The PID-directed shift in PIN polarity has been broadly accepted as one of the essential mechanisms for the regulation of auxin transport polarity. We verified that both PID functionality and its... Show moreThe PID-directed shift in PIN polarity has been broadly accepted as one of the essential mechanisms for the regulation of auxin transport polarity. We verified that both PID functionality and its subcellular localization do not depend on PDK1 function. However, by detailed analysis on these phenotypes and the expression of the auxin response reporter, we found the pdk1 pdk2 double mutant to be impaired in auxin transport in vascular tissues. Together with other mutant phenotypes, we suspect that PDK1 may be the master regulator of AGC1 kinases. The pdk1 pdk2 short root phenotype caused by phloem differentiation defects phenocopied the pax mutant. Complementation results of wild type and phosphomimic PAX in the pdk1 pdk2 background suggest that PDK1-dependent PAX phosphorylation and activation are essential for its full biological function. We also explain the molecular basis of PDK1 basal localization and the unnecessity of this polarity for vascular development. In addition, we investigated downstream action after PID phosphorylation. Several conserved tyrosine residues close to serine phosphorylation sites in the PIN1 and PIN2 HLs are mutated. Two of these tyrosines redundantly affected PIN polarity. However, PID mediated phosphorylation and tyrosine-based PIN trafficking are independent processes. Show less
In this thesis, the role of plant-mediated soil legacy effects in shaping aboveground plant-insect interactions was investigated. This work shows that soil legacy effects on plant-insect... Show moreIn this thesis, the role of plant-mediated soil legacy effects in shaping aboveground plant-insect interactions was investigated. This work shows that soil legacy effects on plant-insect interactions are a common phenomenon in nature, both in individual responding plant species and their associated herbivores, as well as in responding plant communities and associated herbivores. This thesis elucidates two potential mechanisms how soils can influence aboveground insects. First, soils can alter the way plants defend them selves in terms of secondary chemistry and phytohormonal defenses. Second, soils can also directly impact aboveground insects by changing the insect microbiome. This work shows that a large part of the insect microbiome is taken up directly from the soil microbiome, and leads to exciting new research directions on the longterm and evolutionary implications of soils for aboveground insects. Show less
The main aim of my Ph.D. thesis is to further explore the value of using early developing zebrafish larvae (up to 5 dpf) as a model to study anxiety-like behaviour and their pharmacological... Show moreThe main aim of my Ph.D. thesis is to further explore the value of using early developing zebrafish larvae (up to 5 dpf) as a model to study anxiety-like behaviour and their pharmacological modulation with drugs. Several behavioural parameters of larval zebrafish were used to evaluate anxiety-like behaviours, which are locomotion, startle response, and thigmotaxis. In addition to this, behavioural assays are also used to screen synthetic anxiolytics commonly used in the treatment of anxiety disorders. Finally, the toxic effects of the synthetic and herbal-based anxiolytics were also assessed on the developing zebrafish larvae. Show less
My PhD thesis investigates the ecological significance of resistance against drought-induced air bubble formation inside the water conduits of plants (embolism), and the plasticity and... Show moreMy PhD thesis investigates the ecological significance of resistance against drought-induced air bubble formation inside the water conduits of plants (embolism), and the plasticity and functional aspects of stem anatomical traits in woody and herbaceous species. I revealed the impact of temperature, precipitation and soil conditions on wood anatomical variation, as well as the effect of plant height on wood trait variation along the main trunk, in tree populations from two seasonally dry environments in Brazil. The functional significance of embolism resistance was confirmed in shrubby and herbaceous species on Tenerife, Canary Islands, where the more embolism resistant species occur in the drier areas. The anatomical characters that best explained differences in embolism resistance across the Tenerife species were thickness of intervessel pit membranes (TPM) and the amount of stem woodiness. In a daisy clade including derived woody island species and their continental herbaceous counterparts, TPM is the direct functional link explaining the indirect positive relationship between increased wood formation and increased embolism resistance. This result matches with an ongoing global study highlighting the frequent occurrence of derived woody species in dry areas worldwide. These combined anatomical and hydraulic results will contribute to our understanding on plant adaptation to drought. Show less
Streptomyces are bacteria abundant in soil that participate in diverse and complex interactions. These bacteria are the main producers of the antibiotics we currently use in the clinic.... Show moreStreptomyces are bacteria abundant in soil that participate in diverse and complex interactions. These bacteria are the main producers of the antibiotics we currently use in the clinic. Streptomyces are also prolific producers of volatile compounds (VCs) that due to their low molecular weight and vapor pressure can easily travel and distribute through air, water and soil pores. The volatile character of such molecules could suggest that they participate in “long-distance” interactions. This dissertation studies the diversity and functions of VCs produced by Streptomyces. The experiments described here show that even very small VCs such as ammonia can act as antibiotics if they are produced in sufficiently large quantities. it also shows how the perceiver bacteria reacts and responds to antibiotic VCs. The role of other volatile compounds such as terpenes was also studied, in particular 2-methylisoborneol and geosmin. These terpene compounds are highly conserved among Streptomyces and hence may have an important role in the biology of these bacteria. This work includes some results on the possible role of these molecules and opens research questions about the possible regulatory mechanisms for the production of these compounds. Show less
Trigonostemon Blume is a plant genus in the family Euphorbiaceae comprising 59 species. These plants are small trees or shrubs growing in the lowland rainforests in Southeast Asia and adjacent... Show moreTrigonostemon Blume is a plant genus in the family Euphorbiaceae comprising 59 species. These plants are small trees or shrubs growing in the lowland rainforests in Southeast Asia and adjacent areas. The small unisexual flowers with colourful petals and the 3 or 5 united stamens are typical characters of the genus. The present thesis studies four aspects of the genus: the taxonomy, pollen morphology, molecular phylogeny and historical biogeography. The species delimitation has been optimised in the taxonomic revisions and the species complexes can now be clearly recognised by morphological characters. Palynological studies reveal strong correlations between pollen and macromorphology in Trigonostemon and these support the infrageneric classification of the genus. Molecular phylogenetic analyses have demonstrated that Trigonostemon and Dimorphocalyx Thwaites are two monophyletic groups and are not closely related, although they are similar in morphology. Furthermore, Trigonostemon is divided into four sections based on molecular and (pollen) morphological data. Finally, the thesis concludes with the historical biogeography of Trigonostemon: it probably originated on Southeast Asian mainland, one lineage later radiated to the Malay Peninsula, and dispersed further eastwards and diversified before the genus reached its current distribution. Show less
Almost all bacteria are enveloped by a cell wall that provides cellular protection. In this thesis I show that some filamentous actinomycetes have a surprising natural ability to adopt a cell wall... Show moreAlmost all bacteria are enveloped by a cell wall that provides cellular protection. In this thesis I show that some filamentous actinomycetes have a surprising natural ability to adopt a cell wall-deficient life style. The formation of these newly identified S-cells is a transient morphological adaptation in response to hyperosmotic stress conditions. In contrast, prolonged exposure to cell wall-targeting antibiotics or osmotic stress leads to the formation of so-called L-form cells, which are mutants that can proliferate indefinitely without their cell wall. The exciting properties of L-forms were used to discover a new cell-wall biosynthetic enzyme and to study enzyme secretion in the absence of a cell wall. Show less
Increasing the efficiency of gene targeting (GT) as a genome editing tool in plants has been an important goal in plant biotechnology. Improvements have been made using sequence-specific nucleases... Show moreIncreasing the efficiency of gene targeting (GT) as a genome editing tool in plants has been an important goal in plant biotechnology. Improvements have been made using sequence-specific nucleases such as CRISPR/Cas9 to induce DNA double strand breaks at target loci and activate repair via homologous recombination (HR). GT can then be achieved by HR-mediated integration of an artificial repair template, sharing homology with the target locus. Further improvements have been made with the in planta GT method, in which the repair template is pre-inserted in the genome and can be excised by nucleases. Although these improvements led to substantial increases in GT efficiency, GT is still not efficient enough to be feasible for crop biotechnology. This thesis describes strategies to further improve GT efficiency in the model plant Arabidopsis thaliana. One of these strategies was to perform in planta GT in meiocytes, cells that already have a higher rate of HR. Another strategy was to find new Arabidopsis mutants with increased GT frequencies and to identify genes involved in this phenotype. In the end, this may lead to a better understanding of the mechanisms underlying GT and these may be used to realize higher GT frequencies in plants. Show less
Hybrid zones occur where two species meet and produce offspring (hybrids). Typically, hybrids show a considerable reduction in fitness. In this thesis two hybrid zones are treated.Two species of... Show moreHybrid zones occur where two species meet and produce offspring (hybrids). Typically, hybrids show a considerable reduction in fitness. In this thesis two hybrid zones are treated.Two species of banded newts (Ommatotriton nesterovi and O. ophryticus) are thought to meet in a hybrid zone in the north of Turkey. In this thesis I confirm the species status of the two banded newt species based on mitochondrial DNA and two nuclear DNA sequences. The location of the hybrid zone is narrowed down to a 60 km wide region. If a hybrid zone is present between O. nesterovi and O. ophryticus, it is narrower than 60 km and it may be geographically stable. An introduced population of hybrid banded newts in Spain provides evidence that the two species can produce fertile offspring. This increases the likelihood that a hybrid zone indeed exists between the two species.Common and spined toads (Bufo bufo and B. spinosus) meet in an 800 km long hybrid zone that runs diagonally across France. A genetic footprint north of the hybrid zone was previously recorded and linked to southward hybrid zone movement, with B. bufo overtaking B. spinosus. To test hypotheses of hybrid zone movement, a transect in northwest France was studied with 31 nuclear markers. The contrasting results suggest that stronger reproductive isolation on the B. spinosus side of the hybrid zone than on the B. bufo side, may be more likely than hybrid zone movement.To continue the research on the Bufo hybrid zone, two distant transects, one in the northwest and one in the southeast of France, were studied using 1200 nuclear markers. Asymmetries which were previously found in the literature and in this thesis, were confirmed for the hybrid zone in northwest France, but not in the southeast, where the gene flow appears to be symmetric, indicating the hybrid zone is stable here. Barrier markers, genetic regions which may be associated with barrier genes, were identified by their relatively restricted gene flow. The barrier markers present in both transects suggest that the two species have evolved a universal genetic barrier to gene flow. The differences in patterns of gene flow between the transects may be caused by genetic divergence within B. bufo, documented in previous phylogeographical work. We can clearly no longer think of hybrid zones as static upon formation; hybrid zones evolve! With the increasing availability of genome resources, detection of more detailed patterns of differential introgression along the genome in hybrid zone studies will reveal the genetic architecture of speciation and the evolution of hybrid zones. Show less
In this thesis, I targeted the orchid genus Lepanthes, one of the six genera of angiosperms that surpasses 1,000 species in the Neotropics, as a study model to investigate the evolutionary... Show moreIn this thesis, I targeted the orchid genus Lepanthes, one of the six genera of angiosperms that surpasses 1,000 species in the Neotropics, as a study model to investigate the evolutionary processes that promoted species diversifications. To investigate this, we improved the taxonomy of the group integrating a solid phylogenetic framework with morphological evolution, assessing inter-specific relationships in species complexes with hundreds of DNA markers using anchored hybrid enrichment approach, and describing new species. In addition, we addressed the pollination of Trichosalpinx through the study of floral anatomy, pollinator behavior, and floral traits. Trichosalpinx flowers are pollinated exclusively by female biting midges that are attracted by the small quantities of proteins secreted on the flowers. Finally, we inferred the biogeographical history and diversification dynamics of the two largest Neotropical orchid groups (Cymbidieae and Pleurothallidinae), using densely sampled phylogenies coupled with geological datasets and discussed the impact of biogeographical events and orogeny on the species richness of Lepanthes. Species diversification is correlated with Andean orogeny, and multiple migrations and recolonizations across the Andes indicate that mountains do not constrain orchid dispersal over long timescales. This thesis provides new insights into the complex evolution of one of the most species-rich angiosperm. Show less
We showed that constitutive and inducible chemical and morphological defenses against Western flower thrips differ between tomato and chrysanthemum plants. Furthermore, we demonstrated that both... Show moreWe showed that constitutive and inducible chemical and morphological defenses against Western flower thrips differ between tomato and chrysanthemum plants. Furthermore, we demonstrated that both plant species respond differently to bacteria-derived defense elicitors, such as the phytotoxin coronatine. This highlights the plant species-specificity of these interactions and the possible limitation for the use of pathogen-associated molecular patterns to enhance the plant immune system. This study thus provides knowledge and novel strategies for Western flower thrips control. Yet, further comprehensive work is needed to evaluate the influence of these induction strategies on plant fitness. Show less
In this thesis, I aimed at decoding the role of SUMO in the DNA damage repair pathway. The SUMO system is believed to be involved in this process at several levels. I focused on the most inevitable... Show moreIn this thesis, I aimed at decoding the role of SUMO in the DNA damage repair pathway. The SUMO system is believed to be involved in this process at several levels. I focused on the most inevitable DNA obstacle causing DNA replication stress, and the cellular roles of SUMOylation in repairing DNA replication stress caused DNA damage. Post-translational modifications are essential regulators of proteins. PTMs do not only play their roles solo but extensively interact with each other. Our knowledge about proteins modified by a combination of SUMO and ubiquitin, SUMO and phosphate and crosstalk between them is quite limited. This thesis also aimed at deciphering the crosstalk between SUMOylation and phosphorylation and ubiquitination during the DNA damage response and searching for indirect and direct targets for the human STUbL RNF4, which mediates the ubiquitination of SUMOylated target proteins. Lastly, we adopted the strategy described for SUMO and introduced His10-tagged UFM-1-K0 to identify UFM-1 acceptor lysines. We identified and confirmed RPL26 as a key UFM1 target and further confirmed that the UFMylated form of RPL26 can efficiently interact with the Signal Recognition Particle Receptor, implicating that UFMylation could regulate protein transfer to the Endoplasmic Reticulum. Show less
Plants and soils from natural ecosystems harbor great diversity of soil microorganisms, which could potentially contribute to the sustainability of horticulture. The knowledge about using wild... Show morePlants and soils from natural ecosystems harbor great diversity of soil microorganisms, which could potentially contribute to the sustainability of horticulture. The knowledge about using wild plant species and soil from natural ecosystem to improve the crop health will advance the application of ecological knowledge in horticulture. In this thesis, I evaluate the possibilities of using wild plant species and grassland soil to enhance the growth and reduce the disease susceptibility of chrysanthemum through plant-soil feedback principles. Grass-conditioned inocula contributed more to chrysanthemum growth than forb- or legume-conditioned inocula, this also lead to better chrysanthemum performance under Pythium treatment. Moreover, by mixing two plant-conditioned inocula, they interacted synergistically in terms of plant health but antagonistically in terms of plant growth. The influence of plant-conditioned inocula on chrysanthemum were still significant after two successive growth cycles, but their effects tended to converge because of the overriding effect of chrysanthemum growth. Inoculating greenhouse soil conditioned by wild plant species or grassland soil to greenhouse soil did not significantly improve the negative effects of greenhouse soil on chrysanthemum growth. Olpidium brassicae was identified as a main fungal pathogen of chrysanthemum when growing in greenhouse soil. Show less
Plants, are sessile organisms, have developed strategies to adapt to changes in their environment, in part by altering their growth and development. One of the central regulators of this adaptive... Show morePlants, are sessile organisms, have developed strategies to adapt to changes in their environment, in part by altering their growth and development. One of the central regulators of this adaptive plant growth and development is the plant hormone auxin. Auxin is transported by the PIN class of proteins. PIN proteins are membrane localized proteins that transport auxin through the membrane. Plasma membrane localized PIN proteins often show a polarized localization that is regulated by the the AGC kinase PINOID. We investigated the regulation of the PINOID kinase by phosphoinositide-dependent protein kinase 1 (PDK1), a central upstream regulator of AGC kinases. Here we show in arabidopsis protoplasts that PDK1 phosphorylation induces a switch in PINOID subcellular localization from the plasma membrane to endomembrane compartments and the microtubule cytoskeleton (MT). Our results in planta suggest a new role for dynamic PDK1-mediated activation of PINOID in plant development. We reveal a regulatory complex that on the one hand links PINOID to a dynamic process in young tissues that requires high protein turnover and the MT. On the other hand, the complex is not essential for survival and allows much variability within the involved kinesin sequences between plant species. Show less
The use of N fertilizers has increased worldwide in the past century. While this increased input of N has increased food productivity, it has also contributed to decreases in biodiversity, soil... Show moreThe use of N fertilizers has increased worldwide in the past century. While this increased input of N has increased food productivity, it has also contributed to decreases in biodiversity, soil quality and environmental health, including increases in greenhouse gas emissions. These emissions in agricultural soils are largely carried out by the soil microbiome, or the microorganisms living in the soil and transforming N fertilizers to different forms. Here, the overall research aim was to gain detailed insight into the effects of nitrogen fertilizer schemes, including long term fertilization, on soil microbial communities. To do this, I applied next-generation sequencing technology and associated bioinformatics analyses to field experiments in the Netherlands and in Brazil. Show less
Microbiome composition of the spermosphere and the rhizosphere of wild and modern bean accessions grown in an agricultural and a native soil from Colombia was characterized by metagenomics and... Show moreMicrobiome composition of the spermosphere and the rhizosphere of wild and modern bean accessions grown in an agricultural and a native soil from Colombia was characterized by metagenomics and cultivation-dependent approaches. A higher relative abundance of Bacteroidetes, mainly Chitinophagaceae and Cytophagaceae, was observed in the rhizosphere of wild accessions while an increase in relative abundance of Actinobacteria and Proteobacteria was observed in the rhizosphere of modern accessions. These divergences associated with differences in specific root length (SRL). Using 16S-rDNA data from other studies revealed that also wild relatives of other crop plant species presented higher relative abundance of Bacteroidetes. Additionally, bean accessions were grown in a native and an agricultural soil from Colombian. The transition of common bean from a native to an agricultural soil led to a gain of rhizobacterial diversity and to a stronger bean genotype-dependent effect on microbiome assembly. In addition, during seed imbibition and germination, significant differences were detected between the spermosphere microbiomes of wild and modern bean accessions. A domestication effect on microbiome assembly already at this early developmental stage was evidenced. The research presented in this thesis showed that domestication of common bean had a significant effect on the composition of the microbiome. Show less
Streptomyces are multicellular, Gram-positive bacteria in the phylum of actinobacteria which produce a high amount of bioactive natural products of which the expression is tightly coordinated with... Show moreStreptomyces are multicellular, Gram-positive bacteria in the phylum of actinobacteria which produce a high amount of bioactive natural products of which the expression is tightly coordinated with the life cycle. This thesis shows the identification of S. roseifaciens, a novel species with an uncommon, verticillate spore morphology and a unique household of SsgA-like proteins. Analyses of the peptidoglycan composition show that S. coelicolor show a pattern of 3-3 cross-linking befitting a tip-growing organism and change in composition between vegetative mycelium and spores. Kitasatosporae carry meso-DAP in the peptidoglycan of vegetative mycelium and LL-DAP in the peptidoglycan of spores. In line with this difference, the peptidoglycan architecture of these two growth stages undergoes such radical changes that they would seem to be from different species. S. coelicolor is naturally vancomycin resistant, but the addition of D-alanine and disruption in a single gene increases vancomycin sensitivity by a thousandfold. A knockout mutant of the alanine racemase, alr, requires exogenous addition of D-alanine. The Alr crystal structure of S. coelicolor and the D-cycloserine producer S. lavendulae were compared as to look for possible mechanisms for D-cycloserine resistance. Show less
The overall objectives of my thesis are to investigate the dynamics of the sorghum root microbiome and to explore the beneficial effects of the root microbiome on sorghum growth and stress... Show moreThe overall objectives of my thesis are to investigate the dynamics of the sorghum root microbiome and to explore the beneficial effects of the root microbiome on sorghum growth and stress tolerance. In Chapter 2, I describe the differences in rhizobacterial community composition of seven different sorghum cultivars grown in the greenhouse in two different soil types at four different plant growth stages. in Chapter 3 I present the co-occurrence of bacterial and fungal communities in the rhizosphere of different sorghum cultivars. Chapter 4 addresses the effects of different rhizobacterial community compositions on growth and drought tolerance of sorghum. In Chapter 5, I described the effects of five endophytic bacterial strains on the growth of four sorghum cultivars. In Chapter 6, I provide a general discussion of the main findings of this thesis and highlight the importance of sorghum-microbiome interactions. Show less
