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The advent of large-scale and high-throughput technologies has recently caused a shift in focus in contemporary biology from decades of reductionism towards a more systemic view. Alongside the availability of genome sequences the exploration of organisms utilizing such approach should give rise to a more comprehensive understanding of complex systems. Domestication and intensive breeding of crop plants has led to a parallel narrowing of their genetic basis. The potential to improve crops by conventional breeding using elite cultivars is therefore rather limited and molecular technologies, such as marker assisted selection (MAS) are currently being exploited to re-introduce allelic variance from wild species. Molecular breeding strategies have mostly focused on the introduction of yield or resistance related traits to date. However given that medical research has highlighted the importance of crop compositional quality in the human diet this research field is rapidly becoming more important. Chemical composition of biological tissues can be efficiently assessed by metabolite profiling techniques, which allow the multivariate detection of metabolites of a given biological sample. Here, a GC/MS metabolite profiling approach has been applied to investigate natural variation of tomatoes with respect to the chemical composition of their fruits. The establishment of a mass spectral and retention index (MSRI) library was a prerequisite for this work in order to establish a framework for the identification of metabolites from a complex mixture. As mass spectral and retention index information is highly important for the metabolomics community this library was made publicly available. Metabolite profiling of tomato wild species revealed large differences in the chemical composition, especially of amino and organic acids, as well as on the sugar composition and secondary metabolites. Intriguingly, the analysis of a set of S. pennellii introgression lines (IL) identified 889 quantitative trait loci of compositional quality and 326 yield-associated traits. These traits are characterized by increases/decreases not only of single metabolites but also of entire metabolic pathways, thus highlighting the potential of this approach in uncovering novel aspects of metabolic regulation. Finally the biosynthetic pathway of the phenylalanine-derived fruit volatiles phenylethanol and phenylacetaldehyde was elucidated via a combination of metabolic profiling of natural variation, stable isotope tracer experiments and reverse genetic experimentation.
Weltweit versuchen Wissenschaftler, künstliche Viren für den Gentransfer zu konstruieren, die nicht reproduktionsfähig sind. Diese sollen die Vorteile der natürlichen Viren besitzen (effizienter Transport von genetischem Material), jedoch keine Antigene auf ihrer Oberfläche tragen, die Immunreaktionen auslösen. Ziel dieses Projektes ist es, einen künstlichen Viruspartikel herzustellen, dessen Basis eine Polyelektrolytenhohlkugel bildet, die mit einer Lipiddoppelschicht bedeckt ist. Um intakte Doppelschichten zu erzeugen, muss die Wechselwirkung zwischen Lipid und Polyelektrolyt (z.B. DNA) verstanden und optimiert werden. Dazu ist es notwendig, die strukturelle Grundlage der Interaktion aufzuklären. Positiv geladene Lipide gehen zwar starke Wechselwirkungen mit der negativ geladenen DNA ein, sie wirken jedoch toxisch auf biologische Zellen. In der vorliegenden Arbeit wurde daher die durch zweiwertige Kationen vermittelte Kopplung von genomischer oder Plasmid-DNA an zwitterionische oder negativ geladene Phospholipide an zwei Modellsystemen untersucht. 1. Modellsystem: Lipidmonoschicht an der Wasser/Luft-Grenzfläche Methoden: Filmwaagentechnik in Kombination mit IR-Spektroskopie (IRRAS), Röntgenreflexion (XR), Röntgendiffraktion (GIXD), Brewsterwinkel-Mikroskopie (BAM), Röntgenfluoreszenz (XRF) und Oberflächenpotentialmessungen Resultate: A) Die Anwesenheit der zweiwertigen Kationen Ba2+, Mg2+, Ca2+ oder Mn2+ in der Subphase hat keinen nachweisbaren Einfluss auf die Struktur der zwitterionischen DMPE- (1,2-Dimyristoyl-phosphatidyl-ethanolamin) Monoschicht. B) In der Subphase gelöste DNA adsorbiert nur in Gegenwart dieser Kationen an der DMPE-Monoschicht. C) Sowohl die Adsorption genomischer Kalbsthymus-DNA als auch der Plasmid-DNA pGL3 bewirkt eine Reduktion des Neigungswinkels der Alkylketten, die auf einen veränderten Platzbedarf der Kopfgruppe zurückzuführen ist. Durch die Umorientierung der Kopfgruppe wird die elektrostatische Wechselwirkung zwischen den positiv geladenen Stickstoffatomen der Lipidkopfgruppen und den negativ geladenen DNA-Phosphaten erhöht. D) Die adsorbierte DNA weist eine geordnete Struktur auf, wenn sie durch Barium-, Magnesium-, Calcium- oder Manganionen komplexiert ist. Der Abstand zwischen parallelen DNA-Strängen hängt dabei von der Größe der DNA-Fragmente sowie von der Art des Kations ab. Die größten Abstände ergeben sich mit Bariumionen, gefolgt von Magnesium- und Calciumionen. Die kleinsten DNA-Abstände werden durch Komplexierung mit Manganionen erhalten. Diese Ionenreihenfolge stellt sich sowohl für genomische DNA als auch für Plasmid-DNA ein. E) Die DNA-Abstände werden durch die Kompression des Lipidfilms nicht beeinflusst. Zwischen der Lipidmonoschicht und der adsorbierten DNA besteht demnach nur eine schwache Wechselwirkung. Offensichtlich befindet sich die durch zweiwertige Kationen komplexierte DNA als weitgehend eigenständige Schicht unter dem Lipidfilm. 2. Modellsystem: Lipiddoppelschicht an der fest/flüssig-Grenzfläche Methoden: Neutronenreflexion (NR) und Quarzmikrowaage (QCM-D) Resultate: A) Das zwitterionische Phospholipid DMPC (1,2-Dimyristoyl-phosphatidylcholin) bildet keine Lipiddoppelschicht auf planaren Polyelektrolytmultischichten aus, deren letzte Lage das positiv geladene PAH (Polyallylamin) ist. B) Hingegen bildet DMPC auf dem negativ geladenen PSS (Polystyrolsulfonat) eine Doppelschicht aus, die jedoch Defekte aufweist. C) Eine Adsorption von genomischer Kalbsthymus-DNA auf dieser Lipidschicht findet nur in Gegenwart von Calciumionen statt. Andere zweiwertige Kationen wurden nicht untersucht. D) Das negativ geladene Phospholipid DLPA (1,2-Dilauryl-phosphatidsäure) bildet auf dem positiv geladenen PAH eine Lipiddoppelschicht aus, die Defekte aufweist. E) DNA adsorbiert ebenfalls erst in Anwesenheit von Calciumionen in der Lösung an die DLPA-Schicht. F) Durch die Zugabe von EDTA (Ethylendiamintetraessigsäure) werden die Calciumionen dem DLPA/DNA-Komplex entzogen, wodurch dieser dissoziiert. Demnach ist die calciuminduzierte Bildung dieser Komplexe reversibel.
Biogene Amine sind kleine organische Verbindungen, die sowohl bei Wirbeltieren als auch bei Wirbellosen als Neurotransmitter, Neuromodulatoren und/oder Neurohormone wirken können. Sie bilden eine bedeutende Gruppe von Botenstoffen und entfalten ihre Wirkungen über die Bindung an eine bestimmte Klasse von Rezeptorproteinen, die als G-Protein-gekoppelte Rezeptoren bezeichnet werden. Bei Insekten gehören zur Substanzklasse der biogenen Amine die Botenstoffe Dopamin, Tyramin, Octopamin, Serotonin und Histamin. Neben vielen anderen Wirkung ist z.B. gezeigt worden, daß einige dieser biogenen Amine bei der Honigbiene (Apis mellifera) die Geschmacksempfindlichkeit für Zuckerwasser-Reize modulieren können. Ich habe verschiedene Aspekte der aminergen Signaltransduktion an den „Modellorganismen“ Honigbiene und Amerikanische Großschabe (Periplaneta americana) untersucht. Aus der Honigbiene, einem „Modellorganismus“ für das Studium von Lern- und Gedächtnisvorgängen, wurden zwei Dopamin-Rezeptoren, ein Tyramin-Rezeptor, ein Octopamin-Rezeptor und ein Serotonin-Rezeptor charakterisiert. Die Rezeptoren wurden in kultivierten Säugerzellen exprimiert, um ihre pharmakologischen und funktionellen Eigenschaften (Kopplung an intrazelluläre Botenstoffwege) zu analysieren. Weiterhin wurde mit Hilfe verschiedener Techniken (RT-PCR, Northern-Blotting, in situ-Hybridisierung) untersucht, wo und wann während der Entwicklung die entsprechenden Rezeptor-mRNAs im Gehirn der Honigbiene exprimiert werden. Als Modellobjekt zur Untersuchung der zellulären Wirkungen biogener Amine wurden die Speicheldrüsen der Amerikanischen Großschabe genutzt. An isolierten Speicheldrüsen läßt sich sowohl mit Dopamin als auch mit Serotonin Speichelproduktion auslösen, wobei Speichelarten unterschiedlicher Zusammensetzung gebildet werden. Dopamin induziert die Bildung eines völlig proteinfreien, wäßrigen Speichels. Serotonin bewirkt die Sekretion eines proteinhaltigen Speichels. Die Serotonin-induzierte Proteinsekretion wird durch eine Erhöhung der Konzentration des intrazellulären Botenstoffs cAMP vermittelt. Es wurden die pharmakologischen Eigenschaften der Dopamin-Rezeptoren der Schaben-Speicheldrüsen untersucht sowie mit der molekularen Charakterisierung putativer aminerger Rezeptoren der Schabe begonnen. Weiterhin habe ich das ebony-Gen der Schabe charakterisiert. Dieses Gen kodiert für ein Enzym, das wahrscheinlich bei der Schabe (wie bei anderen Insekten) an der Inaktivierung biogener Amine beteiligt ist und im Gehirn und in den Speicheldrüsen der Schabe exprimiert wird.
Die Honigbiene Apis mellifera gilt seit langem als Modell-Organismus zur Untersuchung von Lern- und Gedächtnisvorgängen sowie zum Studium des Sozialverhaltens und der Arbeitsteilung. Bei der Steuerung und Regulation dieser Verhaltensweisen spielt das Indolalkylamin Serotonin eine wesentliche Rolle. Serotonin entfaltet seine Wirkung durch die Bindung an G-Protein-gekoppelte Rezeptoren (GPCRs). In der vorliegenden Arbeit wird der erste Serotonin-Rezeptor aus der Honigbiene molekular charakterisiert. Durch die Anwendung zwei verschiedener Klonierungsstrategien konnten drei cDNA-Sequenzen isoliert werden, die für potentielle Serotonin-Rezeptoren kodieren. Die Sequenzen weisen die größte Ähnlichkeit zu dem 5-HT7- und 5-HT2-Rezeptor von Drosophila melanogaster bzw. dem 5-HT1-Rezeptor von Panulirus interruptus auf. Die isolierten Serotonin-Rezeptoren der Honigbiene wurden dementsprechend Am(Apis mellifera)5-HT1, Am5-HT2 und Am5-HT7 benannt. Das Hydropathieprofil des Am5-HT1-, Am5-HT2- und Am5-HT7-Rezeptors deutet auf das Vorhandensein des charakteristischen heptahelikalen Aufbaus G-Protein-gekoppelter Rezeptoren hin. Die abgeleiteten Aminosäuresequenzen zeigen typische Merkmale biogener Amin-Rezeptoren. Aminosäuren, die eine Bedeutung bei der Bildung der Liganden-Bindungstasche, der Rezeptor-Aktivierung und der Kopplung eines G-Proteins an den Rezeptor haben, sind in allen drei Rezeptoren konserviert. Interessanterweise ist jedoch das in den meisten biogenen Amin-Rezeptoren vorhandene DRY-Motiv in dem Am5-HT2- und Am5-HT7-Rezeptor nicht konserviert. Das Vorhandensein einer PDZ-Domäne in dem Am5-HT1- und Am5-HT7-Rezeptor lässt vermuten, dass diese Rezeptoren als Adapterproteine fungieren, die Signalmoleküle zu einem Signaltransduktionskomplex vereinigen. RT-PCR-Experimente zeigen die Expression der Rezeptoren in verschiedenen Geweben der Honigbiene. Auffallend ist die hohe Expression im Zentralgehirn. Des Weiteren konnte die Expression der Serotonin-Rezeptoren in den optischen Loben, Antennalloben sowie in der Peripherie, d.h. in der Flugmuskulatur und den Malpighischen Gefäßen nachgewiesen werden. Durch in situ Hybridisierungen wurde die Expression in Gefrierschnitten von Gehirnen adulter Sammlerinnen im Detail untersucht. Transkripte der Rezeptoren sind in den Somata von intrinsischen Pilzkörperzellen, Neuronen der optischen Loben und Neuronen der Antennalloben vorhanden. In einem heterologen Expressionssystem wurde der intrazelluläre Signalweg des Am5-HT7-Rezeptors untersucht. Die Aktivierung des stabil exprimierten Rezeptors durch Serotonin führt zur Bildung von cAMP. Der 5-HT7-Rezeptor spezifische Agonist 5-CT zeigt eine mit Serotonin vergleichbare Fähigkeit, die intrazelluläre cAMP-Konzentration zu erhöhen. Am5-HT7 gehört daher funktionell zu der Gruppe der 5-HT7-Rezeptoren. Der EC50-Wert von 1,06~nM (5-HT), ist im Vergleich zu anderen 5-HT7-Rezeptoren äußert niedrig. Des Weiteren wurde gezeigt, dass das basale cAMP-Niveau in den transfizierten Zellen im Vergleich zu nicht transfizierten Zellen deutlich erhöht ist. Das heißt, dass der Rezeptor auch in der Abwesenheit eines Liganden aktiv ist. Diese konstitutive Aktivität ist auch von anderen biogenen Amin-Rezeptoren bekannt. Methiothepin wurde als wirksamer inverser Agonist des Am5-HT7-Rezeptors identifiziert, da es in der Lage ist, der konstitutiven Aktivität entgegenzuwirken. Die Ergebnisse der vorliegenden Arbeit deuten darauf hin, dass die Serotonin-Rezeptoren in verschiedenen Regionen des ZNS der Honigbiene an der Informationsverarbeitung beteiligt sind. Es kann eine Beeinflussung von Lern- und Gedächtnisprozessen sowie des olfaktorischen und visuellen Systems durch diese Rezeptoren vermutet werden. Mit der Klonierung und funktionellen Charakterisierung des ersten Serotonin-Rezeptors der Honigbiene ist eine Grundlage für die Untersuchung der molekularen Mechanismen der serotonergen Signaltransduktion geschaffen worden.
In semi-arid savannas, unsustainable land use can lead to degradation of entire landscapes, e.g. in the form of shrub encroachment. This leads to habitat loss and is assumed to reduce species diversity. In BIOTA phase 1, we investigated the effects of land use on population dynamics on farm scale. In phase 2 we scale up to consider the whole regional landscape consisting of a diverse mosaic of farms with different historic and present land use intensities. This mosaic creates a heterogeneous, dynamic pattern of structural diversity at a large spatial scale. Understanding how the region-wide dynamic land use pattern affects the abundance of animal and plant species requires the integration of processes on large as well as on small spatial scales. In our multidisciplinary approach, we integrate information from remote sensing, genetic and ecological field studies as well as small scale process models in a dynamic region-wide simulation tool. <hr> Interdisziplinäres Zentrum für Musterdynamik und Angewandte Fernerkundung Workshop vom 9. - 10. Februar 2006.
Decisions for the conservation of biodiversity and sustainable management of natural resources are typically related to large scales, i.e. the landscape level. However, understanding and predicting the effects of land use and climate change on scales relevant for decision-making requires to include both, large scale vegetation dynamics and small scale processes, such as soil-plant interactions. Integrating the results of multiple BIOTA subprojects enabled us to include necessary data of soil science, botany, socio-economics and remote sensing into a high resolution, process-based and spatially-explicit model. Using an example from a sustainably-used research farm and a communally used and degraded farming area in semiarid southern Namibia we show the power of simulation models as a tool to integrate processes across disciplines and scales.
Mit der politischen Wende in den Staaten des ehemaligen Ostblockes wurde für viele militärisch genutzte Flächen ein tiefgreifender Nutzungswandel eingeleitet. Truppenübungsplätze als stark gestörte Bestandteile unserer Kulturlandschaft weisen auf großen Flächen naturschutzfachlich wertvolle Habitatmosaike mit speziellen Lebensgemeinschaften auf. Der Nutzungswandel ist mit einer Veränderung der Vegetationsstrukturen (Sukzession) und weiteren landschaftsökologischen Prozessen verbunden. Der ehemalige Truppenübungsplatz Döberitz im Norden der Landeshauptstadt Potsdam kann auf eine lange militärische Nutzungsgeschichte verweisen (erste Manöver des Soldatenkönigs im Jahr 1713). Nach 1992 wurden das NSG Döberitzer Heide (3.415 ha) und das NSG Ferbitzer Bruch (1.155 ha) ausgewiesen. Als Schutzgebiete nach der Vogelschutzrichtlinie sind sie Bestandteile des kohärenten Schutzgebietssystems Natura 2000 der europäischen Gemeinschaft. Trotz des Schutzstatus und der militärischen Altlasten unterliegt das Gebiet als größte zusammenhängende Naturfläche im engeren Verflechtungsraum des Landes Brandenburg einem hohen Nutzungsdruck. <hr> Interdisziplinäres Zentrum für Musterdynamik und Angewandte Fernerkundung Workshop vom 9. - 10. Februar 2006
From its first use in the field of biochemistry, instrumental analysis offered a variety of invaluable tools for the comprehensive description of biological systems. Multi-selective methods that aim to cover as many endogenous compounds as possible in biological samples use different analytical platforms and include methods like gene expression profile and metabolite profile analysis. The enormous amount of data generated in application of profiling methods needs to be evaluated in a manner appropriate to the question under investigation. The new field of system biology rises to the challenge to develop strategies for collecting, processing, interpreting, and archiving this vast amount of data; to make those data available in form of databases, tools, models, and networks to the scientific community. On the background of this development a multi-selective method for the determination of phytohormones was developed and optimised, complementing the profile analyses which are already in use (Chapter I). The general feasibility of a simultaneous analysis of plant metabolites and phytohormones in one sample set-up was tested by studies on the analytical robustness of the metabolite profiling protocol. The recovery of plant metabolites proved to be satisfactory robust against variations in the extraction protocol by using common extraction procedures for phytohormones; a joint extraction of metabolites and hormones from plant tissue seems practicable (Chapter II). Quantification of compounds within the context of profiling methods requires particular scrutiny (Chapter II). In Chapter III, the potential of stable-isotope in vivo labelling as normalisation strategy for profiling data acquired with mass spectrometry is discussed. First promising results were obtained for a reproducible quantification by stable-isotope in vivo labelling, which was applied in metabolomic studies. In-parallel application of metabolite and phytohormone analysis to seedlings of the model plant Arabidopsis thaliana exposed to sulfate limitation was used to investigate the relationship between the endogenous concentration of signal elements and the ‘metabolic phenotype’ of a plant. An automated evaluation strategy was developed to process data of compounds with diverse physiological nature, such as signal elements, genes and metabolites – all which act in vivo in a conditional, time-resolved manner (Chapter IV). Final data analysis focussed on conditionality of signal-metabolome interactions.
The major aim of this work was the identification of new phloem sap proteins and a metabolic characterisation of this transport fluid. The experiments were performed on the three plant species C. sativus, C. maxima and B. napus. To characterise the phloem samples from B. napus, a new model plant for phloem analysis, western blot tests together with metabolite profiling were performed. GC-MS metabolite profiling and enzyme assays were used for measuring metabolites in the phloem of B. napus. Results from the phloem sap measurements showed, as expected, a typical sugar distribution for apoplasmic phloem loaders with sucrose being the predominant sugar. In stem extracts, the most abundant sugar was glucose with much lower fructose and sucrose levels. With the GC-MS approach it was possible to identify a number of metabolites which showed a differential distribution when phloem and stem tissue extracts were compared. For protein identification, two different approaches were employed (i) screening expression libraries with total phloem protein specific antisera and (ii) protein separation on 2 DE gels followed by ESI-MS/MS sequence analyses. For the first approach, three different phloem protein-specific antisera were produced and expression libraries were constructed. Phloem protein antisera were tested for specificity and some attempts to estimate specific epitopes were undertaken. Screening of the libraries resulted in the identification of 14 different proteins from all investigated species. Analyses of B. napus phloem sap proteins from 2 DE with ESI-MS/MS resulted in the identification of 5 different proteins. The phloem localisation of the identified proteins was additionally confirmed by western blot tests using specific antibodies. In order to functionally characterise some selected phloem proteins from B. napus, the group of potential calcium-binding polypeptides was analysed for functional Ca<sup>+2 binding properties and several Ca<sup>+2–binding proteins could be isolated. However, their sequences could as yet not be determined. Another approach used for functional protein characterisation was the analysis of Arabidopsis T-DNA insertion mutants. Four available mutants with insertions in phloem protein-specific genes were chosen from the SALK and GABI-Kat collections and selected homozygous lines were tested for the presence of the investigated proteins. In order to verify if the product of one of the mutated gene (GRP 7) is transported through the phloem, grafting experiments were performed followed by western blot analyses. Although the employed antiserum against GRP 7 protein did not allow distinguishing between the mutant and the wild type plants, successful Arabidopsis grafting could be established as a promising method for further studies on protein translocation through the phloem.
Das Borna Disease Virus (BDV, Bornavirus) besitzt ein einzelsträngiges RNA-Genom negativer Polarität und ist innerhalb der Ordnung Mononegavirales der Prototyp einer eigenen Virusfamilie, die der Bornaviridae. Eine außergewöhnliche Eigenschaft des Virus ist seine nukleäre Transkription und Replikation, eine weitere besteht in seiner Fähigkeit, als neurotropes Virus sowohl in vivo als auch in vitro persistente Infektionen zu etablieren. Die zugrunde liegenden Mechanismen sowohl der Replikation als auch der Persistenz sind derzeit noch unzureichend verstanden, auch deshalb, weil das Virus noch relativ „jung“ ist: Erste komplette Sequenzen des RNA-Genoms wurden 1994 publiziert und erst vor einigen Monaten gelang die Generierung rekombinanter Viren auf der Basis klonierter cDNA. Im Mittelpunkt dieser Arbeit standen das p10 Protein und das Phosphoprotein (P), die von der gemeinsamen Transkriptionseinheit II in überlappenden Leserahmen kodiert werden. Als im Kern der Wirtszelle replizierendes Virus ist das Bornavirus auf zelluläre Importmechanismen angewiesen, um den Kernimport aller an der Replikation beteiligten viralen Proteine zu gewährleisten. Das p10 Protein ist ein negativer Regulator der viralen RNA-abhängigen RNA-Polymerase (L). In vitro Importexperimente zeigten, dass p10 über den klassischen Importin alpha/beta abhängigen Kernimportweg in den Nukleus transportiert wird. Dies war unerwartet, da p10 kein vorhersagbares klassisches Kernlokalisierungssignal (NLS) besitzt und weist darauf hin, dass der zelluläre Importapparat offensichtlich flexibler ist als allgemein angenommen. Die ersten 20 N-terminalen AS vermitteln sowohl Kernimport als auch die Bindung an den Importrezeptor Importin alpha. Durch Di-Alanin-Austauschmutagenese wurden die für diesen Transportprozess essentiellen AS identifiziert und die Bedeutung hydrophober und polarer AS-Reste demonstriert. Die Fähigkeit des Bornavirus, persistente Infektionen zu etablieren, wirft die Frage auf, wie das Virus die zellulären antiviralen Abwehrmechanismen, insbesondere das Typ I Interferon (IFN)-System, unterwandert. Das virale P Protein wurde in dieser Arbeit als potenter Antagonist der IFN-Induktion charakterisiert. Es verhindert die Phosphorylierung des zentralen Transkriptionsfaktors IRF3 durch die zelluläre Kinase TBK1 und somit dessen Aktivierung. Der Befund, dass P mit TBK1 Komplexe bildet und zudem auch als Substrat für die zelluläre Kinase fungiert, erlaubt es, erstmalig einen Mechanismus zu postulieren, in dem ein virales Protein (BDV-P) als putatives TBK1-Pseudosubstrat die IRF3-Aktivierung kompetitiv hemmt.
Die Tailspike Proteine (TSP) der Bakteriophagen P22, Sf6 und HK620 dienen der Erkennung von Kohlenhydratstrukturen auf ihren gram-negativen Wirtsbakterien und zeigen, von den ersten 110 Aminosäuren des N-Terminus abgesehen, keine Sequenzübereinstimmung. Mit Röntgenkristallstrukturanalyse konnte gezeigt werden, dass HK620TSP und Sf6TSP ebenfalls zu einer parallelen, rechtsgängigen beta-Helix falten, wie dies schon für P22TSP bekannt war. Die Kohlenhydratbindestelle ist bei Sf6TSP im Vergleich zu P22TSP zwischen die Untereinheiten verschoben.
This thesis aimed to investigate several fundamental and perplexing questions relating to the phloem loading and transport mechanisms of Cucurbita maxima, by combining metabolomic analysis with cell biological techniques. This putative symplastic loading species has long been used for experiments on phloem anatomy, phloem biochemistry, phloem transport physiology and phloem signalling. Symplastic loading species have been proposed to use a polymer trapping mechanism to accumulate RFO (raffinose family oligosaccharides) sugars to build up high osmotic pressure in minor veins which sustains a concentration gradient that drives mass flow. However, extensive evidence indicating a low sugar concentration in their phloem exudates is a long-known problem that conflicts with this hypothesis. Previous metabolomic analysis shows the concentration of many small molecules in phloem exudates is higher than that of leaf tissues, which indicates an active apoplastic loading step. Therefore, in the view of the phloem metabolome, a symplastic loading mechanism cannot explain how small molecules other than RFO sugars are loaded into phloem. Most studies of phloem physiology using cucurbits have neglected the possible functions of vascular architecture in phloem transport. It is well known that there are two phloem systems in cucurbits with distinctly different anatomical features: central phloem and extrafascicular phloem. However, mistaken conclusions on sources of cucurbit phloem exudation from previous reports have hindered consideration of the idea that there may be important differences between these two phloem systems. The major results are summarized as below: 1) O-linked glycans in C.maxima were structurally identified as beta-1,3 linked glucose polymers, and the composition of glycans in cucurbits was found to be species-specific. Inter-species grafting experiments proved that these glycans are phloem mobile and transported uni-directionally from scion to stock. 2) As indicated by stable isotopic labelling experiments, a considerable amount of carbon is incorporated into small metabolites in phloem exudates. However, the incorporation of carbon into RFO sugars is much faster than for other metabolites. 3) Both CO2 labelling experiments and comparative metabolomic analysis of phloem exudates and leaf tissues indicated that metabolic processes other than RFO sugar metabolism play an important role in cucurbit phloem physiology. 4) The underlying assumption that the central phloem of cucurbits continuously releases exudates after physical incision was proved wrong by rigorous experiments including direct observation by normal microscopy and combined multiple-microscopic methods. Errors in previous experimental confirmation of phloem exudation in cucurbits are critically discussed. 5) Extrafascicular phloem was proved to be functional, as indicated by phloem-mobile carboxyfluorescein tracer studies. Commissural sieve tubes interconnect phloem bundles into a complete super-symplastic network. 6) Extrafascicular phloem represents the main source of exudates following physical incision. The major transported metabolites by these extrafacicular phloem are non-sugar compounds including amino acids, O-glycans, amines. 7) Central phloem contains almost exclusively RFO sugars, the estimated amount of which is up to 1 to 2 molar. The major RFO sugar present in central phloem is stachyose. 8) Cucurbits utilize two structurally different phloem systems for transporting different group of metabolites (RFO sugars and non-RFO sugar compounds). This implies that cucurbits may use spatially separated loading mechanisms (apoplastic loading for extrafascicular phloem and symplastic loading for central phloem) for supply of nutrients to sinks. 9) Along the transport systems, RFO sugars were mainly distributed within central phloem tissues. There were only small amounts of RFO sugars present in xylem tissues (millimolar range) and trace amounts of RFO sugars in cortex and pith. The composition of small molecules in external central phloem is very different from that in internal central phloem. 10) Aggregated P-proteins were manually dissected from central phloem and analysed by both SDS-PAGE and mass spectrometry. Partial sequences of peptides were obtained by QTOF de novo sequencing from trypsin digests of three SDS-PAGE bands. None of these partial sequences shows significant homology to known cucurbit phloem proteins or other plant proteins. This proves that these central phloem proteins are a completely new group of proteins different from those in extrafascicular phloem. The extensively analysed P-proteins reported in literature to date are therefore now shown to arise from extrafascicular phloem and not central phloem, and therefore do not appear to be involved in the occlusion processes in central phloem.
Post-translational redox-regulation is a well-known mechanism to regulate enzymes of the Calvin cycle, oxidative pentose phosphate cycle, NADPH export and ATP synthesis in response to light. The aim of the present thesis was to investigate whether a similar mechanism is also regulating carbon storage in leaves. Previous studies have shown that the key-regulatory enzyme of starch synthesis, ADPglucose pyrophosphorylase (AGPase) is inactivated by formation of an intermolecular disulfide bridge between the two catalytic subunits (AGPB) of the heterotetrameric holoenzyme in potato tubers, but the relevance of this mechanism to regulate starch synthesis in leaves was not investigated. The work presented in this thesis shows that AGPase is subject to post-translational redox-regulation in leaves of pea, potato and Arabidopsis in response to day night changes. Light was shown to trigger posttranslational redox-regulation of AGPase. AGPB was rapidly converted from a dimer to a monomer when isolated pea chloroplasts were illuminated and from a monomer to a dimer when preilluminated leaves were darkened. Conversion of AGPB from dimer to monomer was accompanied by an increase in activity due to changes in the kinetik properties of the enzyme. Studies with pea chloroplast extracts showed that AGPase redox-activation is mediated by thioredoxins f and m from spinach in-vitro. In a further set of experiments it was shown that sugars provide a second input leading to AGPase redox activation and increased starch synthesis and that they can act as a signal which is independent from light. External feeding of sugars such as sucrose or trehalose to Arabidopsis leaves in the dark led to conversion of AGPB from dimer to monomer and to an increase in the rate of starch synthesis, while there were no significant changes in the level of 3PGA, an allosteric activator of the enyzme, and in the NADPH/NADP+ ratio. Experiments with transgenic Arabidopsis plants with altered levels of trehalose 6-phosphate (T6P), the precursor of trehalose synthesis, provided genetic evidence that T6P rather than trehalose is leading to AGPase redox-activation. Compared to Wt, leaves expressing E.coli trehalose-phosphate synthase (TPS) in the cytosol showed increased activation of AGPase and higher starch level during the day, while trehalose-phosphate phosphatase (TPP) overexpressing leaves showed the opposite. These changes occurred independently of changes in sugar and sugar-phosphate levels and NADPH/NADP+ ratio. External supply of sucrose to Wt and TPS-overexpressing leaves led to monomerisation of AGPB, while this response was attenuated in TPP expressing leaves, indicating that T6P is involved in the sucrose-dependent redox-activation of AGPase. To provide biochemical evidence that T6P promotes redox-activation of AGPase independently of cytosolic elements, T6P was fed to intact isolated chloroplasts for 15 min. incubation with concentrations down to 100 µM of T6P, but not with sucrose 6-phosphate, sucrose, trehalose or Pi as controls, significantly and specifically increased AGPB monomerisation and AGPase activity within 15 minutes, implying T6P as a signal reporting the cytosolic sugar status to the chloroplast. The response to T6P did not involve changes in the NADPH/NADP+ ratio consistent with T6P modulating redox-transfer to AGPase independently of changes in plastidial redox-state. Acetyl-CoA carboxylase (ACCase) is known as key-regulatory enzyme of fatty acid and lipid synthesis in plants. At the start of the present thesis there was mainly in vitro evidence in the literature showing redox-regulation of ACCase by DTT, and thioredoxins f and m. In the present thesis the in-vivo relevance of this mechanism to regulate lipid synthesis in leaves was investigated. ACCase activity measurement in leaf tissue collected at the end of the day and night in Arabidopsis leaves revealed a 3-fold higher activation state of the enzyme in the light than in the dark. Redox-activation was accompanied by change in kinetic properties of ACCase, leading to an increase affinity to its substrate acetyl-CoA . In further experiments, DTT as well as sucrose were fed to leaves, and both treatments led to a stimulation in the rate of lipid synthesis accompanied by redox-activation of ACCase and decrease in acetyl-CoA content. In a final approach, comparison of metabolic and transcript profiling after DTT feeding and after sucrose feeding to leaves provided evidence that redox-modification is an important regulatory mechanism in central metabolic pathways such as TCA cycle and amino acid synthesis, which acts independently of transcript levels.
In den letzten 20 Jahren hat sich der Maiszünsler (Ostrinia nubilalis HÜBNER), aus der Schmetterlingsfamilie der Pyralidae oder Zünsler, zum bedeutendsten tierischen Schädling des Maises (Zea mays) entwickelt. Eine Möglichkeit den Befall des Maiszünslers abzuwenden, bietet der Anbau von Bacillus thuringiensis-Mais (Bt-Mais). Mit Hilfe der Gentechnik wurden Gene des Bakteriums Bacillus thuringiensis übertragen, die einen für Fraßinsekten giftigen Wirkstoff bilden, wodurch die Pflanzen während der kompletten Vegetation vor den Larven des Maiszünslers geschützt sind. Ziel des vorliegenden Projektes war es, in einer 3-jährigen Studie die Auswirkungen des großflächigen Anbaus von Bt-Mais auf die ökologische Situation und den Handlungsrahmen des integrierten Pflanzenschutzes komplex zu untersuchen. Dazu wurden in Betrieben im Oderbruch, das als permanentes Befallsgebiet des Maiszünslers gilt, in den Jahren 2002 bis 2004 jährlich zwei Felder mit jeweils einer Bt-Sorte und einer konventionellen Sorte angelegt. Zusätzlich wurden biologische und chemische Maiszünsler-Bekämpfungsvarianten geprüft. Durch verschiedene Methoden wie Bonituren, Ganzpflanzenernten, Bodenfallenfänge und Beobachtungen des Wahlverhaltens von (Flug-)insekten konnten Aussagen zum Vorkommen von Insekten und Spinnentieren getroffen werden, wobei hierfür Daten aus Untersuchungen der Jahre 2000 und 2001 im Oderbruch ergänzend herangezogen werden konnten. Durch Ertragsmessungen, Energie- und Qualitätsermittlungen, sowie Fusarium- und Mykotoxinanalysen konnte der Anbau von Bt-Mais als neue Alternative zur Bekämpfung des Maiszünslers bewertet werden. Bezüglich des Auftretens von Insekten und Spinnentieren wurden im Mittel der fünfjährigen Datenerhebung beim Vergleich der Bt-Sorte zur konventionellen Sorte, mit Ausnahme der fast 100 %igen Bekämpfung des Maiszünslers, keine signifikanten Unterschiede festgestellt. Hierfür wurde ein besonderes Augenmerk auf Thripse, Wanzen, Blattläuse und deren Fraßfeinde, sowie mittels Bodenfallenfängen auf Laufkäfer und Spinnen gerichtet. Die erwarteten ökonomischen Vorteile wie etwa Ertragsplus oder bessere Nährstoff- und Energiegehalte durch geringeren Schaden beim Anbau von Bt-Mais als Silomais blieben in den Untersuchungsjahren aus. Allerdings zeigten Fusarium- und Mykotoxinanalysen eine geringere Belastung des Bt-Maises, was möglicherweise auf den geringeren Schaden zurückzuführen ist, da beschädigte Pflanzen für Fusarium und Mykotoxine anfälliger sind. Desweiteren konnten erste methodische Ansätze für ein auf EU-Ebene gefordertes, den Anbau von Bt-Mais begleitendes Monitoring, erarbeitet werden. So konnten Vorschläge für geeignete Methoden, deren Umfang sowie des Zeitpunktes der Durchführungen gemacht werden.
Arabidopsis thaliana trichomes exhibit strong birefringence under polarized light, a characteristic of cell walls containing large amounts of highly ordered cellulose microfibrils. The tbr1 mutant of Arabidopsis lacks trichome birefringence and is deficient in secondary cell wall cellulose synthesis (Potikha and Delmer, 1995). The TBR gene was identified by recombinational mapping, candidate gene sequencing and molecular complementation using genomic cosmid clones, as well as a p35S:TBR genomic DNA construct, fully rescuing the mutant phenotype in both cases. The only mutant allele available (tbr-1) carries a substitution (G to E) in a conserved aminoacid domain of the protein. TBR gene structure was proved to have a longer size than the one found to be annotated at the time of identification in the data-base. A full cDNA clone containing the full transcript was available and also complementation experiments using different gene fragments (annotated and suggested) leaded to the result that TBR gene is indeed, longer. TBR encodes a novel plant-specific protein with predicted plasma membrane localization, therefore being consistent with idea that is required for-, or is a novel component of a functional cellulose synthase complex. TBR is part of an Arabidopsis gene/protein family, (TBL-trichome birefringence like) which, depending on homology, comprises up to 20 members, none of which has a biological or biochemical function attributed. T-DNA insertion lines in TBR gene and two close homologues have been screened by PCR, but no homozygous were found and no trichomes phenotype was identified. Promoter-GUS lines were produced for TBR, as well as for its two closest homologues (one being a segmentally duplicated gene on chromosome III), using 1.6-2 kb of promoter sequence upstream of the annotated start codons. The TBR promoter was the only one of the three that yielded trichome expression, this probably explaining the phenotype of the TBR mutant. Moreover, TBR is expressed in leaves, in growing lateral roots, and in vascular tissues of young Arabidopsis seedlings and plantlets. Later on, the expression appears in inflorescens, stems, flowers and green siliques. This expression pattern is largely overlapping with those of the two analyzed homologues and it corresponds with data of RT-PCR expression profiling performed for TBR and the two analyzed homologues in different tissues, at different developmental stages. Biochemical analysis of cell wall (leaves and trichomes), as GC and MALDI-TOF, were performed, but revealed no major differences between tbr1 and wild type plants. Scanning electron microscopy analysis and cell wall polysaccharides antibody labeling showed a clear difference in the trichomes cell wall structure between mutant plant and wild type.
Nitrogen is often a limiting factor for plant growth due to its heterogenous distribution in the soil and to seasonal and diurnal changes in growth rates. In most soils, NH4+ and NO3 – are the predominant sources of inorganic nitrogen that are available for plant nutrition. In this context, plants have evolved mechanisms that enable them to optimize nitrogen acquisition, which include transporters specialized in the uptake of nitrogen and susceptible to a regulation that responds to nitrogen limiting or excess conditions. Although the average NH4+ concentrations of soils are generally 100 to 1000 times lower than those of NO3 – (Marschner, 1995), most plants preferentially take up NH4+ when both forms are present because unlike NO3– , NH4+ has not to be reduced prior to assimilation and thus requires less energy for assimilation (Bloom et al., 1992). Apart from high uptake rates in roots, high intracellular ammonium concentrations also result from quantitatively important internal breakdown of amino acids (Feng et al., 1998), and originates in high quantities during photorespiration (Mattson et al., 1997, Pearson et al., 1998). Thus, NH4+ is a key component of nitrogen metabolism for all plants and can accumulate to varying concentrations in all compartments of the cell, including the cytosol, the vacuole and in the apoplast (Wells and Miller, 2000; Nielsen and Schjoerring, 1998). Two related families of ammonium transporters (AMT1 and AMT2), containing six genes which encode transporter proteins that are specific for ammonium had been identified prior to this thesis and some genes had partially been characterised in Arabidopsis (Gazzarrini et al., 1999; Sohlenkamp et al. 2002; Kaiser et al., 2002). However, these studies were not sufficient to assign physiological functions to the individual transporters and AMT1.4 and AMT1.5 had not been studied prior to this thesis. Given this background, it was considered desirable to acquire a deeper knowledge of the physiological functions of the six Arabidopsis ammonium transporters. To this end, tissue specific expression profiles of the individual wildtype AtAMT genes were performed by quantitative real time PCR (qRT-PCR) and promoter-GUS expression. Modern approaches such as the use of T-DNA insertional mutants and RNAi hairpin constructs were employed to reduce the expression levels of AMT genes. Transcript levels were determined, and physiological, biochemical and developmental analysis such as growth tests on different media and 14C-MA and NH4+ uptake studies with the isolated insertional mutants and RNAi lines were performed to deepen the knowledge of the individual functions of the six AMTs in Arabidopsis. In addition, double mutants of the insertional mutants were created to investigate the extent in which homologous genes could compensate for lost transporter functions. The results described in this thesis show that the six AtAMT genes display a high degree of specifity in their tissue specific expression and are likely to play complementary roles in ammonium uptake into roots, in shoots, and in flowers. AtAMT1.1 is likely to be a ‘work horse’ for cellular ammonium transport and reassimilation. A major role is probably the recapture of photorespiratory NH3/NH4+ escaping from the cytosol. In roots, it is likely to transport NH4+ from the apoplast into cortical cells. AtAMT1.3 and AtAMT1.5 appear to be specialised in the acquisition of external NH4+ from the soil. Furthermore, AtAMT1.5 plays an additional role in the reassimilation of NH3/NH4+ released during the breakdown of storage proteins in the cotyledons of germinating seedlings. It was difficult to distinguish a specialisation between the transporters AtAMt1.2 and AtAMt1.1, however the root and flower specific expression patterns are different and indicate alternative functions of both. AtAMT1.4 has a very distinct expression which is restricted to the vascular bundels of leaves and to pollen only, where it is likely to be involved in the loading of NH4+ into the cells.The AtAMT2.1 expression pattern is confined to vascular bundels and meristematic active tissues in leaves where ammonium concentrations can reach very high levels. Additionally, the Vmax of AtAMT2 increases with increasing external pH, contrasting to AtAMT1.1. Thus, AtAMT2.1 it might be specialised in ammonium transport in ammonium rich environments, where the functions of other transporters are limited, enabling cells to take up NH4+ over a wide range of concentrations. The root hair expression ascribes an additional role in NH3/NH4+ acquisition where it possibly serves as a transporter that is able to acquire ammonium from basic soils where other transporters become less effective.RNAi lines showing a reduction in AtAMT gene mRNA levels and NH4+ transport kinetics, grew slower and flowering time was delayed. This indicates that NH4+ is a crucial and limiting factor for plant growth.
The external dispersal ("epizoochory") of vascular plant diaspores (seeds and fruits) by roe deer and wild boar, i.e. the most common wild large mammals with a large home range in central Europe, was investigated in a 6.5-km² forest area in NE Germany dominated by mesic deciduous forests. The study involved brushing out the diaspores from the coats and hooves of 25 shot roe deer and nine wild boar. The results were compared with the forest vegetation of the study area. Whilst wild boar transported large amounts of various diaspores in the coat, the significance of roe deer for epizoochory was low due to their sleek fur and different behaviour compared to wild boar. Altogether, 55 vascular plant species were transported externally. Since only a limited number of seeds came from woodland habitats, the open landscape was at least as important as a source of attached seeds as the forest vegetation. Thus, most plant species occurring in the studied forest area, especially characteristic woodland herbs, showed no adaptations to epizoochorous dispersal, although being very abundant in the herb layer. We conclude that hoofed game play a particular role concerning the dispersal of ruderal and grassland species in the agricultural landscape of central Europe. However, the actual spread of some herb species in forests of northern Germany, e.g. Agrostis capillaris, Brachypodium sylvaticum, Deschampsia flexuosa, Galium aparine and Urtica dioica, may be mainly facilitated by wild ungulates. Though dispersal by large mammals is an important mechanism for long-distance dispersal of plants in general, our results suggest that most of the characteristic herb species of mesic deciduous forests have only low epizoochorous dispersal potentials. The implications for nature conservation and silviculture are discussed.
A seed sowing experiment was conducted in a mixed secondary woodland on acidic soils in NE Germany with Melampyrum pratense, an annual ant-dispersed forest herb which lacks a natural population in the study area, but is abundant in similar habitats. Each set of 300 seeds was sown within one square metre at three sites in 1997, and the development of the populations was recorded from 1998 onward. Additionally, seed fall patterns were studied in a natural population by means of adhesive cardboard. All trials resulted in the recruitment of populations, which survived and increased in both individual number and area, up to the year 2001. Thus, local distribution of Melampyrum pratense is dispersallimited. Total individual number increased from 105 to 3,390, and total population area from 2.07 to 109.04 m². Migration occurred in all directions. Mean migration rate was 0.91 m per year, and the highest migration rate was 6.48 m. No individual was recorded beyond 7.63 m from the centres of the sawn squares after three years, suggesting exclusive short-distance dispersal. As primary dispersal enables only distances of up to 0.25 m, ants are presumed to be the main dispersal vectors. Despite differences in individual number and colonization patterns, migration rates did not differ significantly between the populations, but were significantly higher in 2001 due to an increased population size. Colonization patterns were characterized by a rapid, negative exponential decrease of population density with increasing distance from the sown plot, suggesting a colonization by establishment of more or less isolated outposts of individuals and a subsequent gradual infill of the gaps between. My results resemble myrmecochorous dispersal distances in temperate woodlands, and migration rates and patterns across ecotones from ancient to recent deciduous forests. They may function as a colonization model of Melampyrum pratense after accidental long-distance dispersal.
Structure and reactivity of a biological soil crust from a xeric sandy soil in Central Europe
(2004)
The investigation was designed to explore the structure, composition and activity of a biological soil crust on an acidic, sandy soil from a temperate climate. The crust covers several hundreds of square meters on the hilltop of a large terminal moraine. The conjugate alga Zygogonium ericetorum forms the essential matrix for the crust, a dense web of algal filaments with interspersed lichens and mosses. The crust is composed of three layers, with an uppermost layer consisting nearly entirely of a dense algal mat. In lower layers, a parasitic fungus, penetrating the algal cells, is another important component of the crust community. In this soil crust, photosynthetic and respiratory activity is stabilized at low water activities.