Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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MADS evolution : insights into evolutionary changes in transcription factors and their binding sites
Bruijn, Suze-Annigje de - \ 2017
University. Promotor(en): Gerco Angenent. - Wageningen : Wageningen University - ISBN 9789463436700 - 195
plants - evolution - mads-box proteins - transcription factors - flowers - molecular biology - planten - evolutie - mads-box eiwitten - transcriptiefactoren - bloemen - moleculaire biologie

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.

Systems analysis of Mycoplasma hyopneumoniae to improve vaccine production
Kamminga, Tjerko - \ 2017
University. Promotor(en): Vitor Martins dos Santos, co-promotor(en): Peter Schaap; J.J.E. Bijlsma. - Wageningen : Wageningen University - ISBN 9789463436519 - 152
interdisciplinary research - molecular biology - mycoplasma hyopneumoniae - vaccines - systems biology - systems analysis - pigs - pneumonia - animals - interdisciplinair onderzoek - moleculaire biologie - vaccins - systeembiologie - systeemanalyse - varkens - longontsteking - dieren

Mycoplasma hyopneumoniae (M. hyopneumoniae) is a bacterial pathogen that has evolved from a gram-positive ancestor and specifically colonizes the lower respiratory tract of pigs where it causes enzootic pneumonia and plays a major role in the development of respiratory disease in pigs. Whole-cell inactivated vaccines are available that lower the severity of disease and are widely applied in pig industry to prevent clinical signs and improve pig herd health. However, production of these vaccines is challenging because it is not known which bacterial components are needed for protection and complex cultivation media are needed because growth requirements are not completely understood. The aim of this thesis was to understand growth and survival strategies of M. hyopneumoniae during infection, to integrate this knowledge with metabolic modeling under conditions used for vaccine production and apply this knowledge to improve the current production process for M. hyopneumoniae vaccines.

Chapter 1 provides a general introduction into the disease, treatment and prevention methods with a focus on vaccines. I then introduce the characteristics of the M. hyopneumoniae genome, transcriptome and review the current knowledge on infectious mechanisms and the response of the pig to infection and vaccination. Finally, I discuss the challenges related to vaccine production and introduce systems biology tools that will be applied in the thesis. In chapter 2 we define a strategy for risk-based process development of bacterial vaccines which provided the framework for future studies performed during this thesis. We propose to integrate the academic workflow for rational strain design with the industry standard for process design. Systems biology tools, especially genome-scale metabolic models, play an essential role in this strategy because application of these tools reduces process risks and increases process understanding. Therefore, in line with this strategy, we created a manually curated genome-scale metabolic model of M. hyopneumoniae which we applied to dynamically model the cultivation step in the vaccine production process (chapter 3). We found that only 16% of cellular energy in a standard fermentation was used for growth and 84% was used for non-growth associated maintenance. By model-driven experimentation we were able to increase the fraction of cellular energy used for growth by addition of pyruvate to the production medium, and showed in dedicated fermentor experiments that the improved process reached a 2.3 times higher biomass yield. Although the metabolic model helped to increase process yield, it did not allow prediction of a defined cultivation medium without components from porcine origin. Therefore, to better understand the dependency of M. hyopneumoniae on host derived components, we performed a functional comparison of 80 mycoplasma genomes and used multivariate and machine-learning algorithms to relate functional capability to the specific host and niche of mycoplasma species (chapter 4). This analysis allowed us to identify protein domains possibly needed for growth and survival in the pig lung. In addition, we found that protein domains expected to be essential for bacterial growth were not persistently present in mycoplasma genomes suggesting that alternative domain configurations exist that bypass their essentiality. To better understand whether the proteins we identified as possibly important for survival in pigs actually play a role during M. hyopneumoniae infection, we sequenced the bacterial mRNA during infection in chapter 5 and compared the in vivo transcriptome to that of broth grown mycoplasma. We found 22 up-regulated and 30 down-regulated genes during infection (FDR<0.01 and fold change >2LOG2) and identified differentially expressed ncRNAs. In chapter 6 we build upon our mycoplasma basis to further analyse the role of ncRNAs in bacterial genomes. We identified an exponential relationship between the AT content of genomes and the number of ncRNAs and propose that this relation is the result of spurious transcription, which is more likely to occur in AT rich genomes. This hypothesis is further substantiated by showing that spurious transcription demands minimal cellular energy and that overexpression of cis-binding ncRNAs in M. pneumoniae did not influence the level of proteins translated from their overlapping mRNAs. Finally, in chapter 7 I discuss four system strategies, identified in this thesis and derived from recent literature, and discuss how these strategies could be integrated in the metabolic model of M. hyopneumoniae. Lastly, I provide an outlook on the next steps needed for improvement of the production process for M. hyopneumoniae vaccines.

In conclusion, this work provided novel insight in the metabolic capability of M. hyopneumoniae based on the proteome domain content, captured in a genome-scale metabolic model and studied under in vitro and in vivo conditions. Biomass yield of the cultivation step for vaccine production was increased and the basis was laid to further improve the production process for M. hyopneumoniae vaccines using model-based experimentation.

Functional analyses of plant-specific histone deacetylases : Their role in root development, stress responses and symbiotic interactions
Li, Huchen - \ 2017
University. Promotor(en): Ton Bisseling, co-promotor(en): Olga Kulikova. - Wageningen : Wageningen University - ISBN 9789463436816 - 188
plants - histones - enzymes - roots - development - symbiosis - gene expression - molecular biology - root nodules - mycorrhizas - planten - histonen - enzymen - wortels - ontwikkeling - symbiose - genexpressie - moleculaire biologie - wortelknolletjes - mycorrhizae

Plants have a sessile lifestyle. To ensure survival, they develop a potential to respond to environmental cues to set up an adaptive growth and development. This adaptation involves transcriptional reprogramming of the genome through chromatin-based mechanisms relying on the dynamic interplay of transcription factors (TFs), post-translational modification of histones, the deposition of histone variants, DNA methylation, and nucleosome remodeling. This thesis is focused on a role of one group of histone post-translational modifiers, plant-specific histone deacetylases (HDTs), in plant development under control condition and variable stresses/symbiotic interactions.

It is well known that HDTs are involved in plant responses to environmental stresses. However, whether they play a role in regulating plant growth and development is elusive. In this thesis it is shown that Arabidopsis thaliana AtHDT1/2 regulate the cell fate switch from division to expansion in the Arabidopsis root. Knock-down of AtHDT1/2 (hdt1,2i) causes that this switch occurs earlier and results in less cells in the root meristem. This process slows down root growth. One target of AtHDT1/2, AtGA2ox2, is identified here. Its overexpression displays the same root phenotype as hdt1/2i , and its knock-out partially rescues hdt1,2i root meristem phenotype. AtGA2ox2 inactivates gibberellin (GA4) whose application increases root meristem cell number in WT, but not in hdt1,2i. Based on these data, we conclude that AtHDT1/2 repress the transcription of AtGA2ox2, and likely fine-tunes GA homeostasis to regulate the switch from cell division to expansion in root tips.

HDTs respond to salt stress in Arabidopsis seedlings. Halotropism is a novel reported tropism allowing roots to avoid a saline environment. Whether the AtHDT1/2-AtGA2ox2 module is operational in halotropism is studied here. We show that hdt1,2i mutants respond more severe in halotropism. AtHDT1/2, as well as AtGA2ox2 display asymmetric localization patterns in halotropism with AtHDT1/2 reduced and AtGA2ox2 induced at high salt side of root tips. Our data indicate that their asymmetric patterns likely results in less GA at high salt side of root tips and this is required for halotropism establishment. In line with this, both constitutive expression of AtHDT2 and exogenous GA application reduce halotropic response. A reduction of GA in root tips causes an earlier switch from cell division to expansion. We discuss that this earlier switch enables roots rapidly to bend away from saline environment.

It has been shown that HDTs play a role under biotic stress in rice and tobacco leaves. We demonstrate that they are also involved in response to biotic stress in Arabidopsis leaves. Arabidopsis hdt2 mutants are more susceptible to virulent Pseudomonas syringae pv. tomato PstDC3000, whereas AtHDT2 overexpression mutants are more resistant. In addition, we detected a translocation of AtHDT2 from nucleolus to nucleoplasm after the perception of flagellin22 in Arabidopsis leaf cells. This translocation is not observed under abiotic stress. A mechanism controlling this translocation is identified. AtMPK3 is activated under biotic stress, it interacts with and phosphorylates AtHDT2. This leads to the accumulation of AtHDT2 in nucleoplasm where it contributes to the repression of defense genes.

During the interaction with symbiotic microorganisms, plants could develop a symbiotic organ/structure. For example, legumes of which Medicago truncatula is a model, can form root nodules or arbuscules by interacting with rhizobia or arbuscular mycorrhiza.

We show that nodule-specific knock-down of MtHDT1/2/3 (MtHDTs RNAi) blocks nodule primordia development and affects the function of nodule meristem. This is consistent with their roles in controlling cell division during root development and suggests that the function of nodule and root meristems is closely related. However, MtHDT2 gains a new sub-nuclear localization pattern in nodule meristem by using a not yet known mechanism, different from that in root meristem. This suggests that these two meristems have different transcriptional landscapes. In the nodule infection zone MtHDTs are also expressed and in MtHDTs RNAi the intracellular release of rhizobia is markedly reduced. Expression of MtHMGR1 and its paralogs, encoding 3-hydroxy-3-methylglutaryl-coenzyme A reductases are down-regulated in MtHDTs RNAi. It has been shown MtHMGR1 interacts with MtDMI2, a component of Nod factor signalling pathway, to control rhizobial infection. Knock-down of MtHMGR1/MtDMI2, as well as inhibiting MtHMGRs enzymatic activity blocks nodule primordia development and rhizobial infection in nodule primordia/mature nodules. This phenotype partially resembles MtHDTs RNAi phenotype. We discuss that MtHDTs regulate expression of MtHMGRs and in this way affect Nod factor signalling and control nodule development.

Similar to nodule symbiosis, during arbuscular mycorrhizal symbiosis cells in the cortex are also intracellularly infected. We show that MtHDT2 is also induced in these arbuscule containing cells. Knock-down of MtHDT2 (MtHDT2i) significantly reduces the intracellular infection of the hyphae on the mycorrhized root segments, indicating that MtHDT2 control mycorrhizal intracellular infection. We discuss whether MtHDTs can regulate mycorrhizal/rhizobial infection in a similar way.

The data obtained in this thesis and the published information related to these subjects are discussed at the end. HDTs are key players in plant responses to environmental cues, whereas they respond to abiotic factors and biotic factors differently. They are also key regulators of plant growth and development that is clearly demonstrated in this thesis on examples of root and nodule development. I also propose a role of AtHDT1/2 in response to salt signal to fine-tune the switch from cell division to expansion in root tips during halotropism.

Plant cortical microtubule dynamics and cell division plane orientation
Chakrabortty, Bandan - \ 2017
University. Promotor(en): Ben Scheres; Bela Mulder. - Wageningen : Wageningen University - ISBN 9789463431828 - 124
microtubules - plant cell biology - cell division - plant development - molecular biology - morphogenesis - simulation - microtubuli - plantencelbiologie - celdeling - plantenontwikkeling - moleculaire biologie - morfogenese - simulatie

This thesis work aimed at a better understanding of the molecular basis of oriented cell division in plant cell. As, the efficiency of plant morphogenesis depends on oriented cell division, this work should contribute towards a fundamental understanding of the molecular basis of efficient plant morphogenesis. We describe a modelling framework that allows us to simulate microtubule dynamics on the surface of arbitrary shapes. We further explored the generic role of microtubule regulatory effects such as shape anisotropy, edge-catastrophe and enhanced microtubule stabilization on the orientation of the microtubule array. Through a combined approach of experimental observations of cell division patterns and simulation of microtubule dynamics, we describe a possible molecular basis of oriented cell division during Arabidopsis early embryogenesis. We also infer the necessity of incorporating anisotropic growth/stress response of microtubules towards understanding division plane orientation in the growing epidermal root cells of Arabidopsis.

Ambient temperature‐directed flowering time regulation : the role of alternative splicing
Verhage, Dina Sara Leonie - \ 2017
University. Promotor(en): Gerco Angenent, co-promotor(en): Richard Immink; Guusje Bonnema. - Wageningen : Wageningen University - ISBN 9789462579705 - 161
plants - flowering date - flowering - temperature - alternative splicing - molecular biology - genes - planten - bloeidatum - bloei - temperatuur - alternatieve splitsing - moleculaire biologie - genen

As a consequence of a sessile lifestyle, plants are constantly facing a fluctuating environment. In order to both profit maximally and protect themselves from these environmental cues, plants evolved ways to sense and respond to signals.

Ambient temperature is one of the cues for which plants have acquired a strategy to enhance their chance of survival and reproduction. Small changes in ambient temperature can have major effects on plant architecture and development, such as the transition from the vegetative to the reproductive flowering phase. The moment of flowering is an important event in the life cycle of a plant, since reproductive success depends on it.

In Chapter 1, I introduced the concept of alternative splicing, a molecular mechanism with a pivotal role in ambient temperature regulation of flowering time. In the model plant Arabidopsis thaliana, approximately 60% of the intron-containing genes show alternative splicing. Gene splicing varies depending on developmental stage and tissue type, but also environmental changes trigger differential splicing. Splicing is conducted by a large cellular machinery called the spliceosome, which recognizes intron-defining sequences and other cis-regulatory elements acting as splicing enhancers or silencers. Moreover, factors like chromatin structure, histone marks, RNA polymerase II (polII) elongation speed and the secondary structure of the pre-mRNA all play a role in the splicing outcome. Due to alternative splicing, a single gene can yield various transcripts. However, this does not cause an equal expansion of the proteome. Part of the transcripts are targeted for nonsense-mediated decay, or will be translated into unstable proteins. This is a way of regulating gene expression at the post-transcriptional or –translational level. Other transcripts will be translated into functional proteins that may be structurally and functionally different. Hence, alternative splicing creates additional complexity in the transcriptome, providing plants with molecular tools to respond to their environment, including the translation of ambient temperature alterations into a flowering time response.
In Chapter 2, we reviewed the current knowledge on molecular mechanisms that control the ambient-temperature directed flowering time pathway in the plant model species Arabidopsis thaliana. Several different mechanisms have been proposed, like alternative splicing of FLOWERING LOCUS M (FLM) (described in Chapter 4) and protein degradation of SHORT VEGETATIVE PHASE (SVP), two mechanisms that probably work in a cooperative manner to release floral repression at higher ambient temperatures. Another mechanism that is involved at high ambient temperature is the replacement of the canonical histone H2A by the variant H2A.Z. As a consequence of this replacement, chromatin becomes less tightly wrapped around the nucleosomes, which allows transcription of flowering time activators, such as PHYTOCHROME INTERACTING FACTOR 4 (PIF4). Lastly, we discuss microRNAs (miRNA) that can either repress or activate flowering (miR156 and miR172, respectively). These miRNAs have been proposed to be regulated by low and high ambient temperature. However, due to the lack of mutant analyses, more research is necessary to show the true involvement of these factors. Altogether, there are several mechanisms acting partly in cooperation to regulate thermosensitive floral timing.
In Chapter 3, we analysed ambient temperature-directed alternative splicing events that occur after a temperature shift by RNAseq. We performed the experiment in two different accessions of A. thaliana, and in one variant of B. oleracea (cauliflower). We showed that flowering time genes are overrepresented amongst the ambient temperature induced alternatively spliced genes, but also genes encoding components of the splicing machinery itself, indicating that alternative splicing is one of the potential mechanisms by which plants are able to sense temperature and adapt floral timing. Analysis of a mutant for one of these alternatively-spliced splicing related factors, ATU2AF65A, showed a temperature-dependent flowering time phenotype, confirming its proposed role in the flowering time response upon temperature fluctuations. Based on these findings, we proposed a two-step model in which splicing related genes are targeted for differential splicing upon ambient temperature fluctuations, which results in changes in the composition of the spliceosome, causing differential splicing of downstream genes that affect the development and architecture of the plant, including flowering time.
In Chapter 4, we investigated the molecular mode-of-action of FLM, one of the differentially spliced flowering time regulating genes that we identified in Chapter 3. We showed that in A. thaliana Col-0, the main splice forms of FLM are FLMβ and FLMδ. FLMβ forms an obligate heterodimer with SVP, and this complex represses floral integrators like SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1) by binding to the regulatory regions of these genes. FLMδ also dimerizes with SVP, but this complex is not able to bind to DNA. When temperature rises, more FLMδ is produced at the cost of FLMβ. Hence, less repressive complexes can be formed. However, the fact that FLMδ is still able to binds SVP makes it function as a dominant negative form, titrating out SVP and preventing repressive SVP/FLMβ-complex formation.
Chapter 5 is a short comment written to clarify the concept of thermoplasticity in flowering time control. Occasionally, this concept is confused with adaptation to different ambient temperature environments on the long term. Thermoplasticity is the ability to adapt flowering time to fluctuations in ambient temperature within one life cycle. Furthermore, some genes have been marked as players in the ambient temperature response, whereas these appear to be general flowering repressors or activators, affecting flowering time in a similar manner at low and high ambient temperature. In order to interpret novel findings on thermosensitive flowering time control, it is essential to distinguish between these various concepts.
In chapter 6, we unveiled the first indications that differential splicing of FLM can be caused by differences in polymerase II elongation rate. We mimicked a situation in which FLM is transcribed at a higher rate, by expressing the genomic FLM gene under a strong artificial promoter. Preliminary results showed that plants harbouring this construct have altered flowering time and temperature-responsiveness, which can be explained by the altered FLMβ/FLMδ ratio that we observed.
In chapter 7, we assessed the functional conservation between FLM and closely related genes at the intraspecific level in A. thaliana. FLM (also called MAF1) is a member of the FLC-clade, that consists of FLC, FLM and MAF2-5. FLC is widely known for its function in the vernalization pathway, whereas MAF2 has been shown to regulate flowering time through alternative splicing in a way very similar to FLM. For the other MAF genes, not much is known. We showed that all of these genes produce splicing isoforms that function in a more or less similar way to FLM and MAF2. Despite the high functional conservation at the intraspecific level, FLM and MAF orthologues are not widely present. Through synteny analysis, we showed that FLM and MAF2 are very recent genes, which are only present in a small group of Brassicaceae species. MAF3-5 originated less recently, but are not present outside the Brassicaceae. For FLC, it was previously shown that it originated from an ancestor of the seed plants, and in many plant species belonging to other families, presence of more than one FLC-like gene has been reported. This raises the question what the function of these genes is. In tomato, we showed that the FLC-like gene MBP8 becomes differentially spliced upon temperature changes, suggesting a function in the ambient temperature pathway. A binding assay showed high similarities of the different MBP8 isoforms to FLM and MAF isoforms, but suggests a slightly different functionality, since all three isoforms showed binding to the DNA. Further research is necessary to confirm the role of MBP8 in thermosensitive flowering time control, and elucidate the functionality of the different splice forms.
In Chapter 8, I discussed the finding of this thesis in a broader perspective, and make suggestions for future research. Over the last few years, several mechanisms that act in the temperature-directed floral pathway have been revealed. In this thesis, we showed that alternative splicing plays an important role, and we demonstrated how temperature may affect the splicing outcome directly through the effect of temperature on transcription elongation rate. It is becoming clear that most likely a single thermosensor does not exist in plants, and a model in which temperature is sensed through thermodynamic properties of DNA, RNA and proteins, is gaining support. Future research is assigned to the exiting task to elucidate the exact mechanisms by which temperature-sensing is achieved in different plant species and to determine how conserved the currently identified molecular mechanisms are.

Adventitious root formation in Arabidopsis : underlying mechanisms and applications
Massoumi, Mehdi - \ 2016
University. Promotor(en): Richard Visser, co-promotor(en): Geert-Jan de Klerk; Frans Krens. - Wageningen : Wageningen University - ISBN 9789462578524 - 191
arabidopsis thaliana - adventitious roots - formation - plant development - quantitative traits - etiolation - auxins - explants - molecular biology - gene expression - dna methylation - rooting - ontogeny - plant breeding - adventiefwortels - formatie - plantenontwikkeling - kwantitatieve kenmerken - etiolering - auxinen - explantaten - moleculaire biologie - genexpressie - dna-methylering - beworteling - ontogenie - plantenveredeling

Adventitious root (AR) formation is indispensable in vegetative propagation and is widely used. A better understanding of the underlying mechanisms is needed to improve rooting treatments. We first established a system to study rooting in Arabidopsis, the model organism in plant biology but only occasionally used to study adventitious rooting. Inhibition of polar auxin transport reduced AR formation. The role of auxin transporter proteins (several PIN-proteins) was found to be tissue-specific. Maturation (the transition from juvenile to adult) negatively influenced AR formation. Maturation was associated with increased DNA methylation and decreased miR156 level. 5-Azacytidine, a drug that reduces DNA methylation, increased rooting. We also examined the effect of two donor plant pre-treatments, etiolation and flooding, on rooting. Both increased AR formation.

Applied and fundamental aspects of BABY BOOM-mediated regeneration
Heidmann, I.A. - \ 2015
University. Promotor(en): Gerco Angenent, co-promotor(en): Kim Boutilier. - Wageningen : Wageningen University - ISBN 9789462574663 - 180
nicotiana tabacum - capsicum annuum - verjonging - transcriptiefactoren - somatische embryogenese - auxinen - moleculaire biologie - regeneration - transcription factors - somatic embryogenesis - auxins - molecular biology

Keywords: Somatic embryogenesis, Transcription factor, AINTEGUMENTA-LIKE, BABY BOOM, BBM, Sweet Pepper Transformation

Title: Applied and Fundamental Aspects of BBM-mediated Regeneration

Author: Iris Heidmann

Catergories: Plant regeneration, Plant transformation, transcription factor, somatic embryogenesis

Plant regeneration from tissues or single cells is essential for plant propagation. Efficient regeneration can be archieved through somatic embryogenesis using the plant growth regulator auxin or overexpression of specific transcription factors, but the underlying mechanisms are poorly understood. The potency of the BABY BOOM (BBM) AINTEGUMENTA-LIKE transcription factor to induce somatic embryogenesis in crop (sweet pepper) and model species (tobacco) was investigated. It was found that the introduction of BBM into sweet pepper, which is recalcitrant for transformation, enhanced the regeneration of transgenic plants. Exogenous cytokinin was necessary to induce somatic embryogenesis in both tobacco and sweet pepper. The mechanism underlying BBM-mediated somatic embryogenesis was studied in Arabidopsis by identifying BBM target genes (ChIPSeq). Genes controlling zygotic embryo identity and maturation (LAFL), as well as auxin biosynthesis (TAA1, YUCCA) and transport (PIN) are BBM targets. Mutant analysis and chemical inhibition studies showed that these genes play positive roles in BBM-induced somatic embryogenesis.

Biochemical, physiological and molecular responses of Ricinus communis seeds and seedlings to different temperatures: a multi-omics approach
Ribeiro de Jesus, P.R. - \ 2015
University. Promotor(en): Harro Bouwmeester, co-promotor(en): Henk Hilhorst; Wilco Ligterink. - Wageningen : Wageningen University - ISBN 9789462574700 - 203
ricinus communis - zaden - zaailingen - plantenfysiologie - temperatuur - moleculaire biologie - genexpressie - zaadkieming - zaadopkomst - seeds - seedlings - plant physiology - temperature - molecular biology - gene expression - seed germination - seedling emergence

Biochemical, physiological and molecular responses of Ricinus communis seeds and seedlings to different temperatures: a multi-omics approach

by Paulo Roberto Ribeiro de Jesus

The main objective of this thesis was to provide a detailed analysis of physiological, biochemical and molecular-genetic responses of Ricinus communis to temperature during seed germination and seedling establishment.

In Chapter 2, I describe the assessment of 17 candidate reference genes across a diverse set of samples, including several tissues, various developmental stages and environmental conditions, encompassing seed germination and seedling growth in R. communis. These genes were tested by RT-qPCR and ranked according to the stability of their expression using two different approaches: GeNorm and NormFinder. Both GeNorm and Normfinder indicated that ACT, POB and PP2AA1 represent the optimal combination for normalization of gene expression data in inter-tissue studies. I also describe the optimal combination of reference genes for a subset of samples from root, endosperm and cotyledonary tissues. The selection of reference genes was validated by normalizing the expression levels of three target genes involved in energy metabolism with the identified optimal reference genes. This approach allowed me to identify stably expressed genes, and, thus, reference genes for use in RT-qPCR studies in seeds and seedlings of R. communis.

In Chapter 3, a thermo-sensitive window is identified during seed germination in which high temperatures compromise subsequent seedling development. I assessed the biochemical and molecular requirements of R. communis germination for successful seedling establishment at varying temperatures. For that, I performed metabolite profiling (GC-TOF-MS) and measured transcript levels of key genes involved in several energy-generating pathways such as storage oil mobilization, β-oxidation of fatty acids and gluconeogenesis of seeds germinated at three different temperatures. Transient overexpression of genes encoding for malate synthase (MLS) and glycerol kinase (GK) resulted in higher starch levels in N. benthamiana leaves, which highlights the likely importance of these genes in energy-generating pathways for seedling establishment. Additionally, I showed that γ-aminobutyric acid (GABA), which is a stress-responsive metabolite, accumulated in response to the water content of the seeds during the initial phase of imbibition.

In Chapter 4 I undertook a genomics approach using microarray analysis to determine transcriptome changes in three distinct developmental stages during seed germination at 20, 25 and 35ºC that could explain the thermo-sensitive window that is described in Chapter 3. Most of the differences in the R. communis transcriptome occurred between 6 hours of imbibition and the commencement of germination, i.e. radicle protrusion. This coincides with the thermo-sensitive window identified during seed germination in which high temperatures compromise seedling development. The transcriptome data was used to identify heat-stress responsive genes that might be involved in thermotolerance of R. communis during germination. Temperature had a major effect on genes involved in energy generating pathways, such as the Calvin-Benson-Bassham cycle, gluconeogenesis, and starch- and triacylglycerol degradation. Transcripts coding for ATP binding proteins, DNA binding proteins, RNA binding proteins, DNA-directed RNA polymerases I, II, and III, heat shock factor proteins, multiprotein-bridging factor proteins, and zinc finger proteins were also affected by temperature suggesting the whole transcriptional regulatory machinery was altered in response to temperature. Among the downregulated transcripts under high temperature, only three were shared by all three stages: an oxidation-related zinc finger 2, an F-box and wd40 domain protein, and a DNA binding protein/MYB-like transcription factor. Among the upregulated transcripts, nine were shared by all three stages: a BET1P/SFT1P-like protein, 14BB, a low-molecular-weight cysteine-rich protein LCR78, a WD-repeat protein, a GAST1 protein, an adenylate kinase 1/P-loop containing nucleoside triphosphate hydrolases superfamily protein, and four conserved hypothetical proteins. These genes constitute good candidates for further characterization of temperature-responsive genes in R. communis.

In Chapter 5, I studied the genetic variation in the effect of temperature on growth of young R. communis seedlings and measured primary and secondary metabolites in roots and cotyledons of three R. communis genotypes, varying in stress tolerance. Seedling biomass was strongly affected by the temperature, with the lowest total biomass observed at 20ºC. The response in terms of biomass production for the genotype MPA11 was clearly different from the other two studied genotypes: genotype MPA11 produced heavier seedlings at all temperatures but the root biomass of this genotype decreased with increasing temperature, reaching the lowest value at 35ºC. In contrast, root biomass of genotypes MPB01 and IAC80 was not affected by temperature, suggesting that the roots of these genotypes are less sensitive to changes in temperature. A shift in carbon-nitrogen metabolism towards the accumulation of nitrogen-containing compounds seems to be the main biochemical response to support growth at higher temperatures. Carbohydrate content was reduced in response to increasing temperature in both roots and cotyledons, whereas amino acids accumulated to higher levels. The results in this chapter show that a specific balance between amino acids, carbohydrates and organic acids in the cotyledons and roots of genotype MPA11 seems to be an important trait for faster and more efficient growth of this genotype at higher temperatures.

In Chapter 6, I decided to focus on the differential ability of genotypes MPA11 and IAC80 to sustain root biomass production at higher temperatures. Biomass allocation was assessed by measuring dry weight of roots, stems, and cotyledons of seedlings grown at three different temperatures. Seedlings grown at 25ºC and 35ºC showed greater biomass than seedlings grown at 20ºC. Cotyledon and stem dry weight increased for both genotypes with increasing temperature, whereas root biomass allocation showed a genotype-dependent behaviour. Genotype MPA11 showed a continuous increase in root dry weight with increasing temperature, while genotype IAC80 was not able to sustain further root growth at higher temperatures. Metabolite and gene expression profiles of genotype MPA11 demonstrated an increase in the levels of osmoprotectant molecules, such as galactinol and transcripts of genes encoding antioxidant enzymes and heat shock proteins, to a higher extent than in genotype IAC80.

In Chapter 7 I raised the question whether carbohydrate accumulation in R. communis leaves, roots, and seeds, grown at low temperatures, as compared to higher temperatures, results from up-regulation of biosynthetic pathways, from down-regulation of catabolic pathways, or both. To answer this question, transcript levels were measured of genes encoding enzymes involved in starch biosynthesis, starch catabolism, and gluconeogenesis in leaves, roots, and seeds grown at 20ºC and 35ºC. Transcript levels of genes involved in starch catabolism were higher in leaves grown at 20ºC than at 35ºC, but up-regulation of genes involved in starch biosynthesis seems to compensate for this and, therefore, is the likely explanation for higher levels of starch in leaves grown at 20ºC. Higher levels of soluble carbohydrates in leaves grown at 20ºC may have been caused by a coordinated up-regulation of starch catabolism and gluconeogenesis pathways. In roots, starch catabolism and gluconeogenesis seem to be enhanced at elevated temperatures. Higher levels of starch in seeds germinated at low temperatures is associated with higher transcript levels of genes involved in starch biosynthesis. Similarly, higher transcript levels of RcPEPCK and RcFBPase are most likely causal for fructose and glucose accumulation in seeds germinated at 20ºC.

This thesis provides important insights in the understanding of the plasticity of R. communis in response to temperature. The knowledge obtained may apply to other species as well. Additionally, based on the transcriptomics data, we selected several candidate genes that are potentially involved in, or required for, proper seed germination and seedling establishment under different temperatures, such as a number of transcription factors, a zinc finger protein, heat-shock proteins, malate synthase and glycerol kinase. Overexpressing Arabidopsis lines transformed with these R. communis genes, as well as Arabidopsis T-DNA lines, in which Arabidopsis homologs of these genes are knocked-out, are being generated for further phenotypical analysis. These overexpression and T-DNA lines should help us to understand the molecular requirements for vigorous seedling growth of R. communis under different environmental conditions.

This work was performed at the Laboratory of Plant Physiology, Wageningen University. This research was financially supported by the Brazilian Government through the National Counsel of Technological and Scientific Development (CNPq grant number 200745/2011-5).

Synthetische biologie komende jaren in het vizier
Sikkema, A. ; Martins dos Santos, V.A.P. - \ 2015
Resource: weekblad voor Wageningen UR 9 (2015)20. - ISSN 1874-3625 - p. 12 - 13.
genetische modificatie - moleculaire biologie - bacteriën - industriële microbiologie - systeembiologie - biobased economy - genetic engineering - molecular biology - bacteria - industrial microbiology - systems biology
Wageningen UR gaat de komende jaren investeren in synthetische biologie. Het is benoemd als een van de investeringsthema’s in het strategisch plan. Een jong vakgebied, waarbij onderzoekers bacteriën verbouwen en ontwerpen voor industriële toepassingen. Hoogleraar Martins dos Santos, die onlangs twee grote EU-projecten binnenhaalde, gaat een actieplan schrijven.
Root and nodule : lateral organ development in N2-fixing plants
Xiao, T.T. - \ 2015
University. Promotor(en): Ton Bisseling, co-promotor(en): Rene Geurts; Henk Franssen. - Wageningen : Wageningen University - ISBN 9789462572768 - 198
medicago - wortelknolletjes - endosymbiose - symbiose - mycorrhizae - stikstoffixatie - plantenontwikkeling - moleculaire biologie - root nodules - endosymbiosis - symbiosis - mycorrhizas - nitrogen fixation - plant development - molecular biology

Plants are sessile organisms. This characteristic severely limits their ability of approaching nutrients. To cope with this issue, plants evolved endosymbiotic relationships with soil fungi to extend their interface with surrounding environment. In case of arbuscular mycorrhizae (AM) fungi this occurred about 400 million years ago. The AM fungi can interact with most angiosperms. In this symbiotic relationship, the plant get nutrients, especially phosphate, from the fungi, and plants provide carbohydrates to the fungi in return. About 60 million years ago, a group of plants evolved N2-fixing nodule symbiosis. This includes interactions of legumes plants with rhizobium bacteria and actinorhizal plants with Frankia bacteria. Currently, all plant species that are able to establish a nodule symbiosis belong to the Rosid I clade. In the nodule symbioses the bacteria produce ammonia and the plant provides carbohydrates to the bacteria.

In the root nodule symbiosis, the nitrogen fixing bacteria are hosted in the cell of the root nodule. Although the function and structure of the root nodule are different from the other plant organs, it does share some features with other organs, especially the lateral root. To get further insight into the similarities and differences between root nodule and lateral root, I made use of the model legume (Medicago truncatula) and the non-legume Parasponia (Parasponia andersonii) that is the only genus outside the legumes that forms nodules with rhizobium.

In Chapter 1, I will give a general introduction on the process of root nodule formation in legume plants. I will mainly focus on nodule organogenesis and the plant hormones that are known to be important for this process. Root nodules are supposed to have a close relationship with lateral roots. Therefore a comparison between lateral root and root nodule development will be included in this introduction.

Lateral root development has especially been studied in in Arabidopsis. To be able to compare the root and root nodule developmental process, especially at the early stages, a Medicago lateral root development fate map has been made. This will be described in Chapter 2 and showed that in addition to the pericycle, endodermis and cortex are also mitotically activated during lateral root formation. Pericycle derived cells only form part of the stem cell niche as endodermis derived cells also contribute to this.

In Chapter 3, a Medicago root nodule fate map is presented. In this Chapter, the contribution of different root cell layers to the mature nodule will be described. A set of molecular markers for root tissue, cell cycle and rhizobial infection have been used to facilitate this analysis. The fate map showed that nodule meristem originates from the third cortical layer and many cell layers of the base of the nodule are directly derived from cells of the inner cortical layers, root endodermis and pericycle. The inner cortical cell layers form about 8 cell layers of infected cells while the root endodermis and pericycle derived cells forms the uninfected tissues that are located at the base of the mature nodule. Nodule vascular is formed from the part of the primordium derived from the cortex. The development of primordia was divided in 6 stages. To illustrate the value of this fate map, a few published mutant nodule phenotypes are re-analyzed.

In Chapter 4, the role of auxin at early stages of Medicago nodule formation is studied. In this chapter auxin accumulation is studied during the 6 stages of primordium development. It is studied by using DR5::GUS as an auxin reporter. Auxin accumulation associates with mitotic activity within the primordium. Previously, it has been postulated by theoretical modelling that the accumulation of auxin during nodulation is induced by a local reduction of PIN (auxin efflux carriers) levels. We tested this theory, but this was hampered due to the low level of PIN proteins in the susceptible zone of the root. It is still possible that auxin accumulation is initiated by a decrease of PIN levels. However, the level of 2 PIN already increase before the first divisions are induced. In young primordia they accumulate in all cells. At later stages PINs mainly accumulate at the nodule periphery and the future nodule meristem. The subcellular position of PINs strongly indicates they play a key role in the accumulation of auxin in primordia.

Previous studies showed that a group of root apical meristem regulators is expressed in the nodule meristem. In Chapter 5, we tested whether the Medicago nodule meristem expresses PLETHORA genes that are expressed in the root meristem. These PLETHORAs were functionally analysed, by using RNAi approach using a nodule specific promoter. Knockdown of PLETHORAs expression hampers primordium formation and meristem growth. Hence, we conclude rhizobium recruited key regulators of root development for nodule development.

In Chapter 6, we first introduced the non-legume lateral root and nodule fate maps by using Parasponia. In Parasponia nodules the nodule central vascular bundle is completely derived from the pericycle similar as its lateral roots. The nodule infected cells were shown to be derived from cortex. Together with the data obtained in this thesis, this Chapter further discussed several developmental aspects of the different lateral root organs. Especially, it focused on the vasculature and meristem formation of legume and non-legume nodules.

Dick de Ridder over de bioloog die steeds meer een datawetenschapper wordt
Ridder, D. de - \ 2015
Wageningen UR
bio-informatica - gegevensverwerking - informatica - beroepen - moleculaire biologie - bioinformatics - data processing - informatics - occupations - molecular biology
De biologie krijgt snel het karakter van een datawetenschap. Miljarden gegevens over genomen, genen, eiwitten en andere moleculen worden in grote bestanden bij elkaar gebracht en systematisch onderzocht. Dit moet leiden tot meer basiskennis en begrip van levende organismen waarvan gewassen en vee aan de basis staan van de voedselvoorziening van de wereldbevolking. Dat zegt Dick de Ridder in zijn inaugurele rede bij de aanvaarding van het ambt van hoogleraar Bioinformatica aan Wageningen University op 30 april.
MADS dynamics : gene regulation in flower development by changes in chromatin structure and MADS-domain protein binding
Pajoro, A. - \ 2015
University. Promotor(en): Gerco Angenent, co-promotor(en): Kerstin Kaufmann. - Wageningen : Wageningen University - ISBN 9789462572669 - 219
bloemen - plantenontwikkeling - genregulatie - chromatine - apicale meristemen - arabidopsis - moleculaire biologie - flowers - plant development - gene regulation - chromatin - apical meristems - molecular biology


During the life cycle, a plant undergoes a series of developmental phase changes. The first phase change is the transition from the initial juvenile vegetative stage into the adult vegetative phase. During the juvenile phase plants produce leaves and axillary buds, whereas during the adult phase the initiation of reproductive structures occurs. The next developmental change is the switch from vegetative to reproductive growth, when the shoot apical meristem acquires the identity of an inflorescence meristem that will then produce floral meristems. Arabidopsis floral meristems produce four concentric whorls of floral organs: sepals, petals, stamens and carpels. Each developmental change is controlled by coordinated network of regulators, known as gene regulatory networks (GRNs), which determine the transcription of a specific set of genes. The aim of the study presented in this thesis was to understand the dynamics of GRNs during floral organ development in Arabidopsis and correlate the binding of key regulatory MADS domain transcription factors with the accessibility of the chromatin in a genome-wide context.

In chapter 1 and 2 we reviewed the current knowledge on the regulation of transcription in the model plant Arabidopsis thaliana. In chapter 1 we mainly focus on how the view of the GRN underling flower development has changed during the last decades, while in chapter 2 we more broadly revised the mechanisms that control developmental switches in plants. The recent introduction of next-generation sequencing and genome-wide approaches has changed our view on gene regulation and GRNs. We moved from linear genetic interactions towards global highly connected gene networks. The high numbers of interactions that were detected in protein-DNA binding profiles revealed a much higher network complexity than previously anticipated and demonstrated that master regulators of development not only control another layer of regulators, but also genes encoding structural proteins, enzymes and signalling proteins. Moreover, most transcription factors bind to their own locus, highlighting that auto-regulatory loops are a common mechanism of regulation.

The discovery of interactions between transcriptional master regulators with epigenetic factors provides new insights into general transcriptional regulatory mechanisms. Switches of developmental programmes and cell fates in complex organisms are controlled at the level of gene expression by the combined action of chromatin regulators and transcription factors.

Although many master regulators of meristem and organ identities have been identified, it is still not well understood how they act at the molecular level and how they can switch an entire developmental program in which thousands of genes are involved. Using flower development as a model system, in chapters 3 and 4 we investigated general concepts of transcription regulation by analysing the dynamics of protein-DNA binding, chromatin accessibility and gene expression.

Using an inducible system for synchronised flower formation, we characterised DNA-binding profiles of two MADS-domain transcription factors, APETALA1 (AP1) and SEPALLATA3 (SEP3), at three stages of flower development. Our study revealed that these MADS-domain proteins, select their binding sites, and thereby their target genes, in a partly stage-specific fashion. By combining the information from DNA-binding and gene expression data, we proposed models of stage-specific GRNs in flower development. Since developmental control of gene expression is tightly linked with dynamic changes in chromatin accessibility, we identified DNase I hypersensitive sites (DHSs, chapter 3) and we characterised nucleosome occupancy (chapter 4) at different stages of flower development. We observed dynamics in chromatin landscape manifested in increasing and decreasing DHSs as well as in changes in nucleosome occupancy and position.

Next, we addressed the question how MADS-domain protein stage-specific binding is achieved at the molecular level in a chromatin context. In the nucleus the DNA is wrapped around histone octamers to form nucleosomes, which are then packed into highly dense structures, and hence transcription factor binding sites may not be easily accessible. A result of the combined analysis of MADS-domain binding and chromatin dynamics is that MADS-domain proteins bind prevalently to nucleosome depleted regions, and that binding of AP1 and SEP3 to DNA precedes opening of the chromatin, which suggests that these MADS-domain transcription factors may act as so-called “pioneer factors”.

The isolation and analysis of developing flowers of specific stages increased the specificity of our genome-wide experiments, enabling the identification of novel actors in the GRN that regulates flower development. In this thesis we characterised the role of some novel regulators in more detail: in chapter 3 we focussed on the GROWTH REGULATING FACTOR (GRF) family genes; in chapter 5 we investigated the action of STERILE APETALA (SAP); and in chapter 6 we elucidated the regulation and the role of a member of the WUSCHEL-related homeobox (WOX) family, WOX12. GRF family genes are dynamically bound by AP1 and SEP3 at the different stages of flower development. All family members are bound by SEP3, while only a subset of the genes is bound by AP1. The defects in floral organs observed upon down-regulation of these genes highlight their role down-stream of MADS-domain transcription factors. In addition to AP1 and SEP3, SAP is also a target of other MADS-domain proteins, such as APETALA3 (AP3), PISTILLATA (PI), and AGAMOUS (AG). SAP is strongly expressed in meristems and loss of function of SAP causes strong aberrations in flowers, such as a reduction in petal and stamen numbers. We found that SAP interacts with proteins of the SCF ubiquitin ligase complex, suggesting that SAP could act in the ubiquitination pathway.

WOX12 down-regulation leads to defects in floral organ identity specification with the formation of stamenoid-petals, while ectopic expression of WOX12 leads to an opposite effect: it causes the formation of petaloid-stamens in the third whorl. WOX12 acts downstream of AP1. Ectopic expression of WOX12 leads to reduction of AG expression, suggesting a role for WOX12 in the antagonistic interplay between the homeotic genes AP1 and AG.

In chapter 7 we discuss the findings of this thesis. Taken together, the work performed in this thesis increased our knowledge on the GRN that regulates flower development and on the mode of action of MADS-domain transcription factors. We hypothesise that MADS-domain proteins may act as pioneer factors, proteins that access and remodel condensed chromatin. However, differently from other pioneer factors, MADS-domain transcription factors do not actively deplete nucleosomes, but instead they interact with chromatin remodelers to shape chromatin landscape. Given the important roles of MADS-domain proteins as master regulators of developmental switches, their pioneer behaviour represents an intriguing mode of action.

Systems biology of plant molecular networks: from networks to models
Valentim, F.L. - \ 2015
University. Promotor(en): Gerco Angenent, co-promotor(en): Aalt-Jan van Dijk. - Wageningen : Wageningen University - ISBN 9789462572171 - 139
systeembiologie - netwerken - modellen - genetische regulatie - genexpressie - planten - moleculaire biologie - systems biology - networks - models - genetic regulation - gene expression - plants - molecular biology

Developmental processes are controlled by regulatory networks (GRNs), which are tightly

coordinated networks of transcription factors (TFs) that activate and repress gene expression

within a spatial and temporal context. In Arabidopsis thaliana, the key components and network

structures of the GRNs controlling major plant reproduction processes, such as floral transition

and floral organ identity specification, have been comprehensively unveiled. This thanks to

advances in ‘omics’ technologies combined with genetic approaches. Yet, because of the

multidimensional nature of the data and because of the complexity of the regulatory

mechanisms, there is a clear need to analyse these data in such a way that we can understand

how TFs control complex traits. The use of mathematical modelling facilitates the

representation of the dynamics of a GRN and enables better insight into GRN complexity; while

multidimensional data analysis enables the identification of properties that connect different

layers from genotype-to-phenotype. Mathematical modelling and multidimensional data

analysis are both parts of a systems biology approach, and this thesis presents the application of

both types of systems biology approaches to flowering GRNs.

Chapter 1 comprehensively reviews advances in understanding of GRNs underlying plant

reproduction processes, as well as mathematical models and multidimensional data analysis

approaches to study plant systems biology. As discussed in Chapter 1, an important aspect of

understanding these GRNs is how perturbations in one part of the network are transmitted to

other parts, and ultimately how this results in changes in phenotype. Given the complexity of

recent versions of Arabidopsis GRNs - which involves highly-connected, non-linear networks

of TFs, microRNAs, movable factors, hormones and chromatin modifying proteins - it is not

possible to predict the effect of gene perturbations on e.g. flowering time in an intuitive way by

just looking at the network structure. Therefore, mathematical modelling plays an important role

in providing a quantitative understanding of GRNs. In addition, aspects of multidimensional

data analysis for understanding GRNs underlying plant reproduction are also discussed in the

first Chapter. This includes not only the integration of experimental data, e.g. transcriptomics

with protein-DNA binding profiling, but also the integration of different types of networks

identified by ‘omics’ approaches, e.g. protein-protein interaction networks and gene regulatory


Chapter 2 describes a mathematical model for representing the dynamics of key genes in the

GRN of flowering time control. We modelled with ordinary differential equations (ODEs) the

physical interactions and regulatory relationships of a set of core genes controlling Arabidopsis

flowering time in order to quantitatively analyse the relationship between their expression levels

and the flowering time response. We considered a core GRN composed of eight TFs: SHORT



FLOWERING LOCUS T (FT), LEAFY (LFY) and FD. The connections and interactions

amongst these components are justified based on experimental data, and the model is

parameterised by fitting the equations to quantitative data on gene expression and flowering

time. Then the model is validated with transcript data from a range of mutants. We verify that

the model is able to describe some quantitative patterns seen in expression data under genetic

perturbations, which supported the credibility of the model and its dynamic properties. The

proposed model is able to predict the flowering time by assessing changes in the expression of

the orchestrator of floral transition AP1. Overall, the work presents a framework, which allows

addressing how different quantitative inputs are combined into a single quantitative output, i.e.

the timing of flowering. The model allowed studying the established genetic regulations, and we

discuss in Chapter 5 the steps towards using the proposed framework to zoom in and obtain new

insides about the molecular mechanisms underlying the regulations.

Systems biology does not only involve the use of dynamic modelling but also the development

of approaches for multidimensional data analysis that are able to integrate multiple levels of

systems organization. In Chapter 3, we aimed at comprehensively identifying and characterizing

cis-regulatory mutations that have an effect on the GRN of flowering time control. By using

ChIP-seq data and information about known DNA binding motifs of TFs involved in plant

reproduction, we identified single-nucleotide polymorphisms (SNPs) that are highly

discriminative in the classification of the flowering time phenotypes. Often, SNPs that overlap

the position of experimentally determined binding sites (e.g. by ChIP-seq), are considered

putative regulatory SNPs. We showed that regulatory SNPs are difficult to pinpoint among the

sea of polymorphisms localized within binding sites determined by ChIP-seq studies. To

overcome this, we narrowed the resolution by focusing on the subset of SNPs that are located

within ChIP-seq peaks but that are also part of known regulatory motifs. These SNPs were used

as input in a classification algorithm that could predict flowering time of Arabidopsis accessions

relative to Col-0. Our strategy is able to identify SNPs that have a biological link with changes

in flowering time. We then surveyed the literature to formulate hypothesis that explain the

regulatory mechanism underlying the difference in phenotype conferred by a SNP. Examples

include SNPs that disrupt the flowering time gene FT; in which the mutation presumably disrupts the binding region of SVP. In Chapter 5 we discuss the steps towards extending our approach to obtain a more comprehensive survey of variants that have an effect on the flowering time control.

In Chapter 4, we propose a method for genome-wide prediction of protein-protein interaction

(PPI) sites form the Arabidopsis interactome. Our method, named SLIDERbio, uses features

encoded in the sequence of proteins and their interactions to predict PPI sites. More specifically,

our method mines PPI networks to find over-represented sequence motifs in pairs of interacting

proteins. In addition, the inter-species conservation of these over-represented motifs, as well as

their predicted surface accessibility, are take into account to compute the likelihood of these

motifs being located in a PPI site. Our results suggested that motifs overrepresented in pairs of

interacting proteins that are conserved across orthologs and that have high predicted surface

accessibility, are in general good putative interaction sites. We applied our method to obtain

interactome-wide predictions for Arabidopsis proteins. The results were explored to formulate

testable hypothesis for the molecular mechanisms underlying effects of spontaneous or induced

mutagenesis on e.g. ZEITLUPE, CXIP1 and SHY2 (proteins relevant for flowering time). In

addition, we showed that the binding sites are under stronger selective pressure than the overall

protein sequence, and that this may be used to link sequence variability to functional


Finally, Chapter 5 concludes this thesis and describes future perspectives in systems biology

applied to the study of GRNs underlying plant reproduction processes. Two key directions are

often followed in systems biology: 1) compiling systems-wide snapshots in which the

relationships and interactions between the molecules of a system are comprehensively

represented; and 2) generating accurate experimental data that can be used as input for the

modelling concepts and techniques or multi-dimensional data analysis. Highlighted in Chapter 5

are the limitations in key steps within the systems biology framework applied to GRN studies.

In addition, I discussed improvements and extensions that we envision for our model related to

the GRN underlying the control of flowering time. Future steps for multi-dimensional data

analysis are also discussed. To sum up, I discussed how to connect the different technologies

developed in this thesis towards understanding the interplay between the roles of the genes,

developmental stages and environmental conditions.

Molecular and genetic basis of freezing tolerance in crucifer species
Heo, J. - \ 2014
University. Promotor(en): Eric Schranz, co-promotor(en): P.H. van Tienderen; C.S. Testerink. - Wageningen : Wageningen University - ISBN 9789461738691 - 129
brassicaceae - invriezen - koudetolerantie - moleculaire biologie - plantengenetica - loci voor kwantitatief kenmerk - genen - kouderesistentie - freezing - cold tolerance - molecular biology - plant genetics - quantitative trait loci - genes - cold resistance

Understanding genetic variation for freezing tolerance is important for unraveling an adaptative strategy of species and for finding out an effective way to improve crop productivity to unfavorable winter environments. The aim of this thesis was to examine natural variation for components of freezing tolerance beyond what has been done using the model organism Arabidopsis thaliana. Experiments using B. stricta were carried out to identify potentially novel and beneficial traits, and loci related to cold acclimation, or using a number of related Brassicaceae species were performed to understand mechanism for cold deacclimation. Our results strongly indicate differential regulatory mechanisms are involved in cold acclimation as well as cold deacclimation.Although we are still far from understanding those mechanisms, we have shown that exploiting natural variation using wild species provides new perspectives on ecologically important adaptation to cold, and may contribute to improve tolerance in crucifer species.

Text mining for metabolic reaction extraction from scientific literature
Risse, J.E. - \ 2014
University. Promotor(en): Ton Bisseling; Jack Leunissen, co-promotor(en): P.E. van der Vet. - Wageningen : Wageningen University - ISBN 9789461739001 - 138
metabolomica - gegevensanalyse - databanken - text mining - publicaties - wetenschappelijk onderzoek - moleculaire biologie - thesauri - enzymen - metabolieten - metabolomics - data analysis - databases - publications - scientific research - molecular biology - enzymes - metabolites

Science relies on data in all its different forms. In molecular biology and bioinformatics in particular large scale data generation has taken centre stage in the form of high-throughput experiments. In line with this exponential increase of experimental data has been the near exponential growth of scientific publications. Yet where classical data mining techniques are still capable of coping with this deluge in structured data (Chapter 2), access of information found in scientific literature is still limited to search engines allowing searches on the level keywords, titles and abstracts. However, large amounts of knowledge about biological entities and their relations are held within the body of articles. When extracted, this data can be used as evidence for existing knowledge or hypothesis generation making scientific literature a valuable scientific resource. To unlock the information inside the articles requires a dedicated set of techniques and approaches tailored to the unstructured nature of free text. Analogous to the field of data mining for the analysis of structured data, the field of text mining has emerged for unstructured text and a number of applications has been developed in that field.

This thesis is about text mining in the field of metabolomics. The work focusses on strategies for accessing large collections of scientific text and on the text mining steps required to extract metabolic reactions and their constituents, enzymes and metabolites, from scientific text. Metabolic reactions are important for our understanding of metabolic processes within cells and that information provides an important link between genotype phenotype. Furthermore information about metabolic reactions stored in databases is far from complete making it an excellent target for our text mining application.

In order to access the scientific publications for further analysis they can be used as flat text or loaded into database systems. In Chapter 2we assessed and discussed the capabilities and performance of XML-type database systems to store and access very large collections of XML-type documents in the form of the Medline corpus, a collection of more than 20 million of scientific abstracts. XML data formats are common in the field of bioinformatics and are also at the core of most web services. With the increasing amount of data stored in XML comes the need for storing and accessing the data. The database systems were evaluated on a number of aspects broadly ranging from technical requirements to ease-of-use and performance. The performance of the different XML-type database systems was measured Medline abstract collections of increasing size and with a number of different queries. One of the queries assessed the capabilities of each database system to search the full-text of each abstract, which would allow access to the information within the text without further text analysis. The results show that all database systems cope well with the small and medium dataset, but that the full dataset remains a challenge. Also the query possibilities varied greatly across all studied databases. This led us to conclude that the performances and possibilities of the different database types vary greatly, also depending on the type of research question. There is no single system that outperforms the others; instead different circumstances can lead to a different optimal solution. Some of these scenarios are presented in the chapter.

Among the conclusions of Chapter 2is that conventional data mining techniques do not work for the natural language part of a publication beyond simple retrieval queries based on pattern matching. The natural language used in written text is too unstructured for that purpose and requires dedicated text mining approaches, the main research topic of this thesis. Two major tasks of text mining are named entity recognition, the identification of relevant entities in the text, and relation extraction, the identification of relations between those named entities. For both text mining tasks many different techniques and approaches have been developed. For the named entity recognition of enzymes and metabolites we used a dictionary-based approach (Chapter 3) and for metabolic reaction extraction a full grammar approach (Chapter 4).

In Chapter 3we describe the creation of two thesauri, one for enzymes and one for metabolites with the specific goal of allowing named entity identification, the mapping of identified synonyms to a common identifier, for metabolic reaction extraction. In the case of the enzyme thesaurus these identifiers are Enzyme Nomenclature numbers (EC number), in the case of the metabolite thesaurus KEGG metabolite identifiers. These thesauri are applied to the identification of enzymes and metabolites in the text mining approach of Chapter 4. Both were created from existing data sources by a series of automated steps followed by manual curation. Compared to a previously published chemical thesaurus, created entirely with automated steps, our much smaller metabolite thesaurus performed on the same level for F-measure with a slightly higher precision. The enzyme thesaurus produced results equal to our metabolite thesaurus. The compactness of our thesauri permits the manual curation step important in guaranteeing accuracy of the thesaurus contents, whereas creation from existing resources by automated means limits the effort required for creation. We concluded that our thesauri are compact and of high quality, and that this compactness does not greatly impact recall.

In Chapter 4we studied the applicability and performance of a full parsing approach using the two thesauri described in Chapter 3 for the extraction of metabolic reactions from scientific full-text articles. For this we developed a text mining pipeline built around a modified dependency parser from the AGFL grammar lab using a pattern-based approach to extract metabolic reactions from the parsing output. Results of a comparison to a modified rule-based approach by Czarnecki et al.using three previously described metabolic pathways from the EcoCyc database show a slightly lower recall compared to the rule-based approach, but higher precision. We concluded that despite its current recall our full parsing approach to metabolic reaction extraction has high precision and potential to be used to (re-)construct metabolic pathways in an automated setting. Future improvements to the grammar and relation extraction rules should allow reactions to be extracted with even higher specificity.

To identify potential improvements to the recall, the effect of a number of text pre-processing steps on the performance was tested in a number of experiments. The one experiment that had the most effect on performance was the conversion of schematic chemical formulas to syntactic complete sentences allowing them to be analysed by the parser. In addition to the improvements to the text mining approach described in Chapter 4I make suggestions in Chapter 5 for potential improvements and extensions to our full parsing approach for metabolic reaction extraction. Core focus here is the increase of recall by optimising each of the steps required for the final goal of extracting metabolic reactions from the text. Some of the discussed improvements are to increase the coverage of the used thesauri, possibly with specialist thesauri depending on the analysed literature. Another potential target is the grammar, where there is still room to increase parsing success by taking into account the characteristics of biomedical language. On a different level are suggestions to include some form of anaphora resolution and across sentence boundary search to increase the amount of information extracted from literature.

In the second part of Chapter 5I make suggestions as to how to maximise the information gained from the text mining results. One of the first steps should be integration with other biomedical databases to allow integration with existing knowledge about metabolic reactions and other biological entities. Another aspect is some form of ranking or weighting of the results to be able to distinguish between high quality results useful for automated analyses and lower quality results still useful for manual approaches. Furthermore I provide a perspective on the necessity of computational literature analysis in the form of text mining. The main reasoning here is that human annotators cannot keep up with the amount of publications so that some form of automated analysis is unavoidable. Lastly I discuss the role of text mining in bioinformatics and with that also the accessibility of both text mining results and the literature resources necessary to create them. An important requirement for the future of text mining is that the barriers around high-throughput access to literature for text mining applications have to be removed. With regards to accessing text mining results, there is a long way to go for many applications, including ours, before they can be used directly by biologists. A major factor is that these applications rarely feature a suitable user interface and easy to use setup.

To conclude, I see the main role of a text mining system like ours mainly in gathering evidence for existing knowledge and giving insights into the nuances of the research landscape of a given topic. When using the results of our reaction extraction system for the identification of ‘new’ reactions it is important to go back to the actual evidence presented for extra validations and to cross-validate the predictions with other resources or experiments. Ideally text mining will be used for generation of hypotheses, in which the researcher uses text mining findings to get ideas on, in our case, new connections between metabolites and enzymes; subsequently the researcher needs to go back to the original texts for further study. In this role text mining is an essential tool on the workbench of the molecular biologist.

Molecular regulation of drought tolerance in rice
Haider, I. - \ 2014
University. Promotor(en): Harro Bouwmeester, co-promotor(en): Carolien Ruyter-Spira; P.B.F. Ouwerkerk. - Wageningen : Wageningen University - ISBN 9789461738431 - 183
oryza - oryza sativa - oryza glaberrima - plantenfysiologie - droogte - droogteresistentie - stress - moleculaire biologie - plantengroeiregulatoren - plant physiology - drought - drought resistance - molecular biology - plant growth regulators

Abiotic stresses are the primary cause of crop failure worldwide, reducing average yields by more than 50%. Among the various forms of abiotic stress, drought is the most limiting factor for rice productivity. Drought affects about 20% of the total rice cultivation area in Asia. Understanding the various aspects of drought stress, the response and resistance mechanisms in relation to plant growth is therefore of fundamental importance to improve sustainable agriculture.

Drought tolerance is usually controlled by complex gene networks and engineering of a single gene is unlikely to improve this trait. However, altering the expression of Transcription Factors (TFs) may be a tool for improvement of drought tolerance since they have been shown to activate the expression of multiple genes in a coordinated manner and they are therefore attractive and promising targets for application in molecular breeding or genetic engineering. In addition, studies on TFs will improve our understanding of the physiological and molecular mechanisms of drought tolerance. The overall objective of the work presented in this thesis was to get more detailed insight in the molecular regulation of drought tolerance in rice, with a particular focus on the role of TFs of the homeobox class and two groups of plant hormones, abscisic acid and strigolactones.

In Chapter 2, I described the isolation and characterisation of the rice Oshox22 gene which is an homeobox gene of the HD-Zip I family. I showed that the expression of Oshox22 is strongly induced by salt stress, abscisic acid (ABA) and polyethylene glycol (PEG) treatment, and weakly by cold stress. Trans-activation assays in yeast and transient expression analyses in rice protoplasts demonstrated that Oshox22 is able to bind to the CAAT(G/C)ATTG element and acts as a transcriptional activator that requires both the HD and Zip domains. Rice plants homozygous for a T-DNA insertion in the promoter region of Oshox22 showedreduced Oshox22 expression and ABA content, decreased sensitivity to ABA, and enhanced tolerance to drought and salt stress in the seedling stage. In contrast, transgenic rice over-expressing Oshox22 showed increased sensitivity to ABA, increased ABA content, and decreased drought and salt tolerances. These results support the conclusion that Oshox22 acts as a negative regulator in stress response. Since reporter gene studies in yeast and rice cells suggested that Oshox22 acts as a transcriptional activator, its function as a negative regulator in stress responses might be explained via activation of other repressors.

As Oshox22 is highly expressed in developing panicles and grains, in Chapter 4 I investigated the role of Oshox22 in controlling grain length (GL) in rice. We found a stable quantitative trait locus (QTL) for GL on this position in four mapping populations. Sequence analysis of Oshox22 in rice cultivars Bala, Azucena and Nipponbare revealed an extra A base in the Azucena promoter, which is a long grain type rice. Using a PCR-based insertion/deletion (InDel) CAPS maker assay in rice populations and collections, I found an association between the A InDel in the Oshox22 promoter with GL. Furthermore, expression of Oshox22 under the control of a promoterwith the A InDelin Zhonghua 11 (which does not have the A InDel) resulted in a significant increase in GL in Zhongua 11. Scanning electron microscopy revealed that the enhanced GL was caused by an increased cell length in the inner epidermal cells of the lemma. In addition, the data show that there is a tendency for lower expression of Oshox22 when GL increases which would suggest that Oshox22 functions as a repressor of GL.These findings suggest that natural variation in the Oshox22 promoter can be exploited in breeding programmes to modify GL using molecular marker-assisted selection. However, the exact mechanism of regulation of GL by Oshox22 is still not clear. Since Oshox22 is a homeobox gene, it will exert its function via regulation of downstream target genes which we do not know yet. Therefore, more research is needed to elucidate the genetic and biochemical pathways to understand the molecular mechanisms underlying rice GL development and to determine if there are interactions with other known regulators of GL.

The strigolactones are a relatively new class of plant hormones and a possible role in drought tolerance is unknown. In Chapter 4 of this thesis, I reviewed the various roles that strigolactones (SLs) play both in the rhizosphere and as endogenous plant hormone. In addition, the current knowledge on the SL biosynthetic and downstream signalling pathways and the interactions of SLs with other plant hormones, such as ABA, is described.

It has been reported that there seems to be a functional link between ABA and SLs but the mechanism of that link remained unknown. In Chapter 5, I studied the intimate relationship between ABA and SL biosynthesis through the further characterisation of β-carotene isomerase D27 in rice. The results show that the ABA content was increased in SL-deficient and -insensitive dwarf (d) rice mutants, d10, d17 and d3 compared with wild type, while it was reduced in d27. In addition, this difference was significantly enhanced by exposure to drought. Interestingly, as a consequence of their enhanced ABA levels, d10, d17 and d3 plants displayed an increased tolerance to drought compared with wild-type plants, while the ABA deficient d27 plants were more drought sensitive. Transient over-expression of OsD27 in Nicotiana benthamianaenhanced both ABA and SL production. However, constitutive over-expression of OsD27 in rice plants showed no significant changes in ABA and SL levelsunder normal conditions. Still, OsD27 over-expression did result in higher SL levels, compared with wild-type plants, under phosphate starvation. This suggests that likewise, OsD27over-expression may only result in increased ABA levels during drought stress conditions. I concluded that the OsD27 gene is involved in SL as well as ABA biosynthesis, and that, depending on the environmental conditions, the expression of the more downstream SL and ABA specific biosynthetic genes determines which of the two and how much is being produced.

In Chapter 6, I discussed the main findings of this thesis and presented the future perspective of how the knowledge generated in this thesis can contribute to the improvement of drought tolerance and GL in rice.

Climbing the walls : behavioural manipulation of insects by baculoviruses
Houte, S. van - \ 2014
University. Promotor(en): Just Vlak; Monique van Oers, co-promotor(en): Vera Ros. - Wageningen : Wageningen University - ISBN 9789461738301 - 228
baculoviridae - insecten - rupsen - lepidoptera - gastheer parasiet relaties - diergedrag - gedragsveranderingen - hyperactiviteit - moleculaire biologie - insects - caterpillars - host parasite relationships - animal behaviour - behavioural changes - hyperactivity - molecular biology

Parasites often have severe effects on their hosts by causing developmental and physiological alterations in their hosts. These alterations may contribute to parasite growth, reproduction and survival. For example, host development may be inhibited so that more nutrients become available for the parasite. Parasites can also interfere with host behavior as a strategy to increase parasite survival or transmission. This phenomenon is known as ‘parasitic manipulation’ or ‘behavioural manipulation’. Although many examples of parasitic manipulation are known, the genetic basis underlying such manipulations is largely unexplored. A thorough understanding of how parasites manipulate their hosts’ behavior is therefore lacking, but it can be hypothesized that parasites carry specific genes that induce these behavioural alterations. Such ‘behavioural’ parasite genes likely affect one or more host proteins directly or via the expression of specific target genes in the host, which subsequently leads to altered behaviour. Understanding the details of such interactions between parasite and host is important as parasitic manipulation is thought to be wide spread in nature and to be a strong driver of the co-evolutionary arms race between parasite and host. Furthermore, the strategies employed by parasites to alter behavior likely provide important insights in the molecular mechanism of many behavioural processes. Chapter 2 of this thesis reviews our current understanding of the mechanisms of behavioural manipulation in invertebrates. It discusses known examples of behavioural manipulation and the present knowledge on the mechanistic basis of these manipulations. Furthermore, an overview of host genes and proteins that play a conserved role in behavioural traits in different invertebrate species is given. These genes and proteins are worthwhile to be studied in more detail in the context of parasitic manipulation, as they might be suitable targets for parasites to induce behavioural changes.

This thesis focuses on behavioural manipulation in insect hosts by baculoviruses. Baculoviruses are DNA viruses that infect the larval stages of mainly lepidopteran insects. These viruses alter host behaviour in multiple ways. They induce hyperactivity in the larvae, which likely contributes to virus dispersal over a large area. In addition, baculoviruses alter host climbing behaviour leading to death at elevated positions, a phenomenon known as ‘tree-top disease’ or ‘Wipfelkrankheit’. This latter manipulation is thought to contribute to optimal virus dispersal on plant foliage. In the research described in this thesis baculoviruses and their lepidopteran insect hosts are used as a model system to study molecular mechanisms of behavioural manipulation. In Chapter 3 of this thesis the involvement of the protein tyrosine phosphatase (ptp) gene from the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) in the induction of hyperactivity of the beet armyworm Spodoptera exigua was studied. A homolog of this gene in Bombyx mori nucleopolyhedrovirus (BmNPV) was previously shown to be important in hyperactivity in the silkworm B. mori. The results in Chapter 3 showed that the AcMNPV ptp gene induces hyperactive behaviour in S. exigua larvae and that the phosphatase activity of the encoded PTP enzyme is crucial for this behavioural change. Phylogenetic inference revealed that the baculovirus ptp is presumably transferred from a (ancestral) lepidopteran host to a baculovirus. Within the family Baculoviridae, ptp is only present in group I NPVs, which are a group of phylogenetically related baculoviruses. It is hypothesized that ptp-induced hyperactivity is an evolutionarily conserved strategy of group I NPVs to manipulate host behaviour.

To obtain insights into the target proteins of the baculovirus PTP enzyme to achieve hyperactive behaviour in infected caterpillars, a PTP substrate analysis was performed. Chapter 4 describes host and viral proteins that were found to co-purify with AcMNPV PTP. Many of these host proteins are known to be important in signalling pathways and behavioural traits. For one of these proteins, 14-3-3 z, mRNA transcript levels were found to be significantly higher in wild type AcMNPV-infected S. exigua larvae as compared to larvae infected with a mutant virus from which the ptp gene has been deleted (AcMNPV Δptp). The 14-3-3 protein is a known activator of the enzymes tryptophan hydroxylase and tyrosine hydroxylase, which in turn are required for synthesis of the neurotransmitters serotonin and dopamine. These signalling molecules are both important determinants in hyperactive behaviour in various organisms, and are associated with behavioural manipulation in several parasite-host systems. In Chapter 9 a model is proposed of how the putative interaction between baculovirus PTP and host 14-3-3 zmay lead to hyperactive behaviour.

Within the baculoviruses two different genes that encode protein tyrosine phosphatases, ptp and ptp2, are found. While the ptp gene induces hyperactivity (described in Chapter 3), no function has yet been assigned to the ptp2 gene. Chapter 5 describes the functional

characterization of the baculovirus ptp2 gene. PTP2 protein carries a conserved consensus sequence that is characteristic for mitogen-activated protein kinase (MAPK) phosphatases. SeMNPV ptp2 induced a mild apoptosis and caspase activation in insect cells upon transient expression, which may be related to its putative function as MAPK phosphatase. Several host proteins that co-purified with SeMNPV PTP2 have known functions in apoptosis and/or MAPK signalling, rendering them promising candidate proteins to be involved in SeMNPV PTP2-induced apoptosis and possibly MAPK signalling. Whether PTP2 also has any behavioural effect is unknown, but the data from this chapter indicate that PTP2 likely has a cellular function during virus infection.

Baculoviruses are known to alter host climbing behaviour, commonly leading to death at elevated positions (tree-top disease). In Chapter 6 the hypothesis was tested that baculovirus-induced hyperactive behaviour and tree-top disease are induced by a single baculovirus gene. To this aim the effect of the hyperactivity-inducing ptp gene (Chapter 3) on tree-top disease was investigated. The results demonstrated that AcMNPV ptp, known to cause hyperactive behaviour in S. exigua, is not involved in tree-top disease in this host. This indicates that hyperactivity and tree-top disease induced by baculoviruses are governed by independent mechanisms. Furthermore, a moulting-dependent effect on tree-top disease in S. exigua was found, which may relate to physiological and/or ecological differences between moulted and unmoulted larvae. In the next chapter (Chapter 7) the effect of AcMNPV infection on tree-top disease was investigated for two different host species, Trichoplusia ni and S. exigua. Data show that in T. ni larvae AcMNPV induces tree-top disease, causing death at elevated positions. In contrast, in S. exigua a moulting-dependent effect on the height at death was observed, as was also described in Chapter 6. Furthermore, in this chapter the role of the AcMNPV egt gene, encoding ecdysteroid UDP glucosyl transferase, on tree-top disease in T. ni and S. exigua larvae was analysed. A homolog of this gene causes tree-top disease in Lymantria dispar larvae infected with L. dispar (Ld) MNPV. The results (Chapter 7) show that AcMNPV egt does not play a role in the observed death at elevated positions in the two host systems studied. This indicates that the role of egt in tree-top disease may not be conserved among members of the family Baculoviridae.

In addition to the mechanisms employed by the generalist baculovirus AcMNPV to alter climbing behaviour, the effect of the specialist baculovirus S. exigua (Se) MNPV on tree-top disease in its only known host S. exigua was studied. In Chapter 8 it is shown that SeMNPV induces tree-top disease by triggering an aberrant response to light, and this positive phototaxis leads to death at elevated positions. A hypothesis is put forward that SeMNPV hijacks a host behavioural pathway that is involved in light perception to induce this positive phototactic response.

Overall, the results of this thesis show that hyperactivity and tree-top disease are induced by baculoviruses through independent mechanisms and that distinct baculovirus species presumably use different genes and proximate mechanisms to induce tree-top disease. While the baculovirus ptp gene induces hyperactivity, possibly by targeting host 14-3-3 z, the baculovirus ptp2 gene may function as a pro-apoptotic gene. The baculovirus egt gene does not have a conserved function in tree-top disease, indicating that other viral genes may underlie this host manipulative strategy. This thesis also demonstrates that tree-top disease in SeMNPV-infected caterpillars is the result of a strong attraction to light.

Parasitic manipulation is a fascinating biological phenomenon that can provide crucial information on how behavioural traits are controlled at the molecular level. The research described in this thesis provides several new insights in the mechanisms by which parasites manipulate the behaviour of their hosts.

De bouwstenen van het leven : een introductie tot de moleculaire celbiologie
Prinsen, J.A.M.M. ; Leij, F.R. van der - \ 2014
Wageningen : Wageningen Academic Publishers - ISBN 9789086862412 - 470
cellen - moleculaire biologie - celbiologie - atomen - moleculen - eiwitten - dna - rna - metabolisme - celdeling - studieboeken - cells - molecular biology - cellular biology - atoms - molecules - proteins - metabolism - cell division - textbooks
In De bouwstenen van het leven wordt - ook voor leken - op een toegankelijke wijze een overzicht gegeven van de basale kennis van de biochemie en de moleculaire celbiologie (ook wel Life Sciences genoemd). De wereld van DNA, RNA en eiwitten, van energiemetabolisme tot en met genetica, is door Jan Prinsen en Feike van der Leij op een overzichtelijke manier begrijpelijk gemaakt. Niet alleen voor Bachelor-studenten een goed alternatief naast Engelstalige standaardwerken, maar voor iedereen die wil weten hoe de processen in ons lichaam en in de natuur om ons heen op moleculair niveau verklaard kunnen worden. De auteurs hebben daarbij een klassieke kijk gecombineerd met de laatste inzichten en maken op een heldere manier gebruik van de soms onvermijdelijke vaktaal. Het is daarmee oprecht een introductie tot de moleculaire celbiologie te noemen. De informatie wordt in lagen aangereikt: de lezer kan zelf de mate van diepgang en detail bepalen.
Applications in computer-assisted biology
Nijveen, H. - \ 2013
University. Promotor(en): Ton Bisseling, co-promotor(en): P.E. van der Vet. - Wageningen : Wageningen UR - ISBN 9789461737816 - 106
bio-informatica - moleculaire biologie - computers - databanken - prokaryoten - computeranalyse - informatietechnologie - bioinformatics - molecular biology - databases - prokaryotes - computer analysis - information technology

Biology is becoming a data-rich science driven by the development of high-throughput technologies like next-generation DNA sequencing. This is fundamentally changing biological research. The genome sequences of many species are becoming available, as well as the genetic variation within a species, and the activity of the genes in a genome under various conditions. With the opportunities that these new technologies offer, comes the challenge to effectively deal with the large volumes of data that they produce. Bioinformaticians have an important role to play in organising and analysing this data to extract biological information and gain knowledge. Also for experimental biologists computers have become essential tools. This has created a strong need for software applications aimed at biological research. The chapters in this thesis detail my contributions to this area. Together with molecular biologists, plant breeders, immunologists, and microbiologists, I have developed several software tools and performed computational analyses to study biological questions.

Chapter 2 is about Primer3Plus, a web tool that helps biologists to design DNA primers for their experiments. These primers are typically short stretches of DNA (~20 nucleotides) that direct the DNA replication machinery to copy a selected region of a DNA molecule. The specificity of a primer is determined by several chemical and physical properties and therefore designing good primers is best done with the help of a computer program. Primer3Plus offers a user-friendly task-oriented web interface to the popular primer3 primer design program. Primer3Plus clearly fulfils a need in the biological research community as already over 400 scientific articles have cited the Primer3Plus publication.

Single nucleotide differences or polymorphisms (SNPs) that are present within a species can be used as markers to link phenotypic observations to locations on the genome. Chapter 3 discusses QualitySNPng, which is a stand-alone software tool for finding SNPs in high-throughput sequencing data. QualitySNPng was inspired by the QualitySNP pipeline for SNP detection that was published in 2006 and it uses similar filtering criteria to distinguish SNPs from technical artefacts like sequence read errors. In addition, the SNPs are used to predict haplotypes. QualitySNPng has a graphical user interface that allows the user to run the SNP detection and evaluate the results. It has already been successfully used in several projects on marker detection for plant breeding.

Single nucleotide polymorphisms can lead to single amino acid changes in protein sequences. These single amino acid polymorphisms (SAPs) play a key role in graft-versus-host (GVH) effects that often accompany tissue transplantations. A beneficial variant of GVH is the graft-versus-leukaemia (GVL) effect that is sometimes witnessed after bone marrow transplantation in leukaemia patients. When the GVL effect occurs, the donor’s immune cells actively destroy residual tumour cells in the patient. The GVL effect can already be elicited by a single amino acid difference between the patient and the donor. Currently, a small number of SAPs that can elicit a GVL effect are known and these are used to select the right bone marrow donor for a leukaemia patient. Together with researchers at the Leiden University Medical Center I developed a database to aid in the discovery of more such SAPs. We called this database the “Human Short Peptide Variation database” or HSPVdb. It is described in chapter 4.

The work described in chapter 5 is focused on the regions in bacterial genomes that are involved in gene regulation, the promoters. Intrigued by anecdotal evidence that duplication of bacterial promoters can activate or silence genes, we investigated how often promoter duplication occurs in bacterial genomes. Using the large number of bacterial genomes that are currently available, we looked for clusters of highly similar promoter regions. Since duplication assumes some sort of mobility, we termed the duplicated promoters: putative mobile promoters or PMPs. We found over 4,000 clusters of PMPs in 1,043 genomes. Most of the clusters consist of two members, indicating a single duplication event, but we also found much larger clusters of PMPs within some genomes. A number of PMPs are present in multiple species, even in very distantly related bacterial species, suggesting perhaps that these were subjected to horizontal gene transfer. The mobile promoters could play an important role in the rapid rewiring of gene regulatory networks.

Chapter 6 discusses how current biological research can adapt to make full use of the opportunities offered by the high-throughput technologies by following three different approaches. The first approach empowers the biologists with user-friendly software that allows him to analyse the large volumes of genome scale data without requiring expert computer skills. In the second approach the biologist teams up with a bioinformatician to combine in-depth biological knowledge with expert computational skills. The third approach combines the biologist and the bioinformatician in one person by teaching the biologist computational skills. Each of these three approaches has it merits and shortcomings, so I do not expect any of them to become dominant in the near future. Looking further ahead, it seems inevitable that any biologist will have to learn at least the basics of computational methods and that this should be an integral part of biology education. Bioinformatics might in time cease to exist as a separate field and instead become an intrinsic aspect of most biological research disciplines.

The biochemical basis of plant development
Weijers, D. - \ 2013
Wageningen : Wageningen University - ISBN 9789461736215 - 23
fytochemie - plantenontwikkeling - planten - plantenweefsels - celbiologie - moleculaire biologie - biochemie - plantenembryo's - phytochemistry - plant development - plants - plant tissues - cellular biology - molecular biology - biochemistry - plant embryos
Plants develop highly elaborate structures, ranging from small mosses to large trees. All these structures are made by stem cells and consist of a few basic types of tissue. The field of Biochemistry of Plant Development studies the mechanisms by which regulatory proteins control the formation of stem cells and tissues. The young embryo, developing within the seed, is the simplest model to study these fundamental processes, and to gain understanding of the basis for plant development at cellular, molecular and atomic scale.
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