570 Biowissenschaften; Biologie
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Die Hybridomtechnik zur Produktion von monoklonalen Antikörpern ermöglichte einen großen Schritt in der Entwicklung von Immunoassays für die biochemische Forschung und klinische Diagnostik. Auch die Produktion von Antikörpern gegen niedermolekulare Analyten, Haptene, typische Targets in der Lebensmittel- und Umweltanalytik, erlangte in den letzten Jahren eine immer größere Bedeutung. Im Zuge der Durchführung der Hybridomtechnik werden tausende Antikörper-sezernierende und nicht-sezernierende Zellen generiert. Die Selektion der wenigen antigenselektiven Hybridomzellen zählt dabei zu den herausforderndsten Schritten für die Antikörpergewinnung. Bisherige Selektionsverfahren, wie die Limiting-Dilution-Klonierung in Verbindung mit Enzyme-linked Immunosorbent Assays (ELISAs), garantieren keine Monoklonalität und erlauben nur das Screening von einigen wenigen Zellklonen. Hingegen ermöglichen Hochdurchsatz-Selektionsmethoden, wie die Fluoreszenz-aktivierte Zellsortierung (FACS), einen sehr hohen Probendurchsatz. Eine Einzelzellablage garantiert hierbei Monoklonalität. Jedoch sind die dafür erforderlichen Zellmarkierungen oftmals zellschädigend oder aufwendig zu generieren. Auch ist bisher noch keine Markierungsmethode bekannt, die es ermöglicht, Hapten-selektive Hybridomzellen durchflusszytometrisch zu analysieren und eine FACS-Selektion durchzuführen.
Aus diesem Grund wurden in dieser Arbeit zwei Zellmarkierungsmethoden entwickelt, die dies ermöglichen sollten. Die membranständigen Antikörper von Hybridomzellen sollten entweder direkt oder indirekt immunfluoreszenz-markiert und dadurch für die Durchflusszytometrie und FACS-Selektion zugänglich gemacht werden. Die direkte Markierung wurde mittels eines Hapten-Fluorophor-Konjugats durchgeführt. Sie ermöglichte erstmalig den Anteil an Haptenselektiven Hybridomzellen in einer Hybridomzelllinie zu überprüfen. Dies konnte für zwei Hapten-selektive Hybridomzelllinien, die Antikörper gegen das Hormon 17β-Estradiol und das Cardenolid Digoxigenin bilden, gezeigt werden. Durchflusszytometrie und ELISAs lieferten vergleichbare Ergebnisse. Zellen, die Hapten-selektiv markiert werden konnten, sezernierten ebenfalls Hapten-selektive Antikörper. Des Weiteren konnte die direkte Markierung dazu genutzt werden, zwei Mykotoxin-selektive Hybridomzelllinien, welche Antikörper gegen Aflatoxin und Zearalenon bilden, auf Monoklonalität zu testen. Dies ist mittels ELISA nicht möglich. Die Markierungsmethode eignete sich jedoch nur für fixierte Hybridomzellen. Eine Markierung von lebenden Zellen konnte weder durchflusszytometrisch noch mittels konfokaler Laser-Scanning-Mikroskopie gezeigt werden.
Dies gelang erst mit einer neu entwickelten indirekten Immunfluoreszenzmarkierung. Dabei wurden die Zellen zunächst mit einem Hapten-Peroxidase-Konjugat inkubiert, gefolgt von einem Fluorophor-markierten anti-HRP-Antikörper-Konjugat. Dies wurde für zwei Analyten, das Hormon Estron und das Antiepileptikum Carbamazepin, gezeigt. Die indirekte Markierung wurde erfolgreich dazu verwendet, Carbamazepin-selektive Hybridomzellen aus einem Fusionsansatz für die monoklonale Antikörperproduktion auszusortieren. Damit wurde erstmalig eine Zellmarkierungsmethode entwickelt, die eine Hochdurchsatz-Selektion lebender Hybridomzellen aus einem Fusionsansatz ermöglicht. Sie ist nicht zellschädigend und kann zusätzlich zur Selektion Hapten-selektiver Plasmazellen verwendet werden.
Chloroplast membranes have a unique composition characterized by very high contents of the galactolipids, MGDG and DGDG. Many studies on constitutive, galactolipid-deficient mutants revealed conflicting results about potential functions of galactolipids in photosynthetic membranes. Likely, this was caused by pleiotropic effects such as starvation artefacts because of impaired photosynthesis from early developmental stages of the plants onward. Therefore, an ethanol inducible RNAi-approach has been taken to suppress two key enzymes of galactolipid biosynthesis in the chloroplast, MGD1 and DGD1. Plants were allowed to develop fully functional source leaves prior to induction, which then could support plant growth. Then, after the ethanol induction, both young and mature leaves were investigated over time.
Our studies revealed similar changes in both MGDG- and DGDG-deficient lines, however young and mature leaves of transgenic lines showed a different response to galactolipid deficiency. While no changes of photosynthetic parameters and minor changes in lipid content were observed in mature leaves of transgenic lines, strong reductions in total chlorophyll content and in the accumulation of all photosynthetic complexes and significant changes in contents of various lipid groups occurred in young leaves. Microscopy studies revealed an appearance of lipid droplets in the cytosol of young leaves in all transgenic lines which correlates with significantly higher levels of TAGs. Since in young leaves the production of membrane lipids is lowered, the excess of fatty acids is used for storage lipids production, resulting in the accumulation of TAGs.
Our data indicate that both investigated galactolipids serve as structural lipids since changes in photosynthetic parameters were mainly the result of reduced amounts of all photosynthetic constituents. In response to restricted galactolipid synthesis, thylakoid biogenesis is precisely readjusted to keep the proper stoichiometry and functionality of the photosynthetic apparatus. Ultimately, the data revealed that downregulation of one galactolipid triggers changes not only in chloroplasts but also in the nucleus as shown by downregulation of nuclear encoded subunits of the photosynthetic complexes.
Plastid protein biosynthesis occurs on bacterial-type 70S ribosomes consisting of a large (50S) and a small (30S) subunit. However, since many steps of ribosome biogenesis are not thermodynamically favorable at biological conditions, it requires many assembly factors. One group of assembly factors, circularly permuted GTPases, was implicated in 30S subunit maturation in E. coli, by a protein RsgA. RsgA orthologues are present in bacteria and plastid-containing species and in silico analysis revealed presence of a RsgA-like protein in Arabidopsis thaliana. To functionally characterize the Arabidopsis orthologue, two AtRsgA T-DNA insertion lines were analyzed in this study. The exon line (rsgA-e) led to embryo lethality, while the intron line (rsgA-i) caused severe dwarf, pale green phenotype. Further investigation of rsgA-i mutant line revealed defects in chloroplast biogenesis which led to increased number of chloroplasts, decreased chloroplast size, decreased air space between mesophyll cells and smaller shoot apical meristems, which showed unusual proplastid accumulation. Moreover, rsgA-i plants showed reduction in chlorophyll A and B content, decreased electron transport rate and photosynthetic efficiency. Further analyses revealed that the protein is involved in chloroplast 30S subunit maturation. Interestingly, we observed that while chloroplast-targeted and chloroplast-encoded proteins are generally downregulated in the mutant, a contrasting upregulation of the corresponding transcripts is observed, indicating an elaborate compensatory mechanism. To conclude, the study presented here reveals a ribosome assembly factor and a compensatory mechanism activated during impaired chloroplast function.
Rubisco catalyses the first step of CO2 assimilation into plant biomass. Despite its crucial role, it is notorious for its low catalytic rate and its tendency to fix O2 instead of CO2, giving rise to a toxic product that needs to be recycled in a process known as photorespiration. Since almost all our food supply relies on Rubisco, even small improvements in its specificity for CO2 could lead to an improvement of photosynthesis and ultimately, crop yield. In this work, we attempted to improve photosynthesis by decreasing photorespiration with an artificial CCM based on a fusion between Rubisco and a carbonic anhydrase (CA).
A preliminary set of plants contained fusions between one of two CAs, bCA1 and CAH3, and the N- or C-terminus of RbcL connected by a small flexible linker of 5 amino acids. Subsequently, further fusion proteins were created between RbcL C-terminus and bCA1/CAH3 with linkers of 14, 23, 32, and 41 amino acids. The transplastomic tobacco plants carrying fusions with bCA1 were able to grow autotrophically even with the shortest linkers, albeit at a low rate, and accumulated very low levels of the fusion protein. On the other hand, plants carrying fusions with CAH3 were autotrophic only with the longer linkers. The longest linker permitted nearly wild-type like growth of the plants carrying fusions with CAH3 and increased the levels of fusion protein, but also of smaller degradation products.
The fusion of catalytically inactive CAs to RbcL did not cause a different phenotype from the fusions with catalytically active CAs, suggesting that the selected CAs were not active in the fusion with RbcL or their activity did not have an effect on CO2 assimilation. However, fusions to RbcL did not abolish RbcL catalytic activity, as shown by the autotrophic growth, gas exchange and in vitro activity measurements. Furthermore, Rubisco carboxylation rate and specificity for CO2 was not altered in some of the fusion proteins, suggesting that despite the defect in RbcL folding or assembly caused by the fusions, the addition of 60-150 amino acids to RbcL does not affect its catalytic properties. On the contrary, most growth defects of the plants carrying RbcL-CA fusions are related to their reduced Rubisco content, likely caused by impaired RbcL folding or assembly. Finally, we found that fusions with RbcL C-terminus were better tolerated than with the N-terminus, and increasing the length of the linker relieved the growth impairment imposed by the fusion to RbcL. Together, the results of this work constitute considerable relevant findings for future Rubisco engineering.
Plant cells host two important organelles: mitochondria, known as the cell’s ‘powerhouse’, which act by converting oxygen and nutrients into ATP, and plastids, which perform photosynthesis. These organelles contain their own genomes that encode proteins required for gene expression and energy metabolism. Transformation technologies offer great potential for investigating all aspects of the physiology and gene expression of these organelles in vivo. In addition, organelle transformation can be a valuable tool for biotechnology and molecular plant breeding. Plastid transformation systems are well-developed for a few higher plants, however, mitochondrial transformation has so far only been reported for Saccharomyces cerevisiae and the unicellular alga Chlamydomonas reinhardtii.
Development of an efficient new selection marker for plastid transformation is important for several reasons, including facilitating supertransformation of the plastid genome for metabolic engineering purposes and for producing multiple knock-outs or site-directed mutagenesis of two unlinked genes. In this work, we developed a novel selection system for Nicotiana tabacum (tobacco) chloroplast transformation with an alternative marker. The marker gene, aac(6′)-Ie/aph(2′′)-Ia, was cloned into different plastid transformation vectors and several candidate aminoglycoside antibiotics were investigated as selection agents. Generally, the efficiency of selection and the transformation efficiency with aac(6′)-Ie/aph(2′′)-Ia as selectable marker in combination with the aminoglycoside antibiotic tobramycin was similarly high as that with the standard marker gene aadA and spectinomycin selection. Furthermore, our new selection system may be useful for the development of plastid transformation for new species, including cereals, the world’s most important food crops, and could also be helpful for the establishment of a selection system for mitochondrial transformation.
To date, all attempts to achieve mitochondrial transformation for higher plants have been unsuccessful. A mitochondrial transformation system for higher plants would not only provide a potential for studying mitochondrial physiology but could also provide a method to introduce cytoplasmic male sterility into crops to produce hybrid seeds. Establishing a stable mitochondrial transformation system in higher plants requires several steps including delivery of foreign DNA, stable integration of the foreign sequences into the mitochondrial genome, efficient expression of the transgene, a highly regenerable tissue culture system that allows regeneration of the transformed cells into plants, and finally, a suitable selection system to identify cells with transformed mitochondrial genomes. Among all these requirements, finding a good selection is perhaps the most important obstacle towards the development of a mitochondrial transformation system for higher plants. In this work, two selection systems were tested for mitochondrial transformation: kanamycin as a selection system in combination with the antibiotic-inactivating marker gene nptII, and sulfadiazine as a selection agent that inhibits the folic acid biosynthesis pathway residing in plant mitochondria in combination with the sul gene encoding an enzyme that is insensitive to inhibition by sulfadiazine. Nuclear transformation experiments were considered as proof of the specificity of the sulfadiazine selection system for mitochondria. We showed that an optimized sulfadiazine selection system, with the Sul protein targeted to mitochondria, is much more efficient than the previous sulfadiazine selection system, in which the Sul protein was targeted to the chloroplast. We also showed by systematic experiments that the efficiency of selection and nuclear transformation of the optimized sulfadiazine selection was higher compared to the standard kanamycin selection system. Finally, we also investigated the suitability of this selection system for nuclear transformation of the model alga Chlamydomonas reinhardtii, obtaining promising results. Although we designed several mitochondrial transformation vectors with different expression elements and integration sites in the mitochondrial genome based on the sulfadiazine system, and different tissue culture condition were also considered, we were not able to obtain mitochondrial transformation with this system. Nonetheless, establishing the sul gene as an efficient and specific selection marker for mitochondria addresses one of the major bottlenecks and may pave the way to achieve mitochondrial transformation in higher plants.
The El Nino-Southern Oscillation (ENSO) is the main driver of the interannual variability in eastern African rainfall, with a significant impact on vegetation and agriculture and dire consequences for food and social security. In this study, we identify and quantify the ENSO contribution to the eastern African rainfall variability to forecast future eastern African vegetation response to rainfall variability related to a predicted intensified ENSO. To differentiate the vegetation variability due to ENSO, we removed the ENSO signal from the climate data using empirical orthogonal teleconnection (EOT) analysis. Then, we simulated the ecosystem carbon and water fluxes under the historical climate without components related to ENSO teleconnections. We found ENSO-driven patterns in vegetation response and confirmed that EOT analysis can successfully produce coupled tropical Pacific sea surface temperature-eastern African rainfall teleconnection from observed datasets. We further simulated eastern African vegetation response under future climate change as it is projected by climate models and under future climate change combined with a predicted increased ENSO intensity. Our EOT analysis highlights that climate simulations are still not good at capturing rainfall variability due to ENSO, and as we show here the future vegetation would be different from what is simulated under these climate model outputs lacking accurate ENSO contribution. We simulated considerable differences in eastern African vegetation growth under the influence of an intensified ENSO regime which will bring further environmental stress to a region with a reduced capacity to adapt effects of global climate change and food security.
Orthogonal systems for heterologous protein expression as well as for the engineering of synthetic gene regulatory circuits in hosts like Saccharomyces cerevisiae depend on synthetic transcription factors (synTFs) and corresponding cis-regulatory binding sites. We have constructed and characterized a set of synTFs based on either transcription activator-like effectors or CRISPR/Cas9, and corresponding small synthetic promoters (synPs) with minimal sequence identity to the host’s endogenous promoters. The resulting collection of functional synTF/synP pairs confers very low background expression under uninduced conditions, while expression output upon induction of the various synTFs covers a wide range and reaches induction factors of up to 400. The broad spectrum of expression strengths that is achieved will be useful for various experimental setups, e.g., the transcriptional balancing of expression levels within heterologous pathways or the construction of artificial regulatory networks. Furthermore, our analyses reveal simple rules that enable the tuning of synTF expression output, thereby allowing easy modification of a given synTF/synP pair. This will make it easier for researchers to construct tailored transcriptional control systems.
Exploring the Arabidopsis metabolic landscape by genetic mapping integrated with network analysis
(2017)
Background
The hypopharyngeal gland of worker bees contributes to the production of the royal jelly fed to queens and larvae. The gland consists of thousands of two-cell units that are composed of a secretory cell and a duct cell and that are arranged in sets of about 12 around a long collecting duct.
Results
By fluorescent staining, we have examined the morphogenesis of the hypopharyngeal gland during pupal life, from a saccule lined by a pseudostratified epithelium to the elaborate organ of adult worker bees. The hypopharyngeal gland develops as follows. (1) Cell proliferation occurs during the first day of pupal life in the hypopharyngeal gland primordium. (2) Subsequently, the epithelium becomes organized into rosette-like units of three cells. Two of these will become the secretory cell and the duct cell of the adult secretory units; the third cell contributes only temporarily to the development of the secretory units and is eliminated by apoptosis in the second half of pupal life. (3) The three-cell units of flask-shaped cells undergo complex changes in cell morphology. Thus, by mid-pupal stage, the gland is structurally similar to the adult hypopharyngeal gland. (4) Concomitantly, the prospective secretory cell attains its characteristic subcellular organization by the invagination of a small patch of apical membrane domain, its extension to a tube of about 100 μm in length (termed a canaliculus), and the expansion of the tube to a diameter of about 3 μm. (6) Finally, the canaliculus-associated F-actin system becomes reorganized into rings of bundled actin filaments that are positioned at regular distances along the membrane tube.
Conclusions
The morphogenesis of the secretory units in the hypopharyngeal gland of the worker bee seems to be based on a developmental program that is conserved, with slight modification, among insects for the production of dermal glands. Elaboration of the secretory cell as a unicellular seamless epithelial tube occurs by invagination of the apical membrane, its extension likely by targeted exocytosis and its expansion, and finally the reorganisation of the membrane-associated F-actin system. Our work is fundamental for future studies of environmental effects on hypopharyngeal gland morphology and development.
We studied the short- (12 h) and long-term (144 h) response of Daphnia pulex lipases to quality shifts in diets consisting of different mixtures of the green alga Scenedesmus with the cyanobacterium Synechococcus, two species with contrasting lipid compositions. The lipase/esterase activity in both the gut and the body tissues had fast responses to the diet shift and increased with higher dietary contributions of Synechococcus. When screening the Daphnia genome for TAG lipases, we discovered a large gene-family expansion of these enzymes. We used a subset of eight genes for mRNA expression analyses and distinguished between influences of time and diet on the observed gene expression patterns. We identified five diet-responsive lipases of which three showed a sophisticated short- and long-term pattern of expression in response to small changes in food-quality. Furthermore, the gene expression of one of the lipases was strongly correlated to lipase/esterase activity in the gut suggesting its potentially major role in digestion. These findings demonstrate that the lipid-related enzymatic machinery of D. pulex is finely tuned to diet and might constitute an important mechanism of physiological adaptation in nutritionally complex environments.
Biodiversity and intact ecological interactions form the basis for functional and resilient ecosystems that maintain optimal conditions for life on earth. During the second half of the 20th century, especially land-use changes and an intensification of agricultural management caused an unprecedented loss of biodiversity in agroecosystems worldwide. Concerns have been raised that the ongoing loss of biodiversity would ultimately lead to impaired ecological interactions and ecosystem functioning in agricultural landscapes. In order to stop biodiversity loss while producing enough food for a growing world population, we need to gain detailed knowledge on ecological interactions and the functioning of agroecosystems as a whole.
Bats (Chiroptera) represent an important component of global biodiversity, occupy a variety of ecological niches and fulfill numerous ecosystem services. Especially in temperate zone agroecosystems, bats were repeatedly reported to contribute to the reduction of pest insects above intensively managed arable fields. However, bat populations have been decimated by the consequence of land-use intensification which led to their legal protection status in the European Union (Council of Europe, 1979). The increasing number of wind turbines on arable fields poses an additional threat to bats as they might get injured or killed when flying too close to wind turbine blades. Although a large amount of land area is covered by arable fields, not much is known about how bats use the intensively managed agricultural landscape.
In the present thesis, my general aim was to identify the relevance of factors at different spatiotemporal scales for shaping species-specific bat activity above intensively managed arable fields. Therefore, I repeatedly monitored bat activity above open arable fields in a landscape dominated by agriculture which is located in Northeast Brandenburg, Germany. From 2012 to 2014, I recorded echolocation calls of bats on a total of 113 sites using a passive acoustic approach. I obtained a total of 27,779 recordings, identified the recorded echolocation calls manually to species level and calculated species-specific bat activity measures. Depending on the focus of research, I modeled the obtained species-specific activity measures using generalized linear and additive mixed effect models. In Chapter I, I focused on identifying seasonal patterns in several species-specific activity measures of different functional bat groups. In Chapter II, I investigated small-scale effects of landscape elements, such as hedgerows and forest edges, on the flight and foraging activity of different bat species along the edge-field interface. Additionally, I aimed at identifying whether these effects are influenced by small ponds located within arable field and whether these effects change across seasons. In Chapter III, my aim was to investigate the interaction between factors from different spatiotemporal levels on the flight and foraging activity of bats above arable fields. At the small spatial scale, I focused on prey availability, at a large spatial scale on selected parameters which describe landscape characteristics and at the temporal scale on seasonal effects.
The major findings obtained in each chapter can be summarized in the following three points. The first major finding is that not only landscape elements on a small spatial scale, e.g. a hedgerow at the edge of an arable field, but also landscape characteristics on a large spatial scale, e.g. landscape composition, shaped species-specific bat activity above open arable fields. This activity was also strongly influenced by interactions between landscape characteristics and local prey availability. Second, the influence of landscape elements and characteristics on bat activity above arable fields was not constant over time but changed across seasons with the strongest impact during summer as compared to spring and autumn. Third, I found indications of ecosystem service provided by N. noctula and P. nathusii in all three chapters, as especially these bat species were repeatedly found to forage above arable fields. This foraging activity was positively influenced by the proximity to landscape elements at the edge of the arable field but also by the presence of small ponds within the arable field.
In light of the obtained findings, I strongly recommend protecting and most importantly recreating semi-natural landscape elements in the agricultural landscape. Furthermore, I strongly recommend against the construction of wind turbines close to these linear woody vegetation edges as bats were found to be active close to these landscape elements. Additionally, the operation times for wind turbines should be down-regulated during the mating and migration period in autumn due to high bat activity above arable fields. Since bats are considered being good bioindicators, effective conservation measures for bats might contribute to the protection of species from other taxa leading to an overall support of biodiversity in agricultural landscapes. In their entirety, the findings in this thesis contribute to the knowledge of different aspects of bat ecology and shed light on the complex interplay between factors from different spatiotemporal levels that shape bat activity above arable fields. Additionally, they can serve as a basis for the improvement and development of conservation measures for bats in agricultural landscapes.