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Functional analyses of plant-specific histone deacetylases : Their role in root development, stress responses and symbiotic interactions
Li, Huchen - \ 2017
Wageningen 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.
Differences in transcriptional responses to acute and chronic dietary interventions with fatty acids
Matualatupauw, Juri C. - \ 2017
Wageningen University. Promotor(en): Sander Kersten, co-promotor(en): Lydia Afman; J. Bouwman. - Wageningen : Wageningen University - ISBN 9789463432078 - 172
fatty acids - gene expression - genotyping - phenotypes - nutritional intervention - transcriptomics - fish oils - apolipoprotein e - adipose tissue - microarrays - polymerase chain reaction - vetzuren - genexpressie - fenotypen - maatregel op voedingsgebied - transcriptomica - visoliën - apolipoproteïne e - vetweefsel - polymerase-kettingreactie
Various types of dietary fatty acids have different effects on human health. The aim of this thesis was to increase our understanding of the molecular mechanisms underlying the effects of dietary fatty acids. To do this, we examined changes in whole genome gene expression profiles upon both acute as well as longer term dietary fatty acid interventions. Furthermore, from previous research, it is clear that large inter-individual differences in the response to dietary fatty acids exist. We used whole genome gene expression analyses to increase our understanding of the mechanisms underlying some of these inter-individual differences.
Many modifiable and non-modifiable factors can be the cause of these inter-individual differences. In chapter 2, we reviewed all studies that examined differences in the transcriptional response to dietary interventions based on the presence of one of these factors. These include gender, age, BMI, body composition, blood lipid levels and gut microbial composition. We conclude that transcriptome analyses are well-suited for studying the underlying mechanisms behind these differences in the response to diet. Nevertheless, the number of studies that use this approach remains limited.
Another factor that may modify the response to a dietary intervention is genetics, e.g. the apolipoprotein E4 (APOE4) variant. People who carry the APOE4 allele have an increased risk of cardiovascular disease. Fish-oil supplementation may help in the prevention of cardiovascular disease, though inter-individual differences in the response to n-3 polyunsaturated fatty acids on gene expression profiles have been observed. In chapter 3, we aimed to assess the impact of APOE4 on peripheral blood mononuclear cell (PBMC) whole genome gene expression at baseline and following a 6-month fish-oil intervention. We observed increased gene expression of IFN signaling and cholesterol biosynthesis pathways in APOE4 carriers, which might explain part of the association between APOE4 and CVD. Furthermore, fish-oil supplementation may be beneficial by decreasing interferon signalling-related gene expression in APOE4 carriers.
Another long-term dietary intervention with fatty acids was studied in chapter 4. We examined the effect of a 12-week high medium-chain saturated fatty acid diet on subcutaneous adipose tissue gene expression profiles. We observed increased expression of genes involved in oxidative energy metabolism and decreased inflammation-related gene expression due to the high medium-chain saturated fatty acid intake. Considering the role of the adipose tissue in sustaining the low-grade inflammation that is associated with obesity, these findings may be indicative of a more anti-inflammatory phenotype of the adipose tissue. We concluded that medium-chain saturated fatty acids may potentially have beneficial effects on adipose tissue functioning.
Besides studying the effects of long-term interventions with fatty acids on whole genome gene expression, we also examined the effects of acute high-fat challenges. In chapter 5, we determined the additional value of determining whole genome gene expression changes in response to a high-fat challenge compared to assessment at fasting only. In addition, we aimed to identify whether a 4 week high-fat high-calorie diet can induce a shift in gene expression profiles in healthy subjects towards a metabolic syndrome-like gene expression profile. We found that fasting whole blood whole genome gene expression profiles are highly responsive to a 4-week high-fat high-calorie diet, with changes in in the direction of a metabolic syndrome-like gene expression profile. High-fat challenge responses in healthy subjects show only minimal changes in gene expression upon the dietary intervention and a marginal shift in the direction of the metabolic syndrome. We concluded that fasting gene expression profiles are more responsive compared to high-fat challenge responses to a 4-week high-fat high-calorie diet.
Besides chapter 5, several other studies have also examined changes in whole genome gene expression in blood cells induced by high-fat challenges. In chapter 6, we combined microarray data from four high-fat challenge studies varying in study population, challenge composition and research laboratory. By performing this meta-analysis, we showed a general PBMC whole genome gene expression response to a high-fat challenge. We concluded that a meta-analysis provides added value for the discovery of consistently differentially expressed genes and pathways compared to selecting only those genes and pathways that are identified in all separate studies.
In conclusion, in this thesis we showed differences in the whole genome gene expression response to fish-oil supplementation in PBMCs of APOE4 carriers vs non-carriers. Furthermore, the effects on whole genome gene expression of the two long-term dietary interventions, i.e. the fish-oil supplementation in PBMCs of APOE4 carriers and the high medium-chain saturated fatty acid diet in adipose tissue, may be beneficial by downregulation of gene expression related to inflammation. We also showed that whole genome gene expression responses to high-fat challenges are affected by a 4-week high-fat high-calorie diet, though changes in fasting gene expression profiles are much more pronounced. Finally, we showed the value of meta-analysis of microarray data in high-fat challenge studies for identifying the general response to a high-fat challenge.
The transcriptome as early marker of diet-related health : evidence in energy restriction studies in humans
Bussel, Inge P.G. van - \ 2017
Wageningen University. Promotor(en): Sander Kersten, co-promotor(en): Lydia Afman. - Wageningen : Wageningen University - ISBN 9789463430678 - 194
energy restricted diets - energy intake - gene expression - genomes - proteins - endurance - food composition - human nutrition research - energiearme diëten - energieopname - genexpressie - genomen - eiwitten - uithoudingsvermogen - voedselsamenstelling - voedingsonderzoek bij de mens
Background: Nutrition research is facing several challenges with respect to finding diet related health effects. The effects of nutrition on health are subtle, show high interindividual variations in response, and can take long before they become visual. Recently, the definition of health has been redefined as an organism’s ability to adapt to challenges and ‘this definition’ can be extended to metabolic health. In the metabolic context the ability to adapt has been named ‘phenotypic flexibility’. A potential new tool to magnify the effects of diet on health is the application of challenge tests. Combined with a comprehensive tool such as transcriptomics, the study of challenge tests before and after an intervention might be able to test a change in phenotypic flexibility. A dietary intervention well-known to improve health through weight loss is energy restriction (ER). ER can be used as a model to examine the potential of challenge tests in combination with transcriptomics to magnify diet-induced effects on health. As opposed to ER, caloric restriction (CR) is a reduction in energy intake aimed at improving health and life span in non-obese subjects and not directly aimed at weight loss. In this thesis, we aimed to investigate the use of the transcriptome as an early and sensitive marker of diet-related health.
Methods: First we studied the consequences of age on the effects of CR on the peripheral blood mononuclear cells (PBMCs) transcriptome. For that purpose, we compared the changes in gene expression in PBMCs from old men with the changes in gene expression in PBMCs from young men upon three weeks of 30% CR. To study the effect of a change in dietary composition during ER, we compared the changes in gene expression upon a 12 weeks high protein 25% ER diet with the changes in gene expression upon a 12 weeks normal protein 25% ER diet in white adipose tissue (WAT). Next, we investigated the added value of measuring the PBMC transcriptome during challenge tests compared to measuring the PBMC transcriptome in the fasted state to magnify the effects of ER on health. This was investigated by measuring the changes in gene expression upon an oral glucose tolerance test (OGTT) and upon a mixed meal test (MMT), both before and after 12 weeks of 20% ER. Finally, we determined the differences between a challenge test consisting of glucose alone, the OGTT, or consisting of glucose plus other macronutrients, the MMT, on the PBMC transcriptome in diet-related health.
Results: We observed that the transcriptome of PBMCs of healthy young men had a higher responsiveness in immune response pathways compared to the transcriptome of PBMCs of aged men upon CR (chapter 2). Also, we showed that upon a normal protein-ER diet the transcriptome of WAT showed a decrease in pathways involved in immune response and inflammasome, whereas no such effect was found upon a high protein-ER diet. These effect were observed while parameters such as weight loss, glucose, and waist circumference did not change due to the different protein quantities (chapter 3). 12 weeks of 20% ER was shown to increase phenotypic flexibility as reflected by a faster and more pronounced downregulation of OXPHOS, cell adhesion, and DNA replication during the OGTT compared to the control diet (chapter 4). Finally, two challenge tests consisting of either glucose (OGTT) or glucose plus fat and protein (MMT), were shown to result in a larger overlap than difference in the changes in gene expression of PBMCs (chapter 5).
Conclusions: Based on the differential changes in gene expression upon CR at different ages, we concluded that age is an important modulator in the response to CR. As a high protein ER diet induced transcriptional changes seemed to reflect less beneficial health effects than a normal protein ER diet we concluded that the diet composition is important in the health-effect of ER as measured by the transcriptome. Based on the faster PBMCs changes in gene expression during an OGTT upon 12 weeks of 20% ER, we concluded that the PBMC transcriptome combined with a challenge test can reflect changes in phenotypic flexibility. This makes challenge tests a suitable tool to study diet-related health effects. Finally, based on the changes in gene expression of the MMT and OGTT, we conclude that glucose in a challenge test is the main denominator of the postprandial changes in gene expression in the first two hours. Overall, these results lead to the conclusion that the transcriptome, especially in combination with challenges test, can be used as an early marker of diet-related health. The direct relation to health still needs to be investigated, but the possibility to use the transcriptome as an early marker of diet-related health gives rise to a better understanding of the effects of nutrition on health.
Tuning for light and more : engineering phototrophy and membrane proteins in Escherichia coli
Claassens, Nicolaas J.H.P. - \ 2017
Wageningen University. Promotor(en): John van der Oost; Willem de Vos, co-promotor(en): Vitor Martins dos Santos. - Wageningen : Wageningen University - ISBN 9789463430920 - 328
escherichia coli - phototropism - membranes - proteins - light - photosystem i - gene expression - fototropie - membranen - eiwitten - licht - fotosysteem i - genexpressie
The application of microbial and plant photosynthesis for biobased production on the one hand has a huge potential but on the other hand photosynthesis has serious limitations regarding its efficiency. Hence, studying both fundamental features of photosynthetic processes and engineering of photosystems is of paramount interest, exploring the engineering of photosystems is the overarching aim of this thesis. As described in Chapter 1, natural photosystems may be modified or transplanted to allow for more efficient conversion of solar light energy into biochemical energy. Hereto ambitious proposals to engineer photosystems have been made, and to realize those endeavors the disciplines of synthetic and systems biology are required. To explore how to apply and improve those disciplines hereto, the work described in this thesis has focused on the transplantation of simple photosystems (proton-pumping rhodopsins; PPRs) into the cell membrane of the heterotrophic model bacterium Escherichia coli. Both in silico analyses, including metabolic and thermodynamic modeling (Chapter 3) and a series of experimental studies on transplanting PPR photosystems (Chapters 4,6 and 7) were performed, which identified several challenges, limitations and most importantly opportunities. This thesis also describes the application of novel tools to substantially improve the functional production of PPRs and a variety of other membrane proteins in E. coli.
Chapter 2 provides more details on previously reported examples of heterologous expression of PPRs in several hosts, and on the physiological impact of these transplanted photosystems. Based on this evaluation, some suggestions are made to improve and further exploit the transplantation of these photosystems.
In Chapter 3 a systematic, integrated in silico analysis is made of anaerobic, photo-electro-autotrophic synthetic metabolism in E. coli, consisting of (i) a PPR photosystem for ATP regeneration, (ii) an electron uptake pathway, and (iii) a natural or synthetic carbon fixation pathway. Constraint-based metabolic modelling of E. coli central metabolism is used, in combination with kinetic and thermodynamic pathway analyses. The photo-electro-autotrophic designs are predicted to have a limited potential for anaerobic, autotrophic growth of E. coli, given the relatively low ATP regenerating capacity of the PPR photosystems, and the relatively high ATP consumption due to maintenance. In general these analyses illustrate the potential of in silico analyses to identify potential bottlenecks and solutions in complex designs for autotrophic and photosynthetic metabolism, as a basis for subsequent experimental implementation.
To tackle a main bottleneck of PPR systems: their functional membrane-embedded production level, the heterologous production in E. coli of the proton-pumping rhodopsins from Gloeobacter violaceus (GR) and from Thermus thermophilus JL18 (TR) is quantified and experimentally optimized in Chapter 4. High constitutive production of both rhodopsin proteins is achieved by fine-tuning transcription and translation. Besides the canonical retinal pigment, the GR system has the ability to bind a light-harvesting antennae pigment, echinenone. After optimization of the heterologous pigment biosynthesis pathways for either retinal or echinenone production, appropriate quantities of retinal or echinenone for PPR reconstitution were detected in E. coli. Association of echinenone with GR broadens its absorption spectrum in E. coli, broadening the potential for light-harvesting also to blue light. Optimization of the branched pathway for simultaneous biosynthesis of both retinal and echinenone has been attempted by using a smart library of variable Ribosome Binding Sites (RBSs) with varying strengths (RedLibs). In general, the here described approaches towards improved functional production of rhodopsin photosystems in E. coli and their pigments may prove more widely applicable for heterologous production of more complex photosystems and other systems.
In Chapter 5 an up-to-date overview is provided on how codon usage can influence functional protein production. The fact that all known organisms have an incomplete set of tRNAs, indicates that biased codon usage could act as a general mechanism that allows for fine-tuning the translation speed. Although translation initiation is the key control step in protein production, it is broadly accepted that codon bias, especially in regions further downstream of the start codon, can contribute to the translation efficiency by tuning the translation elongation rate. Modulation of the translation speed depends on a combination of factors, including the secondary structure of the transcript (more or less RNA hairpins), the codon usage landscape (frequent and more rare codons) and for bacteria also RBS-like sequences at which ribosomes can pause. The complex combination of interdependent factors related to codon usage that can influence translation initiation and elongation. This complexity makes that the design of synthetic genes for heterologous expression is still in its infancy, and despite the availability of some codon usage algorithms, it is often as well a matter of trial and error.
In Chapter 6 the effect of different codon usage algorithms (optimization and harmonization) has been experimentally tested for heterologous production of membrane proteins. Apart from the codon usage algorithms also the combined effect of transcriptional fine-tuning in E. coli LEMO21(DE3) was assessed. The overproduction of 6 different membrane-embedded proteins, including 4 PPR variants (from bacteria, archaea and eukaryotes), was tested. For production of tested PPR variants, the different codon usage algorithms hardly influenced production, while transcriptional tuning had a large impact on production levels. Interestingly, for the other two tested non-PPR membrane proteins, some codon usage variants significantly improved production on top of transcriptional tuning. For both these proteins the codon-optimization algorithm reduced functional production below that of the wild-type codon variant, while the harmonization algorithm gave significantly higher production, equal or even higher than for the wild-type variant.
In Chapter 7 it is demonstrated that a translational-tuning system can be used to successfully optimize the expression of several membrane proteins, based on initial findings presented in Chapter 4. The employed, recently developed Bicistronic Design (BCD) system is based on translational coupling of a gene encoding a short leader peptide and the gene of interest that is under control of a variable ribosome binding site. A standardized library of 22 RBSs allows for precise, gene context-independent, fine-tuning of expression of this second gene, here encoding a membrane protein. For all four membrane proteins tested in this study the BCD approach resulted in 3 to 7-fold higher protein levels than those obtained by two other recently developed methods for optimizing membrane protein production. The presented approach allows for inducer-free, constitutive, high-level production of membrane proteins in E. coli, which can be widely applicable for both membrane protein purification studies as well as for synthetic biology projects involving membrane proteins.
In Chapter 8 a broad review and perspectives are provided on the potential of microbial autotrophs for the production of value-added compounds from CO2. Both photoautotrophic and chemolithoautotrophic production platforms are discussed, and recent progress in improving their efficiency and production potential is highlighted. Transplantation efforts for photosystems, but also for CO2 fixation pathways and electron uptake systems are discussed. An overview is provided on novel in silico and experimental approaches to engineer components related to autotrophy in heterotrophic and autotrophic model hosts, including approaches applied in this thesis. Future avenues are discussed for realizing more efficient autotrophic production platforms.
Finally, in Chapter 9 and 10 the work in this thesis is summarized and a general discussion is provided on future avenues for engineering of PPR photosystems, photosystems in general and on the optimization of membrane protein production.
Aspects of rumen adaptation in dairy cattle : morphological, functional, and gene expression changes of the rumen papillae and changes of the rumen microbiota during the transition period
Dieho, Kasper - \ 2017
Wageningen University. Promotor(en): Wouter Hendriks, co-promotor(en): Jan Dijkstra; Andre Bannink; J.Th. Schonewille. - Wageningen : Wageningen University - ISBN 9789463430258 - 248
dairy cattle - rumen - rumen microorganisms - morphology - gene expression - animal nutrition - dry period - lactation - melkvee - pens - pensmicro-organismen - morfologie - genexpressie - diervoeding - gustperiode - lactatie
In dairy cattle the nutrient requirements change rapidly around calving. During the dry period nutrients are required for maintenance, recovery from the previous lactation, and fetal growth. After calving, milk production commences and the energy requirements can increase by a factor 3 to ~184 MJ net energy for lactation during the first weeks of lactation, compared with the dry period, whereas feed intake doubles to ~24 kg dry matter (DM)/d compared with the dry period. In addition, high quality lactation rations are fed, usually containing a sizable portion of concentrate, thereby increasing fermentable organic matter (FOM) intake to ~14 kg/d. As a result, daily volatile fatty acid (VFA) production by the rumen microbiota increases from ~60 mol/d during the dry period to ~132 mol/d during early lactation. To maintain rumen pH at levels favorable for microbial fermentation, and prevent a negative impact on production and health, clearance of the produced VFA is essential. This mainly occurs through absorption over the rumen wall. The increase in capacity of the rumen for absorption of VFA is associated with morphological and functional changes of the rumen papillae which cover the rumen wall. However, current knowledge of these changes as they occur around calving is scarce (Chapter 1). Increasing our understanding of the adaptation of the rumen can provide new insights to optimize dairy cattle nutrition and thereby health, welfare, and production.
The objective of this thesis was to study the adaptation of the rumen to ration changes during the dry period and early lactation. Changes in rumen papillae morphology, fractional absorption rate of VFA (kaVFA), and changes in the composition of the rumen microbiota were the primary targets for study. In addition, the expression of genes and proteins associated with absorption and metabolism of VFA by the rumen epithelium were studied to better understand the relationship between functional changes and morphological changes of the papillae. Uniquely, all these aspects were studied in parallel in the same dairy cows during the dry period and early lactation using a repeated measurement setup. Two experiments were conducted. In the lactation experiment, the effect of transition from the dry period to the subsequent lactation, and the effect of early lactation concentrate build-up strategy on the adaptation of the rumen were studied. In the dry period experiment, the effect of feeding supplemental concentrate during the late dry period in order to ‘prepare’ the rumen for the lactation was studied. Treatments of both experiments were aimed at creating a difference in FOM intake (kg/d) and thereby VFA production (mol/d), as VFA production was hypothesized to affect rumen papillae development and thereby the capacity for VFA absorption.
During the lactation experiment, intake of FOM did not change during the dry period (5.7 kg/d), but increased during the subsequent lactation to 15.0 kg/d at 80 d postpartum (pp). In addition, the rapid increase in concentrate allowance resulted in a temporarily 22% greater FOM intake compared with a gradual increase at 16 d pp (Chapter 2). The total production rate of VFA, measured using an isotope dilution technique (Chapter 3), was affected by these changes in FOM intake and increased 2.3 fold to 123 mol/d after calving, compared with the dry period (53 mol/d). The temporarily greater FOM intake with the rapid increase in concentrate allowance at 16 d pp coincided with a 54% greater propionate production (34 mol/d) compared with a gradual increase in concentrate allowance, whereas acetate (66 mol/d) and butyrate (10 mol/d) production were not affected. Papillae surface area (Chapter 2) decreased by 19% between 50 d antepartum (ap) and 3 d pp to 28.0 mm2, but increased during early lactation to 63.0 mm2. Papillae surface area increased faster with the rapid increase in concentrate allowance and surface area was 38, 34 and 22% larger at 16, 30, and 44 d postpartum respectively, than with a gradual rate of increase of concentrate allowance. Histology (Chapter 2) revealed that rumen papillae and epithelium thickness decreased slightly after calving, but were not affected by the concentrate treatment. Feeding concentrate during the dry period did not affect daily FOM intake (6.0 kg/d) but did increase VFA concentration in the rumen fluid by 21 mM to 121 mM, and increased papillae surface by 29% (Chapter 4). However, the increased papillae surface area in the dry period was not maintained to the subsequent lactation period. After calving, papillae surface area increased by 50% to 58.0 mm2 at 45 d pp. The postpartum development of the rumen papillae was not affected by the treatment during the dry period. These results indicate that rumen papillae respond to changes in FOM and VFA production intake during the dry period and early lactation, and that the magnitude of this response depends on the rate of change in FOM intake.
During both experiments, kaVFA was measured using a buffer incubation technique in an empty washed rumen. During the lactation experiment (Chapter 3), in accordance with the developments in papillae surface area, the kaVFA decreased during the dry period from 0.48/h at 50 d ap to 0.34/h at 3 d pp. During the subsequent lactation, it increased rapidly to 0.56/h at 16 d pp and further to 0.72/h at 80 d pp. However, the greater papillae surface area due to the rapid increase in concentrate did not coincide with a greater kaVFA. During the dry period experiment (Chapter 4), kaVFA increased after calving by 50% to 0.48/h at 45 d pp, but the increase in papillae surface area due to supplemental concentrate during the dry period did not affect the kaVFA during the dry period (0.36/h) or the subsequent lactation. These results indicate that papillae surface area is not the limiting factor for kaVFA.
Changes in the expression of genes were studied at the mRNA level in papillae tissue from both experiments (Chapter 5). The expression of apoptosis related genes was not affected by sampling day or its interaction with treatment for both experiments, suggesting papillae proliferation during the transition period was mainly the result of an increased mitosis rate. The limited changes in the expression of genes associated with rumen epithelial transport and metabolism of VFA in dairy cows during the transition period do not suggest that these capacities of the epithelium increased per unit of surface area. Thus the major response to the increase in daily VFA production after calving was tissue proliferation. In addition, papillae from the lactation experiment were used to study expression at the protein level using immunoblotting. Results showed that expression of several proteins changed during early lactation indicating modulation of intracellular pH regulation and sodium homeostasis, and VFA metabolism. Only for one gene, a significant but weak correlation between the examined mRNA and protein expression levels was observed, indicating that care must be taken when interpreting results obtained at either level.
Ration changes associated with the transition from the dry period to lactation affected the rumen microbiota during the lactation experiment (Chapter 6). The rapid increase in concentrate allowance postpartum temporarily decreased bacterial community richness by as much as 30% compared with a gradual increase in concentrate. This transient depression in bacterial community richness with a rapid, but not a gradual, rate of increase of concentrate allowance pp indicates that the rate of change in ration composition and feed intake has a greater effect than the change in ration composition and feed intake level as such. The relative abundances of most major bacterial taxa were affected by the transition to lactation, but few were affected by the rate of increase of the concentrate allowance. The relative abundances of rumen protozoal taxa changed after calving, and were affected by the concentrate treatment. However, differences between treatments groups disappeared again when concentrate intake became similar. The archaeal community was likewise affected by both the transition to lactation and the treatment. The observed changes in rumen microbiota composition, including changes in bacterial community richness, did not appear to affect the fractional degradation rate of NDF, starch, CP, and OM measured in situ using a nylon bag technique.
The results in the present thesis show that morphologically and functionally the rumen papillae can adapt rapidly to the changes in FOM intake and daily VFA production associated with the transition from the dry period into the subsequent lactation. However, the contrast in response of rumen papillae surface area development and the fractional absorption rate of VFA to the concentrate treatments indicates that papillae surface area is not the limiting factor for VFA absorption. This proposition is further supported by the limited histological changes of the rumen epithelium and limited changes in gene expression. Considering that the capacity for absorption and metabolism of VFA per unit of papillae surface area remains similar, an extra-epithelial factor, likely visceral blood flow, limits VFA absorption. The capacity of the rumen to adapt after calving and the limited beneficial effect of supplementing concentrate during the dry period indicate that dry period feeding strategies can best be optimized for the prevention of periparturient diseases.
Susceptibility pays off: insights into the mlo-based powdery mildew resistance
Appiano, Michela - \ 2016
Wageningen University. Promotor(en): Richard Visser, co-promotor(en): Yuling Bai; Anne-Marie Wolters. - Wageningen : Wageningen University - ISBN 9789462579484 - 265
solanum lycopersicum - tomatoes - disease resistance - susceptibility - oidium neolycopersici - genes - gene expression - genomics - molecular breeding - plant breeding - tomaten - ziekteresistentie - vatbaarheid - genen - genexpressie - genomica - moleculaire veredeling - plantenveredeling
Powdery mildew (PM) is a worldwide-occurring plant disease caused by ascomycete fungi of the order Erysiphales. A conspicuous number of plant species are susceptible to this disease, the occurrence of which is increasing due to the influence of climate change. Symptoms are easy to recognize by the powdery whitish fungal structures growing on the surface of plant organs. Severe infections cause significant losses in crops, such as tomato, cucumber and wheat, as well as in ornamentals, like rose and petunia. Accordingly, breeding crops with a robust immunity to this disease is of great economic importance.
A significant step in this direction was the discovery of mlo (mildew locus o) mutant alleles of the barley HvMlo gene, which are responsible for the non-race specific resistance to the barley PM pathogen, Blumeria graminis f.sp. hordei (Bgh). During the years, this recessively inherited resistance was observed to be durable, contrary to the short life-span of resistances conferred by dominant resistance (R-) genes used in barley breeding programs. Studies on the histological mechanisms of the mlo-based resistance showed that the PM pathogen was stopped during penetration of the cell wall by the formation of a papilla. This structure prevents the formation of the feeding structure of the pathogen, called a haustorium.
After sequencing many plant genomes, we are discovering that MLO genes are not only typical of this cereal, but are ubiquitously present in higher plant species in multiple copies per species, forming a gene family. The impairment of some members of a number of ever increasing plant species lead to broad-spectrum resistance towards their adapted PM pathogens. For example, in tomato the ol-2 gene, naturally harbored by the cherry tomato Solanum lycopersicum var. cerasiforme, represents the loss-of-function allele of the SlMLO1 gene, conferring resistance to the PM pathogen Oidium neolycopersici (On). Consequently, the use of mlo mutants represents a suitable alternative to the classical use of R-genes in breeding programs.
In Chapter 2, we describe the in silico identification of the complete tomato SlMLO gene family using the available information in the SOL genomic network database. In total, 16 tomato SlMLO members were cloned from leaf, root, flower and fruit of the susceptible tomato cv. Moneymaker to confirm the sequences retrieved from the database and to verify their actual expression in these tissues. We observed the presence of various types of splicing variants, although their possible functional meaning has not been investigated. Motif analyses of each of the translated protein sequences and phylogenetic studies highlighted, on one hand, amino acid stretches that characterize the whole MLO family, and, on the other hand, stretches conserved in MLO homologs that are phylogenetically related. Following a gene expression study upon On inoculation, we identified members of the SlMLO family that are upregulated few hours after pathogen challenge. Except SlMLO1, none of the three newly identified homologs in clade V, thus phylogenetically close to SlMLO1, are induced. Interestingly, two homologs, each found in different clades, are upregulated similarly to SlMLO1. Using an RNAi approach, we silenced the additional clade V-SlMLO homologs, namely SlMLO3, SlMLO5 and SlMLO8, to investigate their possible role in PM resistance. We observed that none of these homologs if individually silenced, leads to PM resistance. However, if SlMLO5 and SlMLO8 are silenced together with SlMLO1, a significantly higher level of resistance is achieved compared to plants carrying the ol-2 allele. The role of SlMLO3 could not be verified. We, therefore, concluded that there are three SlMLO genes in tomato unevenly contributing to the PM disease, of which SlMLO1 has a major role.
Chapter 3 focuses on the components of the tomato mlo-based resistance. In Arabidopsis, it is known that four members of the SNARE protein family, involved in membrane fusion, are involved in mlo-based resistance. In this chapter, we focused on the identification of tomato homologs of the Arabidopsis syntaxin PEN1 (AtSYP121). Among the group of syntaxins identified in tomato, two were closely related to each other and also to AtPEN1, denominated SlPEN1a and SlPEN1b. Another Arabidopsis syntaxin that shows a high level of homology with PEN1, called SYP122, was also found to group together with the newly identified SlPEN1 genes. However, the role of SYP122 in plant immunity was not shown in literature. After obtaining individual silencing RNAi constructs, we transformed the resistant ol-2 line, and we challenged the obtained transformants with the adapted PM On, and the non-adapted Bgh. Interestingly, we observed a significant On growth and an enhanced Bgh cell entry only in SlPEN1a silenced plants but not in SlPEN1b silenced ones. We performed a protein alignment of tomato and Arabidopsis functional and non-functional PEN sequences. The presence of three differently conserved non-synonymous amino-acid substitutions is hypothesised to be responsible for the specialization in plant immune function.
In Chapter 4 and Chapter 5, we build up a body of evidence pointing to the fact that the function of the MLO susceptibility genes is highly conserved between monocot and dicot plant species.
In Chapter 4 we started by identifying and functionally characterizing two new MLO genes of Solanaceous crops affected by the PM disease, tobacco (Nicotiana tabacum) and eggplant (Solanum melongena). We named them NtMLO1 and SmMLO1 in the respective species, as they are the closest homologs to tomato SlMLO1. By overexpressing these genes in the resistant ol-2 line, we obtained transgenic plants that were susceptible to the PM pathogen On. This finding demonstrates that both heterologous MLO proteins can rescue the function of the impaired ol-2 allele in tomato. In addition, we found in tobacco NtMLO1 an amino acid (Q198) of critical importance for the susceptibility function of this protein.
In Chapter 5, we used the same approach adopted in Chapter 4 to show that other MLO proteins of more distant dicot species, like pea PsMLO1, can rescue the loss-of-function of the tomato ol-2 allele. And finally, we stretched this concept also to monocot MLO proteins, using barley HvMlo. While performing these experiments, we could verify that the function of the monocot and dicot susceptibility MLO proteins does not rely on the presence of class-specific conservation. The latter can be the reason for the phylogenetic divergence, placing monocot MLO proteins in clade IV and dicot MLO proteins in clade V of the phylogenetic MLO tree. However, functional conservation might depend on crucial shared amino acids of clade IV and V MLO proteins. Therefore, we also conducted a codon-based evolutionary analysis that resulted in the identification of 130 codons under negative selection, thus strongly maintained during evolution.
In Chapter 6 we introduce the PM disease in cucumber caused by Podosphaera xanthii (Px). We cloned the candidate susceptibility gene for PM in cucumber, CsaMLO8, from susceptible and resistant genotypes. The latter was described as an advanced cucumber breeding line characterized by hypocotyl resistance. In this line, we found the presence of aberrant splicing variants of the CsaMLO8 mRNA due to the insertion in its corresponding genomic region of a Class LTR retrotransposon. Heterologous expression of the wild-type cucumber allele in the tomato ol-2 line restored its PM susceptibility, while the heterologous expression of the aberrant protein variant failed to do so. This finding confirms that the resistance of the advanced cucumber breeding line is due to the disruption of the coding region of this gene. We also showed that the expression of CsaMLO8 in the susceptible genotype is induced by Px in hypocotyl tissue, but not in cotyledon or leaf. Finally, by examination of the resequencing data of a collection of 115 cucumber accessions, we found the presence of the TE-containing allele in 31 of them among which a wild cucumber accession that might have been used in breeding programs to obtain resistance to the PM disease in cucumber.
In Chapter 7 a novel loss-of-function allele of the SlMLO1 gene is described, designated m200. This allele was found in a resistant plant (M200) from a mutagenized tomato Micro-Tom (MT) population obtained with the chemical mutagen ethyl methanesulfonate (EMS). The m200 mutation corresponds to a nucleotide transversion (T à A) which results in a premature stop codon. The length of the predicted SlMLO1 protein in the M200 plant is only 21 amino acids, thus much shorter than the predicted protein of the previously described ol-2 allele, consisting of 200 amino acids. Thanks to the development of a High-Resolution Melting (HRM) marker designed to detect the m200 mutation, we observed that this allele confers recessively inherited resistance in backcross populations of the resistant M200 plant with MT and Moneymaker. Histological study showed that the resistance of the m200 mutant is associated with papilla formation. Finally, we compared the rate of On penetration in epidermal cells of m200 plants with the one of plants carrying the ol-2 allele and the transgenic plants in which multiple SlMLO homologs were silenced, generated in Chapter 2.
Ultimately, in Chapter 8 the results of the previous chapters are discussed in the context of 1) practical applications in breeding programs aimed at introducing the mlo-based resistance in new crops, 2) possible research aimed at unraveling the function of the MLO protein and 3) the role of other SNARE proteins.
Building towards a multi-dimensional genetic architecture in Caenorhabditis elegans
Sterken, Mark G. - \ 2016
Wageningen University. Promotor(en): Jan Kammenga, co-promotor(en): Jaap Bakker; Gorben Pijlman. - Wageningen : Wageningen University - ISBN 9789462578692 - 167
caenorhabditis elegans - genetic models - introgression - genetic variation - quantitative trait loci - animal viruses - inheritance - rna interference - viral replication - gene expression - genetische modellen - introgressie - genetische variatie - loci voor kwantitatief kenmerk - dierenvirussen - overerving - rna-interferentie - virusreplicatie - genexpressie
Trait variation within species is shaped by the genotype and the environment an individual is exposed to. Genomic information is inherited from the parents and forms the basis of the phenotype of an organism. The genetic variation between parents becomes differently distributed between their offspring, leading to trait variation in the offspring. Each trait can be affected by many genes, therefore the genetic architecture can be complex. In complex traits, multiple loci contribute to the ultimate trait value. However, complex traits are shaped not only by genetic variation but also by the environment and the interaction between genotype and environment. The interplay between genetic and environmental variation can affect the fitness of an organism.
Chapter 2 discusses how genotype and environment have shaped the phenotype of the nematode Caenorhabditis elegans, the model species used in this thesis, resulting in a laboratory adapted domesticized strain known as Bristol N2. Bristol N2 has been cultivated in the laboratory for over a decade, leading to the fixation of novel mutations in several genes that strongly affect its phenotype. Genotypic variation arisen by novel mutations in the genes npr-1, glb-5, and nath-10 was fixed in N2 due to the laboratory environment. The allelic variation in npr-1 affects the behaviour of this animal in an environment dependent manner, showcasing the interplay between genotype and environment. However, the altered behaviour warrants caution for interpretation of results obtained in the N2 strain.
The genotypic effects on trait variation can be large, and one of the more powerful population designs to study these effects are introgression lines. In chapter 3 the construction of a genome-wide introgression line (IL) panel between the N2 and the CB4856 strain is described. This panel contains loci of N2 introgressed in a homogeneous CB4856 background. It is demonstrated that together with CB4856-in-N2 ILs this new genome-wide introgression line library strongly facilitates the dissection of genetic interactions.
Chapter 4 and 5 investigate natural variation in infection with Orsay virus, a natural pathogen of the nematode C. elegans. In chapter 4 an assay is developed and tested on two wild-type strains (N2 and JU1580) and two mutant strains with mutations in the RNAi pathway. The development of the virus infection in the separate strains can be traced and the influence of genotype and age on the progression of the infection can be quantified. Furthermore, it is demonstrated that heritable RNAi plays a role in the viral load upon Orsay virus infection, an example of an epigenetically inherited environmental influence. In chapter 5 the assay is applied on an N2xCB4856 recombinant inbred line (RIL) population, after observing a lower viral load in CB4856 compared to N2. The RIL analysis resulted in the identification of two QTL on chromosome IV. These quantitative trait loci (QTL) were verified by CB4856-in-N2 ILs, but the IL analysis also indicated that there could be genetic interactions affecting the QTL. By a transcriptome analysis and a candidate gene search, the gene cul-6 was identified as a candidate underlying the allelic variation between the N2 and CB4856 strain.
Chapters 6 and 7 investigate the influence of genetic interactions and the environment on the genetic architecture of gene expression. In chapter 6 a N2xCB4856 RIL population was exposed to heat stress, leading to the identification of a trans-band on the top of chromosome IV. By analysis of candidate genes, cmk-1, egl-4, and eor-1 were implicated as contributing to the heat-stress induced transcriptional response affected by natural variation between N2 and CB4856. Furthermore, the genes with an expression-QTL on the trans-band were indicative of a stress response phenotype. By analysis of CB4856-in-N2 ILs, it was found that this locus leads to increased recovery from stress. In chapter 7 two-loci genetic interactions were mapped for gene expression in a N2xCB4856 RIL panel. These epistatic interactions were confirmed by measuring gene expression in a novel population of inbred line containing the full set of loci combinations. It was found that genetic interactions in gene expression can be identified by clustering and are pervasive. These genetically interacting loci affect evolutionary conserved genes.
In conclusion, this thesis unveils the mechanisms underlying the genetic architecture of complex traits in C. elegans resulting from genotype and interactions between genotype and environment. It provides tools to unravel these interactions in C. elegans, by providing the community with new resources such as the N2-in-CB4856 introgression lines. Although C. elegans has been a very powerful platform for quantitative trait dissection, we need to expand our mechanistic understanding of polygenic traits.
Exploring the genetics underlying the responses to consecutive combinations of biotic stresses and drought in Arabidopsis thaliana
Huang, Pingping - \ 2016
Wageningen University. Promotor(en): Maarten Koornneef, co-promotor(en): Mark Aarts. - Wageningen : Wageningen University - ISBN 9789462578593 - 291
arabidopsis thaliana - genetic models - stress - stress response - drought - botrytis - pieris (lepidoptera) - genetics - gene expression - genetische modellen - stressreactie - droogte - genetica - genexpressie
Plants growing in natural environments are exposed to a broad range of biotic (pathogen attack, insect herbivory, etc.) and abiotic factors (drought, extreme temperatures, UV radiation, salinity, etc.) that are known to cause stress symptoms in many species (Pareek et al., 2010; Robert-Seilaniantz et al., 2010). Biotic and abiotic stress-inducing determinants often adversely impact plant growth and development, frequently leading to severe annual yield losses in agricultural production (Pierik et al., 2013; Pieterse et al., 2012; Stam et al., 2014). In the research endeavors described in this thesis, Arabidopsis thaliana was used as a model organism to study plant responses to different sequential combinations of biotic factors (infection with Botrytis or herbivory by Pieris) and drought. The main objective was to identify genes that contribute to tolerance to the aforementioned sequential stress combinations. Genome-wide association (GWA) mapping and RNA sequencing (RNA-seq) approaches were used to identify combinatorial stress responsive genes. A number of candidate genes to combinatorial stress responses were identified by GWA analysis and RNA-seq. The physiological function of some candidate genes in different stress conditions were characterized using T-DNA insertion mutants and gene expression study. However, the physiological function of many allelic variants in stress conditions remain to be discovered. The study highlights the importance of an array of genes, crucial to the underlying defense processes, as targets for breeding by allele mining, ultimately aimed at improvement of crop tolerance to frequent combinations of stress factors.
An in vitro – in vivo integrated approach for hazard and risk assessment of silver nanoparticles for soil organisms
Makama, S.I. - \ 2016
Wageningen University. Promotor(en): Ivonne Rietjens, co-promotor(en): Nico van den Brink. - Wageningen : Wageningen University - ISBN 9789462578432 - 190 p.
particles - nanotechnology - toxicity - earthworms - gene expression - soil - coatings - deeltjes - nanotechnologie - toxiciteit - aardwormen - genexpressie - bodem - afdeklagen
Owing to their small sizes, nanoparticles (NPs) exhibit completely different and novel characteristics compared to their bulk counterparts of the same chemical composition. These novel properties include increased reactivity due to large specific surface area, fluorescence and colour changes, increased biological barrier crossings and increased material strengthening combined with light-weight. Virtually all fields of human endeavours are exploiting nanotechnology to combat different challenges. This has led to an increase in the production and potential release of NPs into the environment. The novel properties of these NPs however, mean an enhanced potential for interactions with biological systems that are different from the interactions of known conventional chemicals, thus raising environmental and public health/safety concerns. Available literature has reported NP uptake in different organisms along with associated hazards. Therefore, to safeguard human and environmental health and safety, regulatory measures are necessary. Such measures must be based on sound scientific evidence and be risk-based rather than hazard-based. As such, the need to understand the fate of NPs after environmental release and their potential to pose hazards and risks to the environment is critical for a proper risk assessment and further development of policy strategies on the future regulation of the use of NPs.
Some studies have demonstrated different and sometimes conflicting effects of NP properties on their uptake in different organisms. Given that exposure determines whether hazards will turn into risks, there is a critical need for further systematic evaluation of the physico-chemical properties of engineered or manufactured NPs that influence uptake in terrestrial organisms, and also of how soil properties may affect these processes. The objective of this project was to determine the influence of size and surface coating (charge), two important physico-chemical properties of NPs, on their bioavailability, uptake and toxicity. The red earthworm Lumbricus rubellus, common in most parts of Europe, was used as a model soil organism. Silver nanoparticles (AgNPs) have been identified as one of the most commonly used NPs in many products, and their production is expected to continue to increase. Therefore, we selected AgNPs as our model NPs. For our investigations, we applied an integrated in vitro - in vivo approach, utilising high throughput in vitro methods as well as well-established in vivo toxicity end-points in the earthworm. A systematic experimental approach was developed for which AgNPs were synthesized in three sizes: 20, 35 and 50 nm. Surface-coating with bovine serum albumin (AgNP_BSA), chitosan (AgNP_Chit), or polyvinylpyrrolidone (AgNP_PVP) resulted in negative, positive and neutral particles respectively.
Firstly, macrophage cells (RAW 264.7) were exposed to AgNPs at 0 – 200 µg/mL (nominal concentrations) and uptake dynamics, cell viability, as well as induction of tumour necrosis factor (TNF)-α and reactive oxygen species (ROS) were assessed (Chapter 2). Generally, the adverse effects of exposure to the tested AgNPs resulted in reduced overall viability of the cells, which was similar for all AgNPs tested. On adenosine triphosphate (ATP) production and specific mechanisms of toxicity (TNF-α and ROS production) however, we observed that the AgNPs differed significantly, with the negatively charged AgNP_BSA being the most toxic. Significant ROS induction was only observed after exposure to the 20 nm positively charged AgNP_Chit. Effect of size was less prominent than that of surface coating, showing mostly limited differences that were not statistically significant under our experimental conditions. Live confocal imaging of exposed cells allowed the monitoring of the uptake dynamics and subcellular cytoplasmic accumulation of AgNPs. We observed fast uptake of AgNPs within 2.5 hours which is essential in case of exposure durations of 6 and 24 hours, as applied in our experiments. However, similar uptake did not always result in similar effects.
With the insights obtained from the in vitro assessments, we investigated the effects of size and surface coating (charge) of AgNPs on the bioaccumulation in, and toxicity (survival, growth, cocoon production) to the earthworm L. rubellus. Currently, metal engineered NPs in tissues are generally quantified based on total metal concentrations after acid destruction of samples. Such destructive methods are limited in providing information on the speciation and the forms of NPs which is essential for characterising the fate of NPs. In the present thesis, we developed a method using a combination of enzymatic tissue processing and single particle inductively coupled plasma–mass spectrometry (sp-ICP-MS) to characterise and quantify AgNPs in tissues of earthworms (Chapter 3). Subcellular fractionation of tissues was also applied to investigate potential association of AgNPs with the cellular metallothionein (MT) containing fraction of the earthworm tissues. This study provided, to the best of our knowledge, the first estimates of tissue Ag concentrations in both particulate and ionic forms in earthworms exposed in vivo to AgNPs via soil. The results obtained showed fairly low uptake of AgNPs, with earthworms exposed to a commercially obtained PVP-coated AgNP showing approximately 34% of their total Ag tissue burden being in particulate form. This indicates that although AgNPs accumulated in tissues of earthworms in their primary form, the dissolution of Ag in the soil, organism, or both played an important role in determining the ultimate fate of the AgNPs. Although the biological uptake of AgNPs was generally low, the method described in Chapter 3 was still capable of extracting NPs in quantities sufficient for identification, quantification and characterisation. It should be noted however, that the lower size detection threshold for the ICP-MS instrument used for these analyses is approximately 30 nm. Consequently, information on NPs smaller than 30 nm was not available. With the increasing optimisation of analytical systems that combine sp-ICP-MS, or other detection methods with, for example, asymmetric flow field-flow fractionation (AF4) which pre-sort different particle sizes, the potential for application of methods described in this thesis will be even greater.
Having developed a method for extracting Ag from tissues, we exposed earthworms to all nine synthesized AgNPs as well as to AgNO3 at two concentrations below known EC50s to control for ionic effects of Ag in a 28-day sub-chronic reproduction toxicity test in soil in Chapter 4. Uptake was observed to be generally highest for the negatively charged AgNP_BSA especially at the lower exposure concentration ranges. Total Ag concentrations in earthworm tissues reached a plateau level of about 80 mg Ag/kg dry weight (DW) for exposure concentrations between 15 – 100 mg Ag/kg soil DW. Reproduction was impaired at high nominal soil concentrations of all AgNPs tested, with AgNP_BSA particles being the most toxic. Size had an influence on uptake of the AgNP_PVP, showing both uptake and effect on reproduction of the 20 nm sized group to be significantly more than those of the 35 and 50 nm AgNP_PVP. This size effect however, did not hold for AgNP_BSA nor AgNP_Chit. Higher uptake from the soil may consequently lead to a higher potential for toxicity in organisms. Interestingly, internal total Ag tissue concentrations measured after 72 hour exposure were better at predicting the effect on reproduction than tissue concentrations after 28 days exposure. It is likely therefore, that reproduction was affected already in the 72 hour exposure window.
In order to further elucidate the likely mechanisms by which these AgNPs were exerting their effects, we conducted a toxicogenomic study in Chapter 5. Although AgNPs have been increasingly investigated, information regarding their effect on the gene expression profile of especially soil organisms is yet inadequate. Using RNAseq, we investigated the transcriptome and gene expression profiles of the earthworm L. rubellus, following exposure to the nine AgNPs. Overall, exposure to medium sized AgNPs at a concentration close to the EC50 for effects on cocoon production caused most pronounced responses at the transcriptional level. There was a correlation however, between the numbers of differentially expressed genes (DEGs) and internal Ag concentrations in the earthworms. Within the medium size AgNPs, AgNP_BSA caused extensive transcriptional responses, with 684 genes affected. In contrast ionic silver (AgNO3) did not affect gene expression at low as well as higher exposure levels. Only one gene was regulated by all AgNP and Ag+ treatments, indicating that there was hardly any functional overlap between the responses of the organisms to AgNPs with different coatings. Remarkably, this gene was metallothionein, a cysteine-rich peptide known to strongly bind free metal ions for chelation and detoxification, which was strongly up-regulated. Gene ontology enrichment analysis for 35 nm AgNP_BSA exposures revealed a total of 33 significantly enriched gene ontology terms related to biological processes. These included responses to pH, proton transport, cell differentiation, microtubule organisation, and and MT induction. Surface coating (BSA) was important in triggering the AgNP-induced differential gene expression profiles in earthworms. The importance of physicochemical properties of NPs in influencing their fate and toxicity is thus elucidated in the current study.
The studies reported in the current thesis showed that within the range of 20 to 50 nm, effects of the size of AgNPs on toxicokinetics and toxicodynamics are limited. However, effects of surface coating were consistent over the different levels of biological integration. Generally, the negatively charged AgNP_BSA accumulated to a higher extent in the earthworms, especially at lower concentrations. The in vitro uptake was fast for all NPs, but also showed the highest uptake of AgNP_BSA. The negatively charged AgNPs were also the most toxic, likely related to their increased uptake. This was evident at all levels: gene expression, cellular, and individual (population dynamic parameters) levels. At the in vitro level, this applied mostly to effects on specific modes of action (TNF-α induction, ROS production). For more general cytotoxic effects, the effects of surface coatings were less evident. Except in cells exposed to AgNP_Chit 20 nm, where there was a slight increase in ROS production, this set of AgNPs under the experimental conditions applied, did not appear to induce the production of ROS. This was supported by the lack of expression of any ROS-related gene in the gene expression profile analyses.
Based on the results of the current research, it can be concluded that the physico-chemical properties of NPs do influence their environmental fate and toxicity. It should be noted however that general predictions on the outcome of exposure to NPs are difficult to make, and NPs should be evaluated on a case by case basis. Our research supports the use of in vitro models to limit and prioritize further in vivo studies. Studies investigating the fate and effects of NPs for soil organisms are vital for a holistic approach towards a comprehensive and adequate environmental risk assessment (ERA). The studies described in this thesis contribute to this knowledge, thereby improving our understanding of the hazards and risks due to exposure to AgNPs, thus enabling their adequate and comprehensive ERA.
Adventitious root formation in Arabidopsis : underlying mechanisms and applications
Massoumi, Mehdi - \ 2016
Wageningen 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.
Linking growing up and getting old : plastic and evolutionary effects of developmental diet on adult phenotypes and gene expression in the fruit fly
May, C.M. - \ 2016
Wageningen University. Promotor(en): Bas Zwaan, co-promotor(en): Fons Debets. - Wageningen : Wageningen University - ISBN 9789462577633 - 183 p.
drosophila melanogaster - gene expression - nutrition - evolution - diet - lifespan - fecundity - biological development - genexpressie - voeding - evolutie - dieet - levensduur - voortplantingspotentieel - biologische ontwikkeling
Role of MLO genes in susceptibility to powdery mildew in apple and grapevine
Pessina, Stefano - \ 2016
Wageningen University. Promotor(en): Richard Visser, co-promotor(en): Henk Schouten; M. Malnoy; Yuling Bai. - Wageningen : Wageningen University - ISBN 9789462576209 - 222 p.
malus domestica - apples - vitis vinifera - grapes - plant pathogenic fungi - podosphaera leucotricha - erysiphe necator - disease resistance - susceptibility - genes - gene expression - gene knock-out - resistance breeding - appels - druiven - plantenziekteverwekkende schimmels - ziekteresistentie - vatbaarheid - genen - genexpressie - inactivering van genen - resistentieveredeling
Powdery mildew (PM) is a major fungal disease that threatens thousands of plant species. PM is caused by Podosphaera leucotricha in apple and Erysiphe necator in grapevine. Powdery mildew is controlled by frequent applications of fungicides, having negative effects on the environment, and leading to additional costs for growers. To reduce the amount of chemicals required to control this pathogen, the development of resistant apple and grapevine varieties should become a priority.
PM pathogenesis is associated with up-regulation of specific MLO genes during early stages of infection, causing down-regulation of plant defense pathways. These up-regulated genes are responsible for PM susceptibility (S-genes) and their knock-out causes durable and broad-spectrum resistance. All MLO S-genes of dicots belong to the phylogenetic clade V. In grapevine, four genes belong to clade V. VvMLO7, 11 and 13 are up-regulated during PM infection, while VvMLO6 is not.
Chapter 2 reports the genome-wide characterization and sequence analysis of the MLO gene family in apple, peach and woodland strawberry, and the isolation of apricot MLO homologs. Twenty-one homologues were found in apple, 19 in peach and 17 in woodland strawberry. Evolutionary relationships between MLO homologs were studied and syntenic blocks constructed. Candidate genes for causing PM susceptibility were inferred by phylogenetic relationships with functionally characterized MLO genes and, in apple, by monitoring their expression following inoculation with the PM causal pathogen P. leucotricha. In apple, clade V genes MdMLO11 and 19 were up-regulated, whereas the two other members of clade V, MdMLO5 and 7, were not up-regulated. The clade VII gene MdMLO18 was also up-regulated upon P. leucotricha infection.
Chapter 3 reports the knock-down, through RNA interference, of MdMLO11 and 19, as well as complementation of the mutant phenotype by expression of the MdMLO18 gene in the Arabidopsis thaliana triple mlo mutant Atmlo2/6/12. The knock-down of MdMLO19 resulted in a reduction of PM disease severity up to 75%, whereas the knock-down of MdMLO11, alone or combined with MdMLO19, did not cause any reduction or additional reduction of susceptibility compared to MdMLO19 alone. Complementation by MdMLO18 did not restore susceptibility. Cell wall appositions (papillae), a response to PM infection, were found in both susceptible plants and PM resistant plants where MdMLO19 was knocked-down, but were larger in resistant lines. The expression analysis of 17 genes related to plant defense, and quantification of phenolic metabolites in resistant lines revealed line-specific changes compared to the control.
Chapter 4 evaluates the presence of non-functional alleles of the MdMLO19 S-gene in apple germplasm. The screening of the re-sequencing data of 63 apple genotypes led to the identification of 627 SNP in five MLO genes (MdMLO5, MdMLO7, MdMLO11, MdMLO18 and MdMLO19). Insertion T-1201 in MdMLO19 caused the formation of an early stop codon, resulting in a truncated protein lacking 185 amino-acids and the calmodulin-binding domain. The presence of the insertion was evaluated in a collection of 159 apple genotypes: it was homozygous in 53 genotypes, 45 of which were resistant or very resistant to PM, four partially susceptible and four not assessed. These results strongly suggest that this insertion is causative for the observed PM resistance. The absence of a clear fitness cost associated to the loss-of-function of MdMLO19, might have contributed to the high frequency of the mutation in breeding germplasm and cultivars. Among the genotypes containing the homozygous insertion, ‘McIntosh’ and ‘Fuji’ are commonly used in apple breeding. After barley and tomato, apple is the third species with a reported natural non-functional mlo allele in its germplasm, with the important difference that the allele is present in a relatively large number of apple genotypes, most of which not related to each other.
Chapter 5 reports the knock-down through RNA interference of four grapevine MLO genes, all members of clade V. VvMLO7, 11 and 13 are up-regulated in early stages of infection, whereas VvMLO6 is not responsive to the pathogen. Knock-down of VvMLO6, 11 and 13, alone or combined, did not decrease PM severity, whereas the knock-down of VvMLO7, alone or in combination with VvMLO6 and VvMLO11, caused a reduction of severity of 77%. Cell wall appositions (papillae), a response to PM attack, were present in both resistant and susceptible lines, but were larger in resistant lines. Thirteen genes involved in defense were less up-regulated in resistant plants, highlighting the reduction of PM disease severity.
In Chapter 6 we discuss the results presented in this thesis. The pivotal role of MLO genes in the interaction of PM pathogens with apple and grapevine is described and further experiments aimed at addressing open questions are proposed. The results described in this thesis open interesting avenues in MLO genes research, particularly the finding that a natural mlo mutation in apple appeared to be more common than expected. This mutation is directly applicable in marker assisted breeding for durable PM resistance in apple.
Gomphrena claussenii, a Zn and Cd hyperbioindicator species
Tomaz Villafort Carvalho, M. - \ 2016
Wageningen University. Promotor(en): Maarten Koornneef, co-promotor(en): Mark Aarts. - Wageningen : Wageningen University - ISBN 9789462576360 - 196 p.
gomphrena - indicator plants - zinc - cadmium - indicator species - metal tolerance - gene expression - indicatorplanten - zink - indicatorsoorten - metaaltolerantie - genexpressie
Gomphrena claussenii, a Zn and Cd hyperbioindicator species
A small group of plant species called metallophytes have evolved the ability to grow in highly metal-enriched soils that are toxic to other plants. Some of these metal-tolerant species have evolved the ability to accumulate high levels of metals or metalloids, such as nickel (Ni), zinc (Zn), arsenic (As), cadmium (Cd) and lead (Pb). Important progress towards understanding the physiological and molecular basis of metal and metalloid homeostasis in plants has been made by studying these metal hyperaccumulator species, which may also be useful for as the basis of phytoremediation technologies in which plants are used to stabilize or extract metals from soil. Gomphrena claussenii Moq. (Amaranthaceae), is a previously uncharacterized plant species which grows in the metal‑rich soils of a Zn mining area in the state of Minas Gerais, Brazil. This thesis describes the investigation of G. claussenii to determine the molecular basis of its ability to tolerate and accumulate Zn and Cd.
Chapter 2 presents a detailed comparative investigation of the physiological impact of Zn and Cd exposure on G. claussenii and the closely-related non-tolerant species G. elegans Mart. growing in soil or in hydroponic conditions. The impact of Zn/Cd in each species was determined by measuring growth characteristics such as biomass and root elongation. It was found that G. claussenii plants growing in the field in the Zn mining area accumulated up to 10434 µg Zn and 96 µg Cd per gram of shoot dry weight. Under hydroponic conditions, G. claussenii tolerated up to 3000 µM Zn and up to 100 µM Cd, showing only slight metal toxicity symptoms at the highest concentrations and no significant decrease in biomass or root length. In contrast, G. elegans showed significant toxicity symptoms at 100 µM Zn and 5 µM Cd. It was also found that both species accumulated more Zn and Cd in roots than shoots and that metal accumulation in G. claussenii showed a bioindicator-like response. Finally, the concentrations of other minerals such as Fe and Mn were not affected by Zn/Cd in G. claussenii shoots but declined dramatically in G. elegans in the presence of Zn/Cd. Taken together, these results indicated that G. claussenii is extremely tolerant to Zn and Cd and accumulates high levels of these metals in shoots, making it potentially valuable for phytoremediation applications.
Chapter 3 addresses the distribution of Zn/Cd in G. claussenii stem and leaf tissues, and metabolic profiles were used to investigate the involvement of metabolites in the sequestration of Zn/Cd. G. claussenii plants were exposed to high concentrations of Zn/Cd and analysed by scanning electron microscopy using energy dispersive X-ray (SEM-EDX) and micro-proton-induced X‑ray emission (micro-PIXE) technologies. We also investigated the dynamic profiles of primary metabolites in roots and shoots exposed to high levels of Zn/Cd to identify potential ligands for these metals. We observed the presence of abundant calcium oxalate (CaOx) crystals in the stem and leaf tissues of G. claussenii plants exposed to control and high levels of Cd, but intriguingly the number of crystals declined in the presence of Zn. Cd was shown to co‑localize with calcium (Ca) in the CaOx crystals, indicating that Cd sequestration in vacuolar CaOx crystals in G. claussenii is part of a tolerance mechanism to deal with excess Cd accumulation. Furthermore, citrate, malate and oxalate levels all increased in the shoots of G. claussenii exposed to Zn/Cd suggesting these organic acids are involved in metal chelation and contribute to metal tolerance.
Chapter 4 focuses on the molecular genetic aspects of hypertolerance in G. claussenii. The comparative transcriptomics analysis of G. claussenii and G. elegans was used to identify genes potentially responsible for the adaptation of G. claussenii to high Zn/Cd exposure. The transcriptional response of both species to high Zn/Cd concentrations was investigated by RNA-Seq analysis. Transcript sequences were annotated, and differential expression induced by Zn/Cd exposure was analysed in G. claussenii and G. elegans roots and shoots. Orthologous transcript pairs were identified between both species, allowing the direct comparison of gene expression profiles. G. elegans showed a stronger transcriptional response to metal exposure than G. claussenii, featuring the significant modulation of 10–20 times as many genes. Many of these transcripts encode proteins involved in metal homeostasis or stress responses. Metal hypertolerance in G. claussenii therefore appears to be a constitutive expression trait, based on adaptations in the metal homeostasis and general stress response.
Chapter 5 summarizes and evaluates the knowledge gained by the investigation set out in this thesis, focusing on the relevance of the information obtained from G. claussenii and its contribution to our current understanding of metal hypertolerance. It is also discussed the benefits of G. claussenii for phytoremediation applications, as well as its potential for future research activities.
Mapping moves on Arabidopsis : from natural variation to single genes affecting aphid behaviour
Kloth, K.J. - \ 2016
Wageningen University. Promotor(en): Marcel Dicke; Harro Bouwmeester, co-promotor(en): Maarten Jongsma. - Wageningen : Wageningen University - ISBN 9789462576483 - 269 p.
016-3933 - arabidopsis thaliana - insect pests - aphidoidea - pest resistance - genetic mapping - gene expression - quantitative traits - functional genomics - feeding behaviour - insect plant relations - insectenplagen - plaagresistentie - genetische kartering - genexpressie - kwantitatieve kenmerken - functionele genomica - voedingsgedrag - insect-plant relaties
Discovery, characterization and applications of natural DNA transformation in Streptococcus suis
Zaccaria, E. - \ 2015
Wageningen University. Promotor(en): Jerry Wells, co-promotor(en): Peter van Baarlen. - Wageningen : Wageningen University - ISBN 9789462576056 - 171
streptococcus suis - virulence - pathogenesis - gene expression - direct dna uptake - virulence factors - infection - modeling - virulentie - pathogenese - genexpressie - directe dna-opname - virulente factoren - infectie - modelleren
Streptococcus suis is Gram-positive bacterium and its natural habitat is the upper respiratory tract of pigs, and in particular the tonsils and nasal cavity. Although it is considered to be a normal member of the adult pig microbiome, it can cause serious diseases in pigs and humans. S. suis is in fact one of the most important swine pathogens world-wide, causing a wide variety of diseases in pigs including septicemia, arthritis, endocarditis, and meningitis that leads often to a rapid death within 1-2 days. Although most human infections are considered the consequence of occupational exposure, in the last years the number of human cases has increased and isolates with multi-resistance genes have been isolated. Human infection caused by S. suis are characterized by a similar symptomatology as in pigs. Despite the economic loss in the pork industry due to S. suis infection and its importance as emerging zoonotic agent, experimental studies of S. suis virulence and pathology have been hampered by the lack of efficient methods for genetic transformation and the lack of a simple, cost-effective model to investigate S. suis virulence.
In some streptococcal species, genetic transformation can be carried out very efficiently as these species can be experimentally induced to take-up and recombine homologous extracellular DNA. The discovery of natural competence in some streptococci and the potential of opening up new avenues for genetic analysis of S. suis, was the motivation for investigating natural competence in this important pathogen.
In Chapter 2 we showed that a peptide pheromone induces competence in S. suis. The induction was dependent on ComX, a sigma factor that controls the streptococcal late competence regulon; the SigX-inducing peptide (XIP); and ComR, a regulator of comX. XIP was identified as an N-terminally truncated variant of ComS. This has resulted in the development of a novel methodology that will enable diverse research groups to accelerate discovery of novel features of S. suis ecology and pathology, especially with respect to virulence.
In Chapter 3 we investigated the genetic regulation of competence in S. suis and we provided a hypothetical model of the S. suis transformasome. We verified the essential role of the S. suis major pilin, and CinA for efficient competence development, supporting the notion that our predicted multi-protein transformasome indeed appears to function as described for other streptococci. We have also characterised the differential metabolic states that enable competence, and the metabolic state associated with competence exit (Chapter 4).
In Chapter 5 we investigated for the first time the use a zebrafish larvae model to assess the relative virulence of S. suis strains in porcine infections. Because of its convenience and cost-effectiveness, this model may be used to assay virulence of environmental S. suis strains, in particularly those of clinical relevance to infection of pigs and humans. Furthermore, a large number of bacterial mutants and strains can be screened for their virulence and in vivo pathogenicity, opening up new avenues to investigate the so far undiscovered pathways mediating successful host infection by S. suis.
In Chapter 6 we applied these two innovative methods (the competence system and the zebrafish larval model) to characterize two different two-component systems (TCS) of S. suis. TCS are important players in the regulation of bacterial adaptation to changes in environmental conditions, including those encountered in the host during infection. In this study, we studied the role of the two TCS of S. suis 2 strain S10 in virulence and in the survival of the bacteria in the bloodstream and host tissue.
Chapter 7 summarizes and discusses the key results and the future prospective of the thesis research.
Studies on global transcriptional regulator EBR1 and genome-wide gene expression in the fungal plant pathogen Fusarium graminearum
Zhao, C. - \ 2015
Wageningen University. Promotor(en): Pierre de Wit; D. Tang, co-promotor(en): Theo van der Lee. - Wageningen : Wageningen University - ISBN 9789462575998 - 167
plant pathogenic fungi - gibberella zeae - transcription factors - gene expression - gene mapping - genomics - plantenziekteverwekkende schimmels - transcriptiefactoren - genexpressie - genkartering - genomica
Abstract of PhD thesis
Fusarium graminearum is a destructive plant pathogen that causes Fusarium head blight (FHB) on many crops, such as wheat, barley, rye and oat. In the first part of this thesis, we studied a transcription factor EBR1 that is required for radial growth and virulence in F. graminearum. Mutant ebr1 shows reduced apical dominance of the hyphal tip and loses its ability to penetrate the rachis of the spikelets. Subcellular localization analysis showed that EBR1 protein is exclusively localized in the nucleus of both conidia and hyphae. In the second part of thesis, by using RNA-Seq data, we revised 655 incorrectly predicted gene models and identified 231 genes with two or more alternative splice variants in F. graminearum. Furthermore, we analyzed the genome-wide gene expression pattern and found that genes locate in non-conserved regions of chromosomes showed relatively lower expression level. We further provided evidence showing that the non-conserved regions are full of gene relocations in F. graminearum.
Dissecting the seed-to-seedling transition in Arabidopsis thaliana by gene co-expression networks
Silva, A.T. - \ 2015
Wageningen University. Promotor(en): Harro Bouwmeester, co-promotor(en): Henk Hilhorst; Wilco Ligterink. - Wageningen : Wageningen University - ISBN 9789462575929 - 177
arabidopsis thaliana - zaden - zaadkieming - zaailingen - genexpressie - uitdrogingstolerantie - seeds - seed germination - seedlings - gene expression - desiccation tolerance
One of the most important developmental processes in the life-cycle of higher plants is the transition from a seed to a plant and from a generative to a vegetative developmental program. The major hallmark or end-point of the transition from seed to plant is the onset of photosynthesis and the concomitant shift from a heterotrophic to an autotrophic organism. It is advantageous for a species to keep the period of seedling establishment as short as possible since young seedlings are highly sensitive to biotic and abiotic stresses. This implies that the extreme stress tolerance of seeds to i.e. desiccation is lost upon germination. If the regulatory principles of the seed-to-seedling transition are better understood it may become feasible to maintain the seed’s stress tolerance well into the seedling stage.
Despite the profound impact of seedling performance on crop establishment and yield, the seed-to-seedling transition has hardly been studied at the molecular level. This thesis aims at deciphering and understanding the molecular processes that govern this transition in Arabidopsis thaliana. A high-resolution study of the molecular events that occur during these successive transitional stages may provide clues as to the regulatory principles that drive this transition. It may also yield information about the factors that determine the (in)ability to revert to a developmental mode and which features are critical for the maintenance and loss of desiccation tolerance and other stress responses.
In Chapter 1 important processes such as abscisic acid and their regulation are described and it is discussed in what way the seed-to-seedling transition may have links to a trait such as desiccation tolerance. An overview is presented of the current knowledge of the seed-to-seedling phase transition and the existence of a temporal developmental block that can be manipulated by osmotic treatment, the carbon/nitrogen balance and by abscisic acid which results in the re-establishment of desiccation tolerance.
Chapter 2 focuses on comprehensive gene regulation by a detailed transcriptional analysis across seven developmental stages of the seed-to-seedling transition. It describes the inference of a gene co-expression network and several transcriptional modules. I show that such an approach highlights important molecular processes during seedling development, which would not likely be derived from comparative transcript profiling. Moreover, I show that a putative key regulator in one of the transcriptional modules affects late seedling establishment.
In Chapter 3 it is shown how this phase transition is expressed in the primary metabolite profiles in correlation with gene expression. A metabolite-metabolite correlation analysis suggested two profiles, which point at the metabolic preparation of seed germination and of vigorous seedling establishment. Furthermore, a linear correlation between metabolite contents and transcript abundance (Chapter 2) provides a global view of the transcriptional and metabolic changes during the seed-to-seedling transition. It creates new perspectives of the regulatory complexes underlying the seed-to-seedling transition.
Chapter 4 describes the development of a novel method to re-establish desiccation tolerance during the seed-to-seedling transition without adverse effects such as those caused by an osmotic treatment with polyethylene glycol. By using this method, named ‘Mild Air Drying Treatment’ (MADT), I show that the re-establishment of desiccation tolerance is not linked to a reduced ability to accumulate ABA in the desiccation sensitive seeds (germinated seeds at root hair stage). I also present a genetic interaction study of ABSCISIC ACID INSENSITIVE (ABI) genes in their germination response to ABA, and their response to the re-establishment of desiccation tolerance using the MADT. The interaction between ABI3 and ABI4, and between ABI4 and ABI5 act synergistically in the re-establishment of DT, as well as in the germination response to ABA.
In a more in depth study in Chapter 5 I carried out an extensive transcript analysis to infer possible mechanisms of the re-establishment of desiccation tolerance using the MADT protocol. Possible mechanisms underlying the re-establishment of desiccation tolerance were inferred by employing a time-series comparison of germinated desiccation tolerant and -sensitive seeds. Early-response genes of the re-establishment of desiccation tolerance may play a role in events that promote the initial protection to dehydration stress, whereas the late-response genes may play a role in events that help seed to respond to the changes in water dynamics. Moreover, using a gene co-expression network and transcriptional module I concluded that a crosstalk between ABA-dependent and ABA-independent transcription factors regulate the re-establishment of desiccation tolerance.
In Chapter 6 I discuss how the results presented in this thesis contribute to our knowledge of the molecular basis of the seed-to-seedling transition and the re-establishment of desiccation tolerance during its phase changes. Finally, new possibilities for further research are discussed, as well as the further use of the data sets to delineate the mechanisms underlying the seed-to-seedling transition and desiccation tolerance. Possible applications of the results for crop improvement are addressed. Thus, the generation of genetically modified plants that produce seeds with a stress tolerance that extends well into seedling stage may be feasible.
Functional analyses of AtCHR12 and AtCHR23 : plant growth responses upon over-expression of chromatin remodeling ATPase genes
Folta, A. - \ 2015
Wageningen University. Promotor(en): Ton Bisseling, co-promotor(en): Ludmila Mlynarova. - Wageningen : Wageningen University - ISBN 9789462575561 - 144
planten - groei - genexpressie - chromatine - atp - arabidopsis - solanum lycopersicum - zaadkieming - groeiregulatoren - plants - growth - gene expression - chromatin - seed germination - growth regulators
Living organisms have to deal with changing environmental conditions during their whole life cycle. In contrast to animals, plants are sessile organisms. Therefore they have evolved multiple regulatory mechanisms that help them to cope with changing conditions. One of the first responses to stress conditions is reduction or arrest of growth. Therefore regulation of growth and development is essential to successfully complete their life cycle. To correctly time their development, plants need to integrate various environmental signals with intrinsic developmental programs. In this integration, regulation of gene expression plays a major role.
The genetic information of an organism is stored in DNA sequence. DNA forms a complex with histones and other proteins, which is called chromatin. Chromatin is a highly dynamic complex and modification of the chromatin structure makes DNA more or less accessible to the transcriptional machinery and other regulatory proteins. The modification of chromatin organization is called chromatin remodeling and it involves both covalent modifications of DNA and histone tails, and non-covalent modification of chromatin structure by ATP-dependent chromatin remodeling complexes. ATP-dependent chromatin remodeling complexes comprise multiple protein subunits with SNF2 ATPase as a catalytic subunit. Depending on the subunit composition, the complexes can perform different tasks at various places of chromatin, and can be active at different developmental stages (Chapter 1).
The SNF2 ATPases are conserved from yeast to plants. In Arabidopsis, 41 SNF2 ATPases have been identified. The focus of this thesis is on two of those ATPases – AtCHR12 and AtCHR23. It has been shown previously that AtCHR12 is involved in growth responses to environmental cues. We have extended these studies to its paralog AtCHR23 (Chapter 2). In contrast to over-expression of AtCHR12, which affects growth only during reproductive stage of development, over-expression of AtCHR23 leads to smaller seedlings and reduced vegetative growth. Upon application of mild abiotic stress, the growth reduction is stronger than in wild-type plants. Moreover, the transgenic plants manifest increased variability of growth. The increased growth variability correlates with increased expression variability of genes associated with stress. The results indicate that accurate and controlled expression of AtCHR23 is required for stability and robustness of growth, as well as gene expression.
Regulation of growth is important not only during vegetative or reproductive stage of development, but also during embryo development. The growth of the embryo is interrupted during the embryo maturation phase and it was suggested that AtCHR12 may be involved in this temporary growth arrest. Here we have shown that both AtCHR12 and AtCHR23 are expressed during embryo development, and that over-expression of AtCHR12 or AtCHR23 affects the embryo maturation phase with consequences on two important developmental transitions in plant life – germination and transition to flowering.
Over-expression of AtCHR12 or AtCHR23 leads to reduced seed germination, which is more pronounced under stress conditions (Chapter 3). The reduced germination of over-expressing lines is associated with increased transcript levels of seed maturation genes and reduced degradation of their mRNAs in germinating seeds. The results indicate that repression of AtCHR12 and AtCHR23 in germinating seeds is required for full germination.
The connection between embryo development and flowering time control was observed in transgenic lines over-expressing AtCHR12 (Chapter 4). Over-expression of AtCHR12 results in early flowering under both long- and short-day conditions. The early flowering phenotype correlates with reduced expression of the main flowering repressor, FLC. The reduced FLC expression correlates with increased levels of repressive histone mark H3K27me3 on the FLC locus. Additionally, FLC expression was affected already during FLC reprogramming, which takes place during embryo development. This leads to reduced FLC expression in mature embryos. The results show that AtCHR12 over-expression affects flowering time by different mechanisms than other Snf2-subfamily ATPases. In contrast to AtCHR12, BRAHMA was shown to regulate flowering time via the photoperiod pathway, while SPLAYED affects flowering time by repressing FT expression.
We have observed that over-expression of AtCHR12 or AtCHR23 affects plant growth in response to stress, and play a role in germination and transition to flowering. These traits are also important for agriculture, and such genes are potentially interesting targets for breeding programs. To test, if such genes have a similar role in crops, we have studied the effect of the tomato ortholog of AtCHR12 and AtCHR23 on tomato growth.
Tomato (Solanum lycopersicum), as well as other crops, have only one ortholog of AtCHR12 and AtCHR23, which was suggested to possess a role of both ATPases. We have successfully cloned the tomato ortholog and over-expressed it in tomato plants (Chapter 5). The transgenic tomato plants have reduced vegetative growth and compacted reproductive structures, resembling the phenotype of AtCHR23 and AtCHR12 over-expression, respectively. However, in contrast to Arabidopsis, the tomato plants responded to abiotic stress similarly as wild-type, and they flowered later than wild-type plants. The results indicate that modification of expression of AtCHR12 and AtCHR23 orthologs could be used to develop novel methods to control plant growth.
Taken together, the research described in this thesis identifies AtCHR12 and AtCHR23 as regulators of plant growth, especially in response to environment, as well as of the seed maturation program with clear effects on seed germination and flowering time, and we show that such genes can be potentially interesting for agriculture and horticulture practice.
Carboxypeptidase Z : an extracellular protein in zebrafish development
Kessels, M.Y. - \ 2015
Wageningen University. Promotor(en): Sacco de Vries; Johan van Leeuwen, co-promotor(en): Stefan Schulte-Merker; Sander Kranenbarg. - Wageningen : Wageningen University - ISBN 9789462575677 - 134
danio rerio - biologische ontwikkeling - carboxypeptidasen - skelet - eiwitten - eiwitexpressieanalyse - genexpressie - biological development - carboxypeptidases - skeleton - proteins - proteomics - gene expression
Historically the skeleton, in particular bone, was depicted as a rigid, inflexible, lifeless structure that readily breaks upon bending. We now know that bones in living organisms are complex, dynamic organs that combine toughness with flexibility. Astonishing is the fact that after their initial formation, bone structures are continuously remodeled (recycled and renewed), adapting to environmental demands at such a speed that in active healthy humans the distal part of the femur is completely replaced every six to twelve months. The zebrafish is a relatively new model organism in the field of skeletal development and primarily used as a powerful model for the identification of novel gene functions during skeletogenesis. At the molecular level, the biological similarity between zebrafish and humans is striking (Spoorendonk et al., 2010), but the actual skeletal composition of the zebrafish remained largely unknown.
The mechanical properties of the skeleton are largely dependent on the composition of proteins that are secreted into the extracellular matrix (ECM). In order to increase our understanding of normal skeletal development, it is necessary to identify and characterize changes in skeletal composition. The aim of this thesis was to analyze the composition of the extracellular matrix in zebrafish, in order to enable the identification of potential key regulatory proteins. By determination of the protein content in the zebrafish skeletal ECM with major changes in protein abundance during development we were able to identify various components of signaling pathways implicated in skeletogenesis. This first proteomic analysis of the zebrafish skeleton revealed the homology between the zebrafish and the skeleton of other vertebrate species including mammals. Our study provides a solid foundation for future studies on the composition and the regulation of the morphogenesis of the vertebrate skeleton.
After the identification of potential regulatory proteins in the developing zebrafish skeleton during the MS-based approach, the protein carboxypeptidase Z (Cpz) was selected for further analysis. This peptidase has previously been implicated in the Wnt signaling pathway, an elaborate pathway that regulates crucial aspects of development (Nusse and Varmus, 2012). Previous studies implicated a regulatory role for this peptidase through the processing of Wnt (Moeller et al., 2003; Wang et al., 2009). An analysis of the role of this peptidase in zebrafish was still lacking and therefore we mapped the spatio-temporal expression of cpz. We showed that expression of cpz is localized in and around juvenile zebrafish ossified structures. A more thorough analysis showed a complex expression pattern during early developmental stages that partially overlaps with that observed in other species. Furthermore, we provide a comparative view of cpz expression and the expression of its proposed ligand, Wnt4 (zebrafish Wnt4a and Wnt4b). Partial overlap with wnt4 expression provided the first evidence for a potential complementary function in the Wnt signaling pathway as observed in mammalian species.
In order to explore the role of cpz in zebrafish development, a loss-of-function mutant for zebrafish cpz was generated via TALEN-mediated mutagenesis. We show that mutant embryos display a variety of phenotypes during early development, most of which were similar to described defects in the Wnt/Calcium signaling pathway. These morphological phenotypes provide the first evidence for a connection between Cpz and regulation of the β-catenin independent Wnt signaling pathways which places the function of Cpz in a completely new perspective.
As a complement to these genetic studies an alternative approach was employed to examine the role of another pathway, the thyroid hormone system, in skeletogenesis. Thyroid hormones are required for skeletal development (Kim and Mohan, 2013), but to what extent thyroid hormone affects early bone development in zebrafish remained unclear. By exposing zebrafish embryos to the thyroid hormone triiodothyronine (T3) we showed that exposure accelerates ossification of craniofacial elements including the opercle and ceratohyal in a dose-dependent manner. This provides the first histological evidence of increased ossification in zebrafish due to thyroid hormone exposure which can be used as a starting point to explore the mechanism of thyroid hormone on skeletal development at a greater depth.
The zebrafish skeleton displays remarkable resemblance to that of other vertebrate species with regards to composition, regulatory components and hormonal response. The use of zebrafish as a model in future research will undoubtedly increase our understanding of vertebrate skeletal development and disease. In this thesis we provide a first insight in the extracellular protein content of the zebrafish skeleton, identify a role for the Cpz protein in β-catenin independent (non-canonical) Wnt signaling during development and shows the effect of thyroid hormone on ossification during early zebrafish development.
Plant Biotechnology meets Immunology : plant-based expression of immunologically relevant proteins
Wilbers, R.H.P. - \ 2015
Wageningen University. Promotor(en): Jaap Bakker, co-promotor(en): Arjen Schots; Geert Smant. - Wageningen : Wageningen University - ISBN 9789462574335 - 229
plantenbiotechnologie - immunologie - planten - eiwitten - farmaceutische eiwitten - interleukine 10 - ontstekingsremmers - biologische activiteit - cytokinen - genexpressie - transforming growth factor - wormen - recombinant eiwitten - glycoproteïnen - plant biotechnology - immunology - plants - proteins - pharmaceutical proteins - interleukin 10 - antiinflammatory agents - biological activity - cytokines - gene expression - helminths - recombinant proteins - glycoproteins
The incidence of inflammatory disorders in industrialized countries has dramatically increased over the last decennia, which is believed to result from a change in life-style. Treatment of these inflammatory disorders relies on the intervention in immune responses thereby restoring homeostasis. For now, many inflammatory disorders are treated with broad-acting immunosuppressive drugs or monoclonal antibodies that specifically target pro-inflammatory molecules of the immune system. An alternative therapeutic approach would be to use immunomodulatory proteins that are naturally involved in re-establishing immune homeostasis. This thesis describes the plant-based expression of a variety of immunomodulatory cytokines that may be used as biopharmaceutical proteins in the future. Furthermore, this thesis contains a pioneering chapter on the plant-based expression of immunomodulatory helminth-secreted glycoproteins.
In Chapter 2 we describe the plant-based expression of the immune-regulatory cytokine human transforming growth factor β1 (TGF-β1). By co-expressing human furin with latent TGF-β1 we were able to engineer the post-translational proteolytic processing of TGF-β1, which enabled the production of biologically active TGF-β1. In Chapter 3 we reveal that aggregation is a major production bottleneck for the anti-inflammatory cytokine interleukin-10 (IL-10). By protein engineering we were able to prevent aggregation and created a biologically active fusion protein of IL-10. In Chapter 4 we express biologically active IL-22 in plants. We reveal that, in contrast to current literature, its activity is independent of the presence of N-glycans or their composition. This chapter further reveals that plants offer a powerful tool to allow investigation into the role of N-glycans in protein folding and biological activity of glycoproteins. In Chapter 5 we further explore the potential of glyco-engineering in plants by engineering helminth-like N-glycans. We produce large quantities of two major egg antigens from Schistosoma mansoni and successfully engineer Lewis X, LDN and LDNF N-glycan structures. These plant biotechnological research lines are a showcase for the potential of engineering proteins as well as post-translational modifications in plants with special emphasis on N-glycan engineering. Altogether, the results presented in the first four chapters reveal the remarkable flexibility of plants as a production platform for recombinant proteins. It showcases the potential of engineering proteins as well as post-translational modifications in plants, but it especially highlights the engineering of tailor made N-glycans in plants. This, combined with the speed of transient expression by means of agroinfiltration, makes transient expression in Nicotiana benthamiana a powerful tool to study the role of N-glycans on glycoprotein function.
In parallel to these plant biotechnological research lines, we also developed an in vitro model system based on mouse bone marrow-derived cells to study immunological responses. We used this model to obtain clues on why IL-10 therapy has not been as successful as previously anticipated. In Chapter 6 we have set-up biological activity assays based on bone marrow-derived cells and reveal that IL-10 activity is dependent on both IL-10R1 and IL-10R2, but not IL-10R2-associated signalling via Tyk2. We also show that interactions between IL-10R1 and IL-10R2 (both intracellular and extracellular) reduce cellular binding of IL-10, but are crucial to initiate IL-10 mediated signalling. Furthermore, we observed that macrophages and dendritic cells respond differently to IL-10. This was further investigated in Chapter 7 where we reveal that GM-CSF (the cytokine used to differentiate dendritic cells) is responsible for negatively regulating early IL-10-mediated responses. Strikingly, GM-CSF does not strongly affect the IL-10-induced activation of the transcription factor STAT3. Instead, GM-CSF induces strong constitutive phosphorylation of GSK-3β, a signalling component downstream of the PI3K/Akt pathway. These immunological chapters give novel insights on the mechanism of initiating IL-10-induced signalling and on the possible integration of signal transduction pathways elicited by different cytokines. Ultimately this knowledge could provide us with new therapeutic strategies to treat inflammatory disorders.