Developmental Robustness by Obligate Interaction of Class B Floral Homeotic Genes and Proteins

DEF-like and GLO-like class B floral homeotic genes encode closely related MADS-domain transcription factors that act as developmental switches involved in specifying the identity of petals and stamens during flower development. Class B gene function requires transcriptional upregulation by an autoregulatory loop that depends on obligate heterodimerization of DEF-like and GLO-like proteins. Because switch-like behavior of gene expression can be displayed by single genes already, the functional relevance of this complex circuitry has remained enigmatic. On the basis of a stochastic in silico model of class B gene and protein interactions, we suggest that obligate heterodimerization of class B floral homeotic proteins is not simply the result of neutral drift but enhanced the robustness of cell-fate organ identity decisions in the presence of stochastic noise. This finding strongly corroborates the view that the appearance of this regulatory mechanism during angiosperm phylogeny led to a canalization of flower development and evolution

Evolutionary Dynamics of Floral Homeotic Transcription Factor Protein–Protein Interactions

Protein–protein interactions (PPIs) have widely acknowledged roles in the regulation of development, but few studies have addressed the timing and mechanism of shifting PPIs over evolutionary history. The B-class MADS-box transcription factors, PISTILLATA (PI) and APETALA3 (AP3) are key regulators of floral development. PI-like (PIL) and AP3-like (AP3L) proteins from a number of plants, including Arabidopsis thaliana (Arabidopsis) and the grass Zea mays (maize), bind DNA as obligate heterodimers. However, a PIL protein from the grass relative Joinvillea can bind DNA as a homodimer. To ascertain whether Joinvillea PIL homodimerization is an anomaly or indicative of broader trends, we characterized PIL dimerization across the Poales and uncovered unexpected evolutionary lability. Both obligate B-class heterodimerization and PIL homodimerization have evolved multiple times in the order, by distinct molecular mechanisms. For example, obligate B-class heterodimerization in maize evolved very recently from PIL homodimerization. A single amino acid change, fixed during domestication, is sufficient to toggle one maize PIL protein between homodimerization and obligate heterodimerization. We detected a signature of positive selection acting on residues preferentially clustered in predicted sites of contact between MADS-box monomers and dimers, and in motifs that mediate MADS PPI specificity in Arabidopsis. Changing one positively selected residue can alter PIL dimerization activity. Furthermore, ectopic expression of a Joinvillea PIL homodimer in Arabidopsis can homeotically transform sepals into petals. Our results provide a window into the evolutionary remodeling of PPIs, and show that novel interactions have the potential to alter plant form in a context-dependent manner. Key words: PISTILLATA, Poales, APETALA3, convergent molecular evolution, B-class MADS box genes, evolution of flower development

Role of floral organ identity genes in the development of unisexual flowers of Quercus suber L

Supplementary information accompanies this paper at doi:10.1038/s41598-017-10732-0Monoecious species provide an excellent system to study the specific determinants that underlie male and female flower development. Quercus suber is a monoecious species with unisexual flowers at inception. Despite the overall importance of this and other tree species with a similar reproductive habit, little is known regarding the mechanisms involved in the development of their male and female flowers. Here, we have characterised members of the ABCDE MADS-box gene family of Q. suber. The temporal expression of these genes was found to be sex-biased. The B-class genes, in particular, are predominantly, or exclusively (in the case of QsPISTILLATA), expressed in the male flowers. Functional analysis in Arabidopsis suggests that the B-class genes have their function conserved. The identification of sex-biased gene expression plus the identification of unusual protein-protein interactions suggest that the floral organ identity of Q. suber may be under control of specific changes in the dynamics of the ABCDE model. This study constitutes a major step towards the characterisation of the mechanisms involved in reproductive organ identity in a monoecious tree with a potential contribution towards the knowledge of conserved developmental mechanisms in other species with a similar sex habit.This work was funded by FCT/COMPETE/FEDER with the project grants FCOMP-01-0124-FEDER-019461/PTDC/AGR-GPL/118508/2010. “Characterization of Reproductive Development of Quercus suber”. R.S. and MMRC were supported by FCT grants with the references SFRH/BD/84365/2012 and SFRH/BSAB/113781/2015, respectively. A special acknowledgment for the John Innes Centre Bioimaging facility and staff for their contribution to this publication and for Sara Laranjeira and Helena Silva for helping revise the manuscriptinfo:eu-repo/semantics/publishedVersio

Evolution of the interaction of floral homeotic proteins

The flower development of angiosperms is controlled by floral homeotic MIKCC-type MADS-domain transcription factors (MADS-TFs) that activate or repress target genes by forming floral organ specific DNA-bound heterotetrameric complexes termed floral quartets. The ability to form floral quartets highly differs between floral homeotic MADS-TFs of certain subfamilies. However, to date relatively little is known about how these subfamily-specific interaction patterns of floral homeotic proteins evolved during angiosperm evolution and which sequence determinants account for the different interaction capabilities. Based on interaction studies of floral homeotic proteins from early diverging angiosperms I could show that the interactions governing flower development in core eudicots are also present in these distantly related species. However, especially AP3- and PI-like proteins from early diverging angiosperms possess additional interactions compared to their orthologs from core eudicots which form obligate heterodimers only. The more diverse interactions among floral homeotic proteins from early diverging angiosperms suggest a shift from promiscuity to specificity in the protein-protein interaction network during early angiosperm evolution. By comprehensive amino acid sequence analyses of MADS-TFs I demonstrated that the structure of the protein-protein interacting keratin-like domain (K-domain) is most likely highly similar among all subfamilies of floral homeotic proteins. Amino acid substitutions within the K-domain of the floral homeotic hub protein SEP3 revealed that highly conserved leucine residues at interacting sites are essential mediators of floral quartet-like complex formation. The absence of leucine residues at homologous amino acid positions in non-hubs such as AP3- and PI-like proteins probably accounts for their less promiscuous interactions. Beside the highly specific protein-protein interactions among floral homeotic proteins I studied another interaction of the K-domain. The phytoplasma effector protein SAP54 targets the K-domain to specifically bind MADS-TFs of certain subfamilies and destines them for degradation. Based on amino acid sequence analyses and structural predictions I provided preliminary evidence that SAP54 folds into a structure similar to that of the K-domain. Based on my findings I hypothesized that SAP54 evolved via convergent sequence and structural evolution to mimic the K-domain of its MADS-TF targets

From Cellular Components to Living Cells (and Back): Evolution of Function in Biological Networks

Network models pervade modern biology. From ecosystems down to molecular interactions in cells, they provide abstraction and explanation for biological processes. Thus, the relation between structure and function of networks is central to any comprehensive attempt for a theoretical understanding of life. Just as any living system, biological networks are shaped by evolutionary processes. In reverse, artificial evolution can be employed to reconstruct networks and to study their evolution. To this end, I have implemented an evolutionary algorithm specifically designed for the evolution of network models. With the developed evolutionary framework, a study of the evolution of information-processing networks was performed. It is shown that selection favours an organisational structure that is related to function, such that computations can be visualised as transitions between organisations. Furthermore, mathematical modelling is applied to extract reaction-kinetic constants from fluorescence microscopy data, and the model is presented and discussed in detail. Using this approach, a detailed quantitative model of exchange dynamics at PML nuclear bodies (NBs) is created, showing that PML NB components exhibit highly individual exchange kinetics. The FRAP data for PML NBs is additionally used as a test-case for automatic model inference using evolutionary methods, and a set of necessary and sufficient criteria for a good model fit is revealed. In the last part of this thesis, a stochastic analysis of the genetic regulatory system of DEF-like and GLO-like class B floral homeotic genes provides an explanation for their intricate regulatory wiring. The different potential regulatory architectures are investigated using Monte Carlo simulation, a simplified master-equation model, and fixedpoint analysis. It is shown that a positive autoregulatory loop via obligate heterodimerisation of transcription factor proteins reduces noise in cell-fate organ identity decisions.Netzwerkmodelle sind weit verbreitet in der modernen Biologie. In allen Teilgebieten - von der Ökologie bis hin zur Molekularbiologie - bieten sie die Möglichkeit, untersuchte Prozesse und Phänomene zu abstrahieren und damit auf theoretischer Ebene zugänglich zu machen. Es wird ein evolutionärer Algorithmus vorgestellt, der speziell für die Erzeugung von Netzwerkmodellen angepasst ist. Dafür wurde eine Genetische Programmierung der Netzwerkstruktur mit einer Evolutionsstrategie auf den kinetischen Parametern verknüpft. Mit dem neu entwickelten Evolutionären Algorithmus wurde dann eine Studie zur Evolution von informationsverarbeitenden Netzwerken durchgeführt. Selektion erzeugt eine funktionale Organisationsstruktur, in welcher eine Berechnung als Transition zwischen Organisationen abgebildet werden kann. Desweiteren wurden mathematische Modellierungsmethoden verwendet, um kinetische Reaktionskonstanten aus fluoreszenz-mikroskopischen Daten zu gewinnen. Die verwendete Methode wird im Detail vorgestellt und diskutiert. Auf diese Weise entstand ein detailliertes Modell des Proteinaustauschs an PML nuclear bodies (NBs), in welchem die Komponenten der PML NBs sehr differenzierte Austauschverhalten zeigen. Darüber hinaus werden die gewonnenen Daten genutzt, um die automatische Evolution von Netzwerkmodellen in einer realistischen Fallstudie zu testen. Zum Schluss wird eine stochastische Analyse des Zusammenspiels der DEF- und GLO-Gene in der Blütenentwicklung gezeigt, welche eine Erklärung für ihre überraschend komplexe Verschaltung liefert. Die verschiedenen möglichen Regulationsmechanismen werden mithilfe von Monte-Carlo-Simulation, einem Master-Equation-Ansatz und der Fixpunktanalyse verglichen. Es wird gezeigt, dass positive Autoregulation durch obligatorische Heterodimerisierung den Einfluss des Zufalls auf die Organidentität reduziert

The class E floral homeotic protein SEPALLATA3 is sufficient to loop DNA in ‘floral quartet’-like complexes in vitro

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

Continuous-time modeling of cell fate determination in Arabidopsis flowers

<p>Abstract</p> <p>Background</p> <p>The genetic control of floral organ specification is currently being investigated by various approaches, both experimentally and through modeling. Models and simulations have mostly involved boolean or related methods, and so far a quantitative, continuous-time approach has not been explored.</p> <p>Results</p> <p>We propose an ordinary differential equation (ODE) model that describes the gene expression dynamics of a gene regulatory network that controls floral organ formation in the model plant <it>Arabidopsis thaliana</it>. In this model, the dimerization of MADS-box transcription factors is incorporated explicitly. The unknown parameters are estimated from (known) experimental expression data. The model is validated by simulation studies of known mutant plants.</p> <p>Conclusions</p> <p>The proposed model gives realistic predictions with respect to independent mutation data. A simulation study is carried out to predict the effects of a new type of mutation that has so far not been made in <it>Arabidopsis</it>, but that could be used as a severe test of the validity of the model. According to our predictions, the role of dimers is surprisingly important. Moreover, the functional loss of any dimer leads to one or more phenotypic alterations.</p

The evolutionary origin of "floral quartets": clues from molecular interactions of orthologues of floral homeotic proteins from the gymnosperm Gnetum gnemon

The identity of floral organs in angiosperms is specified by multimeric transcription factor complexes composed of floral homeotic MADS-domain proteins that bind to specific cis-regulatory DNA-elements (‘CArG-boxes’) of their target genes, thus constituting floral quartets. Gymnosperms possess orthologues of floral homeotic genes enconding MIKC-type MADS-domain proteins, but when and how the interactions constituting floral quartets were established during evolution has remained unknown. To better understand the ‘abominable mystery’ of flower origin, in this project a comprehensive study was carried out to detect the dimerization and DNA-binding of several classes of MADS-domain proteins from a gymnosperm, Gnetum gnemon of the Gnetales. Determination of protein-protein interactions by pull-down assays revealed complex patterns of heterodimerization among orthologues of class B, class C and class E floral homeotic proteins and Bsister proteins, while homodimerization was not observed. In contrast, electrophoretic mobility shift assays (EMSAs) revealed that all proteins tested except one bind to CArG-boxes also as homodimers, suggesting that homodimerization is relatively weak, but facilitated by DNA-binding. Proteins able of DNA-based homodimerization include orthologues of class B and C proteins; B and C proteins also form heterodimers in vitro and in yeast, which is in sharp contrast to their orthologues from angiosperms, which require class E floral proteins to ‘glue’ them together in multimeric complexes. Remarkably, the heterodimers of B and C proteins from G. gnemon are not capable of binding to CArG-boxes, suggesting that DNA-binding in vivo is based on homodimers, while heterodimerization of B and C proteins may constitute multimeric, DNA-bound complexes by mediating the interaction between two DNA-bound homodimers. EMSAs and DNase I footprint assays indicated that both B with C proteins and C proteins alone but not B proteins alone can induce DNA-looping to form tetrameric protein-DNA complexes similar to floral quartets. These data suggest that at least some of the gymnosperm orthologues of floral homeotic proteins may have the capability of forming higher-order complexes and that gymnosperm B and C proteins control male organ identity and C proteins controls female organ identity, respectively, by forming quartet-like complexes composed of two homodimers, each bound to a CArG-box

The dynamics of flower development in Castanea sativa Mill

The sweet chestnut tree (Castanea sativa Mill.) is one of the most significant Mediterranean tree species, being an important natural resource for the wood and fruit industries. It is a monoecious species, presenting unisexual male catkins and bisexual catkins, with the latter having distinct male and female flowers. Despite the importance of the sweet chestnut tree, little is known regarding the molecular mechanisms involved in the determination of sexual organ identity. Thus, the study of how the different flowers of C. sativa develop is fundamental to understand the reproductive success of this species and the impact of flower phenology on its productivity. In this study, a C. sativa de novo transcriptome was assembled and the homologous genes to those of the ABCDE model for floral organ identity were identified. Expression analysis showed that the C. sativa B- and C-class genes are differentially expressed in the male flowers and female flowers. Yeast two-hybrid analysis also suggested that changes in the canonical ABCDE protein–protein interactions may underlie the mechanisms necessary to the development of separate male and female flowers, as reported for the monoecious Fagaceae Quercus suber. The results here depicted constitute a step towards the understanding of the molecular mechanisms involved in unisexual flower development in C. sativa, also suggesting that the ABCDE model for flower organ identity may be molecularly conserved in the predominantly monoecious Fagaceae family.This work was funded by FCT/COMPETE/FEDER with the project grant POCI-01-0145- FEDER-027980/PTDC/ASP-SIL/27980/2017—“FlowerCAST—Characterisation of genetic and environmental determinants involved in reproductive development of Castanea sativa”. A.T.A. and S.A. were supported by FCT with PhD grants (ref. SFRH/BD/136834/2018 and SFRH/BD/146660/2019, respectively)

Recent advances in understanding the roles of whole genome duplications in evolution

Ancient whole-genome duplications (WGDs)—paleopolyploidy events—are key to solving Darwin’s ‘abominable mystery’ of how flowering plants evolved and radiated into a rich variety of species. The vertebrates also emerged from their invertebrate ancestors via two WGDs, and genomes of diverse gymnosperm trees, unicellular eukaryotes, invertebrates, fishes, amphibians and even a rodent carry evidence of lineage-specific WGDs. Modern polyploidy is common in eukaryotes, and it can be induced, enabling mechanisms and short-term cost-benefit assessments of polyploidy to be studied experimentally. However, the ancient WGDs can be reconstructed only by comparative genomics: these studies are difficult because the DNA duplicates have been through tens or hundreds of millions of years of gene losses, mutations, and chromosomal rearrangements that culminate in resolution of the polyploid genomes back into diploid ones (rediploidisation). Intriguing asymmetries in patterns of post-WGD gene loss and retention between duplicated sets of chromosomes have been discovered recently, and elaborations of signal transduction systems are lasting legacies from several WGDs. The data imply that simpler signalling pathways in the pre-WGD ancestors were converted via WGDs into multi-stranded parallelised networks. Genetic and biochemical studies in plants, yeasts and vertebrates suggest a paradigm in which different combinations of sister paralogues in the post-WGD regulatory networks are co-regulated under different conditions. In principle, such networks can respond to a wide array of environmental, sensory and hormonal stimuli and integrate them to generate phenotypic variety in cell types and behaviours. Patterns are also being discerned in how the post-WGD signalling networks are reconfigured in human cancers and neurological conditions. It is fascinating to unpick how ancient genomic events impact on complexity, variety and disease in modern life