Refine
Has Fulltext
- yes (35) (remove)
Year of publication
Document Type
- Postprint (13)
- Doctoral Thesis (12)
- Article (7)
- Conference Proceeding (2)
- Other (1)
Is part of the Bibliography
- no (35) (remove)
Keywords
- climate change (3)
- Israel (2)
- Klimawandel (2)
- PUFA (2)
- Transkriptionsfaktoren (2)
- fatty acid (2)
- food quality (2)
- metabolism (2)
- transcription factors (2)
- ATP (1)
Institute
- Institut für Biochemie und Biologie (35) (remove)
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.
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.
The contractile vacuole (CV) is an osmoregulatory organelle found exclusively in algae and protists. In addition to expelling excessive water out of the cell, it also expels ions and other metabolites and thereby contributes to the cell's metabolic homeostasis. The interest in the CV reaches beyond its immediate cellular roles. The CV's function is tightly related to basic cellular processes such as membrane dynamics and vesicle budding and fusion; several physiological processes in animals, such as synaptic neurotransmission and blood filtration in the kidney, are related to the CV's function; and several pathogens, such as the causative agents of sleeping sickness, possess CVs, which may serve as pharmacological targets. The green alga Chlamydomonas reinhardtii has two CVs. They are the smallest known CVs in nature, and they remain relatively untouched in the CV-related literature. Many genes that have been shown to be related to the CV in other organisms have close homologues in C. reinhardtii. We attempted to silence some of these genes and observe the effect on the CV. One of our genes, VMP1, caused striking, severe phenotypes when silenced. Cells exhibited defective cytokinesis and aberrant morphologies. The CV, incidentally, remained unscathed. In addition, mutant cells showed some evidence of disrupted autophagy. Several important regulators of the cell cycle as well as autophagy were found to be underexpressed in the mutant. Lipidomic analysis revealed many meaningful changes between wild-type and mutant cells, reinforcing the compromised-autophagy observation. VMP1 is a singular protein, with homologues in numerous eukaryotic organisms (aside from fungi), but usually with no relatives in each particular genome. Since its first characterization in 2002 it has been associated with several cellular processes and functions, namely autophagy, programmed cell-death, secretion, cell adhesion, and organelle biogenesis. It has been implicated in several human diseases: pancreatitis, diabetes, and several types of cancer. Our results reiterate some of the observations in VMP1's six reported homologues, but, importantly, show for the first time an involvement of this protein in cell division. The mechanisms underlying this involvement in Chlamydomonas, as well as other key aspects, such as VMP1's subcellular localization and interaction partners, still await elucidation.
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.
Background: To improve the understanding of consequences of climate change for annual plant communities, I used a detailed, grid-based model that simulates the effect of daily rainfall variability on individual plants in five climatic regions on a gradient from 100 to 800 mm mean annual precipitation (MAP). The model explicitly considers moisture storage in the soil. I manipulated daily rainfall variability by changing the daily mean rain (DMR, rain volume on rainy days averaged across years for each day of the year) by ± 20%. At the same time I adjusted intervals appropriately between rainy days for keeping the mean annual volume constant. In factorial combination with changing DMR I also changed MAP by ± 20%. Results: Increasing MAP generally increased water availability, establishment, and peak shoot biomass. Increasing DMR increased the time that water was continuously available to plants in the upper 15 to 30 cm of the soil (longest wet period, LWP). The effect of DMR diminished with increasing humidity of the climate. An interaction between water availability and density-dependent germination increased the establishment of seedlings in the arid region, but in the more humid regions the establishment of seedlings decreased with increasing DMR. As plants matured, competition among individuals and their productivity increased, but the size of these effects decreased with the humidity of the regions. Therefore, peak shoot biomass generally increased with increasing DMR but the effect size diminished from the semiarid to the mesic Mediterranean region. Increasing DMR reduced via LWP the annual variability of biomass in the semiarid and dry Mediterranean regions. Conclusion: More rainstorms (greater DMR) increased the recharge of soil water reservoirs in more arid sites with consequences for germination, establishment, productivity, and population persistence. The order of magnitudes of DMR and MAP overlapped partially so that their combined effect is important for projections of climate change effects on annual vegetation.
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.
Transcription factors (TFs) are ubiquitous gene expression regulators and play essential roles in almost all biological processes. This Ph.D. project is primarily focused on the functional characterisation of MYB112 - a member of the R2R3-MYB TF family from the model plant Arabidopsis thaliana. This gene was selected due to its increased expression during senescence based on previous qRT-PCR expression profiling experiments of 1880 TFs in Arabidopsis leaves at three developmental stages (15 mm leaf, 30 mm leaf and 20% yellowing leaf). MYB112 promoter GUS fusion lines were generated to further investigate the expression pattern of MYB112. Employing transgenic approaches in combination with metabolomics and transcriptomics we demonstrate that MYB112 exerts a major role in regulation of plant flavonoid metabolism. We report enhanced and impaired anthocyanin accumulation in MYB112 overexpressors and MYB112-deficient mutants, respectively. Expression profiling reveals that MYB112 acts as a positive regulator of the transcription factor PAP1 leading to increased anthocyanin biosynthesis, and as a negative regulator of MYB12 and MYB111, which both control flavonol biosynthesis. We also identify MYB112 early responsive genes using a combination of several approaches. These include gene expression profiling (Affymetrix ATH1 micro-arrays and qRT-PCR) and transactivation assays in leaf mesophyll cell protoplasts. We show that MYB112 binds to an 8-bp DNA fragment containing the core sequence (A/T/G)(A/C)CC(A/T)(A/G/T)(A/C)(T/C). By electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation coupled to qPCR (ChIP-qPCR) we demonstrate that MYB112 binds in vitro and in vivo to MYB7 and MYB32 promoters revealing them as direct downstream target genes. MYB TFs were previously reported to play an important role in controlling flavonoid biosynthesis in plants. Many factors acting upstream of the anthocyanin biosynthesis pathway show enhanced expression levels during nitrogen limitation, or elevated sucrose content. In addition to the mentioned conditions, other environmental parameters including salinity or high light stress may trigger anthocyanin accumulation. In contrast to several other MYB TFs affecting anthocyanin biosynthesis pathway genes, MYB112 expression is not controlled by nitrogen limitation, or carbon excess, but rather is stimulated by salinity and high light stress. Thus, MYB112 constitutes a previously uncharacterised regulatory factor that modifies anthocyanin accumulation under conditions of abiotic stress.
Germination rates and germination fractions of seeds can be predicted well by the hydrothermal time (HTT) model. Its four parameters hydrothermal time, minimum soil temperature, minimum soil moisture, and variation of minimum soil moisture, however, must be determined by lengthy germination experiments at combinations of several levels of soil temperature and moisture. For some applications of the HTT model it is more important to have approximate estimates for many species rather than exact values for only a few species. We suggest that minimum temperature and variation of minimum moisture can be estimated from literature data and expert knowledge. This allows to derive hydrothermal time and minimum moisture from existing data from germination experiments with one level of temperature and moisture. We applied our approach to a germination experiment comparing germination fractions of wild annual species along an aridity gradient in Israel. Using this simplified approach we estimated hydrothermal time and minimum moisture of 36 species. Comparison with exact data for three species shows that our method is a simple but effective method for obtaining parameters for the HTT model. Hydrothermal time and minimum moisture supposedly indicate climate related germination strategies. We tested whether these two parameters varied with the climate at the site where the seeds had been collected. We found no consistent variation with climate across species, suggesting that variation is more strongly controlled by site-specific factors.