Functional divergence within the APETALA3/PISTILLATA floral homeotic gene lineages.
ABSTRACT: Changes in homeotic gene expression patterns or in the functions of the encoded proteins are thought to play a prominent role in the evolution of new morphologies. The floral homeotic APETALA3 (AP3) and PISTILLATA (PI) genes encode MADS domain-containing transcription factors required to specify petal and stamen identities in Arabidopsis. We have previously shown that perianth expression of AP3 and PI homologs varies in different groups of angiosperms with diverse floral structures, suggesting that changes in expression may contribute to changing morphology. We have investigated the possibility that changes in the functions of the encoded gene products may also have played a role in the evolution of different floral morphologies. AP3 and PI are members of paralogous gene lineages and share extensive similarity along the length of the protein products. Genes within these lineages encode products with characteristic C-terminal motifs that we show are critical for functional specificity. In particular, the C terminus of AP3 is sufficient to confer AP3 functionality on the heterologous PI protein. Furthermore, we have shown that the evolution of the divergent AP3 C-terminal domain in the core eudicots is correlated with the acquisition of a role in specifying perianth structures. These results suggest that divergence in these sequence motifs has contributed to the evolution of distinct functions for these floral homeotic gene products.
Project description:BACKGROUND AND AIMS: According to the floral ABC model, B-function genes appear to play a key role in the origin and diversification of the perianth during the evolution of angiosperms. The basal angiosperm Hedyosmum orientale (Chloranthaceae) has unisexual inflorescences associated with a seemingly primitive reproductive morphology and a reduced perianth structure in female flowers. The aim of this study was to investigate the nature of the perianth and the evolutionary state of the B-function programme in this species. METHODS: A series of experiments were conducted to characterize B-gene homologues isolated from H. orientale, including scanning electron microscopy to observe the development of floral organs, phylogenetic analysis to reconstruct gene evolutionary history, reverse transcription-PCR, quantitative real-time PCR and in situ hybridization to identify gene expression patterns, the yeast two-hybrid assay to explore protein dimerization affinities, and transgenic analyses in Arabidopsis thaliana to determine activities of the encoded proteins. KEY RESULTS: The expression of HoAP3 genes was restricted to stamens, whereas HoPI genes were broadly expressed in all floral organs. HoAP3 was able to partially restore the stamen but not petal identity in Arabidopsis ap3-3 mutants. In contrast, HoPI could rescue aspects of both stamen and petal development in Arabidopsis pi-1 mutants. When the complete C-terminal sequence of HoPI was deleted, however, no or weak transgenic phenotypes were observed and homodimerization capability was completely abolished. CONCLUSIONS: The results suggest that Hedyosmum AP3-like genes have an ancestral function in specifying male reproductive organs, and that the activity of the encoded PI-like proteins is highly conserved between Hedyosmum and Arabidopsis. Moreover, there is evidence that the C-terminal region is important for the function of HoPI. Our findings indicate that the development of the proposed perianth in Hedyosmum does not rely on the B homeotic function.
Project description:How different organs are formed from small sets of undifferentiated precursor cells is a key question in developmental biology. To understand the molecular mechanisms underlying organ specification in plants, we studied the function of the homeotic selector genes APETALA3 (AP3) and PISTILLATA (PI), which control the formation of petals and stamens during Arabidopsis flower development. To this end, we characterized the activities of the transcription factors that AP3 and PI encode throughout flower development by using perturbation assays as well as transcript profiling and genomewide localization studies, in combination with a floral induction system that allows a stage-specific analysis of flower development by genomic technologies. We discovered considerable spatial and temporal differences in the requirement for AP3/PI activity during flower formation and show that they control different sets of genes at distinct phases of flower development. The genomewide identification of target genes revealed that AP3/PI act as bifunctional transcription factors: they activate genes involved in the control of numerous developmental processes required for organogenesis and repress key regulators of carpel formation. Our results imply considerable changes in the composition and topology of the gene network controlled by AP3/PI during the course of flower development. We discuss our results in light of a model for the mechanism underlying sex-determination in seed plants, in which AP3/PI orthologues might act as a switch between the activation of male and the repression of female development.
Project description:Molecular variation in genes that regulate development provides insights into the evolutionary processes that shape the diversification of morphogenetic pathways. Intraspecific sequence variation at the APETALA3 and PISTILLATA floral homeotic genes of Arabidopsis thaliana was analyzed to infer the extent and nature of diversity at these regulatory loci. Comparison of AP3 and PI diversity with three previously studied genes revealed several features in the patterning of nucleotide polymorphisms common between Arabidopsis nuclear loci, including an excess of low-frequency nucleotide polymorphisms and significantly elevated levels of intraspecific replacement variation. This pattern suggests that A. thaliana has undergone recent, rapid population expansion and now exists in small, inbred subpopulations. The elevated intraspecific replacement levels may thus represent slightly deleterious polymorphisms that differentiate distinct ecotypes. The distribution of replacement and synonymous changes in AP3 and PI core and noncore functional domains also indicates differences in the patterns of molecular evolution between these interacting floral regulatory genes.
Project description:In Arabidopsis, two floral homeotic genes APETALA2 (AP2) and AGAMOUS (AG) specify the identities of perianth and reproductive organs, respectively, in flower development. The two genes act antagonistically to restrict each other to their proper domains of action within the floral meristem. In addition to AG, which antagonizes AP2, miR172, a microRNA, serves as a negative regulator of AP2. In this study, we showed that AG and miR172 have distinct functions in flower development and that they largely act independently in the negative regulation of AP2. We uncovered functions of miR172-mediated repression of AP2 in the regulation of floral stem cells and in the delineation of the expression domain of another class of floral homeotic genes. Given the antiquity of miR172 in land plants, our findings have implications for the recruitment of a microRNA in the building of a flower in evolution.
Project description:Characterization of the activities of the transcription factors that AP3 and PI encode throughout flower development using perturbation assays in combination with a floral induction system (FIS) that allows a stage-specific analysis of flower development. The series contains two types of perturbation experiments, static permutations (null alleles pi-1 and ap3-3, respectively) and dynamic perturbations (temperature-sensitive ap3-1 allele). Two conditions (i.e. genotypes: ap3-3 or pi-1 homozygous in the FIS vs ap3-3 or pi-1 heterozygous in the FIS, respectively / ap3-1 plants vs AP3 plants shifted from 16degrees to 27 degrees ) at three developmental stages each
Project description:Characterization of the activities of the transcription factors that AP3 and PI encode throughout flower development using perturbation and ChIPSeq assays in combination with a floral induction system (FIS) that allows a stage-specific analysis of flower development. Examination of genomic regions bound by fully functional AP3-GFP and PI-GFP proteins at approx floral stage 4-5 as compared to a negative control sample
Project description:In Arabidopsis thaliana expression of the B-class MADS-box genes APETALA3 (AP3) and PISTILLATA (PI) is confined to petals and stamens but in other plant species these genes are also transcribed in non-flower tissues; in Solanum tuberosum they are transcribed specifically in vascular bundles leading to petals and stamens. Transcription analysis of B-class genes in Eranthis hyemalis using reverse transcribed in situ PCR revealed that both AP3 and PI are expressed in developing vascular bundles in the tuberous rhizome, flowering stem and floral primordia. In addition, AP3 and PI transcripts are also found in stems and leaves. These results suggest a more complex role of B-class genes in Eranthis and possible involvement in the development of vascular tissue.
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:UNLABELLED: BACKGROUND:The Hawaiian endemic genus Clermontia (Campanulaceae) includes 22 species, 15 of which, the double-corolla species, are characterized by an extra whorl of organs that appear to be true petals occupying what is normally the sepal whorl. Previous research has shown that the presence of homeotic petaloid organs in some other plant groups correlates with ectopic expression of B-function MADS box genes, but similar core eudicot examples of apparent groundplan divergence remain unstudied. B-function genes, which are not normally expressed in the sepal whorl, are required for determination and maintenance of petal identity. Here, we investigate the potential role of altered B-function gene expression contributing to the morphological diversity of this island genus. RESULTS:We examined the morphology and developmental genetics of two different species of Clermontia, one of which, C. arborescens, has normal sepals while the other, C. parviflora, has two whorls of petal-like organs. Scanning electron microscopy of cell surface morphologies of first and second whorl organs in the double-corolla species C. parviflora revealed conical epidermal cells on the adaxial surfaces of both first and second whorl petaloid organs, strongly suggesting a homeotic conversion in the former. Phylogenetic analysis of Clermontia species based on 5S ribosomal DNA non-transcribed spacer sequences indicated a probable single and geologically recent origin of the double-corolla trait within the genus, with numerous potential reversals to the standard sepal-petal format. Quantitative polymerase chain reaction analysis of homologs of the B-function genes PISTILLATA (PI), APETALA3 and TOMATO MADS 6 indicated ectopic expression of two PI paralogs in the first whorl of C. parviflora; no such homeotic expression was observed for the other two genes, nor for several other MADS box genes involved in various floral and non-floral functions. In the standard sepal-petal species C. arborescens, ectopic expression of PI homologs was not observed. In C. parviflora, the upregulation of PI homologs was precisely restricted to the perianth and stamen whorls, excluding a simple overexpression phenotype. In situ hybridization analysis of C. parviflora material similarly showed first and second whorl PI homolog expression in developing flower buds. CONCLUSIONS:Our morphological and gene expression data strongly suggest that a drastic and heritable phenotypic change, at the level of floral groundplan, can originate from a homeotic mutation that is likely regulatory, being under precise spatiotemporal control as opposed to having pleiotropic characteristics. The uniqueness of this trait among core eudicots could be linked to increased ecological viability in an unstable island environment, a chance event which need not have posed any immediate adaptive benefit. We argue that the evolutionarily young morphological radiation of Clermontia may form a model system for general understanding of mechanisms of larger-scale angiosperm diversification in past, similarly unstable environments, in which small regulatory changes may have been responsible for modern-day groundplan differences. TRIAL REGISTRATION:Clinical Trials.gov- NCT01710735 SIGNIFICANCE AND INNOVATIONS: The present investigation is one of the first to examine the hypothesis of gross muscle contractile inhibition due to the presence of diagnostically relevant MFTrPs. Individuals suffering from clinically relevant levels of self-reported pain are able to tolerate maximum voluntary contraction testing, but delayed onset muscle soreness (DOMS) is a likely side-effect irrespective of symptom status. As a consequence, its confounding effect during subsequent testing must be taken into account.
Project description:The Dasymaschalon alliance within the early divergent angiosperm family Annonaceae comprises c. 180 species in four genera (Dasymaschalon, Desmos, Friesodielsia, and Monanthotaxis). The alliance offers an excellent opportunity for investigating perianth evolution and functional adaptations because of the presence of different numbers of petal whorls and contrasting floral chamber morphologies. The absence of the inner petal whorl in Dasymaschalon renders it distinctive in the family: previous studies have suggested that its three outermost stamens might be homologous with the inner petals of the sister genus, Friesodielsia, reflecting a homeotic shift of floral organ identify from inner petals to stamens. To investigate this hypothesis and general perianth evolution in the alliance, we (i) compared the floral vascularization of selected Dasymaschalon and Friesodielsia species using paraffin serial sectioning, and (ii) mapped selected perianth characters of inferred functional significance onto a molecular phylogenetic framework of the Dasymaschalon alliance (46 accessions; five cpDNA, and two nrDNA markers). The results indicate that the vasculature of the outermost stamen whorl of Dasymaschalon does not fuse with the perianth cortical vascular system, but instead splits from the basal traces of the free stamen bundles, contradicting previous inferences of homology with the inner corolla whorl of other Annonaceae. The loss of the inner petal whorl in Dasymaschalon is less likely to be due to a homeotic mutation, and instead possibly involved either the loss of genes that are responsible for determining inner petals or else the expression failure of these genes. Optimizations of perianth characters indicate that the absence of the inner petal whorl and the connivence of outer petals during anthesis are synapomorphic for Dasymaschalon. Circadian trapping of pollinators is inferred either to be derived in the stem lineage of the Dasymaschalon-Friesodielsia clade, or else to have evolved in parallel in the Dasymaschalon and Friesodielsia lineages. Subsequent changes in the remaining petals of Dasymaschalon flowers (which do not fully separate during anthesis) are likely to have enabled perpetuation of the circadian trapping mechanism, lessening the adverse impacts of inner petal loss.