570 Biowissenschaften; Biologie
Refine
Year of publication
- 2011 (25) (remove)
Document Type
- Doctoral Thesis (17)
- Postprint (6)
- Article (1)
- Master's Thesis (1)
Language
- English (25) (remove)
Is part of the Bibliography
- yes (25)
Keywords
- Dictyostelium (2)
- Evolution (2)
- Koexpression (2)
- Systembiologie (2)
- Acinonyx jubatus (1)
- Aggression (1)
- Aktomyosin (1)
- Alzheimer (1)
- Amyloid beta (1)
- Arabidopsis (1)
A systems biological approach towards the molecular basis of heterosis in Arabidopsis thaliana
(2011)
Heterosis is defined as the superiority in performance of heterozygous genotypes compared to their corresponding genetically different homozygous parents. This phenomenon is already known since the beginning of the last century and it has been widely used in plant breeding, but the underlying genetic and molecular mechanisms are not well understood. In this work, a systems biological approach based on molecular network structures is proposed to contribute to the understanding of heterosis. Hybrids are likely to contain additional regulatory possibilities compared to their homozygous parents and, therefore, they may be able to correctly respond to a higher number of environmental challenges, which leads to a higher adaptability and, thus, the heterosis phenomenon. In the network hypothesis for heterosis, presented in this work, more regulatory interactions are expected in the molecular networks of the hybrids compared to the homozygous parents. Partial correlations were used to assess this difference in the global interaction structure of regulatory networks between the hybrids and the homozygous genotypes. This network hypothesis for heterosis was tested on metabolite profiles as well as gene expression data of the two parental Arabidopsis thaliana accessions C24 and Col-0 and their reciprocal crosses. These plants are known to show a heterosis effect in their biomass phenotype. The hypothesis was confirmed for mid-parent and best-parent heterosis for either hybrid of our experimental metabolite as well as gene expression data. It was shown that this result is influenced by the used cutoffs during the analyses. Too strict filtering resulted in sets of metabolites and genes for which the network hypothesis for heterosis does not hold true for either hybrid regarding mid-parent as well as best-parent heterosis. In an over-representation analysis, the genes that show the largest heterosis effects according to our network hypothesis were compared to genes of heterotic quantitative trait loci (QTL) regions. Separately for either hybrid regarding mid-parent as well as best-parent heterosis, a significantly larger overlap between the resulting gene lists of the two different approaches towards biomass heterosis was detected than expected by chance. This suggests that each heterotic QTL region contains many genes influencing biomass heterosis in the early development of Arabidopsis thaliana. Furthermore, this integrative analysis led to a confinement and an increased confidence in the group of candidate genes for biomass heterosis in Arabidopsis thaliana identified by both approaches.
Non-mycorrhizal fungal endophytes are able to colonize internally roots without causing visible disease symptoms establishing neutral or mutualistic associations with plants. These fungi known as non-clavicipitaceous endophytes have a broad host range of monocot and eudicot plants and are highly diverse. Some of them promote plant growth and confer increased abiotic-stress tolerance and disease resistance. According to such possible effects on host plants, it was aimed to isolate and to characterize native fungal root endophytes from tomato (Lycopersicon esculentum Mill.) and to analyze their effects on plant development, plant resistance and fruit yield and quality together with the model endophyte Piriformospora indica. Fifty one new fungal strains were isolated from desinfected tomato roots of four different crop sites in Colombia. These isolates were roughly characterized and fourteen potential endophytes were further analyzed concerning their taxonomy, their root colonization capacity and their impact on plant growth. Sequencing of the ITS region from the ribosomal RNA gene cluster and in-depth morphological characterisation revealed that they correspond to different phylogenetic groups among the phylum Ascomycota. Nine different morphotypes were described including six dark septate endophytes (DSE) that did not correspond to the Phialocephala group. Detailed confocal microscopy analysis showed various colonization patterns of the endophytes inside the roots ranging from epidermal penetration to hyphal growth through the cortex. Tomato pot experiments under glass house conditions showed that they differentially affect plant growth depending on colonization time and inoculum concentration. Three new isolates (two unknown fungal endophyte DSE48, DSE49 and one identified as Leptodontidium orchidicola) with neutral or positiv effects were selected and tested in several experiments for their influence on vegetative growth, fruit yield and quality and their ability to diminish the impact of the pathogen Verticillium dahliae on tomato plants. Although plant growth promotion by all three fungi was observed in young plants, vegetative growth parameters were not affected after 22 weeks of cultivation except a reproducible increase of root diameter by the endophyte DSE49. Additionally, L. orchidicola increased biomass and glucose content of tomato fruits, but only at an early date of harvest and at a certain level of root colonization. Concerning bioprotective effects, the endophytes DSE49 and L. orchidicola decreased significantly disease symptoms caused by the pathogen V. dahliae, but only at a low dosis of the pathogen. In order to analyze, if the model root endophytic fungus Piriformospora indica could be suitable for application in production systems, its impact on tomato was evaluated. Similarly to the new fungal isolates, significant differences for vegetative growth parameters were only observable in young plants and, but protection against V. dahliae could be seen in one experiment also at high dosage of the pathogen. As the DSE L. orchidicola, P. indica increased the number and biomass of marketable tomatoes only at the beginning of fruit setting, but this did not lead to a significant higher total yield. If the effects on growth are due to a better nutrition of the plant with mineral element was analyzed in barley in comparison to the arbuscular mycorrhizal fungus Glomus mosseae. While the mycorrhizal fungus increased nitrogen and phosphate uptake of the plant, no such effect was observed for P. indica. In summary this work shows that many different fungal endophytes can be also isolated from roots of crops and, that these isolates can have positive effects on early plant development. This does, however, not lead to an increase in total yield or in improvement of fruit quality of tomatoes under greenhouse conditions.
Actin-based directional motility is important for embryonic development, wound healing, immune responses, and development of tissues. Actin and myosin are essential players in this process that can be subdivided into protrusion, adhesion, and traction. Protrusion is the forward movement of the membrane at the leading edge of the cell. Adhesion is required to enable movement along a substrate, and traction finally leads to the forward movement of the entire cell body, including its organelles. While actin polymerization is the main driving force in cell protrusions, myosin motors lead to the contraction of the cell body. The goal of this work was to study the regulatory mechanisms of the motile machinery by selecting a representative key player for each stage of the signaling process: the regulation of Arp2/3 activity by WASP (actin system), the role of cGMP in myosin II assembly (myosin system), and the influence of phosphoinositide signaling (upstream receptor pathway). The model organism chosen for this work was the social ameba Dictyostelium discoideum, due to the well-established knowledge of its cytoskeletal machinery, the easy handling, and the high motility of its vegetative and starvation developed cells. First, I focused on the dynamics of the actin cytoskeleton by modulating the activity of one of its key players, the Arp2/3 complex. This was achieved using the carbazole derivative Wiskostatin, an inhibitor of the Arp2/3 activator WASP. Cells treated with Wiskostatin adopted a round shape, with no of few pseudopodia. With the help of a microfluidic cell squeezer device, I could show that Wiskostatin treated cells display a reduced mechanical stability, comparable to cells treated with the actin disrupting agent Latrunculin A. Furthermore, the WASP inhibited cells adhere stronger to a surface and show a reduced motility and chemotactic performance. However, the overall F-actin content in the cells was not changed. Confocal microscopy and TIRF microscopy imaging showed that the cells maintained an intact actin cortex. Localized dynamic patches of increased actin polymerization were observed that, however, did not lead to membrane deformation. This indicated that the mechanisms of actin-driven force generation were impaired in Wiskostatin treated cells. It is concluded that in these cells, an altered architecture of the cortical network leads to a reduced overall stiffness of the cell, which is insufficient to support the force generation required for membrane deformation and pseudopod formation. Second, the role of cGMP in myosin II dynamics was investigated. Cyclic GMP is known to regulate the association of myosin II with the cytoskeleton. In Dictyostelium, intracellular cGMP levels increase when cells are exposed to chemoattractants, but also in response to osmotic stress. To study the influence of cyclic GMP on actin and myosin II dynamics, I used the laser-induced photoactivation of a DMACM-caged-Br-cGMP to locally release cGMP inside the cell. My results show that cGMP directly activates the myosin II machinery, but is also able to induce an actin response independently of cAMP receptor activation and signaling. The actin response was observed in both vegetative and developed cells. Possible explanations include cGMP-induced actin polymerization through VASP (vasodilator-stimulated phosphoprotein) or through binding of cGMP to cyclic nucleotide-dependent kinases. Finally, I investigated the role of phosphoinositide signaling using the Polyphosphoinositide-Binding Peptide (PBP10) that binds preferentially to PIP2. Phosphoinositides can recruit actin-binding proteins to defined subcellular sites and alter their activity. Neutrophils, as well as developed Dictyostelium cells produce PIP3 in the plasma membrane at their leading edge in response to an external chemotactic gradient. Although not essential for chemotaxis, phosphoinositides are proposed to act as an internal compass in the cell. When treated with the peptide PBP10, cells became round, with fewer or no pseudopods. PH-CRAC translocation to the membrane still occurs, even at low cAMP stimuli, but cell motility (random and directional) was reduced. My data revealed that the decrease in the pool of available PIP2 in the cell is sufficient to impair cell motility, but enough PIP2 remains so that PIP3 is formed in response to chemoattractant stimuli. My data thus highlights how sensitive cell motility and morphology are to changes in the phosphoinositide signaling. In summary, I have analyzed representative regulatory mechanisms that govern key parts of the motile machinery and characterized their impact on cellular properties including mechanical stability, adhesion and chemotaxis.
Background
In many species males face a higher predation risk than females because males display elaborate traits that evolved under sexual selection, which may attract not only females but also predators. Females are, therefore, predicted to avoid such conspicuous males under predation risk. The present study was designed to investigate predator-induced changes of female mating preferences in Atlantic mollies (Poecilia mexicana). Males of this species show a pronounced polymorphism in body size and coloration, and females prefer large, colorful males in the absence of predators.
Results
In dichotomous choice tests predator-naïve (lab-reared) females altered their initial preference for larger males in the presence of the cichlid Cichlasoma salvini, a natural predator of P. mexicana, and preferred small males instead. This effect was considerably weaker when females were confronted visually with the non-piscivorous cichlid Vieja bifasciata or the introduced non-piscivorous Nile tilapia (Oreochromis niloticus). In contrast, predator experienced (wild-caught) females did not respond to the same extent to the presence of a predator, most likely due to a learned ability to evaluate their predators' motivation to prey.
Conclusions
Our study highlights that (a) predatory fish can have a profound influence on the expression of mating preferences of their prey (thus potentially affecting the strength of sexual selection), and females may alter their mate choice behavior strategically to reduce their own exposure to predators. (b) Prey species can evolve visual predator recognition mechanisms and alter their mate choice only when a natural predator is present. (c) Finally, experiential effects can play an important role, and prey species may learn to evaluate the motivational state of their predators.
Genetic variation is crucial for the long-term survival of the species as it provides the potential for adaptive responses to environmental changes such as emerging diseases. The Major Histocompatibility Complex (MHC) is a gene family that plays a central role in the vertebrate’s immune system by triggering the adaptive immune response after exposure to pathogens. MHC genes have become highly suitable molecular markers of adaptive significance. They synthesize two primary cell surface molecules namely MHC class I and class II that recognize short fragments of proteins derived respectively from intracellular (e.g. viruses) and extracellular (e.g. bacteria, protozoa, arthropods) origins and present them to immune cells. High levels of MHC polymorphism frequently observed in natural populations are interpreted as an adaptation to detect and present a wide array of rapidly evolving pathogens. This variation appears to be largely maintained by positive selection driven mainly by pathogenic selective pressures. For my doctoral research I focused on MHC I and II variation in free-ranging cheetahs (Acinonyx jubatus) and leopards (Panthera pardus) on Namibian farmlands. Both felid species are sympatric thus subject to similar pathogenic pressures but differ in their evolutionary and demographic histories. The main aims were to investigate 1) the extent and patterns of MHC variation at the population level in both felids, 2) the association between levels of MHC variation and disease resistance in free-ranging cheetahs, and 3) the role of selection at different time scales in shaping MHC variation in both felids. Cheetahs and leopards represent the largest free-ranging carnivores in Namibia. They concentrate in unprotected areas on privately owned farmlands where domestic and other wild animals also occur and the risk of pathogen transmission is increased. Thus, knowledge on adaptive genetic variation involved in disease resistance may be pertinent to both felid species’ conservation. The cheetah has been used as a classic example in conservation genetics textbooks due to overall low levels of genetic variation. Reduced variation at MHC genes has been associated with high susceptibility to infectious diseases in cheetahs. However, increased disease susceptibility has only been observed in captive cheetahs whereas recent studies in free-ranging Namibian cheetahs revealed a good health status. This raised the question whether the diversity at MHC I and II genes in free-ranging cheetahs is higher than previously reported. In this study, a total of 10 MHC I alleles and four MHC II alleles were observed in 149 individuals throughout Namibia. All alleles but one likely belong to functional MHC genes as their expression was confirmed. The observed alleles belong to four MHC I and three MHC II genes in the species as revealed by phylogenetic analyses. Signatures of historical positive selection acting on specific sites that interact directly with pathogen-derived proteins were detected in both MHC classes. Furthermore, a high genetic differentiation at MHC I was observed between Namibian cheetahs from east-central and north-central regions known to differ substantially in exposure to feline-specific viral pathogens. This suggests that the patterns of MHC I variation in the current population mirrors different pathogenic selective pressure imposed by viruses. Cheetahs showed low levels of MHC diversity compared with other mammalian species including felids, but this does not seem to influence the current immunocompetence of free-ranging cheetahs in Namibia and contradicts the previous conclusion that the cheetah is a paradigm species of disease susceptibility. However, it cannot be ruled out that the low MHC variation might limit a prosperous immunocompetence in the case of an emerging disease scenario because none of the remaining alleles might be able to recognize a novel pathogen. In contrast to cheetahs, leopards occur in most parts of Africa being perhaps the most abundant big cat in the continent. Leopards seem to have escaped from large-scale declines due to epizootics in the past in contrast to some free-ranging large carnivore populations in Africa that have been afflicted by epizootics. Currently, no information about the MHC sequence variation and constitution in African leopards exists. In this study, I characterized genetic variation at MHC I and MHC II genes in free-ranging leopards from Namibia. A total of six MHC I and six MHC II sequences were detected in 25 individuals from the east-central region. The maximum number of sequences observed per individual suggests that they likely correspond to at least three MHC I and three MHC II genes. Hallmarks of MHC evolution were confirmed such as historical positive selection, recombination and trans-species polymorphism. The low MHC variation detected in Namibian leopards is not conclusive and further research is required to assess the extent of MHC variation in different areas of its geographic range. Results from this thesis will contribute to better understanding the evolutionary significance of MHC and conservation implications in free-ranging felids. Translocation of wildlife is an increasingly used management tool for conservation purposes that should be conducted carefully as it may affect the ability of the translocated animals to cope with different pathogenic selective pressures.
AM symbiosis has a positive influence on plant P-nutrition and growth, but little is known about the molecular mechanism of the symbiosis adaptation to different phosphate conditions. The recently described induction of several pri-miR399 transcripts in mycorrhizal shoots and subsequent accumulation of mature miR399 in mycorrhizal roots indicates that local PHO2 expression must be controlled during symbiosis, presumably in order to sustain AM symbiosis development, in spite of locally increased Pi-concentration. A reverse genetic approach used in this study demonstrated that PHO2 and thus the PHR1-miR399-PHO2 signaling pathway, is involved in certain stages of progressive root colonization. In addition, a transcriptomic approach using a split-root system provided a comprehensive insight into the systemic transcriptional changes in mycorrhizal roots and shoots of M. truncatula in response to high phosphate conditions. With regard to the transcriptional responses of the root system, the results indicate that, although the colonization is drastically reduced, AM symbiosis is still functional at high Pi concentrations and might still be beneficial to the plant. Additionally, the data suggest that a specific root-borne mycorrhizal signal systemically induces protein synthesis, amino acid metabolism and photosynthesis at low Pi conditions, which is abolished at high Pi conditions. MiRNAs, such as miR399, are involved in long-distance signaling and are therefore potential systemic signals involved in AM symbiosis. A deep-sequencing approach identified 243 novel miRNAs in the root tissue of M. truncatula. Read-count analysis, qRT-PCR measurements and in situ hybridizations clearly indicated a regulation of miR5229a/b, miR5204, miR160f*, miR160c, miR169 and miR169d*/l*/m*/e.2* during arbuscular mycorrhizal symbiosis. Moreover, miR5204* represses a GRAS TF, which is specifically transcribed in mycorrhizal roots. Since miR5204* is induced by high Pi it might represent a further Pi status-mediating signal beside miR399. This study provides additional evidence that MtNsp2, a key regulator of symbiosis-signaling, is regulated and presumably spatially restricted by miR171h cleavage. In summary, a repression of mycorrhizal root colonization at high phosphate status is most likely due to a repression of the phosphate starvation responses and the loss of beneficial responses in mycorrhizal shoots. These findings provide a new basis for investigating the regulatory network leading to cellular reprogramming during interaction between plants, arbuscular mycorrhizal fungi and different phosphate conditions.
Indirect resource competition and interference are widely occurring mechanisms of interspecific interactions. We have studied the seasonal expression of these two interaction types within a two-species, boreal small mammal system. Seasons differ by resource availability, individual breeding state and intraspecific social system. Live-trapping methods were used to monitor space use and reproduction in 14 experimental populations of bank voles Myodes glareolus in large outdoor enclosures with and without a dominant competitor, the field vole Microtus agrestis. We further compared vole behaviour using staged dyadic encounters in neutral arenas in both seasons. Survival of the non-breeding overwintering bank voles was not affected by competition. In the spring, the numbers of male bank voles, but not of females, were reduced significantly in the competition populations. Bank vole home ranges expanded with vole density in the presence of competitors, indicating food limitation. A comparison of behaviour between seasons based on an analysis of similarity revealed an avoidance of costly aggression against opponents, independent of species. Interactions were more aggressive during the summer than during the winter, and heterospecific encounters were more aggressive than conspecific encounters. Based on these results, we suggest that interaction types and their respective mechanisms are not either-or categories and may change over the seasons. During the winter, energy constraints and thermoregulatory needs decrease direct aggression, but food constraints increase indirect resource competition. Direct interference appears in the summer, probably triggered by each individual's reproductive and hormonal state and the defence of offspring against conspecific and heterospecific intruders. Both interaction forms overlap in the spring, possibly contributing to spring declines in the numbers of subordinate species.
Indirect resource competition and interference are widely occurring mechanisms of interspecific interactions. We have studied the seasonal expression of these two interaction types within a two-species, boreal small mammal system. Seasons differ by resource availability, individual breeding state and intraspecific social system. Live-trapping methods were used to monitor space use and reproduction in 14 experimental populations of bank voles Myodes glareolus in large outdoor enclosures with and without a dominant competitor, the field vole Microtus agrestis. We further compared vole behaviour using staged dyadic encounters in neutral arenas in both seasons. Survival of the non-breeding overwintering bank voles was not affected by competition. In the spring, the numbers of male bank voles, but not of females, were reduced significantly in the competition populations. Bank vole home ranges expanded with vole density in the presence of competitors, indicating food limitation. A comparison of behaviour between seasons based on an analysis of similarity revealed an avoidance of costly aggression against opponents, independent of species. Interactions were more aggressive during the summer than during the winter, and heterospecific encounters were more aggressive than conspecific encounters. Based on these results, we suggest that interaction types and their respective mechanisms are not either–or categories and may change over the seasons. During the winter, energy constraints and thermoregulatory needs decrease direct aggression, but food constraints increase indirect resource competition. Direct interference appears in the summer, probably triggered by each individual’s reproductive and hormonal state and the defence of offspring against conspecific and heterospecific intruders. Both interaction forms overlap in the spring, possibly contributing to spring declines in the numbers of subordinate species.
Regulation of gene transcription plays a major role in mediating cellular responses and physiological behavior in all known organisms. The finding that similar genes are often regulated in a similar manner (co-regulated or "co-expressed") has directed several "guilt-by-association" approaches in order to reverse-engineer the cellular transcriptional networks using gene expression data as a compass. This kind of studies has been considerably assisted in the recent years by the development of high-throughput transcript measurement platforms, specifically gene microarrays and next-generation sequencing. In this thesis, I describe several approaches for improving the extraction and interpretation of the information contained in microarray based gene expression data, through four steps: (1) microarray platform design, (2) microarray data normalization, (3) gene network reverse engineering based on expression data and (4) experimental validation of expression-based guilt-by-association inferences. In the first part test case is shown aimed at the generation of a microarray for Thellungiella salsuginea, a salt and drought resistant close relative to the model plant Arabidopsis thaliana; the transcripts of this organism are generated on the combination of publicly available ESTs and newly generated ad-hoc next-generation sequencing data. Since the design of a microarray platform requires the availability of highly reliable and non-redundant transcript models, these issues are addressed consecutively, proposing several different technical solutions. In the second part I describe how inter-array correlation artifacts are generated by the common microarray normalization methods RMA and GCRMA, together with the technical and mathematical characteristics underlying the problem. A solution is proposed in the form of a novel normalization method, called tRMA. The third part of the thesis deals with the field of expression-based gene network reverse engineering. It is shown how different centrality measures in reverse engineered gene networks can be used to distinguish specific classes of genes, in particular essential genes in Arabidopsis thaliana, and how the use of conditional correlation can add a layer of understanding over the information flow processes underlying transcript regulation. Furthermore, several network reverse engineering approaches are compared, with a particular focus on the LASSO, a linear regression derivative rarely applied before in global gene network reconstruction, despite its theoretical advantages in robustness and interpretability over more standard methods. The performance of LASSO is assessed through several in silico analyses dealing with the reliability of the inferred gene networks. In the final part, LASSO and other reverse engineering methods are used to experimentally identify novel genes involved in two independent scenarios: the seed coat mucilage pathway in Arabidopsis thaliana and the hypoxic tuber development in Solanum tuberosum. In both cases an interesting method complementarity is shown, which strongly suggests a general use of hybrid approaches for transcript expression-based inferences. In conclusion, this work has helped to improve our understanding of gene transcription regulation through a better interpretation of high-throughput expression data. Part of the network reverse engineering methods described in this thesis have been included in a tool (CorTo) for gene network reverse engineering and annotated visualization from custom transcription datasets.
Although horses and donkeys belong to the same genus, their genetic characteristics probably result in specific proteomes and post-translational modifications (PTM) of proteins. Since PTM can alter protein properties, specific PTM may contribute to species-specific characteristics. Therefore, the aim of the present study was to analyse differences in serum protein profiles of horses and donkeys as well as mules, which combine the genetic backgrounds of both species. Additionally, changes in PTM of the protein transthyretin (TTR) were analysed. Serum protein profiles of each species (five animals per species) were determined using strong anion exchanger ProteinChips (R) (Bio-Rad, Munich, Germany) in combination with surface-enhanced laser desorption ionisation-time of flight MS. The PTM of TTR were analysed subsequently by immunoprecipitation in combination with matrix-assisted laser desorption ionisation-time of flight MS. Protein profiling revealed species-specific differences in the proteome, with some protein peaks present in all three species as well as protein peaks that were unique for donkeys and mules, horses and mules or for horses alone. The molecular weight of TTR of horses and donkeys differed by 30Da, and both species revealed several modified forms of TTR besides the native form. The mass spectra of mules represented a merging of TTR spectra of horses and donkeys. In summary, the present study indicated that there are substantial differences in the proteome of horses and donkeys. Additionally, the results probably indicate that the proteome of mules reveal a higher similarity to donkeys than to horses.