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Moleculaire basis van MADS-domein eiwit-eiwit interacties ,,
No full textScientific summary (English)
In this study we aimed to understand the complex protein network interactions of one of the largest plant transcription factors families, the MADS-box genes. Through high throughput screening via yeast two/three hybrid and electro mobility shift assays, we were able to establish a good understanding of the interaction specificity in extant protein networks.
To obtain this understanding, we have used resurrected ancestral MADS-box proteins at different evolutionary time points, around key whole genome duplication events. This highlighted the role of hub proteins in complex protein interaction networks. The evolutionary data resulted in a better understanding of key protein-protein interactions at the molecular level, where the effect of a mutation/s along the evolutionary trajectory could be traced back. By a number of mutagenesis assays, we were able to confirm the effect of these mutations, when introduced into extant proteins in the network. For the hub protein SEP3, we were able to illustrate that this hub protein has gained specificity during its evolution through proline accumulation, which was associated with a reduction in flexibility in the extant protein compared to its ancestors. This approach of testing protein-protein interaction in ancestral networks was quite novel. When combined with the reciprocal swapping experiment of certain protein members between networks of different ages, it proved to be a very useful and powerful technique to understand network evolution. This approach can be applied to any protein network providing that there are enough sequenced genes of its members to reconstruct their ancestral proteins.
We augmented our wet lab results with several in silico simulations to illustrate the folding dynamics at the molecular level of the dimerization domain of the hub protein SEP3. This enabled us to make more solid conclusions regarding the effect of protein flexibility on protein affinity and specificity and illustrated how flexibility affects the conformational space and the folding energy landscape. We were able to illustrate that these protein-protein interactions follow an induced fit model rather than key-lock model, the dimerization process is dynamic, and mutations that affect the energy landscape between different conformers can contribute to the interaction specificity and affinity equally as mutation on the interaction interface itself.
By studying a second case of protein evolution in the MADS-box protein network, the SHORT VEGETATIVE PHASE (SVP) protein, we were able to recognize a different molecular mechanism that was selected during evolution. A new subdomain through insertion has enabled SVP to gain more interaction in the protein network. This shows how complex and diverse are the protein-protein interactions even in a family of close paralogs such as the MADS-box proteins.
This combination of wet-lab/dry-lab techniques will become more necessary and feasible in the future. In addition to large-scale high throughput mutational scans and protein-protein interaction assays. Enabling us to achieve a holistic understanding from the molecular to phenotype level.status: Publishe
Het effect van omgevingstemperatuur op de ontwikkeling van Brachypodium distachyon en mRNA bindende eiwitten in bloemplanten
No full textGlobal temperature has been predicted to increase by 1.8°C to 4°C by the end of this century. This minor change in temperature could cause an average 4% decrease in crop yield. Therefore, understanding how crops react to this minor temperature change is important to predict the impact on crop yield. Plant research based on the model plant Arabidopsis thaliana has broadened our knowledge of molecular biology in plants, but Arabidopsis has limitations when used as a temperate grass model when one wants to explore grass specific characteristics. Brachypodium distachyon has become a model for temperate grasses such as wheat and barley because of its smaller genome, relative ease to manipulate in the lab and available mutants. Therefore, in this work, we used Brachypodium as a model to explore the temperature effects on plant development.
Firstly, we explored the ambient temperature effects on five different Brachypodium accessions through growing the plants at 14°C, 18°C and 22°C. The flowering time, leaf and branch numbers when flowering, seed set period, seed weight and size, and seed dormancy were recorded. We have shown that the seed development stage is more sensitive to the changing temperature than flowering time, and seed weight is negatively correlated to elevated temperatures.
Secondly, the mutant lines of FLC homologs and SVP homologs in Brachypodium were phenotyped at different temperature conditions because of the important roles of FLM and SVP genes in ambient temperature regulation in Arabidopsis. We found that one mutant of the SVP homologs in Brachypodium, BdVRT2 T-DNA line, has delayed flowering at higher ambient temperature.
Thirdly, the function of BdVRT2 in Brachypodium was studied more deeply in this work. We found that BdVRT2 is upregulated during cold treatment and the mutant line delayed flowering in vernalized condition. This indicated that BdVRT2 promotes the transition to flowering in vernalization. In addition, BdVRT2 showed similar expression patterns as the key floral promoter BdVRN1. The interaction of BdVRT2 and BdVRN1 were shown through yeast-two hybrid assay, GSP pull-down assay and BiFC in leaf mesophyll protoplasts.
Fourthly, one side project was to explore the possible function of one of FLC homologs, BdODDSOC1, in Brachypodium. The mutant lines were phenotyped in different conditions combining daylength and vernalization, but no interesting phenotype has been found except a minor difference in leaf numbers when flowering. The leaf development in the same conditions was phenotyped, and the mutant line of the BdODDSOC1 T-DNA showed a faster rate of leaf development. It appears the single function of BdODDSOC1 is minor. The freezing tolerance experiment was tested survival rate of BdODDSOC1 T-DNA and WT in cold temperature, which resulted in the possible role of BdODDSOC1 to protect the seedlings from frost stress.
The last project I did was to capture the RNA binding proteins with a modified RNA interactome capture method in Brachypodium. This method was applied to Brachypodium shoot and leaf mesophyll protoplasts successfully, and we identified 405 RBPs. However, the validation of these captured proteins remained an issue.
In conclusion, the results indicate that the reproductive stage in Brachypodium development is more sensitive to different temperatures than the floral transition stage. We showed that BdVRT2 is a floral promoter in vernalization regulation. Also, we captured 405 RBPs in Brachypodium shoot and leaf mesophyll protoplasts and pointed out the limitation of RNA interactome capture method.status: Publishe
De functionele evolutie van MADS-box genen aangedreven door genduplicatie en mRNA-gebonden proteomen in planten
No full textSummary
During my entire PhD career, one of my main focus was the research of the impact of whole genome duplications on gene functional innovation. Specifically, we reconstructed two ancestral MADS-domain protein interaction networks (PINs) at different time points by use of phylogenetic and biochemical approaches. One network was reconstructed just before the ancient whole genome gamma triplication (γ triplication) which occurred at the origin of core eudicots, the largest group of flowering plants. A second one was reconstructed after the γ triplication and positioned at the Asterid-Rosid split. As a reference for comparison, present-day MADS-domain PINs in Arabidopsis thaliana and Solanum lycopersicum, which are two model plants representative of Rosids and Asterids respectively, were set up by use of same methods. The chosen MADS-box gene subfamilies are major key regulators for plant reproductive development and the γ triplication has been long considered as the main force for driving the divergence of core eudicots. Through direct comparison among ancestral and present-day networks, we revealed a set of innovations driven by the γ triplication at the network level. The γ triplication generated a dramatically innovated network that strongly rewired through the addition of many new interactions. These new interactions displayed new redundancy that many of these interactions were established between paralogous proteins and a new interaction partner. The simulation models demonstrated that both node and edge addition through the γ triplication were important to maintain modularity in the network. Preferential attachment occurred only for the conserved interactions through triplication and was not observed in gained interactions. After triplication, the hubs gradually lost more interactions. Because the network density was observed to be almost constant, low degree nodes that became new hubs in the network gained these interactions lost by hubs. The protein hubs become more specific in comparison to the relative promiscuity at their ancestral state. This was the underlying mechanism behind the observed network rewiring. Furthermore, several putative MADS-domain protein complexes with novel protein interactions were observed originated through γ triplication, which are mainly involved in the regulation of inflorescence meristem specification, floral meristem specification and floral organ identity related to the increased robustness of floral development in core eudicots or Pentapetalae.
During the second and third year of PhD, I was also involved in one interesting substudy contributing to understand the evolution of flower in which the experiments were designed by Dr. Philip Ruelens. The sudden radiation of angiosperms approximately c.a. 130 million years ago was considered by Charles Darwin as an “abominable mystery”. The gymnopserms, which are the closest angiosperm’ relatives, are morphologically different. Angiosperm flowers are bisexual while gymnosperm male and female reproductive organs develop separately. Because of the large morphological difference between gymnosperms and angisoperms and the absence of a fossil records with transitional plant species, the origin of angiosperm flower remains a mystery. However, one approach to understand this transition in evolution can be found in the evolution of several regulatory type II MADS-box gene subfamilies (mainly B-, C/D- and E-class genes), which are phylogenetically related between gymnosperms and angiosperms lineages. MADS-box genes encode MADS-domain transcription factors, which contain a highly conserved DNA-binding domains (the MADS domains). Over the recent two decades, studies demonstrate that the different combination of these transcription factors through forming quaternary complexes and binding the cis-regulatory CArG box of their target genes, specifies the identification of male and female reproductive organs in gymnosperms and angiosperms. Such quaternary complex are also known as “floral quartets” in flowering plants. Therefore, by reconstructing ancestral MADS-domain proteins of B-, C/D- and E-class genes, we can trace the evolution of “floral quartets” prior to the speciation of angiosperms. Our studies demonstrated that just before the origin of flowering plants, a compositional shift occurred in the floral quartets specifying male organ identity from ancestral complexes with two types of subunits through gene duplication to four types of subunits with the integration of SEPALLATA proteins. The protein interaction changes were the result of a gradual and reversible evolutionary trajectory. The simulation model shows that the compositional shifts can facilitate the evolution of the bisexual flower with the male organ in the basal regions and the female organ at the top regions. Our studies provided new insights in how the floral quartets evolved before the rise of angiosperms, which could lead to better understand the origin of angiosperm flowers.
Furthermore, I also participated in another study to understand how SEPALLATA3 (AtSEP3), a protein with hub and glue properties in Arabidopsis, originated and evolved in network organization by use of reconstructed ancestral and extant plant MADS-domain transcription factors from the previous two studies. After γ triplication, many novel protein interactions were mediated and the network density and complexity were extended by SEP3 due to its glue property. By swapping SEPALLATA3 and its ancestors between networks of different ages, it was discovered that the protein lost the capacity of promiscuous interaction and acquired specificity. This was accompanied by constraints on conformations through proline residue accumulation in linker regions between domains, which made the protein less flexible. This suggests that the proteins can evolve towards increased specificity with reduced flexibility when the complexity of the protein interaction network requires specificity.
During the last two years of my PhD, another main study concerned bio-molecular functions, different from the previous studies about evolution. In molecular biology, the complexity of RNA regulation is one of the current frontiers in animal and plant molecular biology research. RNA-binding proteins (RBPs) are characteristically involved in post-transcriptional gene regulation (PTGR) through interaction with RNA. In plants, e.g. in the Arabidopsis genome, more than 200 RBP genes have been predicted based on well-defined sequence motifs. However, when compared to recent studies of mammalian RBPs, experimental evidence for most of these predicted plant RBPs is rather scarce. Recently, the mRNA-bound proteome of mammalian cell lines has been successfully cataloged using a new method called interactome capture. This method relies on in vivo UV crosslinking of proteins to RNA, purifying the mRNA using complementary oligo(dT) beads and identifying the crosslinked proteins using mass spectrometry. We mainly describe an optimized system of mRNA interactome capture for Arabidopsis thaliana and Brachypodium distachyon leaf mesophyll protoplasts, a cell type often used in functional cellular assays. We established the conditions for optimal protein yield and demonstrated high efficiency of optimized interactome capture for plant cells. Furthermore, the discovered leaf mesophyll protoplast mRNA interactome provided in vivo experimental evidence for the first time for a number of candidate RBPs (non-classical RBPs) in plants, contributing a function for these candidate RBPs in the regulation of mRNAs.status: Publishe
Evolutie en diversificatie van MADS-box genen
No full textThis PhD is focused around the deep evolutionary history of floral-organ identity MADS-box genes. MADS-box genes encode for transcription factors functioning in a diverse array of distinct processes throughout the development of flowering plants, from seed germination to seed set. This functional variation showcases their potential to diversify and evolve novel functions, making this gene family profoundly suitable to study the evolutionary forces that produced the awe-inspiring diversity of flowering plants. To that end, by studying their evolution and diversification around the time angiosperms originated, we provide a better understanding what role they played in the evolution of angiosperms or flowering plants. To do this, we utilize a multidisciplinary approach, which is a combination of phylogenetics, paleogenetics, modelling and experimental techniques.status: Publishe
Functionele en moleculaire analyse van Flowering Locus C (FLC) homologen in Brachypodium distachyon ,,
No full textWinter genotypes of temperate cereals require prolonged cold to transition from vegetative to reproductive development. This process, referred to as vernalization, has been extensively studied in Arabidopsis thaliana where it is regulated by a key flowering repressor called FLOWERING LOCUS C or FLC. By contrast, in cereals, vernalization is known to be regulated by the combined function of VERNALIZATION genes VRN1 and VRN2, which converge on flowering integrator FLOWERING LOCUS T (or VRN3 in cereals). These VRN genes are not orthologous to FLC. The function of recently identified grass FLC-like genes, has only been partially characterized. Here, we investigated the role of three FLC homologs (BdODDSOC1, BdODDSOC2 and BdMADS37) in the model grass Brachypodium distachyon. Our results revealed that all three FLC homologs are cold responsive under short days in different Brachypodium accessions. Moreover, in a winter accession, BdODDSOC2 responded to cold before BdVRN1. Functional characterization of FLC homologs by using transgenics shows that BdODDSOC2 indeed functions as a repressor of flowering because constitutive expression of BdODDSOC2 renders plants insensitive to vernalization and RNAi-mediated knockdown plants exhibit early flowering when grown without a vernalization treatment. However, we could not find any effect of vernalization on flowering time for BdODDSOC1 and BdMADS37 transgenic overexpression or RNAi plants. Furthermore, similar to FLC in Arabidopsis, we found that stable downregulation of BdODDSOC2 in response to vernalization is associated with the enrichment of H3K27me3 at BdODDSOC2 chromatin. Taken together, our study suggests that ODDSOC2 is not only phylogenetically related to FLC in eudicots, but also functions as a flowering repressor in the vernalization pathway of Brachypodium and likely other temperate grasses. These novel insights could prove useful in breeding efforts to refine the vernalization requirement of temperate cereals and adapt varieties to local climates, which is an important goal when these climates are rapidly changing.status: Publishe
Evolutie van ancestrale MADS-gen functies.
No full textDit proefschrift bestudeert de evolutie van de functie van verschillende subfamilies binnen de MADS-box genfamilie. MADS-box genen functioneren als transcriptiefactoren en spelen een belangrijke rol in verschillende ontwikkelingsprocessen van bloeiende planten. Studies over de evolutie v an deze genfamilie hebben reeds aangetoond dat deze genfamilie gekenmerk t wordt door herhaalde en frequente genduplicatie, waarbij de transcript iefactoren na duplicatie behouden worden in het genoom. Een dergelijk pa troon van genduplicatie creëert een potentieel voor functionele diversif icatie via wijzigingen in de spatiotemporele expressie of door veranderi ngen in eiwit-eiwit interactiespecificiteit.. De eerste twee hoofdstukken bestuderen het patroon van genduplicatie en genverlies in twee verschillende MADS-box subfamilies, PISTILLATA en AGL 6. In een eerste hoofdstuk reconstrueren we de evolutie van PISTILLATA e n twee van zijn interactiepartners, TM6 and APETALA3, in de asteriden. H iervoor hebben we nieuwe B-klasse genen geïsoleerd uit basale asteriden en gecombineerd met EST-data. Onze resultaten suggereren dat zowel gedur ende de evolutie van de basale asteriden als doorheen de evolutie van de kernasteriden, oude en recente duplicaties zijn opgetreden in de PISTIL LATA-subfamilie. Bovendien correleren deze duplicaties niet met gelijkaa rdige duplicaties in de evolutie van de interactiepartners TM6 en APETAL A3. We hebben ook de evolutie na duplicatie bestudeerd voor een aantal v an deze gedupliceerde genen via RT-PCR, in situ hybridisatie en yeast-2- hybrid. Samen met informatie over andere soorten uit de groep van de ker nasteriden suggereren deze data dat, gedurende de evolutie van ast eriden, verschillende PISTILLATA duplicaties zijn opgetreden en dat deze gedupliceerde genen kunnen aangewend worden voor nieuwe functies in de bloem. In een tweede hoofdstuk wordt de evolutie van de AGL6-subfamilie doorhee n de bloemplanten beschreven. In totaal werden vier nieuwe duplicaties g eïdentificeerd; één aan de basis van de kerneudicotylen die resulteerde in een AGL6-like en een euAGL6 groep, een tweede gedurende de evolutie v an de basale angiospermen en twee extra duplicaties gedurende de evoluti e van de monocotylen. We hebben de expressiepatronen van deze genfamilie bepaald via kwantitatieve real time PCR om na te geen hoe deze subfamil e is gediversifieerd na duplicatie. Een van beide clades die ontstaan is na de duplicatie aan de basis van de kerneudicotylen (AGL6-like) komt s terk tot expressie in vegetatief weefsel, terwijl de andere clade (euAGL 6) vooral tot expressie komt in reproductieve weefsels. We suggereren da t, na duplicatie aan de basis van de kerneudicotylen, AGL6 een functie h eeft verkregen in de transitie van vegetatieve scheuten, naast de gekend e voorouderlijke functie in reproductieve structuren. In een derde hoofdstuk hebben we de evolutie en functie van een derde MA DS-box subfamile bestudeerd, TM8. Deze subfamilie heeft geen vertegenwoo rdigers in de modelplanten Arabidopsis en rijst en bovendien werd er nog geen enkele TM8-vertegenwoordiger functioneel gekarakteriseerd. In een eerste fase hebben we de reeds beschikbare TM8-vertegenwoordigers in ang iospermen geïdentificeerd en stellen als eerste een reconstructie van de evolutie van deze subfamilie voor. Deze resultaten suggereren dat genve rlies vermoedelijk is opgetreden in de evolutionaire lijn die leidt naar de monocotylen en de Brassicales. Vervolgens hebben we door middel van virus induced gene silencing de functie van TM8 gekarakteriseerd in Nico tiana benthamiana. De interactie van TM8 met SVP (SHORT VEGETATIVE PHASE ), een ander MADS-box gen dat een rol speelt in de controle van bloeitij d in Arabidopsis thaliana, speelt hierin een cruciale rol. Zo hebben we TM8 geïdentificeerd als een bloeirepressor die zijn functie vervult door een gekende promoter van bloei, FLOWERING LOCUS T (FT), te onderdrukken. De effecten op bloeijtijd zouden ook tot stand kunnen kome n via twee andere kleine RNA-molecules, miR156 en miR172. Voor beide is reeds aangetoond dat ze zowel de juveniele-adulte overgang als de bloei- inductie beïnvloeden in Arabidopsis. Onze data suggereren inderdaad dat TM8 (en vermoedelijk ook SVP) een rol spelen in de controle van de juven iele-adulte overgang in Nicotiana benthamiana. Tenslotte hebben we ook v ia virus induced gene silencing in Papaver somniferum aangetoond dat TM8 eveneens beide microRNAs reguleert, wat suggereert dat dit mechanisme geconserveerd is doorheen de evolutie.status: Publishe
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
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