpel transformation in the transgenic plants. Protein interaction assays in yeast cells indicated that PaAG could interact directly with PaAP3 (a B-class MADS-box protein in P. acerifolia), and also PaSEP1 and PaSEP3 (E-class MADS-box proteins in P. acerifolia). This study performed the functional analysis of AG orthologous genes outside core eudicots and monocots. Our findings demonstrate a conserved functional role of AG homolog in London plane tree, which also represent a contribution towards understanding the molecular mechanisms of flower development in this monoecious tree species.
Project description:Perennial woodland herbs in the genus Thalictrum exhibit high diversity of floral morphology, including four breeding and two pollination systems. Their phylogenetic position, in the early-diverging eudicots, makes them especially suitable for exploring the evolution of floral traits and the fate of gene paralogs that may have shaped the radiation of the eudicots. A current limitation in evolution of plant development studies is the lack of genetic tools for conducting functional assays in key taxa spanning the angiosperm phylogeny. We first show that virus-induced gene silencing (VIGS) of a PHYTOENE DESATURASE ortholog (TdPDS) can be achieved in Thalictrum dioicum with an efficiency of 42% and a survival rate of 97%, using tobacco rattle virus (TRV) vectors. The photobleached leaf phenotype of silenced plants significantly correlates with the down-regulation of endogenous TdPDS (P<0.05), as compared to controls. Floral silencing of PDS was achieved in the faster flowering spring ephemeral T. thalictroides. In its close relative, T. clavatum, silencing of the floral MADS box gene AGAMOUS (AG) resulted in strong homeotic conversions of floral organs. In conclusion, we set forth our optimized protocol for VIGS by vacuum-infiltration of Thalictrum seedlings or dormant tubers as a reference for the research community. The three species reported here span the range of floral morphologies and pollination syndromes present in Thalictrum. The evidence presented on floral silencing of orthologs of the marker gene PDS and the floral homeotic gene AG will enable a comparative approach to the study of the evolution of flower development in this group.
Project description:Molecular genetic studies of floral development have concentrated on several core eudicots and grasses (monocots), which have canalized floral forms. Basal eudicots possess a wider range of floral morphologies than the core eudicots and grasses and can serve as an evolutionary link between core eudicots and monocots, and provide a reference for studies of other basal angiosperms. Recent advances in genomics have enabled researchers to profile gene activities during floral development, primarily in the eudicot Arabidopsis thaliana and the monocots rice and maize. However, our understanding of floral developmental processes among the basal eudicots remains limited.Using a recently generated expressed sequence tag (EST) set, we have designed an oligonucleotide microarray for the basal eudicot Eschscholzia californica (California poppy). We performed microarray experiments with an interwoven-loop design in order to characterize the E. californica floral transcriptome and to identify differentially expressed genes in flower buds with pre-meiotic and meiotic cells, four floral organs at preanthesis stages (sepals, petals, stamens and carpels), developing fruits, and leaves.Our results provide a foundation for comparative gene expression studies between eudicots and basal angiosperms. We identified whorl-specific gene expression patterns in E. californica and examined the floral expression of several gene families. Interestingly, most E. californica homologs of Arabidopsis genes important for flower development, except for genes encoding MADS-box transcription factors, show different expression patterns between the two species. Our comparative transcriptomics study highlights the unique evolutionary position of E. californica compared with basal angiosperms and core eudicots.
Project description:Homeotic MADS box genes encoding transcription factors specify the identity of floral organs by interacting in a combinatorial way. The 'floral quartet model', published several years ago, pulled together several lines of evidence suggesting that floral homeotic proteins bind as tetramers to two separated DNA sequence elements termed 'CArG boxes' by looping the intervening DNA. However, experimental support for 'floral quartet' formation remains scarce. Recently, we have shown that the class E floral homeotic protein SEPALLATA3 (SEP3) is sufficient to loop DNA in floral-quartet-like complexes in vitro. Here, we demonstrate that the class B floral homeotic proteins APETALA3 (AP3) and PISTILLATA (PI) do only weakly, at best, form floral-quartet-like structures on their own. However, they can be incorporated into such complexes together with SEP3. The subdomain K3 of SEP3 is of critical importance for the DNA-bound heterotetramers to be formed and is capable to mediate floral quartet formation even in the sequence context of AP3 and PI. Evidence is presented suggesting that complexes composed of SEP3, AP3 and PI form preferentially over other possible complexes. Based on these findings we propose a mechanism of how target gene specificity might be achieved at the level of floral quartet stability.
Project description:Sexual systems are highly variable in flowering plants and an important contributor to floral diversity. The ranunculid genus Thalictrum is especially well-suited to study evolutionary transitions in sexual systems. Homeotic transformation of sexual organs (stamens and carpels) is a plausible mechanism for the transition from hermaphroditic to unisexual flowers in this lineage because flowers of dioecious species develop unisexually from inception. The single-copy gene PISTILLATA (PI) constitutes a likely candidate for rapid switches between stamen and carpel identity. Here, we first characterized the expression pattern of all B class genes in the dioecious species T. dioicum. As expected, all B class orthologs are expressed in stamens from the earliest stages. Certain AP3 lineages were also expressed late in sepal development. We then tested whether orthologs of PI could potentially control sexual system transitions in Thalictrum, by knocking-down their expression in T. dioicum and the hermaphroditic species T. thalictroides. In T. dioicum, we found that ThdPI-1/2 silencing caused stamen primordia to develop into carpels, resulting in male to female flower conversions. In T. thalictroides, we found that ThtPI silencing caused stamen primordia to develop into supernumerary carpels, resulting in hermaphroditic to female flower conversions. These phenotypes illustrate the ability for homeotic mutations to bring about sudden and potentially adaptive changes by altering the function of a single gene. We propose a two-step evolutionary model where transitions from hermaphroditic to unisexual plants in Thalictrum result from two independent mutations at a B class gene locus. Our PI knockdown experiments in T. thalictroides recapitulate the second step in this model: the evolution of female plants as a result of a loss-of-function mutation in a B class gene.
Project description:The organs of a eudicot flower are specified by four functional classes, termed class A, B, C and E, of MADS domain transcription factors. The combinatorial formation of tetrameric complexes, so called 'floral quartets', between these classes is widely believed to represent the molecular basis of floral organ identity specification. As constituents of all complexes, the class E floral homeotic proteins are thought to be of critical relevance for the formation of floral quartets. However, experimental support for tetrameric complex formation remains scarce. Here we provide physico-chemical evidence that in vitro homotetramers of the class E floral homeotic protein SEPALLATA3 from Arabidopsis thaliana bind cooperatively to two sequence elements termed 'CArG boxes' in a phase-dependent manner involving DNA looping. We further show that the N-terminal part of SEPALLATA3 lacking K3, a subdomain of the protein-protein interactions mediating K domain, and the C-terminal domain, is sufficient for protein dimerization, but not for tetramer formation and cooperative DNA binding. We hypothesize that the capacity of class E MADS domain proteins to form tetrameric complexes contributes significantly to the formation of floral quartets. Our findings further suggest that the spacing and phasing of CArG boxes are important parameters in the molecular mechanism by which floral homeotic proteins achieve target gene specificity.
Project description:Four classes of floral homeotic MADS domain proteins specify the identities of the four organ types in an Arabidopsis flower. While the activities of the MADS domain proteins are essentially confined to the flower or to the inflorescence, several genes, such as APETALA2, HUA1 and HUA2, also act outside the flower in addition to their organ identity functions inside the flower. We identified a new gene, HUA ENHANCER 1 (HEN1) from a sensitized genetic screen in the hua1-1 hua2-1 background that is compromised in floral homeotic C function. We showed that HEN1, like the C function gene AGAMOUS, acts to specify reproductive organ identities and to repress A function. HEN1 also shares AG's non-homeotic function in controlling floral determinacy. HEN1 may achieve these functions by regulating the expression of AG. hen1 single mutants exhibit pleiotropic phenotypes such as reduced organ size, altered rosette leaf shape and increased number of coflorescences, during most stages of development. Therefore, HEN1, like the A function gene AP2, plays multiple roles in plant development as well as acting in organ identity specification in the flower. HEN1 codes for a novel protein and is expressed throughout the plant.
Project description:Grasses represent a major family of monocots comprising mostly cereals. When compared to their eudicot counterparts, cereals show a remarkable morphological diversity. Understanding the molecular basis of floral organ identity and inflorescence development is crucial to gain insight into the grain development for yield improvement purposes in cereals, however, the exact genetic mechanism of floral organogenesis remains elusive due to their complex inflorescence architecture. Extensive molecular analyses of Arabidopsis and other plant genera and species have established the ABCDE floral organ identity model. According to this model, hierarchical combinatorial activities of A, B, C, D, and E classes of homeotic genes regulate the identity of different floral organs with partial conservation and partial diversification between eudicots and cereals. Here, we review the developmental role of A, B, C, D, and E gene classes and explore the recent advances in understanding the floral development and subsequent organ specification in major cereals with reference to model plants. Furthermore, we discuss the evolutionary relationships among known floral organ identity genes. This comparative overview of floral developmental genes and associated regulatory factors, within and between species, will provide a thorough understanding of underlying complex genetic and molecular control of flower development and floral organ identity, which can be helpful to devise innovative strategies for grain yield improvement in cereals.
Project description:Eudicots, the most diverse of the three major clades of living angiosperms, are first recognized in the latest Barremian-earliest Aptian. All Early Cretaceous forms appear to be related to species-poor lineages that diverged before the rise of core eudicots, which today comprise more than 70% of angiosperm species. Here, we report the discovery of a well-preserved flower, Caliciflora mauldinensis, from the earliest Late Cretaceous, with unequivocal core eudicot features, including five sepals, five petals and two whorls of stamens borne on the rim of a floral cup containing three free carpels. Pollen is tricolporate. Carpels mature into follicular fruitlets. This character combination suggests a phylogenetic position among rosids, but more specific assignment is precluded by complex patterns of character evolution among the very large number of potentially relevant extant taxa. The whorled floral organization is consistent with ideas that this stable pattern evolved early and was a prerequisite for more integrated patterns of floral architecture that evolved later. However, limited floral synorganization in Caliciflora and all earlier eudicot flowers recognized so far, calls into question hypotheses that substantial diversification of core eudicots had already occurred by the end of the Early Cretaceous.