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Although most flowers follow a conserved 'bauplan' consisting of sepals, petals, stamens and carpels, there is a remarkable amount of morphological diversity. Interestingly, all flowers are specified by the conserved (A)BCE-model. Most of the transcription factors in this model belong to the MADS-domain family. We examined how these transcription factors and their binding sites in the genome evolved, as a first step to elucidate how diversity in flower morphology has been created. We analyzed the evolution of transcription factor binding sites by comparing binding sites of the major floral regulator SEPALLATA3 between two closely related Arabidopsis species, as well as between A. thaliana ecotypes. We found substantial overlap in transcription factor binding profiles between ecotypes, but limited overlap between the related species. We also assessed how transcription factors themselves can change in their properties by analyzing the divergence between paralogs. We examined how the PISTILLATA paralogs in Tarenaya hassleriana diverged, as this species occupies an interesting position in the eudicot phylogeny. We also studied whether divergence of the APETALA3 paralogs in Aquilegia could explain the specification of an additional floral organ in this genus. In both cases, we conclude that the paralogs diverged from each other in their biochemical properties. In the future, it would be interesting to assess how these changes in transcription factors and their binding sites affect floral regulatory networks and ultimately floral shape.

Encrypted in the DNA lays most information needed for the development of an organism. The transcription of this information into precise patterns of gene activity results in the development of different cell types, organs, and developmental structures. Moreover, transcriptional regulation enables an organism to respond to changing environmental conditions. Essential for the regulation of transcription are DNA-binding transcription factors (TFs). TFs bind the DNA in a sequence-specific fashion. Upon binding of a TF to its DNA binding site, TFs typically activate or repress the transcription of nearby genes. To better understand transcriptional regulation it is essential to study DNA binding specificity of TFs. In the last decades, technological advances allowed the development of high-throughput methods to study protein-DNA interactions. Traditional in vitro methods study one or a few interactions, while new high-throughput methods can determine TF specificity by measuring relative DNA-binding affinities against a large collection or even all possible binding sites. Several high-throughput techniques to study TF-DNA interactions are discussed in Chapter 1 of this thesis. These new technologies and methods resulted in a fast growing number of studies on DNA binding specificities of TFs, expanding the knowledge about TF specificity. A review on the current knowledge of TF DNA binding specificity is described in Chapter 1. One aspect that influences DNA binding of TFs are differences in ability to form protein-protein interactions. The aim of this thesis was to study the role of protein-protein interactions in determining DNA binding specificity of a developmental regulatory MADS domain TF in Arabidopsis thaliana. While the members of the MADS-box protein family have many, diverse in vivo functions, all members bind in vitro to a 10-bp motif called the CArG-box. Moreover, studies demonstrated that closely related MADS proteins are expressed in the same cells, therefore encountering the same DNA accessibility and DNA methylation patterns, but bind different in vivo targets. Interestingly, MADS domain proteins bind DNA obligatorily as homo- and heterodimers and the interactions between MADS domain proteins are highly protein specific. Hence, MADS domain proteins are a perfect model system to study the influence of intra-family protein interactions on DNA binding specificity. To study the influence of protein-protein interactions on DNA-binding specificity this work focusses on one specific MADS domain protein, FRUITFULL (FUL). FUL is expressed at two stages during flower development and, in both stages FUL has highly diverse functions. In Chapter 2 we demonstrate using RNA-seq that FUL regulates different sets of target genes in the two stages. Moreover, using ChIP-seq we show that FUL genomic DNA binding is partly tissue-specific. These tissue-specifically bound and regulated genes are in line with the known dual functions of FUL during development. Interestingly, using protein complex immunoprecipitation for the two studied tissues/stages we show that the interactions of FUL with other MADS domain proteins are also tissue-specific. To determine whether the tissue-specific in vivo binding pattern are due to differences in DNA binding specificity of the FUL-MADS dimers, we studied the DNA binding specificities of the different protein complexes using SELEX-seq. The SELEX-seq results show that although all tested dimers preferably bind the canonical binding motif of MADS domain proteins, different dimers have different preferences for nucleotides within and surrounding the canonical binding site. Hence, different MADS domain dimers have different in vitro DNA binding specificities. By mapping the SELEX-seq affinities to the genome we were able to compare these results with in vivo tissue-specific ChIP-seq data. This analysis revealed a strong correlation between tissue-specific dimer affinities and tissue-specific genomic binding sites of FUL. Hence, we show that the choice of MADS dimerization partner influences DNA binding specificity, highlighting the role of intra-family protein interactions in defining DNA binding specificity. To allow other researchers to determine genome-wide DNA binding of TFs Chapter 3 provides a step-by-step guide for ChIP-seq experiments and computational analysis. The protocol is designed for wet-lab biologists to perform ChIP-seq experiments and analyse their own ChIP-seq data. Using the genome-wide DNA binding patterns determined by ChIP-seq, Chapter 4 and Chapter 5 take a more detailed look at some of the genes directly bound by FUL. In Chapter 4, we demonstrate a connection between developmentally and environmentally regulated growth programs. We studied a gene directly bound by FUL in pistil tissue, SMALL AUXIN UPREGULATED RNA 10 (SAUR10). SAUR10 expression is regulated by FUL in multiple tissues, among others cauline leaves, stems, and branches. The results show that the expression of SAUR10 at the abaxial side of branches is influenced by a combination of environmental and developmental regulated growth programs: hormones, light conditions, and FUL binding. This spatial regulation possibly affects the angle between the side branches and the main inflorescence stem. Additionally, we discuss several other FUL target genes involved in hormone pathways and light conditions. Chapter 5 focusses on the putative direct targets of FUL in IM tissue. Among the putative direct targets two genes involved in flavonoid synthesis were identified, FLAVONOID SYNTHESE 1 (FLS1) and UDP-GLUCOSYL TRANSFERASE 78D3 (UGT78D3). Interestingly, similar to the ful-7 mutant, the fls1 mutant is late flowering. Moreover, expression data exposed an increased gene expression for both FLS1 and UGT78D3 in developing meristems and showed FLS1 expression to be influenced by light conditions. We report the first link between the MADS domain protein FUL and flavonoid synthesis in Arabidopsis. Moreover, our results indicate a possible link between flavonoids and flowering time. In Chapter 6 I discuss the findings of this thesis and make suggestions for further research. Taken together, the work in this thesis shows that intra-family protein interactions can influence DNA-binding specificity of a protein. Thereby these protein-protein interactions can influence genome-wide binding patterns and, as a result, the function of a protein. Moreover, by studying several putative direct targets of FUL in more detail, we demonstrated a connection between development and environment in growth-regulated programs. Interestingly, the FUL target SAUR10 is repressed by FUL in several tissues, including cauline leaves, inflorescence stems, and branches. However, no influence of FUL on SAUR10 expression could be detected in the pistil. So, despite the binding of FUL to the promotor of SAUR10 in the pistil, this binding does not result in gene regulation. This finding reflects the complex relation between TF occupancy and gene regulation, further research is needed to better understand this relation. Moreover, besides MADS domain protein interactions, we found FUL to interact with several proteins of other families. The role of these cross-family protein interactions in cooperative gene regulation is not fully understood and will be an important research topic in the coming years.

Unlike animals, plants produce new organs throughout their life cycle using pools of stem cells that are organized in meristems. Although many key regulators of meristem and organ identities have been identified, it is still not well understood how they function at the molecular level and how they can switch an entire developmental programme in which thousands of genes are involved. Recent advances in the genome-wide identification of target genes controlled by key plant transcriptional regulators and their interactions with epigenetic factors provide new insights into general transcriptional regulatory mechanisms that control switches of developmental programmes and cell fates in complex organisms.

Protein-protein and protein-DNA interactions are essential for the molecular action of transcription factors. By combinatorial binding to target gene promoters, transcription factors are able to up- or down-regulate the expression of these genes. MADS-domain proteins comprise a large family of transcription factors present in all eukaryotes. In plants, and especially in seed plants, this family has significantly expanded. For example, more than 100 representatives are found in the Arabidopsis genome. MADS-box genes have initially been shown to play major roles in flower development, however their emerging functional characterization revealed functions in almost all developmental processes throughout the plant life cycle. How MADS-domain transcription factors acquire their functional specificity remains unresolved. The goal of this thesis was to characterize some of the molecular mechanisms by which MADS-domain proteins act in Arabidopsis. Chapter 1 comprehensively reviews functions of MADS-domain transcription factors in flowering plants, with a main focus on Arabidopsis. Major classes of MADS-domain proteins are introduced, and their modular structures are described. Additionally, it is shown that several distinctive subfamilies of MADS-box genes can be inferred from the phylogenetic analysis of the whole gene family. By compiling recent studies on MADS-domain protein-protein and protein-DNA interactions, we present a hypothetical model of MADS-domain protein action that combines higher-order protein complex formation and active chromatin remodeling by large transcriptional machineries. Chapter 2 describes MADS-domain protein complexes that are potentially formed during Arabidopsis flower development. By using a targeted proteomics approach we were able to characterize the protein interactome of major floral homeotic MADS-domain proteins (APETALA1, APETALA3, PISTILLATA, AGAMOUS, SEPALLATA3 and FRUITFULL) in native plant tissues, confirming interactions suggested in the ‘floral quartet’ model. Additionally, we discovered transcription factors from other families and chromatin-associated proteins as possible interaction partners of MADS-domain proteins. These interactions shed light on the combinatorial modes of action of MADS-domain transcription factors and suggest that they can act by recruiting or redirecting the chromatin remodeling machinery to control the expression of their target genes. In Chapter 3 we review recent advances in proteomics approaches used to study cellular signaling and developmental processes in plants. We mention the emerging tools for of whole plant proteome characterization as well as sub-cellular protein localization. The major focus, though, is on the description of complete cellular signaling cascades in plants, starting from the characterization of signaling mobile molecules (e.g. peptide or protein), through identification of receptors and receptor protein complexes, ending with identification of intermediate signaling pathway members. Two examples of biochemical procedures used to identify complexes of membrane-bound receptors and transcriptional regulators from nuclei are described in Chapter 4. In our optimized method we make use of fluorophore-tagged single step affinity purification of protein complexes and label-free mass spectrometry-based protein quantification to distinguish true complex partners from non-specifically precipitated proteins. The exact molecular mechanisms of DNA sequence recognition by MADS-domain transcription factors are still unknown. Particularly intriguing is the question whether various MADS-domain protein complexes possess different DNA-binding specificities. We address this question in Chapter 5. We used systematic evolution of ligands by exponential enrichment (SELEX) followed by high-throughput sequencing (seq) approach to discriminate DNA-binding specificities of several MADS-domain protein homo- and heterodimers. Finally, in Chapter 6, we aimed to identify the molecular features of different DNA-binding specificities of MADS-domain transcription factors.With help of bioinformatics tools and in vitro DNA-binding assays we found that structural characteristics of the DNA play an important role in DNA-binding of MADS-domain proteins. Taken together, research described in this thesis advances our knowledge on the molecular mechanisms of MADS-domain transcription factor action in plants. Chapter 7 concludes the thesis and describes future perspectives in MADS-domain protein research. Highlighted are the advances of high-throughput (proteomics and genomics) technologies that could be used to unravel not only the static characteristics of transcriptional regulation but also the dynamic and stoichiometric changes of complex protein and gene regulatory networks during plant development.

Protein-protein and protein-DNA interactions are essential for the molecular action of transcription factors. By combinatorial binding to target gene promoters, transcription factors are able to up- or down-regulate the expression of these genes. MADS-domain proteins comprise a large family of transcription factors present in all eukaryotes. In plants, and especially in seed plants, this family has significantly expanded. For example, more than 100 representatives are found in the Arabidopsis genome. MADS-box genes have initially been shown to play major roles in flower development, however their emerging functional characterization revealed functions in almost all developmental processes throughout the plant life cycle. How MADS-domain transcription factors acquire their functional specificity remains unresolved. The goal of this thesis was to characterize some of the molecular mechanisms by which MADS-domain proteins act in Arabidopsis. Chapter 1 comprehensively reviews functions of MADS-domain transcription factors in flowering plants, with a main focus on Arabidopsis. Major classes of MADS-domain proteins are introduced, and their modular structures are described. Additionally, it is shown that several distinctive subfamilies of MADS-box genes can be inferred from the phylogenetic analysis of the whole gene family. By compiling recent studies on MADS-domain protein-protein and protein-DNA interactions, we present a hypothetical model of MADS-domain protein action that combines higher-order protein complex formation and active chromatin remodeling by large transcriptional machineries. Chapter 2 describes MADS-domain protein complexes that are potentially formed during Arabidopsis flower development. By using a targeted proteomics approach we were able to characterize the protein interactome of major floral homeotic MADS-domain proteins (APETALA1, APETALA3, PISTILLATA, AGAMOUS, SEPALLATA3 and FRUITFULL) in native plant tissues, confirming interactions suggested in the ‘floral quartet’ model. Additionally, we discovered transcription factors from other families and chromatin-associated proteins as possible interaction partners of MADS-domain proteins. These interactions shed light on the combinatorial modes of action of MADS-domain transcription factors and suggest that they can act by recruiting or redirecting the chromatin remodeling machinery to control the expression of their target genes. In Chapter 3 we review recent advances in proteomics approaches used to study cellular signaling and developmental processes in plants. We mention the emerging tools for of whole plant proteome characterization as well as sub-cellular protein localization. The major focus, though, is on the description of complete cellular signaling cascades in plants, starting from the characterization of signaling mobile molecules (e.g. peptide or protein), through identification of receptors and receptor protein complexes, ending with identification of intermediate signaling pathway members. Two examples of biochemical procedures used to identify complexes of membrane-bound receptors and transcriptional regulators from nuclei are described in Chapter 4. In our optimized method we make use of fluorophore-tagged single step affinity purification of protein complexes and label-free mass spectrometry-based protein quantification to distinguish true complex partners from non-specifically precipitated proteins. The exact molecular mechanisms of DNA sequence recognition by MADS-domain transcription factors are still unknown. Particularly intriguing is the question whether various MADS-domain protein complexes possess different DNA-binding specificities. We address this question in Chapter 5. We used systematic evolution of ligands by exponential enrichment (SELEX) followed by high-throughput sequencing (seq) approach to discriminate DNA-binding specificities of several MADS-domain protein homo- and heterodimers. Finally, in Chapter 6, we aimed to identify the molecular features of different DNA-binding specificities of MADS-domain transcription factors.With help of bioinformatics tools and in vitro DNA-binding assays we found that structural characteristics of the DNA play an important role in DNA-binding of MADS-domain proteins. Taken together, research described in this thesis advances our knowledge on the molecular mechanisms of MADS-domain transcription factor action in plants. Chapter 7 concludes the thesis and describes future perspectives in MADS-domain protein research. Highlighted are the advances of high-throughput (proteomics and genomics) technologies that could be used to unravel not only the static characteristics of transcriptional regulation but also the dynamic and stoichiometric changes of complex protein and gene regulatory networks during plant development.

Background 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. Results 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 Arabidopsis thaliana. 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. Conclusions 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 Arabidopsis, 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.

Background The WRKY transcription factor gene family has a very ancient origin and has undergone extensive duplications in the plant kingdom. Several studies have pointed out their involvement in a range of biological processes, revealing that a large number of WRKY genes are transcriptionally regulated under conditions of biotic and/or abiotic stress. To investigate the existence of WRKY co-regulatory networks in plants, a whole gene family WRKYs expression study was carried out in rice (Oryza sativa). This analysis was extended to Arabidopsis thaliana taking advantage of an extensive repository of gene expression data. Results The presented results suggested that 24 members of the rice WRKY gene family (22% of the total) were differentially-regulated in response to at least one of the stress conditions tested. We defined the existence of nine OsWRKY gene clusters comprising both phylogenetically related and unrelated genes that were significantly co-expressed, suggesting that specific sets of WRKY genes might act in co-regulatory networks. This hypothesis was tested by Pearson Correlation Coefficient analysis of the Arabidopsis WRKY gene family in a large set of Affymetrix microarray experiments. AtWRKYs were found to belong to two main co-regulatory networks (COR-A, COR-B) and two smaller ones (COR-C and COR-D), all including genes belonging to distinct phylogenetic groups. The COR-A network contained several AtWRKY genes known to be involved mostly in response to pathogens, whose physical and/or genetic interaction was experimentally proven. We also showed that specific co-regulatory networks were conserved between the two model species by identifying Arabidopsis orthologs of the co-expressed OsWRKY genes. Conclusion In this work we identified sets of co-expressed WRKY genes in both rice and Arabidopsis that are functionally likely to cooperate in the same signal transduction pathways. We propose that, making use of data from co-regulatory networks, it is possible to highlight novel clusters of plant genes contributing to the same biological processes or signal transduction pathways. Our approach will contribute to unveil gene cooperation pathways not yet identified by classical genetic analyses. This information will open new routes contributing to the dissection of WRKY signal transduction pathways in plants.