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Mathematical modeling of biological systems is a powerful tool to systematically investigate the functions of biological processes and their relationship with the environment. To obtain accurate and biologically interpretable predictions, a modeling framework has to be devised whose assumptions best approximate the examined scenario and which copes with the trade-off of complexity of the underlying mathematical description: with attention to detail or high coverage. Correspondingly, the system can be examined in detail on a smaller scale or in a simplified manner on a larger scale. In this thesis, the role of photosynthesis and its related biochemical processes in the context of plant metabolism was dissected by employing modeling approaches ranging from kinetic to stoichiometric models. The Calvin-Benson cycle, as primary pathway of carbon fixation in C3 plants, is the initial step for producing starch and sucrose, necessary for plant growth. Based on an integrative analysis for model ranking applied on the largest compendium of (kinetic) models for the Calvin-Benson cycle, those suitable for development of metabolic engineering strategies were identified. Driven by the question why starch rather than sucrose is the predominant transitory carbon storage in higher plants, the metabolic costs for their synthesis were examined. The incorporation of the maintenance costs for the involved enzymes provided a model-based support for the preference of starch as transitory carbon storage, by only exploiting the stoichiometry of synthesis pathways. Many photosynthetic organisms have to cope with processes which compete with carbon fixation, such as photorespiration whose impact on plant metabolism is still controversial. A systematic model-oriented review provided a detailed assessment for the role of this pathway in inhibiting the rate of carbon fixation, bridging carbon and nitrogen metabolism, shaping the C1 metabolism, and influencing redox signal transduction. The demand of understanding photosynthesis in its metabolic context calls for the examination of the related processes of the primary carbon metabolism. To this end, the Arabidopsis core model was assembled via a bottom-up approach. This large-scale model can be used to simulate photoautotrophic biomass production, as an indicator for plant growth, under so-called optimal, carbon-limiting and nitrogen-limiting growth conditions. Finally, the introduced model was employed to investigate the effects of the environment, in particular, nitrogen, carbon and energy sources, on the metabolic behavior. This resulted in a purely stoichiometry-based explanation for the experimental evidence for preferred simultaneous acquisition of nitrogen in both forms, as nitrate and ammonium, for optimal growth in various plant species. The findings presented in this thesis provide new insights into plant system's behavior, further support existing opinions for which mounting experimental evidences arise, and posit novel hypotheses for further directed large-scale experiments.
For the first time the transcriptional reprogramming of distinct root cortex cells during the arbuscular mycorrhizal (AM) symbiosis was investigated by combining Laser Capture Mirodissection and Affymetrix GeneChip® Medicago genome array hybridization. The establishment of cryosections facilitated the isolation of high quality RNA in sufficient amounts from three different cortical cell types. The transcript profiles of arbuscule-containing cells (arb cells), non-arbuscule-containing cells (nac cells) of Rhizophagus irregularis inoculated Medicago truncatula roots and cortex cells of non-inoculated roots (cor) were successfully explored. The data gave new insights in the symbiosis-related cellular reorganization processes and indicated that already nac cells seem to be prepared for the upcoming fungal colonization. The mycorrhizal- and phosphate-dependent transcription of a GRAS TF family member (MtGras8) was detected in arb cells and mycorrhizal roots. MtGRAS shares a high sequence similarity to a GRAS TF suggested to be involved in the fungal colonization processes (MtRAM1). The function of MtGras8 was unraveled upon RNA interference- (RNAi-) mediated gene silencing. An AM symbiosis-dependent expression of a RNAi construct (MtPt4pro::gras8-RNAi) revealed a successful gene silencing of MtGras8 leading to a reduced arbuscule abundance and a higher proportion of deformed arbuscules in root with reduced transcript levels. Accordingly, MtGras8 might control the arbuscule development and life-time. The targeting of MtGras8 by the phosphate-dependent regulated miRNA5204* was discovered previously (Devers et al., 2011). Since miRNA5204* is known to be affected by phosphate, the posttranscriptional regulation might represent a link between phosphate signaling and arbuscule development. In this work, the posttranscriptional regulation was confirmed by mis-expression of miRNA5204* in M. truncatula roots. The miRNA-mediated gene silencing affects the MtGras8 transcript abundance only in the first two weeks of the AM symbiosis and the mis-expression lines seem to mimic the phenotype of MtGras8-RNAi lines. Additionally, MtGRAS8 seems to form heterodimers with NSP2 and RAM1, which are known to be key regulators of the fungal colonization process (Hirsch et al., 2009; Gobbato et al., 2012). These data indicate that MtGras8 and miRNA5204* are linked to the sym pathway and regulate the arbuscule development in phosphate-dependent manner.
Lakes are increasingly being recognized as an important component of the global carbon cycle, yet anthropogenic activities that alter their community structure may change the way they transport and process carbon. This research focuses on the relationship between carbon cycling and community structure of primary producers in small, shallow lakes, which are the most abundant lake type in the world, and furthermore subject to intense terrestrial-aquatic coupling due to their high perimeter:area ratio. Shifts between macrophyte and phytoplankton dominance are widespread and common in shallow lakes, with potentially large consequences to regional carbon cycling. I thus compared a lake with clear-water conditions and a submerged macrophyte community to a turbid, phytoplankton-dominated lake, describing differences in the availability, processing, and export of organic and inorganic carbon. I furthermore examined the effects of increasing terrestrial carbon inputs on internal carbon cycling processes. Pelagic diel (24-hour) oxygen curves and independent fluorometric approaches of individual primary producers together indicated that the presence of a submerged macrophyte community facilitated higher annual rates of gross primary production than could be supported in a phytoplankton-dominated lake at similar nutrient concentrations. A simple model constructed from the empirical data suggested that this difference between regime types could be common in moderately eutrophic lakes with mean depths under three to four meters, where benthic primary production is a potentially major contributor to the whole-lake primary production. It thus appears likely that a regime shift from macrophyte to phytoplankton dominance in shallow lakes would typically decrease the quantity of autochthonous organic carbon available to lake food webs. Sediment core analyses indicated that a regime shift from macrophyte to phytoplankton dominance was associated with a four-fold increase in carbon burial rates, signalling a major change in lake carbon cycling dynamics. Carbon mass balances suggested that increasing carbon burial rates were not due to an increase in primary production or allochthonous loading, but instead were due to a higher carbon burial efficiency (carbon burial / carbon deposition). This, in turn, was associated with diminished benthic mineralization rates and an increase in calcite precipitation, together resulting in lower surface carbon dioxide emissions. Finally, a period of unusually high precipitation led to rising water levels, resulting in a feedback loop linking increasing concentrations of dissolved organic carbon (DOC) to severely anoxic conditions in the phytoplankton-dominated system. High water levels and DOC concentrations diminished benthic primary production (via shading) and boosted pelagic respiration rates, diminishing the hypolimnetic oxygen supply. The resulting anoxia created redox conditions which led to a major release of nutrients, DOC, and iron from the sediments. This further transformed the lake metabolism, providing a prolonged summertime anoxia below a water depth of 1 m, and leading to the near-complete loss of fish and macroinvertebrates. Pelagic pH levels also decreased significantly, increasing surface carbon dioxide emissions by an order of magnitude compared to previous years. Altogether, this thesis adds an important body of knowledge to our understanding of the significance of the benthic zone to carbon cycling in shallow lakes. The contribution of the benthic zone towards whole-lake primary production was quantified, and was identified as an important but vulnerable site for primary production. Benthic mineralization rates were furthermore found to influence carbon burial and surface emission rates, and benthic primary productivity played an important role in determining hypolimnetic oxygen availability, thus controlling the internal sediment loading of nutrients and carbon. This thesis also uniquely demonstrates that the ecological community structure (i.e. stable regime) of a eutrophic, shallow lake can significantly influence carbon availability and processing. By changing carbon cycling pathways, regime shifts in shallow lakes may significantly alter the role of these ecosystems with respect to the global carbon cycle.
Measuring the metabolite profile of plants can be a strong phenotyping tool, but the changes of metabolite pool sizes are often difficult to interpret, not least because metabolite pool sizes may stay constant while carbon flows are altered and vice versa. Hence, measuring the carbon allocation of metabolites enables a better understanding of the metabolic phenotype. The main challenge of such measurements is the in vivo integration of a stable or radioactive label into a plant without perturbation of the system. To follow the carbon flow of a precursor metabolite, a method is developed in this work that is based on metabolite profiling of primary metabolites measured with a mass spectrometer preceded by a gas chromatograph (Wagner et al. 2003; Erban et al. 2007; Dethloff et al. submitted). This method generates stable isotope profiling data, besides conventional metabolite profiling data. In order to allow the feeding of a 13C sucrose solution into the plant, a petiole and a hypocotyl feeding assay are developed. To enable the processing of large numbers of single leaf samples, their preparation and extraction are simplified and optimised. The metabolite profiles of primary metabolites are measured, and a simple relative calculation is done to gain information on carbon allocation from 13C sucrose. This method is tested examining single leaves of one rosette in different developmental stages, both metabolically and regarding carbon allocation from 13C sucrose. It is revealed that some metabolite pool sizes and 13C pools are tightly associated to relative leaf growth, i.e. to the developmental stage of the leaf. Fumaric acid turns out to be the most interesting candidate for further studies because pool size and 13C pool diverge considerably. In addition, the analyses are also performed on plants grown in the cold, and the initial results show a different metabolite pool size pattern across single leaves of one Arabidopsis rosette, compared to the plants grown under normal temperatures. Lastly, in situ expression of REIL genes in the cold is examined using promotor-GUS plants. Initial results suggest that single leaf metabolite profiles of reil2 differ from those of the WT.
Background: Leishmania tarentolae, a unicellular eukaryotic protozoan, has been established as a novel host for recombinant protein production in recent years. Current protocols for protein expression in Leishmania are, however, time consuming and require extensive lab work in order to identify well-expressing cell lines. Here we established an alternative protein expression work-flow that employs recently engineered infrared fluorescence protein (IFP) as a suitable and easy-to-handle reporter protein for recombinant protein expression in Leishmania. As model proteins we tested three proteins from the plant Arabidopsis thaliana, including a NAC and a type-B ARR transcription factor. Results: IFP and IFP fusion proteins were expressed in Leishmania and rapidly detected in cells by deconvolution microscopy and in culture by infrared imaging of 96-well microtiter plates using small cell culture volumes (2 μL - 100 μL). Motility, shape and growth of Leishmania cells were not impaired by intracellular accumulation of IFP. In-cell detection of IFP and IFP fusion proteins was straightforward already at the beginning of the expression pipeline and thus allowed early pre-selection of well-expressing Leishmania clones. Furthermore, IFP fusion proteins retained infrared fluorescence after electrophoresis in denaturing SDS-polyacrylamide gels, allowing direct in-gel detection without the need to disassemble cast protein gels. Thus, parameters for scaling up protein production and streamlining purification routes can be easily optimized when employing IFP as reporter. Conclusions: Using IFP as biosensor we devised a protocol for rapid and convenient protein expression in Leishmania tarentolae. Our expression pipeline is superior to previously established methods in that it significantly reduces the hands-on-time and work load required for identifying well-expressing clones, refining protein production parameters and establishing purification protocols. The facile in-cell and in-gel detection tools built on IFP make Leishmania amenable for high-throughput expression of proteins from plant and animal sources.
Im Rahmen des ersten Teils der vorliegenden Doktorarbeit konnten zwei nicht-essentielle (rps15, rpl36) und fünf essentielle (rps3, rps16, rpl22, rpl23, rpl32) im Plastom von Nicotiana tabacum kodierte Proteine des plastidären Ribosoms bezüglich ihrer Essentialität charakterisiert werden. Diese Gene wurden durch gezielte Knockout-Experimente inaktiviert und die resultierenden Effekte untersucht. Die Ergebnisse lassen einen Rückschluss auf die Lokalisation der Gene der insgesamt sieben untersuchten ribosomalen Proteine zu, die im Plastom mehrerer parasitischer, Plastiden-besitzender Spezies nicht mehr nachweisbar sind. Im Fall von rps15 könnte tatsächlich ein Verlust des Genes stattgefunden haben, im Fall der restlichen Gene ist eher mit einem Transfer in den Nukleus zu rechnen (rpl36 ausgenommen). Dies würde bedeuten, dass die Geschwindigkeit der erfolgreichen Etablierung eines Gentransfers in vielen parasitischen Spezies gegenüber grünen Pflanzen stark erhöht ist. Alle in E. coli nicht-essentiellen Proteine mit Homologen in Plastiden (rps15, rpl33, rpl36) sind auch dort, trotz ~1,5 Milliarden Jahren getrennter Evolution, nicht essentiell. Dieses Ergebnis bestätigt den schon früher festgestellten hohen Konservierungsgrad der bakteriellen und plastidären Translationsmaschinerien. Die Phänotypen der KO-Pflanzen der nicht-essentiellen Gene (rps15, rpl36) weisen auf eine interessante Rolle von S15 während der Ribosomenassemblierung hin und im Fall von L36 auf eine wichtige funktionelle Rolle im Plastiden-Ribosomen sowie auf eine Involvierung der Plastidentranslation in der Generierung eines retrograden Signals, welches die Blattform zu beeinflussen im Stande ist. Des Weiteren konnte eine Verbindung der Translationsaktivität mit der Ausbildung von Seitentrieben hergestellt werden, die vermutlich auf veränderte Auxinsynthese im Chloroplast zurückzuführen ist. Aus dem Folgeprojekt, bei dem Doppel-KO-Pflanzen nicht-essentieller ribosomaler Proteine erzeugt wurden, lässt sich auf eine relativ große Plastizität der Architektur von Plastidenribosomen schließen. Im zweiten Teil der Arbeit konnte erfolgreich ein Hochdurchsatz-Screeningsystem zur semiquantitativen Analyse von 192 verschiedenen miRNAs aus Chlamydomonas reinhardtii etabliert werden. Es gelang durch die Untersuchung von 23 verschiedenen Wachstums- und Stressbedingungen sowie Entwicklungsstadien mehrere miRNAs zu identifizieren, die eine differenzielle Expression zeigen sowie unter allen untersuchten Bedingungen konstant bleibende miRNAs nachzuweisen. Dadurch konnten mehrere vielversprechende Kandidaten-miRNAs ausgemacht werden, die nun eingehender untersucht werden können.
Körpermaße 2000
(2001)