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- Institut für Biochemie und Biologie (3471) (remove)
Domestication syndrome has resulted in the large loss of genetic variation of crop plants. Because of such genetic loss, productivity of various beneficial secondary (specialized) metabolites that protect against abiotic/biotic stresses, has been narrowed in many domesticated crops. Many key regulators or structural genes of secondary metabolic pathways in the domesticated as well as wild tomatoes are still largely unknown. In recent studies, metabolic quantitative trait loci (mQTL) analysis using the population of introgression lines (ILs), each containing a single introgression from Solanum pennellii (wild tomato) in the genetic background of domesticated tomato (M82, Solanum lycopersicum), has been used for investigation of metabolic regulation and key genes involved in both primary and secondary metabolism. In this thesis, three research projects, i) understanding of metabolic linkage between branched chain amino acids (BCAAs) and secondary metabolism using antisense lines of BCAAs metabolic genes, ii) investigation of novel key genes involved in tomato secondary metabolism and fruit ripening, iii) mapping of drought stress responsive mQTLs in tomato, are presented and discussed. In the first part, metabolic linkage between leucine and secondary metabolism is investigated by analyzing antisense lines of four key genes (ketol-acid reductoisomerase, KARI; dihydroxy-acid dehydratase, DHAD; isopropylmalate dehydratase, IPMD and branched chain aminotransferases1, BCAT1) found previously in mQTL of leucine contents. Obtained results indicate that KARI might be a rate limiting enzyme for iC5 acyl-sucrose synthesis in young leaf but not in red ripe fruits. By integrating obtained results with previous reports, inductive metabolic linkage between BCAAs and other secondary metabolic pathways at DHAD transcriptional levels in fruit is proposed. In the second part, candidate genes that are involved in secondary metabolism and fruit ripening in tomato were found by the approach of expression quantitative trait loci (eQTL) analysis. To predict functions of those candidate genes, functional validation by virus induced gene silencing and transient overexpression were performed. Results obtained by analyzing T0 overexpression and artificial miRNA lines for some of those candidates confirm their predicted functions, for example involved in fruit ripening (WD40, Solyc04g005020) and iC5 acyl-sucrose synthesis (P450, Solyc03g111940). In the third part, mapping of drought stress responsive mQTLs was performed using 57 S. pennellii ILs population. Evaluation of genetic architecture of mQTL analysis resulted in identifying drought responsive ILs (11-2, 8-3-1, 10-1-1 and 3-1). Location of well characterized regulators in these ILs helped to filter potential new key genes involved in drought stress tolerance. Obtained results suggests us our approaches could be viable for narrowing down potential candidates involved in creating interspecific variation in secondary metabolite content and at the level of fruit ripening.
Systems biology aims at investigating biological systems in its entirety by gathering and analyzing large-scale data sets about the underlying components. Computational systems biology approaches use these large-scale data sets to create models at different scales and cellular levels. In addition, it is concerned with generating and testing hypotheses about biological processes. However, such approaches are inevitably leading to computational challenges due to the high dimensionality of the data and the differences in the dimension of data from different cellular layers.
This thesis focuses on the investigation and development of computational approaches to analyze metabolite profiles in the context of cellular networks. This leads to determining what aspects of the network functionality are reflected in the metabolite levels. With these methods at hand, this thesis aims to answer three questions: (1) how observability of biological systems is manifested in metabolite profiles and if it can be used for phenotypical comparisons; (2) how to identify couplings of reaction rates from metabolic profiles alone; and (3) which regulatory mechanism that affect metabolite levels can be distinguished by integrating transcriptomics and metabolomics read-outs.
I showed that sensor metabolites, identified by an approach from observability theory, are more correlated to each other than non-sensors. The greater correlations between sensor metabolites were detected both with publicly available metabolite profiles and synthetic data simulated from a medium-scale kinetic model. I demonstrated through robustness analysis that correlation was due to the position of the sensor metabolites in the network and persisted irrespectively of the experimental conditions. Sensor metabolites are therefore potential candidates for phenotypical comparisons between conditions through targeted metabolic analysis.
Furthermore, I demonstrated that the coupling of metabolic reaction rates can be investigated from a purely data-driven perspective, assuming that metabolic reactions can be described by mass action kinetics. Employing metabolite profiles from domesticated and wild wheat and tomato species, I showed that the process of domestication is associated with a loss of regulatory control on the level of reaction rate coupling. I also found that the same metabolic pathways in Arabidopsis thaliana and Escherichia coli exhibit differences in the number of reaction rate couplings.
I designed a novel method for the identification and categorization of transcriptional effects on metabolism by combining data on gene expression and metabolite levels. The approach determines the partial correlation of metabolites with control by the principal components of the transcript levels. The principle components contain the majority of the transcriptomic information allowing to partial out the effect of the transcriptional layer from the metabolite profiles. Depending whether the correlation between metabolites persists upon controlling for the effect of the transcriptional layer, the approach allows us to group metabolite pairs into being associated due to post-transcriptional or transcriptional regulation, respectively. I showed that the classification of metabolite pairs into those that are associated due to transcriptional or post-transcriptional regulation are in agreement with existing literature and findings from a Bayesian inference approach.
The approaches developed, implemented, and investigated in this thesis open novel ways to jointly study metabolomics and transcriptomics data as well as to place metabolic profiles in the network context. The results from these approaches have the potential to provide further insights into the regulatory machinery in a biological system.
Microbial processing of organic matter (OM) in the freshwater biosphere is a key component of global biogeochemical cycles. Freshwaters receive and process valuable amounts of leaf OM from their terrestrial landscape. These terrestrial subsidies provide an essential source of energy and nutrients to the aquatic environment as a function of heterotrophic processing by fungi and bacteria. Particularly in freshwaters with low in-situ primary production from algae (microalgae, cyanobacteria), microbial turnover of leaf OM significantly contributes to the productivity and functioning of freshwater ecosystems and not least their contribution to global carbon cycling.
Based on differences in their chemical composition, it is believed that leaf OM is less bioavailable to microbial heterotrophs than OM photosynthetically produced by algae. Especially particulate leaf OM, consisting predominantly of structurally complex and aromatic polymers, is assumed highly resistant to enzymatic breakdown by microbial heterotrophs. However, recent research has demonstrated that OM produced by algae promotes the heterotrophic breakdown of leaf OM in aquatic ecosystems, with profound consequences for the metabolism of leaf carbon (C) within microbial food webs. In my thesis, I aimed at investigating the underlying mechanisms of this so called priming effect of algal OM on the use of leaf C in natural microbial communities, focusing on fungi and bacteria.
The works of my thesis underline that algal OM provides highly bioavailable compounds to the microbial community that are quickly assimilated by bacteria (Paper II). The substrate composition of OM pools determines the proportion of fungi and bacteria within the microbial community (Paper I). Thereby, the fraction of algae OM in the aquatic OM pool stimulates the activity and hence contribution of bacterial communities to leaf C turnover by providing an essential energy and nutrient source for the assimilation of the structural complex leaf OM substrate. On the contrary, the assimilation of algal OM remains limited for fungal communities as a function of nutrient competition between fungi and bacteria (Paper I, II). In addition, results provide evidence that environmental conditions determine the strength of interactions between microalgae and heterotrophic bacteria during leaf OM decomposition (Paper I, III). However, the stimulatory effect of algal photoautotrophic activities on leaf C turnover remained significant even under highly dynamic environmental conditions, highlighting their functional role for ecosystem processes (Paper III).
The results of my thesis provide insights into the mechanisms by which algae affect the microbial turnover of leaf C in freshwaters. This in turn contributes to a better understanding of the function of algae in freshwater biogeochemical cycles, especially with regard to their interaction with the heterotrophic community.
Mouse aldehyde oxidases (mAOXs) have a homodimeric structure and belong to xanthine oxidase family of molybdo-flavoenzymes. In general, each dimer is characterized by three subdomains: a 20 kDa N-terminal 2x[2Fe2S] cluster containing domain, a 40 kDa central FAD-containing domain and an 85 kDa C-terminal molybdenum cofactor (Moco) containing domain. Aldehyde oxidases have a broad substrate specificity including the oxidation of different aldehydes and N-heterocyclic compounds. AOX enzymes are present in mainly all eukaryotes. Four different homologs of AOX were identified to be present with varying numbers among species and rodents like mice and rats contain the highest number of AOX isoenzymes. There are four identified homologs in mouse named mAOX1, mAOX3, mAOX2, and mAOX4. The AOX homologs in mice are expressed in a tissue-specific manner. Expression of mAOX1 and mAOX3 are almost superimposable and predominantly synthesized in liver, lung, and testis. The richest source of mAOX4 is the Harderian gland, which is found within the eye's orbit in tetrapods. Expression of mAOX2 is strictly restricted to the Bowman’s gland, the main secretory organ of the nasal mucosa.
In this study, the four catalytically active mAOX enzymes were expressed in a heterologous expression system in Escherichia coli and purified in a catalytically active form. Thirty different structurally related aromatic, aliphatic and N-heterocyclic compounds were used as substrates, and the kinetic parameters of all four mAOX enzymes were directly compared. The results showed that all enzymes can catalyze a broad range of substrates. Generally, no major differences between mAOX1, mAOX3 and mAOX2 were identified and the substrate specificity of mAOX1, mAOX3, and mAOX2 was broader compared to that of mAOX4 since mAOX4 showed no activity with substrates like methoxy-benzaldehydes, phenanthridine, N1-methyl-nicotinamide, and cinnamaldehyde and 4-(dimethylamino)cinnamaldehyde.
We investigated differences at the flavin site of the mAOX enzymes by measuring the ability of the four mAOX enzymes to oxidize NADH in the absence of oxygen. NADH was able to reduce only mAOX3. The four mouse AOXs are also characterized by quantitative differences in their ability to produce superoxide radicals. mAOX2 is the enzyme generating the largest rate of superoxide radicals of around 40% in relation to moles of substrate converted and it is followed by mAOX1 with a ratio of 30%.
To understand the factors that contribute to the substrate specificity of mAOX4, site-directed mutagenesis was applied to substitute amino acids in the substrate-binding funnel by the ones present in mAOX1, mAOX3, and mAOX2. The amino acids Val1016, Ile1018 and Met1088 were selected as targets. An increase in activity was obtained by the amino acid exchange M1088V in the active site identified to be specific for mAOX4, to the amino acid identified in mAOX3.
The complete mitochondrial genome of a European fire-bellied toad (Bombina bombina) from Germany
(2019)
The European fire-bellied toad, Bombina bombina, is a small aquatic toad belonging to the family Bombinatoridae. The species is native to the lowlands of Central and Eastern Europe, where population numbers have been in decline in recent past decades. Here, we present the first complete mitochondrial genome of the endangered European fire-bellied toad from Northern Germany recovered using iterative mapping. Phylogenetic analyses including other representatives of the Bombinatoridae placed our German specimen as sister to a Polish B. bombina sequence with high support. This finding is congruent with the postulated Pleistocene history of the species. Our complete mitochondrial genome represents an important resource for further population analysis of the European fire-bellied toad, especially those found within Germany.
The complete mitochondrial genome of a European fire-bellied toad (Bombina bombina) from Germany
(2018)
The European fire-bellied toad, Bombina bombina, is a small aquatic toad belonging to the family Bombinatoridae. The species is native to the lowlands of Central and Eastern Europe, where population numbers have been in decline in recent past decades. Here, we present the first complete mitochondrial genome of the endangered European fire-bellied toad from Northern Germany recovered using iterative mapping. Phylogenetic analyses including other representatives of the Bombinatoridae placed our German specimen as sister to a Polish B. bombina sequence with high support. This finding is congruent with the postulated Pleistocene history of the species. Our complete mitochondrial genome represents an important resource for further population analysis of the European fire-bellied toad, especially those found within Germany.
Characterization of altered inflorescence architecture in Arabidopsis thaliana BG-5 x Kro-0 hybrid
(2018)
A reciprocal cross between two A. thaliana accessions, Kro-0 (Krotzenburg, Germany) and BG-5 (Seattle, USA), displays purple rosette leaves and dwarf bushy phenotype in F1 hybrids when grown at 17 °C and a parental-like phenotype when grown at 21 °C. This F1 temperature-dependent-dwarf-bushy phenotype is characterized by reduced growth of the primary stem together with an increased number of branches. The reduced stem growth was the strongest at the first internode. In addition, we found that a temperature switch from 21 °C to 17 °C induced the phenotype only before the formation of the first internode of the stem. Similarly, the F1 dwarf-bushy phenotype could not be reversed when plants were shifted from 17 °C to 21 °C after the first internode was formed. Metabolic analysis showed that the F1 phenotype was associated with a significant upregulation of anthocyanin(s), kaempferol(s), salicylic acid, jasmonic acid and abscisic acid. As it has been previously shown that the dwarf-bushy phenotype is linked to two loci, one on chromosome 2 from Kro-0 and one on chromosome 3 from BG-5, an artificial micro-RNA approach was used to investigate the necessary genes on these intervals. From the results obtained, it was found that two genes, AT2G14120 that encodes for a DYNAMIN RELATED PROTEIN3B and AT2G14100 that encodes a member of the Cytochrome P450 family protein CYP705A13, were necessary for the appearance of the F1 phenotype on chromosome 2. It was also discovered that AT3G61035 that encodes for another cytochrome P450 family protein CYP705A13 and AT3G60840 that encodes for a MICROTUBULE-ASSOCIATED PROTEIN65-4 on chromosome 3 were both necessary for the induction of the F1 phenotype. To prove the causality of these genes, genomic constructs of the Kro-0 candidate genes on chromosome 2 were transferred to BG-5 and genomic constructs of the chromosome 3 candidate genes from BG-5 were transferred to Kro-0. The T1 lines showed that these genes are not sufficient alone to induce the phenotype. In addition to the F1 phenotype, more severe phenotypes were observed in the F2 generations that were grouped into five different phenotypic classes. Whilst seed yield was comparable between F1 hybrids and parental lines, three phenotypic classes in the F2 generation exhibited hybrid breakdown in the form of reproductive failure. This F2 hybrid breakdown was less sensitive to temperature and showed a dose-dependent effect of the loci involved in F1 phenotype. The severest class of hybrid breakdown phenotypes was observed only in the population of backcross with the parent Kro-0, which indicates a stronger contribution of the BG-5 allele when compared to the Kro-0 allele on the hybrid breakdown phenotypes. Overall, the findings of my thesis provide a further understanding of the genetic and metabolic factors underlying altered shoot architecture in hybrid dysfunction.
Die Folgen einer lebensmittelbedingten Erkrankung sind zum Teil gravierend, insbesondere für Kinder und immunsupprimierte Menschen. Hierbei gehören Salmonella und Campylobacter zu den häufigsten Erregern, die verantwortlich für gastrointestinale Erkrankungen in Deutschland sind. Trotz umfassender Maßnahmen der EU zur Prävention und Bekämpfung von Salmonellen in Geflügelbeständen und der Lebensmittel-Industrie, wird von einem stagnierenden Trend von Infektionszahlen berichtet. Zoonose-Erreger wie Salmonellen können über Nutztiere in die Nahrungskette des Menschen gelangen, wodurch sich Infektionsherde schnell ausbreiten können. Dabei sind bestehende Präventionsstrategien für Geflügel vorhanden, die aber nicht auf den Menschen übertragbar sind. Folglich sind Diagnostik und Prävention in der Lebensmittelindustrie essentiell. Deshalb besteht ein hoher Bedarf für spezifische, sensitive und zuverlässige Nachweismethoden, die eine Point-of-care Diagnostik gewährleisten. Durch ein wachsendes Verständnis der wirtsspezifischen Faktoren von S. enterica Serovaren kann die Entwicklung sowohl neuartiger diagnostischer Methoden, als auch neuartiger Therapien und Impfstoffe maßgeblich vorangetrieben werden.
Infolgedessen wurde in dieser Arbeit ein infektionsähnliches in vitro Modell für S. Enteritidis etabliert und darauf basierend eine umfassende Untersuchung zur Identifizierung neuer Zielstrukturen für den Erreger durchgeführt. Während einer Salmonellen-Infektion ist die erste zelluläre Barriere im Wirt die Epithelschicht. Dementsprechend wurde eine humane Zelllinie (CaCo 2, Darmepithel) für die Pathogen-Wirt-Studie ausgewählt. Das Salmonellen-Transkriptom und morphologische Eigenschaften der Epithelzellen wurden in verschiedenen Phasen der Salmonellen-Infektion untersucht und mit bereits gut beschriebenen Virulenzfaktoren und Beobachtungen in Bezug gesetzt. Durch dieses Infektionsmodell konnte ein spezifischer Phänotyp für die intrazellulären Salmonellen in den Epithelzellen nachgewiesen werden. Zudem wurde aufgezeigt, dass bereits die Kultivierung in Flüssigmedium einen invasionsaktiven Zustand der Salmonellen erzeugt. Allerdings wurde durch die Kokultivierung mit Epithelzellen eine zusätzliche Expression relevanter Gene induziert, um eine effiziente Adhäsion und Transmembran-Transport zu gewährleisten. Letzterer ist charakteristisch für die intrazelluläre Limitierung von Nährstoffen und prägt den infektionsrelevanten Status. Unter Berücksichtigung dieser Faktoren ergab sich ein Phänotyp, der eindeutig Mechanismen zur Wirtsadaptation und möglicherweise auch Pathogenese aufzeigt. Die intrazellulären Bakterien müssen vom Wirt separiert werden, was ein wesentlicher Schritt für Pathogen-bestimmende Analysen ist. Hierbei wurde mithilfe einer Detergenz-basierten Lyse der eukaryotischen Zellmembran und differentieller Zentrifugation, der eukaryotische Eintrag minimal gehalten. Unter Verwendung der Virulenz-adaptierten Salmonellen wurden Untersuchungen in Hinblick auf die Identifizierung neuer Zielstrukturen für S. Enteritidis durchgeführt. Mithilfe eines immunologischen Screenings wurden neue potentielle Antigene entdeckt. Zu diesem Zweck wurden bakterielle cDNA-basierte Expressionsbibliotheken hergestellt, die durch eine vereinfachte Microarray-Anwendung ein Hochdurchsatzscreening von Proteinen als potentielle Binder ermöglichen. Folglich konnten neue unbeschriebene Proteine identifiziert werden, die sich durch eine Salmonella-Spezifität oder Membranständigkeit auszeichnen. Ebenso wurde ein Vergleich der im Screening identifizierten Proteine mit der Regulation der kodierenden Gene im infektionsähnlichen Modell durchgeführt. Dabei wurde deutlich, dass die Häufigkeit von Transkripten einen Einfluss auf die Verfügbarkeit in der cDNA-Bibliothek und folglich auch auf die Expressionsbibliothek nimmt. Angesichts eines Ungleichgewichts zwischen der Gesamtzahl protein-kodierender Gene in S. Enteritidis zu möglichen Klonen, die während des Microarray-Screenings untersucht werden können, besteht der Bedarf einer Anreicherung von Proteinen in der Expressionsbibliothek. Das infektionsähnliche Modell zeigte, dass nicht nur Virulenz-assoziierte, sondern auch Stress- und Metabolismus-relevante Gene hochreguliert werden. Durch die Konstruktion dieser spezifischen cDNA-Bibliotheken ist die Erkennung von charakteristischen molekularen Markern gegeben.
Weiterhin wurden anhand der Transkriptomanalyse spezifisch hochregulierte Gene identifiziert, die relevant für das intrazelluläre Überleben von S. Enteritidis in humanen Epithelzellen sind. Hiervon wurden drei Gene näher untersucht, indem ihr Einfluss im infektionsähnlichen Modell mittels entsprechender Gen-Knockout-Stämme analysiert wurde. Dabei wurde für eine dieser Mutanten ein reduziertes Wachstum in der späten intrazellulären Phase nachgewiesen. Weiterführende in vitro Analysen sind für die Charakterisierung des Knockout-Stamms notwendig, um den Einsatz als potenzielles Therapeutikum zu verifizieren.
Zusammenfassend wurde ein in vitro Infektionsmodell für S. Enteritidis etabliert, wodurch neue Zielstrukturen des Erregers identifiziert wurden. Diese sind für diagnostische oder therapeutische Anwendungen interessant. Das Modell lässt sich ebenso für andere intrazelluläre Pathogene übertragen und gewährleistet eine zuverlässige Identifizierung von potentiellen Antigenen.
