Institut für Biologie

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Uptake of pathogenic Escherichia coli into crop plants
(2023) Detert, Katharina Margarete; Schmidt, Herbert
Contaminations of fresh produce by enterohemorrhagic Escherichia coli (EHEC) have been an increasing source of human disease outbreaks in the past few decades. In particular, the large disease outbreak in 2011 in Germany has left many questions about the survival of EHEC in soil and the colonization of plants unanswered. Agricultural soil as a reservoir for EHEC strains represents an important contamination source for crop plants. The use of cattle manure for soil fertilization or contaminated irrigation water can result in the introduction of pathogens in fields. To prevent the transfer of pathogens into the food chain, the German fertilizer ordinance disallows the use of organic fertilizer 12 weeks before harvesting the crop plants. However, it is expected that EHEC survives for longer and therefore continue to pose a risk for crop plant contamination. In this study, the overall survival ability of the pathogenic E. coli O104:H4 strain C227/11Φcu, which represents a stx2a-negative derivative of the 2011 outbreak strain C227/11, in agricultural soil was investigated. Thereby, different environmental conditions, soil types and genetic factors of the bacterial strain were identified as influencing factors. Furthermore, the ability of E. coli O104:H4 strain C227/11Φcu to colonize lamb’s lettuce via the root system was investigated to demonstrate that contamination of soil can result in crop plant contamination. In the first publication, the survival of E. coli O104:H4 strain C227/11Φcu in soil microenvironments containing either diluvial sand or alluvial loam was investigated. Two different temperatures (4°C and 22°C) were used and the samples were incubated for more than 12 weeks. The study aimed to analyze whether cattle manure addition prolongs EHEC survival in the soil microenvironments. In the last step, the survival studies were performed using ΔrpoS and ΔfliC deletion mutant strains of C227/11Φcu. The results demonstrated that E. coli O104:H4 strain C227/11Φcu survived for at least 12 weeks in the soil microenvironment model. The survival rate was influenced by the soil type and the temperature. In more detail, the incubation at lower temperature prolonged the survival rate and pathogens were detected up to 20 weeks after inoculation. The application of contaminated cattle manure increased the survival ability at 22°C. Sigma factor RpoS was recognized as an important factor for soil survival. The rpoS deletion mutants showed significant reduction of the survival period while FliC did not influence the overall survival ability in these experiments. To investigate the influence of further genetic factors of the bacterial strain in more detail and thereby characterize all transcription activities, transcriptome analysis was performed in the second publication. Since the strain showed the best survival in alluvial loam at 4°C, this condition was used for the next experiments. To decrease the amount of competing soil microbiota, the soil samples were autoclaved prior inoculation. After inoculation and after 1 and 4 weeks, samples were taken for RNA isolation. The differential expression analysis was performed using the sample from time point 0 as control. The analysis revealed that stress response genes and genes of the primary metabolism were upregulated after 1 and 4 weeks. In addition, genes and gene sets for the uptake of various carbohydrates or amino acids were upregulated, indicating adaption to an environment with low nutrient availability. Moreover, the results of the second publication demonstrated that persistence of C227/11Φcu in soil is related with a complex interface of metabolic networks. The third paper focused on the colonization of lamb’s lettuce by E. coli O104:H4 strain C227/11Φcu via the root system. Surface-sterilized seeds were cultivated on Murashige-Skoog agar or in autoclaved agricultural soil and migration into the edible portions of the plants was analyzed after 2, 4 and 8 weeks. The results of this publication demonstrated that migration into the edible parts occurred when the surrounding agar or soil was contaminated. This highlighted the threat of plant contamination with pathogenic E. coli on the field as a result of soil contamination.
Integrative taxonomy, systematics and biogeography of geometrid moths in a Middle Eastern biodiversity hotspot
(2023) Wanke, Dominic; Krogmann, Lars
Iran is an important biodiversity hotspot in the world. Recent studies have shown that two of the 36 global biodiversity hotspots are located in Iran: The Irano-Anatolian and the Caucasian hotspots. These two hotspots include parts of the two mountain ranges in Iran, the Alborz Mountains and the Zagros Mountains, which are crucial for the biodiversity, hosting a large number of endemic species. However, climate change and anthropogenic activities threaten its diversity. This study uses geometrid moths as a model group to better understand general patterns of biodiversity and zoogeography in Iran. Geometridae are suitable for such studies and scientifically interesting for several reasons: The family is species-rich with nearly 24,000 known species worldwide (539 known species in Iran), the species have short life-cycles and thus react quickly to environmental changes, and they occupy specialized ecological niches. Knowledge of Palearctic geometrid moths is rather advanced compared to other regions. The Western Palearctic, in particular, has been the target of considerable research. However, this is not the case for regions in the Middle East and Central Asia, where much is still unknown and further research is crucial. To fill this gap for geometrid moths in this region, data on their species richness and distribution patterns were collected to reveal regions with special faunal elements. Therefore, this dissertation consists of three parts, each of which contributes an essential element to achieve these goals. The first part deals with the taxonomic problems of partially species-rich and morphologically very difficult genera within the three subfamilies Sterrhinae (Problepsis, Scopula, Cinglis, Pseudocinglis, Scopuloides, Glossotrophia, Zygophyxia); Geometrinae (Xenochlorodes); and Ennominae (Nychiodes, Synopsia, Synopsidia, Eumera). Type specimens and original descriptions were used for critical revisions to understand the diagnostic characters of the species. Additionally, large series of specimens from many different museums and private collections were examined to highlight morphological variations. Using an integrative taxonomic approach that includes morphological and molecular data, a total of one new genus and four new species were described and 37 taxonomic changes (e.g., new synonyms, new combinations) were made. The second part addresses genera with uncertain tribal affiliation or questionable taxonomic status, which were also partially targets of the taxonomic revisions. A multi-gene phylogenetic analysis was performed using one mitochondrial gene and up to nine nuclear genes, sequences generated as part of this work and sequences from published phylogenetic studies were taken to run the analyses. As a result, the genus Eumera was determined to belong to the tribe Prosopolophini, the genera Cinglis and Scopuloides were removed from synonymy with Scopula, two genera were synonymized (Glossotrophia, Pseudocinglis), and two species were transferred to a different genus (Problepsis wiltshirei, Aphilopota tyttha). In the third part, distribution data of Iranian Geometridae was used to identify biodiversity hotspots and regions of high endemism. In addition, a network-based method was used to divide the country into unique bioregions and highlight areas with specific faunal elements. As a result, an exceptional species richness was found along the two main mountain ranges, Zagros in the west and south and Alborz in the north. Considering only the endemic species, the southern mountain areas were identified as the most species-rich regions. The bioregionalization analysis also identified six main bioregions. Most of these bioregions reflect specific faunal structures and are in accordance with previous studies. This highlights the complex species composition in Iran and demonstrates the exceptional biodiversity of the country. In addition, our results indicated two transition zones between zoogeographical realms. Of the six zoogeographical realms defined by Wallace, three occur in Iran meeting in the south of the country: The Palearctic and Saharo-Arabian along the foothills of the Zagros Mountains and the Palearctic and Oriental in southeastern Iran. At these transition zones, Iran has very specific faunal elements of the Geometridae, which makes these zones important for conservation. Overall, this work contributes to a better understanding of the biodiversity of geometrids in Iran and neighboring countries. It serves as a resource for the identification of species, their distribution and habitats, which are of great interest for conservation efforts in Iran and neighboring countries.
Reproductive success and escape behaviour in urban greylag geese (Anser anser)
(2023) Mai, Sabrina; Steidle, Johannes
Urbanisation and its effect on animals and plants is an important factor to analyse in behavioural studies. Warmer temperatures and an increased availability of food provide benefits to animals occurring in urban habitats. In contrast, these animals also have to tolerate the disturbance caused by artificial lights or noise. This work studies an urban local population of greylag geese Anser anser. Greylag geese live in wetland landscapes, but also in urban parks with wide lawns and water bodies. Since the nineties, the city of Stuttgart in southwest Germany is home to a breeding population of greylag geese. As a result of a long-term ringing project, a large percentage of the population is ringed. This data shows that the population is non-migratory, as the geese generally remain in the area year-round. The individual identification offers the opportunity of tracing data, such as reproduction or behaviour, for the same animal over a longer period. This data can then be individually connected to genetic information, which is a large benefit in behavioural studies. The first chapter of this work focuses on reproductive success as one of the two key parameters which influence population change. As reproductive success itself is influenced by a variety of factors, this study analysed the effect of factors such as population size, brood size or dispersal by using two different measures of reproductive success: fledging success (the relation between hatched and fledged young of a brood ) and hatchling survival (the likelihood of a hatchling to survive to fledging). Fledging success of pairs initially increased with the number of times pairs bred together but decreased again in later broods. While the experience of a pair is therefore beneficial for their reproductive success, the subsequent decrease may be caused by the increasing age of the parents. The brood size also influenced reproductive success, as fledging success was higher in larger broods and hatchling survival was also positively influenced by brood size. In some years, several goose families dispersed from the breeding ground to a different brood rearing area. Hatchling survival was higher in these families, though this effect decreased again when the broods were large. The total population size had a negative effect on hatchling survival, indicating density dependence. To determine if there is a genetic basis for escape behaviour in greylag geese, the second chapter of this work compares Stuttgart’s urban local population of greylag geese with a rural local population in the countryside near Ludwigsburg. Animals occurring in urban habitats need a higher tolerance towards disturbance. This tolerance may be due to the animal’s personality. Animal personality is defined as consistent behaviour across time or context and is expressed for example through risk-prone behaviour. Urban animals are likely more risk-prone than their rural counterparts and thus have a bolder personality. There is increasing evidence that personality and thus behaviour have a genetic basis. This study analyses the dopamine D4 receptor (DRD4) as a candidate gene associated with boldness / shyness and fear. The gene showed a high variation with eleven alleles and 35 genotypes being detected in the analysed greylag geese. Allele and genotype frequencies were skewed, with one allele and two genotypes occurring more often than the others. Based on the detected frequencies, these two genotypes were defined as common and the remaining 33 as rare genotypes. Common genotypes were found more frequently in geese from urban areas, while rare genotypes were found more frequently in geese from rural areas. To determine if the detected genotypes could be associated with behaviour, the flight initiation distance (FID) of individual geese was measured. As the geese are ringed, FID measurements could be directly assigned to a specific goose and thus to its genotype. A high FID indicates geese which are more cautious, while a low FID indicates boldness. Indeed, greylag geese of the rural location show increased FID, while those in urban areas in Stuttgart have lower FIDs and thus a bolder personality. Despite these results, there was no significant correlation between DRD4 genotype frequency and FID. This may be explained by methodological effects. Alternatively, DRD4 has also been associated with other behaviours such as novelty seeking and exploratory behaviour. These behaviours may also differ between urban and rural animals and may thus be the reason for the detected frequencies. It is therefore likely that local habitat selection may be influenced by the DRD4 genotype. Overall, this work demonstrates that different factors, including genetics, should be taken into account when managing urban populations of wild animals, as their behaviour can differ significantly from their rural counterparts, even within a single species.
Mobile signals in plant parasitism
(2024) Greifenhagen, Anne; Schaller, Andreas
Close to two percent of all flowering plants evolved parasitism, with some parasitic species, like Striga spp. from the Orobanchaceae family, posing a prevailing threat to crop yield. Parasitic weed management is challenging and requires a deeper understanding of the complex parasite-host relationship (Section 1.1). Parasitic plants infect and parasitize host plants through a multicellular feeding organ, the haustorium. This organ may either develop from the root tip as a single terminal haustorium or emerge multiple times along the growing roots, called lateral haustoria. In both cases, protohaustoria develop into mature haustoria that enable the withdrawal of water and nutrients. Parasitism depends on parasite and host endogenous signaling but also on communication between both partners (Section 1.2). This is similar to the development of other plant organs like lateral roots and symbiotic nodules, whose number is adjusted by an autoregulation of nodulation (AON) system. The induction of parasitic organs by pathogenic nematodes, but in particular also by parasitic plants, involves the manipulation, neofunctionalization, and interspecific exchange of mobile signals (Section 1.3). However, most of these molecular cues remain elusive in the parasitic plant-host plant interaction. This work aimed to address the biogenesis and function of parasite-derived endogenous and interspecific mobile signals involved in early till late stages of parasitism in the model system Phtheirospermum japonicum infecting Arabidopsis thaliana (Section 1.4). Transcriptome and genome studies on parasitic plants paved the way to unravel signaling cues contributing to parasitism. The evolution of parasitism correlates with the expansion of certain gene families followed by their parasitism-related neofunctionalization as seen for the KARRIKIN-INSENSITIVE 2 ‘divergent’-type (KAI2d) gene family in parasitic plants of the Orobanchaceae. Likewise, subtilisin-like serine protease (subtilase, SBT) genes in P. japonicum and Striga underwent an expansion, and some show haustorium tip-specific expression. The proteolytic activity of PjSBTs is required for proper haustorium formation and development. Despite their importance, no substrates of PjSBTs have been identified. In this work, PjSBT1.2.3 was found to be co-expressed with CLAVATA3(CLV)/EMBRYO-SURROUNDING REGION-related 3 (PjCLE3) during infection in the same domain of the haustorium. PjSBT1.2.3 cleaved PjCLE3 in vitro, thereby releasing the bioactive mature PjCLE3 peptide (Section 2.1, Fig.2.1.1). Sensing host-derived haustorium-inducing factors (HIFs) initiates haustorium organogenesis. In the absence of a host, the synthetic mature PjCLE3 induced protohaustorium formation similar to a host-derived benzoquinone HIF. Combined treatment of both HIFs potentiated their activity (Section 2.1, Fig.2.1.2). Pj cle3 knock-out hairy roots (HRs) formed fewer haustoria, particularly due to the absence of secondary protohaustoria (Section 2.1, Fig.2.1.3). These data demonstrate the existence of an autoregulation of haustoria formation (AOH) system as part of which the PjSBT1.2.3-PjCLE3 module, in analogy to AON, regulates the number of P. japonicum lateral haustoria. During the early stages of parasitism, the parasitism-related PjSBT1.2.3-PjCLE3 module promotes protohaustorium formation by sensitizing the parasite root for host-derived HIFs (Section 3.1). A homologous SBT-CLE module also exists in Striga, even though the parasite forms a terminal haustorium. Striga CLE2s are identical to host CLEs and this mimicry might improve nutrient allocations from the host during later stages of parasitism (Section 2.1, Fig.S2.1.2; Section 3.2). Similarly, parasite-derived cytokinin (CK) translocates through the haustorium inducing host hypertrophy, a swelling of host tissue above the infection site, thereby potentially benefiting parasite nutrient acquisition. In agrobacteria and plants, isopentenyltransferases (IPTs) synthesize CK precursors. Similar to SBTs and KAI2ds from parasitic plants, P. japonicum and S. hermonthica IPT1 genes exist as multiple copies, with one copy, PjIPT1a showing specific expression at the tip of haustoria (Section 2.2, Fig.2.2.1, Fig.2.2.2). Bioinformatic tools predicted a chloroplast transit peptide (CTP) for PjIPT1s, but PjIPT1-GFP fusions localized to cytoplasm and nucleus suggesting the CTP to be non-functional (Section 2.2, Fig.2.2.2, Fig.S2.2.2). To test substrate specificity and activity of both PjIPT1 copies, isoprenylation-activity was probed in vitro. PjIPT1b used both AMP and ATP as substrates, whereas PjIPT1a displayed a higher affinity for AMP, indicating that PjIPT1b may be the canonical, whereas PjIPT1a is the parasitism-related IPT (Section 2.2, Fig.2.2.4). This is further supported by the observation that CRISPR/Cas9-mediated mutation of PjIPT1a abolishes CK responses in the infected host (Section 3.3, Section 2.2, Fig.2.2.3). SBT-CLE, IPT-CK together with KAI2ds all have in common that their parasitism-related function may evolutionally result from gene duplication combined with neofunctionalization. Targeting duplicated and neofunctionalized genes may prove to be a promising strategy to combat parasitic weeds. (Sections 3.4, 3.5).
Adaptation of model organisms and environmental bacilli to glyphosate gives insight to species-specific peculiarities of the shikimate pathway
(2024) Schwedt, Inge; Commichau, Fabian
Glyphosate (GS), the active ingredient of the popular herbicide Roundup, inhibits the 5-enolpyruvyl shikimate-3-phosphate (EPSP) synthase of the shikimate pathway, which is present in archaea, bacteria, Apicomplexa, algae, fungi, and plants. In these organisms, the shikimate pathway is essential for de novo synthesis of aromatic amino acids, folates, quinones and other metabolites. Therefore, the GS-dependent inhibition of the EPSP synthase results in cell death. Previously, it has been observed that isolates of the soil bacteria Burkholderia anthina and Burkholderia cenocepacia are resistant to high amounts of GS. In the framework of this PhD thesis, it could be demonstrated that B. anthina isolates are not intrinsically resistant to GS. However, B. anthina rapidly adapts to the herbicide at the genome level and the characterization of GS-resistant suppressor mutants led to the discovery of a novel GS resistance mechanism. In B. anthina, the acquisition of loss-of-function mutations in the ppsR gene increases GS resistance. The ppsR gene encodes a regulator of the phosphoenolpyruvate (PEP) synthetase PpsA. In the absence of a functional PpsR protein, the bacteria synthesize more PEP, which competes with GS for binding in the active site of the EPSP synthase, increasing GS resistance. The EPSP synthase in B. anthina probably does not allow changes in the amino acid sequence as it is the case in other organisms. Indeed, the Gram-negative model organism Escherichia coli evolves GS resistance by the acquisition of mutations that either reduce the sensitivity of the EPSP synthase or increase the cellular concentration of the enzyme. Unlike E. coli, the EPSP synthase is also critical for the viability of Gram-positive model bacterium Bacillus subtilis. This observation is surprising because the enzyme belongs to the class of GS-insensitive EPSP synthases. In fact, the EPSP synthase is essential for growth of B. subtilis. The determination of the nutritional requirements allowing the growth of B. subtilis and E. coli mutants lacking EPSP synthase activity revealed that the demand for shikimate pathway intermediates is higher in the former organism. This finding explains why laboratory as well as environmental Bacilli exclusively adapt to GS by the mutational inactivation of glutamate transporter genes. Here, it was also shown that a B. subtilis mutant lacking EPSP synthase activity grows in minimal medium only when additional mutations accumulate in genes involved in the regulation of aerobic/anaerobic metabolism and central carbon metabolism. The characterization of these additional mutants will help to elucidate the peculiarities of the shikimate pathway in B. subtilis. Moreover, the mutants could be useful to identify the aromatic amino acid transporters that still await their discovery.
Speciation and isolating barriers in a parasitoid wasp focusing on the role of reproductive isolation caused by endosymbionts
(2023) Pollmann, Marie; Steidle, Johannes
Hymenoptera are one of the most speciose animal taxa, presumably caused by a high rate of speciation. The investigation of evolutionary processes is predominantly based on the biological species concept (BSC), which defines species as groups of interbreeding individuals which are reproductively isolated from individuals of other groups. As an alternative approach, species are delimitated by predetermined threshold distances in the so-called barcode segment of the mitochondrial COI gene. Many arthropods are infected with endosymbiotic bacteria, some of which have been shown to manipulate their hosts’ reproduction via mechanisms like cytoplasmic incompatibility (CI). It occurs between infected males and uninfected females and results in the reduction or absence of diploid offspring. Due to their ability to interfere with the reproduction of their hosts, endosymbionts have been suggested to be potential drivers of their hosts’ speciation processes. Lariophagus distinguendus is a parasitoid wasp which uses larvae of multiple coleopteran species as hosts. In past studies, two distinct species of L. distinguendus have been discovered. They are separated by different barriers including unidirectional CI caused by an unknown bacterium. Here, the nature of the CI inducer in crosses between untreated males and tetracycline-treated females of the L. distinguendus strain STU was investigated. Additionally, diversity and speciation processes were investigated within the L. distinguendus species complex by constructing phylogenetic trees with COI and nuclear genes as well as by conducting crossing experiments with different strain combinations. The bacterium Spiroplasma, termed sDistinguendus (sDis) for its L. distinguendus host, was identified to be the causative agent of this CI. Despite being known for multiple types of interactions with various hosts, Spiroplasma had not been demonstrated to cause CI before. None of the other bacteria identified within STU were associated with CI and none of the known CI inducers and other bacteria manipulating reproduction were found. The potential for maternal transmission was shown by the presence of sDis in the ovaries of STU females. Transferring sDis and CI from infected to uninfected STU females via hemolymph microinjection solidified the connection between CI and sDis. The simplicity of this transfection technique could prove to be valuable for applicable purposes in future. CI strength varying between male offspring of the injected females was not linked to the titer of sDis. All available L. distinguendus strains were analyzed with the barcode segment of COI as well as five nuclear genes to infer their phylogenetic relationships. Furthermore, five pairs of strains covering a gradient of genetic divergence were selected for crossing experiments investigating their reproductive isolation. The nuclear genes recovered two clusters identical to the previously known species, whereas three distinct clusters were found based on barcoding. These three clusters were revealed as separate species due to very high to complete reproductive isolation in crossing experiments between strains representing each of them. Threshold values of COI distances commonly used for species delimitation relying predominantly on barcodes, i.e. 2% to 2.2%, were shown to be not applicable for L. distinguendus due to the divergence between strains of the same species exceeding these thresholds. This calls the faith in these pre-set thresholds and the barcode-only approach to species delimitation into question and suggests the consideration of additional data along with those obtained by barcoding for species delimitation. Reproductive isolation between the strains was found to span a continuum of divergence from no reproductive isolation and low genetic divergence to complete isolation and high genetic divergence. The first barrier, present in all strain pairs, was sexual isolation, ranging from weak and unidirectional to complete and near complete in both directions. In the untreated cross between BIR females and STU males, sDis-induced CI was present along with sexual isolation. The reduced fertility of hybrid males was the next barrier to appear in the three more distantly related strain pairs along with behavioral sterility of hybrid females in the one strain pair among them where it was measured. These barriers were followed by the behavioral sterility, inviability, and physiological sterility of hybrid males and as last barrier by the reduced fertility of hybrid females. Due to the majority of barriers affecting hybrid males, but not females, speciation in L. distinguendus follows Haldane’s rule, stating that a fitness loss present in the hybrids of only one sex will be affecting the heterogametic sex, i.e. the males in L. distinguendus. In conclusion, the CI between infected males and uninfected females of L. distinguendus was found to be caused by Spiroplasma, termed sDis, marking the first time for CI to be demonstrated as phenotype of Spiroplasma. It was also one reproductive barrier present in crossings of different L. distinguendus strains, apparently contributing to their divergence. The diversity within the L. distinguendus species complex was shown to be greater than detected before, as seen by the discovery of a third species. The studied pairs of strains covered a wide range of both genetic divergence and strength of reproductive isolation, suggesting L. distinguendus to be currently in the process of ongoing speciation. The commonly used threshold distances in COI were not applicable for species delimitation in L. distinguendus, challenging their unconditional use.
Cellular stress regulates fibroblast growth factor 23 (FGF23) und αklotho
(2023) Münz, Sina; Föller, Michael
Cellular stress is defined as the impairment of regular cell function by internal or external stimuli including critical temperatures, energy deficiency, infections, mechanic injury, or chemical noxae. The present thesis aims to investigate the influence of cellular stress on the expression of FGF23 and αklotho. FGF23 is predominantly produced in bone and regulates the phosphate excretion in the kidney. Thereby, αklotho functions as a co-receptor for FGF23. By binding to the FGF receptor-αklotho complex, FGF23 reduces the reabsorption of phosphate from the tubular lumen by decreasing the abundance of sodium-phosphate co-transporters. Furthermore, FGF23 decreases the synthesis of 1,25(OH)2D3, active vitamin D, and increases its degradation. 1,25(OH)2D3 is a regulator of intestinal phosphate absorption and therefore, FGF23 additionally reduces dietary phosphate uptake. Chronically elevated FGF23 is associated with numerous disorders such as kidney disease or CVD. Beside its function as a co-receptor of FGFR, αklotho has many beneficial FGF23-independent functions. It has originally been identified as an anti-aging hormone, as a loss-of-function mutation in the αklotho gene causes numerous aging-like symptoms such as vascular and tissue calcification, osteoporosis, sterility, and an early death. The present papers investigated the influence of cytostatic drugs cisplatin, paclitaxel, and doxorubicin as well as apoptosis inducers PAC-1 and serum depletion on the regulation of FGF23 and αklotho. In UMR106 rat osteoblast-like osteosarcoma cells, a 24 or 48 h-treatment with cisplatin, doxorubicin, PAC-1, or serum reduction and depletion significantly up-regulated Fgf23 expression. Under serum depletion, also FGF23 protein secretion was increased. In addition to FGF23, cisplatin and doxorubicin also increased gene expression of pro-inflammatory cytokine Il6 hinting at the presence of necrotic cell death. By inhibiting Il-6 membrane receptor gp130 it has been shown, that FGF23 stimulation partially depended on IL-6 signaling. The stimulation of FGF23 by inflammatory mediators including IL-6, TNFα, TGF-β, or IL-1β has already been reported by others. Furthermore, inflammatory diseases such as rheumatoid arthritis, CKD, or inflammatory bowel disease are associated with excess FGF23 serum concentrations. In this regard, we investigated gene expression and activation of the transcription factor NFκB, which regulates numerous inflammatory functions. Cisplatin and doxorubicin increased the expression of NFκB subunit Rela and cisplatin also stimulated the phosphorylation of NFκB. Independently, NFκB inhibitors wogonin and withaferin A attenuated cisplatin-mediated stimulation of FGF23 indicating, that FGF23 excess was in part promoted by NFκB signaling. These investigations confirmed a strong impact of cisplatin or doxorubicin-induced inflammation on FGF23 synthesis, whereas PAC-1 and serum depletion have reported to directly induce apoptosis, which is commonly not associated with inflammation. Known factors, induced by all cytotoxic substances used here, are the formation of ROS and activation of HIF1α. Both are positive regulators of FGF23, leading to the conclusion, that cellular stress might regulate FGF23 via HIF1α or oxidative stress. FGF23 excess results in increased bone resorption and suppressed bone formation. Likewise, also chemotherapeutic drugs and serum deficiency reduce bone density. Therefore, the stimulation of FGF23 may cause or further stimulate bone resorption. In paper 2, the influence of the cytostatic drugs cisplatin, paclitaxel, and doxorubicin as well as apoptosis inductors PAC-1 or serum depletion on αklotho expression in renal MDCK, NRK-52E, and HK-2 cells has been investigated. In fact, all cytotoxic compounds stimulated gene expression of αklotho while decreasing cell proliferation and viability. By using a combined apoptosis and necrosis assay, we confirmed the induction of apoptosis but also necrosis to a variable extent. Additionally, the transcriptional regulation of apoptotic proteins of the BCL-2 family was assessed and confirmed apoptosis stimulation. Transcription factor PPARγ is a known positive regulator of αklotho. In MDCK cells, we detected a significant influence of cisplatin-mediated stimulation of PPARγ mRNA on the αklotho increase. Furthermore, cisplatin, doxorubicin, PAC-1, and serum deprivation also up-regulated FGFR production in MDCK cells. In cancer cells, overexpression of FGFR is associated with enhanced resistance against chemotherapeutic drugs. Consequently, αklotho and FGFR1 stimulation may be a protective mechanism to prevent hyperphosphatemia during diseases. However, human HK-2 cells treated with cisplatin, paclitaxel, doxorubicin, or serum depletion significantly down-regulated αklotho expression and protein secretion. PAC-1 did not change the expression or production of αklotho in HK-2 cells, which might be explained by the minor effect of PAC-1 on non-carcinogenic cells lacking an overexpression of procaspase-3. The differential regulation of αklotho in MDCK and NRK-52E versus HK-2 cells by cytotoxic stress might have numerous causes. For instance, there is evidence of an increased sensitivity of HK-2 cells to stress stimuli but a better comparability to the animal model. However, immortalized cell lines can not completely reflect the conditions of native tissue especially with regard to cell death. Furthermore, the species, sex or age of the donor organism as well as passage number of the cells and drug transporter expression might impact αklotho regulation. Additionally, the mode of cell death determined by intracellular ATP homeostasis and its regulation of AMPK might play an important role in αklotho regulation. However, all these theories need to be further addressed. In summary, inflammation, ROS formation, or the activation of HIF1α are all reported to correlate in a negative manner with αklotho production or serum levels. αklotho down-regulation may be a tool to increase cell proliferation or prevent hypophosphatemia. In contrast, AMPK activation by intracellular ATP restriction may positively regulate αklotho to promote cell protection and avoid hyperphosphatemia.
Integrative taxonomy of platygastrine wasps of Germany
(2023) Awad, Jessica; Krogmann, Lars
In the context of the sixth mass extinction, our understanding of insect decline is severely limited by a lack of information on biodiversity. Many highly abundant and diverse insect groups remain unidentifiable to species or even to genus. The parasitoid wasp superfamily Platygastroidea is one such “dark taxon”. The taxonomic impediment is especially severe in the Palearctic Platygastrinae due to the proliferation of names with vague concepts. Platygastrine wasps parasitize the hyper-diverse Cecidomyiidae (Diptera), suggesting that their species richness may be correspondingly high. Revisionary work is needed to identify named species and to discover new ones. This work applies integrative taxonomic methods to address the most pressing problems within the subfamily. The research presented in this dissertation focuses on Europe, particularly Germany, which is the current center of insect decline research as well as a historical center of platygastrine taxonomy. As part of this work, historical descriptions and their associated type material were examined and clarified so that further revisionary work can occur. The dissertation is structured in five chapters, of which two are published and three are unpublished. The first chapter reviews 18 genera of Platygastridae described by Arnold Förster (1856), most of which represent distinct and recognizable lineages. The study provides their taxonomic history, diagnostic remarks, English translations, and illustrations of important specimens from the Förster collection in the Natural History Museum Vienna. The collection also includes original exemplar specimens of European species whose types have been lost. Potential neotypes and lectotypes from this material are noted, with the aim of improving nomenclatural stability in the group. Potential neotypes were found for Amblyaspis forticornis (Nees, 1834), Isocybus grandis (Nees, 1834), Platygaster striolata Nees, 1834, and Trichacis tristis (Nees, 1834). Lectotypes will be designated for Platygaster spinigera Nees, 1834, which will be transferred to Leptacis, and for Platygaster corvina Förster, 1861, with Platygaster henkvlugi Buhl, 1996 treated as a junior synonym. Synopeas mutica (Nees, 1834) should be transferred back to Platygaster. The second chapter addresses generic concepts within Platygastrinae sensu Ashmead. Part of Platygastrinae (former Inostemmatinae sensu Ashmead) was reviewed and keyed by Masner & Huggert (1989). However, more than half of the genera in the subfamily, including the vast majority of species, have not been revised. A working key to 14 major world genera, the first of its kind, is provided. An additional six genera and three subgenera of uncertain placement are discussed. The third chapter focuses on nomenclatural problems in the genus Platygaster Latreille, which is the type genus for its subfamily, family, and superfamily. It is also the largest genus in Platygastroidea with nearly 700 species, and recent evidence indicates that it is not monophyletic. It is necessary to establish the identity of the type specimen, as well as the identities of the 13 generic junior synonyms and four subgenera, in order to proceed with taxonomic divisions. The type specimen Scelio ruficornis Latreille, 1805, lost to science for 192 years, was rediscovered. However, it does not belong to the prevailing concept of Platygaster, but to Isocybus Förster, which has extreme taxonomic ramifications. The proposed solution would replace the type species of each genus in order to retain prevailing usage and prevent widespread confusion. A petition is presented to designate Epimeces ensifer Westwood as the type of Platygaster and Scelio ruficornis as the type of Isocybus. In the fourth chapter, the genus Trichacis Förster is revised for Europe. Examination of historical and modern collections combined with DNA barcoding revealed the presence of only a single species in Europe, Trichacis tristis (Nees, 1834), redescribed here. The results suggest that T. tristis has 14 junior synonyms: T. abdominalis (Thomson, 1859); T. bidentiscutum Szabó, 1981; T. didas (Walker, 1835); T. fusciala Szabó, 1981; T. hajduica Szabó, 1981; T. illusor Kieffer, 1916; T. nosferatus Buhl, 1997; T. pisis (Walker, 1835); T. persicus Asadi & Buhl, 2021; T. pulchricornis Szelényi, 1953; T. quadriclava Szabó, 1981; T. remulus (Walker, 1835); T. vitreus Buhl, 1997; and T. weiperti Buhl, 2019. Four species should be transferred to Amblyaspis Förster: T. afurcata Szabó, 1977, T. hungarica Szabó, 1977, T. pannonica Szabó, 1977, and T. tatika Szabó, 1977. Intraspecific variation, biological associations, and taxonomic history are discussed. DNA barcodes are provided and analyzed in the context of worldwide Trichacis and its sister genus Isocybus Förster. The fifth chapter examines platygastrid diversity in Germany in the context of insect decline. DNA barcodes indicate the presence of 178 observed species, with an estimated total of 290. GBOL sampling captured an estimated 93.7% of total species richness, but only 45.8% of rare species. A case study from Isocybus compares historical specimens, DNA barcode vouchers, and ecological data to illustrate the possible decline of a wetland parasitoid species. Altogether, the research demonstrates the importance of historical material, especially type specimens, in understanding biodiversity through time, and provides an essential taxonomic foundation for much-needed modern identification resources for Platygastrinae in the western Palearctic and beyond.
Intracellular regulation of Wnt and FGF signal transduction by the late endosomal compartment in Xenopus laevis
(2022) Kreis, Jennifer; Feistel, Kerstin
The endosomal network depicts a vast playground of multiple processing capabilities in terms of signaling. Distinct compartments of the endosomal machinery exert specific functions and thus contribute in signal termination, transduction, attenuation or amplification. Initially, these functions were attributed to early endosomes but recent research likewise considers late endosomes to be just as relevant in mediating such processes. Functionality as well as the molecular identity of these intracellular membranous platforms are orchestrated by a large superfamily of small Ras like GTPases. The collected data of this study particularly highlight the involvement of late endosomes and its associated regulator Rab7 in the early development of the African clawed frog Xenopus laevis. In particular, the first two chapters address the Rab7-dependent specification of the mesodermal germ layer by regulating intracellular pathway activity of Wnt and FGF/MAPK signaling. After fertilization formation of the germ layers is one of the first processes to be initiated. An essential part of mesoderm development comprises subdivision into different mesodermal regions, thus clustering it into ventrolateral and dorsal mesoderm. This patterning is crucial to promote further differentiation into various tissues arising from the mesodermal germ layer. It turned out, Rab7 regulates ventrolateral fates in a Wnt-dependent manner. The small GTPase exerts its function upstream of the Wnt co-transcription factor Ctnnb1 to ensure its nuclear relocalization. In addition to that, Rab7-positive endosomes are likewise required to mediate intracellular FGF/MAPK signal transduction in order to specify dorsal mesoderm. Here, Rab7 regulates proper signaling at the level or downstream of Ras and upstream of Erk/Mapk1. The last chapter then elicits further regulative properties of the late endosomal platform, concerning Cd63 function. The tetraspanin Cd63, which constitutes a transmembrane protein, associates with late endolysosomal compartments and exhibits a similar expression pattern like the small GTPase Rab7 in Xenopus laevis. Contrary to Rab7, function of Cd63 seems to be dispensable whilst gastrulation. However, the presented studies in this chapter suggest a vital function of the tetraspanin Cd63 during axial elongation and correct eye development. Therefore, these investigations regarding Cd63 demonstrated an involvement of the regulative function of late endosomes as signaling platforms for embryonic development beyond mesoderm specification and gastrulation. Overall, the summarized data of this study provides further insights into the determining capacity of Rab7-positive endosomal platforms in intracellular signal transduction of different pathways during early embryonic development.
The formation of an apoplastic diffusion barrier in Arabidopsis seeds is regulated by peptide hormone signaling
(2022) Royek, Stefanie; Schaller, Andreas
Diffusion barrier formation is a critical factor in plant development. The most well described diffusion barriers in Arabidopsis are the Casparian strip and the cuticle. They function in the formation of organ boundaries, prevent water and molecule loss, and protect the plant against environmental stresses. The Casparian strip surrounds the root vascular tissue, whereas the cuticle covers aerial plant organs and is formed de novo during seed development. Embryonic cuticle formation is regulated by a peptide hormone signaling pathway, involving the leucine rich repeat receptor like kinases GASSHO1 (GSO1), GASSHO2 (GSO2) (Tsuwamoto et al. 2008) and the subtilisin-like serine protease ABNORMAL LEAF SHAPE 1 (ALE1). Whereas the latter pathway components have been identified in 2001 and 2008, the peptide hormone mediating the signaling has remained elusive. One aim of this work was to identify the missing pathway element. It was hypothesized that the peptide hormone is released from a larger precursor by ALE1 protease activity to trigger cuticle formation via interaction with the GSO receptors. To uncover the unknown element, the signaling pathway for Casparian strip formation, prooved to be a useful lead. Remarkably, Casparian strip and embryonic cuticle formation employ the same receptor (GSO1), and for Casparian strip formation the GSO1 ligands are known to be members of the CASPARIAN STRIP INTEGRITY FACTOR (CIF) protein family (Doblas et al. 2017, Nakayama et al. 2017). Based on its similarity to the mature CIF peptides and on its phenotypic appearance, it was speculated that a seed expressed protein, called TWISTED SEED1 (TWS1), could serve as the sought ALE1 substrate. As it can be challenging to link proteases to their physiological substrates, this work describes methods how to identify protease specific cleavage sites. One of them was applied to test if TWS1 serves as ALE1 substrate. GFP-tagged TWS1 was transiently coexpressed with ALE1 in Nicotiana benthamiana via agroinfiltration. An ALE1-specific TWS1 cleavage product was detected in the protein extract of coinfiltrated leaves. It was identified by pull down via GFP immunoprecipitation, subsequent separation by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry (MS) analysis. Another method, described in this work, is the identification of protease cleavage sites by in-gel reductive dimethylation: cleavage product-containing gel bands are treated with formaldehyde and cyanoborohydride, prior to in-gel tryptic digest, to achieve a dimethylation of N-terminal free amino groups. The chemically modified N-termini can rapidly be identified and assigned to previous cleavage by the protease of interest. With the method described above, it was found that TWS1 is c-terminally cleaved by ALE1. The two amino acids directly flanking the cleavage site were found to be important for ALE1 cleavage site selection, as their substitution caused a loss of ALE1- dependent cleavage. Our cooperation partners demonstrated an interaction of mature TWS1 with the GSO receptors. The binding affinity of mature TWS1 was reduced by a 3 amino acid C-terminal extension, demonstrating the biological relevance of ALE1-mediated TWS1 processing. Like the CIFs, TWS1 contains a DY tyrosine sulfation motive at its N-terminal processing site. The role of tyrosine sulfation in precursor processing is largely unexplored and was addressed in this work by comparing in-vitro cleavage of different sulfated versus nonsulfated TWS1 precursors. SBT1.8 was found to cleave TWS1 at the N-terminal processing site, and cleavage site selection was influenced by the sulfation state of TWS1 P2´ tyrosine. A homology based 3D model of SBT1.8 was created, which suggested that SBT1.8 interacts with the negatively charged sulfate via a positively charged arginine residue (R302). The role of R302 in substrate binding and recognition was confirmed by in-vitro cleavage assays with mutated SBT1.8 versions, in which R302 was replaced. N-terminal TWS1 cleavage was no longer observed when R302 was substituted. Likewise, no N-terminal cleavage was observed for two other seed expressed Arabidopsis subtilases (SBT1.1 and SBT5.4) that feature an arginine at the corresponding position, indicating that the sole presence of R302 is not sufficient for N terminal cleavage site recognition.
Adaptations of maize to low phosphate availability : establishing regulatory networks from large-scale quantitative proteomic profiling
(2022) He, Mingjie; Schulze, Waltraud
Maize (Zea mays) is an important crop in global for human food, animal feed and industrial usage. Suboptimal phosphorus (P) availability is one of the primary constraints for maize growth and productivity (Jianbo Shen et al., 2011; L.pez-Arredondo et al., 2014). Over 70% arable land suffers from P-deficiency, and plants can take up small amounts of P from the soil due to P-fixation. However, over-application of P fertilizer has frequently happened in last decades and resulted in environmental pollution (L.pez- Arredondo et al., 2014). Modern agriculture calls for maintaining productivity while reducing synthetic-P fertilizer inputs and losses, thus, requiring breeding of novel cultivars to increase phosphate use efficiency (PUE) (Balemi and Negisho, 2012; X., Li, Mang, et al., 2021; Mardamootoo et al., 2021). Understanding the regulation of maize to low phosphate(LP)-availability at the molecular level will offer unlimited potential for the development of selection markers and engineering targets in breeding programs. Nowadays, “OMIC” approaches and computational science are developing rapidly. They are advanced tools for investigation of molecular adaptations on a large-scale and in a systemic view. Thereby, the major research task within this thesis is to reveal P-deficiency induced responsive components and regulations at protein level based on proteomic profiles, aiming to provide promising candidate genes/proteins for research on the molecular mechanisms of adaptation to LP-stress, and potentially to provide promising candidate gene/proteins for development of selection markers and engineering targets to obtain desired traits, in the long term goal of improving PUE in novel cultivars. In Chapter 1, we focused on six genotypes (EP1, F2, F142, F160, SF1, SM1) with close genetic background but several contrasting traits to LP-stress, such as PUE (X., Li, Mang, et al., 2021). They were cultured in pot with either sufficient or inefficient P-fertilizer in a climate chamber for one month. The young seedlings were sampled by root and shoot for analysis of multiple traits, transcriptome and proteome. Firstly, we constructed the co-expression network of proteins and transcripts separately using WGCNA method (Langfelder and Horvath, 2008), which predicted potential protein-protein interactions or their co-regulations. Secondly, we categorized proteins/transcripts to modules according to their different coexpression patterns, thus, identified potential determining relationships of modules-traits. Thirdly, we compared the responses between transcripts and proteins, presenting their responses being concordant or dis-concordant. Fourthly, we identified common and genotype-specific P-starvation response modules and biological processes. Finally, we focused on protein kinases, which play roles as regulators, to demonstrated protein kinases-centered network and validated protein interactions between mitogenactivated protein kinase-kinase 1 (MEK1, Zm00001d043609) either with sucrose synthase1 (SH1,Zm00001d045042) or translation elongation factor 1-gamma 3 (eEF1B-γ, Zm00001d046352). MEK1 is a potential genotype-specific regulator via sucrose metabolism and translation elongation process. In Chapter 2, we aimed to adapted an experimental workflow for phosphoproteome analysis in maize, addressing the interference to phosphoproteome quantification by fibers, secondary metabolites and low abundant of phosphorylated proteins. In this manuscript, we described a rapid and universal protocol for both proteome and phosphoproteome analysis that is suitable for cereal crops. The results of phosphoproteome in maize root testing samples showed that proteins within kinase-centered network in Chapter 1 can be largely quantified based on this workflow. It provides a possible way to analyze phosphorylation dynamics to P-starvation responses, it allows further investigation for kinase-centered 1 network in Chapter 1 to identify phosphorylation pairs of “protein kinase – protein substrate”, which will largely expand a view on P-starvation regulations through posttranslational modifications.
Elucidating the megadiversity of Chalcidoidea (Hymenoptera) with a multi-taxonomic approach
(2022) Haas, Michael; Krogmann, Lars
With over 22,500 described and up to 500,000 estimated species, the jewel wasps (Chalcidoidea: Hymenoptera) are among the most species-rich insect lineages. Their evolutionary success is tightly linked to their parasitoid biology, having evolved to utilize a wide array of different arthropod hosts. Additionally, secondary phytophagy evolved several times within this superfamily. Although new approaches are employed in integrative taxonomic research, progress to decipher the megadiversity of this taxon, including their evolution, is still limited. With this work, the diversity of the superfamily is studied at two evolutionary key points in time. The evolutionary origin of Chalcidoidea is investigated in the Cretaceous and the resulting diversity since then is examined in the present. Different systematic levels will be elucidated with the help of integrative taxonomic methods. In the first chapter, the fossil origins of jewel wasps are addressed, around the middle of the Cretaceous period 110 million years ago. The morphology of a putative early chalcidoid specimen is studied, as it is highly informative for chalcidoid evolution due to its age. Based on those results, its phylogenetic placement is critically examined. The specimen is assumed to be one of the oldest described chalcidoid fossils, Parviformosus wohlrabeae Barling et al., 2013. It is a key fossil because of its age and putative assignment to the polyphyletic family Pteromalidae and could therefore be a valuable voucher for dating modern phylogenies. A precise redescription of the fossil was conducted and its morphology and phylogenetic position was discussed. No synapomorphic characters could be identified, warranting an inclusion in an already established chalcidoid family. In fact, none of the autapomorphies for Chalcidoidea could be recognized, necessitating a revised systematic placement in the Proctotrupomorpha. In the second chapter, several fossils in amber are described that grant insights in the early evolution of Chalcidoidea and the morphological diversity of Cretaceous lineages. Morphological characters are studied to answer the question of plesiomorphic character states in Chalcidoidea, aiding to understand their early evolution. The phylogenetic placement of these fossils is discussed, to provide hypotheses on the diversification of the superfamily, which so far has only few fossil representatives described from this time. Four fossils are made scientifically available that were found in 99 million year old Burmese amber. Those specimens are described in a new, extinct family, the Diversinitidae. This family exhibits a unique combination of plesiomorphic characters, not present in any other chalcidoid taxon, but lacks apomorphic characters. In total, three new genera and three new species are delimited and described. Phylogenetically relevant characters like the fully developed funicular segments, possessing multiporous plate sensilla, or the peg like cerci that improve our understanding of the early evolution of Chalcidoidea, are discussed based on the newly established family. A phylogenetic analysis based on morphological characters was performed. This analysis supported the monophyly of Diversinitidae, but left its exact systematic position within Chalcidoidea open. In the third chapter the focus shifts from the early evolution of Chalcidoidea towards the extant fauna, representing the diversity evolved since the Cretaceous. Exemplary, in the speciose family Pteromalidae the unknown diversity is examined to better understand the undiscovered species richness of parasitoid wasps. DNA barcoding is used to record and help identify previously unknown genera and species in Germany. Compared to the already known pteromalid fauna, 17 genera and 41 species are added as new records for Germany and the males of two species are described anew. The identified DNA barcodes were made available to enable the genetic identification of those species that have a high potential as indicators for nature conservation efforts due to their high host specificity. In the fourth chapter, the pertinent problem of cryptic diversity in Chalcidoidea is investigated. Via an extensive integrative taxonomic approach, the morphological species hypothesis is tested for one of the most abundant pteromalid species in Europe, Spintherus dubius. In this example, the benefit of combining different methods for species discovery and delimitation is highlighted. Genetic analyses of S. dubius reveal discrepancies between the morphological species concept and molecular data, indicating two potential species instead of one. The usage of an advanced morphological method, the multivariate ratio analysis, results in a confirmation of the molecular results, also exposing distinctive morphological characters per taxonomic unit. The examination of the host spectrum through rearing experiments further substantiates these findings, by revealing different host parasitoid affiliations. Altogether, this thesis showed that it is necessary to combine methods and examine different evolutionary points in time, to better understand the diversity of parasitoid lineages. Fossil taxa are important study subjects to examine the character evolution of any taxon, laying the base for phylogenetic research. The study of Diversinitidae highlights the plasticity of character states in Chalcidoidea, also providing evidence for plesiomorphic states. Their encompassing description and the redescription of P. wohlrabeae allow their incorporation into phylogenetic studies, to serve as solid anchor points in dating lineages and morphological evolution on the way towards extant diversity. Examining the extant fauna of Pteromalidae revealed the amount of diversity of species, for which the biology is often unknown. It is shown that molecular methods aid in the discovery of this diversity, opening possibilities for further research. It is affirmed that hidden diversity is even pertinent in abundant, well known species, with S. dubius being an example of cryptic diversity unveiled by integrative taxonomy.
Investigations into the secondary metabolite profile and bioactivity characteristics of various Achillea species grown under different environmental conditions
(2022) Salomon, Lysanne; Kammerer, Dietmar R.
Various Achillea species, including common yarrow Achillea millefolium L., are known to be rich in bioactive substances and are important medicinal plants in modern phytotherapy. In contrast, the alpine species Achillea atrata L. has hardly been studied so far regarding its bioactive compounds and the concomitant potential for human health. Having this in mind, the present work combined studies on the cultivation of A. atrata in different habitats with the phytochemical characterization of its secondary metabolite profile aiming at the generation of plant material with a defined and consistent phenolic profile suitable for pharmaceutical application. Particular emphasis was put on potential relationships between phytochemical profile and bioactivity, and these parameters were considered in comparison with other Achillea species. The results presented in this thesis reveal that the cultivation of A. atrata can be realized at sites differing significantly in altitude and, consequently, in their climatic conditions. Environmental conditions were found to have only a minor impact on the secondary metabolite profile. Rather, differences of various chemotypes might be more decisive. However, the contents of individual phenolic components, especially phenolic acids, is strongly dependent on environmental factors and reach their maximum at increasing altitude levels. Significant differences in the occurrence of the identified phenolic compounds were found between flowers and leaves. In addition to the previously mentioned studies, A. millefolium, Achillea moschata Wulfen, and A. atrata were compared with respect to their phenolic profile and antibacterial activity against gram-positive bacterial strains (Staphylococcus, Propionibacterium). A. atrata showed more similarities in the secondary metabolite profile with the alpine A. moschata than with A. millefolium. The two alpine Achillea species only differed in the occurrence of four compounds. All three aforementioned Achillea species reduced the growth of the tested bacteria. A. atrata showed the highest activity against Propionibacterium acnes and Staphylococcus epidermidis, which are involved in the development of the skin disease Acne vulgaris. In addition, A. atrata exhibited pronounced anti-MRSA potential. Bioassay-guided fractionation of the corresponding extracts revealed that the high antimicrobial activity was due to the flavonoids apigenin, centaureidin, and nevadensin, which were detected exclusively in the most polar fraction and were present in high amounts in A. atrata. Additional studies on the bioactivity and phytochemistry of A. atrata proved that this species exhibits higher antioxidant activity compared to A. millefolium, which was significantly correlated with its total phenolic content. Further bioassay-guided fractionation confirmed the correlation between the mentioned flavonoids in the most polar fraction and the high antioxidant capacity of A. atrata. In conclusion, A. atrata may be an alternative source for the development of novel herbal drugs, based on its compound and bioactivity characteristics.
Energy conservation in anaerobic Prevotella bryantii and Prevotella bivia : the role of membrane bound electron transfer complexes
(2022) Schleicher, Lena; Fritz-Steuber, Julia
Members of the family Prevotellaceae are Gram-negative, obligate anaerobic bacteria found in animal and human microbiomes, where they participate in the degradation of carbohydrates and peptides. Some Prevotella species are also opportunistic pathogens. In this study, growth requirements and central catabolic reactions of two different Prevotella strains were characterized. First, the energy conservation by Prevotella bryantii was analyzed. P. bryantii is a dominant species in the ruminal microbiome. It was demonstrated, that P. bryantii ferments glucose mainly to acetate and succinate. Furthermore, enzymatic and biochemical studies revealed that P. bryantii membranes harbor fully functional Na+ -translocating NADH:quinone oxidoreductase and quinol:fumarate reductase. It was shown, that electron transfer between these two enzymes occurs in native membranes. The enzymatic activities increased significantly by anoxic membrane preparations. Electron transfer in membrane vesicles was coupled to the build-up of a sodium motive force in P. bryantii. A respiratory chain composed of NQR and QFR in P. bryantii was proposed, which links succinate formation to NADH oxidation and SMF formation. Thus, P. bryantii does not rely solely on substrate-level phosphorylation for energy conservation, but gains additional energy utilizing the Na+-pump, NQR. This increases the overall yield of ATP per consumed glucose molecule. By gel electrophoresis and size exclusion chromatography, the existence of a supercomplex composed of NQR and QFR in P. bryantii membranes was demonstrated, which operates as sodium-translocating NADH:fumarate oxidoreductase. The understanding of the catabolic reactions in the rumen by the ruminal microbiota is important for the optimal nutrition of the ruminant. Our results indicate that P. bryantii plays an important role in the ruminal microbiota. P. bryantii extrudes mainly acetate and succinate as fermentative end-products into the rumen. The latter can be used by other organisms of the ruminal microbiome to metabolize propionate, which is an important nutrient for the ruminant since it enters the pathway of gluconeogenesis, yielding glucose. Prevotella bivia is considered to act as causative agent of human bacterial vaginosis. Growth of P. bivia on glucose was dependent on CO2 and resulted in the production of succinate, malate and acetate. With the help of optical spectroscopy and enzymatic measurements, the presence and activity of NQR and QFR in P. bivia were demonstrated. Electron transfer in membrane vesicles of P. bivia resulted in the build-up of a SMF. Similar to P. bryantii, P. bivia operates NQR and QFR for energy conservation in its membrane, resulting in succinate formation and SMF generation. P. bivia also exhibits high L-asparaginase and aspartate ammonia lyase activities in vitro, catalyzing the conversion of L-asparagine to fumarate and NH4+. These results were confirmed in vivo by growth experiments. Additional L-asparagine in the growth medium led to an elevated production of NH4+ and succinate from fumarate obtained during degradation of L-asparagine. At the same time an inhibitory effect of NH4+ on growth of P. bivia was observed. It is proposed, that amino acid degradation by P. bivia in microbial consortia associated with BV depends on the consumption of ammonium by Gardnerella vaginalis, another typical pathogen found in BV. At the same time, G. vaginalis could provide L-asparagine to P. bivia, strengthening their symbiotic relationship and triggering BV
Expression and functional domains of Arabidopsis and tobacco NIM1-INTERACTING (NIMIN) genes
(2021) Saur, Mathias; Pfitzner, Artur J. P.
Systemic acquired resistance (SAR) is an important defense mechanism in plants initiated after exposure to biotrophic pathogens. SAR is characterized by accumulation of PR proteins in non-infected tissues, as well as increased concentrations of the phytohormone salicylic acid (SA). SA is directly perceived by NPR1, the key regulator of SAR. Through interaction with TGA transcription factors and NIM1-INTERACTING (NIMIN) proteins, NPR1 mediates the SA-dependent induction of PR1 gene expression. The Arabidopsis genome contains four NIMIN genes – NIMIN1 (N1), N1b, N2, and N3 – but members of the NIMIN family can also be found in other higher plants. While NIMIN proteins share their general domain architecture and a C-terminal EAR motif, they differ in other aspects. NIMIN genes are expressed differentially during pathogen infection and development. NIMIN proteins can be subdivided based on their NPR1-interaction motifs, the DXFFK and the EDF motif. N1-type proteins harbor both domains, while N2-type and N3-type proteins carry only the DXFFK or the EDF motif, respectively. Accordingly, NIMIN proteins interact differentially with NPR1: N1, N1b and N2 bind to the C-terminal moiety while N3 binds to the N-terminus. Overexpression studies revealed a role for the N1 and N3 proteins in the transcriptional repression of PR1 gene induction. Strikingly, infiltrated plants overexpressing Arabidopsis N1 and N3 or tobacco N2c also manifest significantly accelerated cell death. These numerous differences indicate diverse functions of NIMIN proteins during SAR establishment and beyond. The objective of this work was to further characterize differences between NIMIN proteins from Arabidopsis and tobacco regarding biochemical properties and biological functions with special emphasis on their cell death promoting activity. For this purpose, reporter constructs harboring promoter and coding regions from Arabidopsis and tobacco NIMIN genes were analyzed in transient gene expression experiments in Nicotiana benthamiana and in transgenic tobacco plants. Functional domains were examined using the introduction of targeted mutations to study their significance for NIMIN protein function. The following results were obtained: 1. The N1b 1135 promoter region is functional and two reporter genes under its control, GUS and the proapoptotic Bax, are active during transient overexpression. In transgenic tobacco plants the N1b promoter is not responsive to chemical induction by SA or its functional analog BTH and phenotypical studies showed no expression during plant development. To what extent the N1b gene is expressed in plants must therefore remain open. 2. Transient overexpression of Arabidopsis N1 and N3 and tobacco N2 type genes N2c and N2-like (FS) results in accelerated cell death. This enhanced emergence of cell death is associated with strong protein accumulation. In transgenic tobacco plants overexpression of the N1, N2c and FS genes is also accompanied by emergence of cell death, especially in the flower area, and low seed production. The affected plants often display defects in growth and leaf morphology. 3. The ability to promote cell death requires the C-terminal EAR motif, a transcriptional repression domain. Mutation of the EAR motif in N1, N2c and FS significantly reduces the emergence of cell death. In yeast the EAR motifs of N1 and N3 interact with a N-terminal fragment of the transcriptional co-repressor TOPLESS (TPL). Transient overexpression of this TPL1/333 fragment also induces cell death but coexpression with N1 or N3 reduces cell death emergence, indicating that NIMIN proteins not only affect NPR1 but also modulate the activity of TPL. 4. The enhanced emergence of cell death mediated by overexpression of NIMIN genes and Bax interferes with measurement of SA induced activity of the PR1 promoter. However, using EAR motif mutans with reduced cell death emergence, like the N1 F49/50S E94A D95V EAR mutant, which is also unable to bind NPR1, allows the analysis of the transcriptional repression of the PR1 promoter mediated by cell-death promoting NIMIN proteins. 5. N1 contains a conserved N-terminal domain (N1nT) of 15 amino acids which regulates its accumulation. In N-terminal position, this domain functions autonomously with other NIMIN proteins and Venus, increasing their accumulation. Mutational analysis has not yet revealed reliance on certain sequences. Presence of the N-terminal methionine is not required for function of the N1nT domain hinting at a function at the mRNA level. NIMIN proteins are multifunctional and could perform different functions through their conserved domains. The results indicate that NIMIN proteins, through their interaction with TOPLESS, could also affect other hormone-dependent signal pathways. While the exact mechanism remains unclear, the enhanced protein accumulation bestowed by the N1nT domain of N1 could allow for more effective study of poorly accumulating proteins.
The role of the actin binding protein Calponin2 during embryonic development of Xenopus laevis
(2021) Mantino, Sabrina Maria; Feistel, Kerstin
Despite the abundant variability among adult vertebrate body plans, the developmental steps transforming the single zygote into a multicellular organism of remarkable complexity, are evolutionary highly conserved. Morphogenetic processes such as gastrulation, neural tube closure, body axis extension, neural crest cell migration and organogenesis are thereby at the heart of embryogenesis. Especially the formation of a closed neural tube, which gives rise to the central nervous system, constitutes a fundamental event. Neural tube closure is achieved by convergent extension movements and by apical constriction of neuroepithelial cells. Along with proceeding neurulation, cranial neural cells start to delaminate from the neuroepithelial border. In order to initiate directed migration movements, neural crest cells require polarised cell protrusions and mediate mechanical forces. Changes in cell shape and motility underlying neural tube closure and neural crest cell migration are controlled by specific regulation of the actin cytoskeleton. How these actin dynamics and the myosin-mediated contraction of actin networks are precisely coordinated is not fully understood. In this context, actin filament-associated proteins play an important role for the structural organisation of different actin network types. Calponins constitute an evolutionary highly conserved family of F-actin binding proteins, which are able to influence actin-myosin dynamics and to stabilise actin filaments. Previous studies already demonstrated a role of Calponin proteins in smooth muscle contraction, cell motility and phagocytosis. Vertebrates possess three Calponin isoforms, each displaying specific expression patterns and functions. Calponin2 is expressed in a variety of cell types and several studies performed in vitro indicated that Calponin2 is important for mechanical tension mediation during the course of cell migration. In the early embryo of Xenopus laevis, calponin2 is expressed in tissues that undergo extensive morphogenetic movements and cell migration. This implies an elemental role of Calponin2 for respective morphogenetic steps during embryonic development of this well-established model organism. Within the scope of the present work, the specific function of Calponin2 for dynamic regulation of the actin cytoskeleton was analysed more closely. Localisation of the protein, by utilising a tagged construct, was shown in neural plate cells as well as in migrating neural crest cells. In both cell types, regulated protein degradation occurred, which led to specific expression restricted to the apex of constricting neural plate cells or to forming lamellipodia. Thus, tagged Calponin2 localised to regions of the actin cortex. Loss of Calponin2 function led to defects in neural crest cell specification and migration as well as in convergent extension and apical constriction within the neural plate. All induced phenotypes were rescued by additional calponin2 mRNA injection. In summary, these data demonstrated a specific function of Calponin2 for correct formation of the neural crest as well as for neural tube closure. Furthermore, the precise regulation of protein expression levels, which directly correlated with correct Calponin2 function, was dependent on specific domains that potentially mediate actin-binding. Clik1, Clik2 and the C-terminus were identified as a critical unit regulating protein degradation, both in neural crest cells and neural plate cells. Additionally, it was shown that Calponin2 function for neural apical constriction depends on each of these domains as well. Overall, the degradation of Calponin2, regulated via its F-actin binding, implies a filament stabilising function. Thus, a temporospatial coordination of protein degradation would be necessary to enable dynamic changes of the actin cytoskeleton by a regulated release of actin filaments and to allow the association of other structural effectors during morphogenetic processes of early vertebrate development.
PEP7 is a ligand for receptor kinase SIRK1 to regulate aquaporins and root growth
(2021) Wang, Jiahui; Schulze, Waltraud
Receptor kinases constitute the largest protein family in regulating various responses to external and internal biotic and abiotic signals. Functional characterization of this large protein family and particularly the identification of their ligands remains a major challenge in plant biology. Previously, we identified SIRK1 and QSK1 as a receptor / co-receptor pair involved in regulation of aquaporins in response to osmotic changes induced by sucrose. Here, we now identify a member of the Elicitor Peptide (PEP) family, namely PEP7, as a ligand to receptor kinase SIRK1. PEP7 was shown to bind to the extracellular domain of SIRK1 with a binding constant of 19 µM. PEP7 was secreted to the apoplasm specifically in response to sucrose. Formation of a signaling complex involving SIRK1, QSK1 as well as aquaporins as substrates was induced by sucrose or external PEP7 treatment. PEP7 induced aquaporin phosphorylation and water influx activity. The knock-out mutant of receptor SIRK1 was not responsive to external PEP7 treatment. Binding to receptor SIRK1 and induction of physiological responses was specific to PEP7, neither other members of the PEP-family (PEP6, PEP4), nor other small signaling peptides (CLEs, IDA, RALFs) induced SIRK1 kinase activity, aquaporin phosphorylation, or protoplast water influx activity.
Establishment of the body axes in Xenopus laevis through goosecoid, myosin 1d and bicaudal c
(2021) Maerker, Markus Ferdinand; Schweickert, Axel
The bilaterian body plan consists of three body axes: the anteroposterior (AP; head-trunk/tail), the dorsoventral (DV; back-belly) and the left-right (LR; placement of inner organs) axis. Axis formation occurs during early embryogenesis and is critical for further development and viability of the embryo. In this comprehensive study three highly conserved determinants were functionally analyzed in the context of axis development. The first chapter of this work covers the autoregulatory, homeodomain containing, repressor gene goosecoid (gsc), whose most prominent expression marks the Spemann-(Mangold) organizer (SO). The SO is the primary dorsal signaling center and is instructive for tissue patterning along the DV and AP axes. Transplanting the SO or misexpressing gsc on the opposite ventral side of an embryo is sufficient to establish a new/secondary AP axis. However, its function during normal development in the SO remained enigmatic as the gsc loss of function (LOF) lead to no severe early developmental defects. To elucidate the function of gsc, timed gain of function (GOF) experiments were performed. Gsc efficiently repressed the planar cell polarity (PCP)/Wnt signaling pathway leading to severe gastrulation and neurulation defects. This novel Gsc function was correlated with two vertebrate specific domains, suggesting an evolutionary new function of Gsc with the emergence of jaws/neural crests in vertebrates. The second chapter of this study addresses the functions of Myosin1d (Myo1d) and Bicaudal c1 (Bicc1) during the LR axis determination in vertebrates. In this group LR symmetry breakage takes place at a ciliated epithelium called LR organizer (LRO). The initial cue for the asymmetric LR axis development is a cilia-driven leftward fluid flow. These cilia have to be correctly polarized through PCP/Wnt signaling. Interestingly, the invertebrate Drosophila melanogaster also displays a distinct LR axis but uses a cilia independent, yet not fully understood, mechanism. It depends on a myo1d homologous gene, myo31DF, and PCP. To unravel a potential common evolutionary origin of the bilaterian LR axis myo1d was analyzed during Xenopus laevis lateralization. Myo1d LOF experiments disturbed LR axis formation by compromising PCP dependent outgrowth and polarization of LRO cilia. These experiments link the PCP/Myosin based mechanism of flies to the newly evolved cilia/flow dependent mode of vertebrate LR axis determination suggesting actomyosin as common ancestral LR determinant. Contrary to Myo1d, Bicc1 was already described for its function during polarization of flow producing LRO cilia. However bicc1s expression is most prominent in the sensory LRO cells (sLRO). These cells detect the fluid flow and translate it into left-sided signaling of the morphogen Nodal1 and consequently asymmetric LR axis formation. These cells downregulate the expression of the secreted Nodal1 antagonist DAN domain family member 5 (dand5) in response to flow. Bicc1s function was re-evaluated with respect to its function in sLRO cells. Ex vivo and in vivo experiments involving GOF as well as LOF experiments showed that Bicc1 regulates both dand5 and nodal1 via a direct and indirect post-transcriptional mechanism, respectively. In the process of dand5 regulation several other LR determinants and regulatory events were linked with the Bicc1 dependent mechanism: Dicer1 dependent microRNA repression of dand5 and a proposed cation channel Polycystin 2 mediated Bicc1 modification. These results highlight the importance of a tightly controlled Dand5 protein level as decisive for the overall outcome of the LR symmetry breakage in vertebrates.
Die Insertion des „minor coat“ Proteins G3P des Bakteriophagen M13 in die innere E. coli Membran benötigt die Insertase YidC und die Translokase SecYEG.
(2021) Kleinbeck, Farina; Kuhn, Andreas
The membrane of every cell forms a spacial limitation for this smallest unit of a life form. Such a very simple unicellular life form is also the Gram-negative bacterium Escherichia coli (E. coli) and is therefore a valid model organism for a living cell. Due to the inner membrane the cellular components are held together in close proximity and are separated from the extracellular environment. Most substrates cannot pass the lipid bilayer, which forms the membrane, so an import and export system had to be developed to accomplish this. For these import and export systems, very complex, polytopic transmembrane protein complexes are needed. Examples are ion channels, ion pumps or large complexes through which energy production, secretion of toxins and the transfer of nutrients are catalysed. Moreover, proteins with functions in the periplasm or outer membrane must also travel from their site of synthesis in the cytoplasm to their destination. For these different processes proteins must be inserted into or translocated across the inner membrane. Of the total proteome in prokaryotes approximately 25 to 30% is either inserted into or secreted across the inner membrane. This work identified several components required for the insertion of the "minor coat" protein G3P of M13 bacteriophage. This protein is important for the assembly of the phage particle that occurs in the inner membrane. The outermost C-terminus of G3P is anchored in the inner membrane via a single transmembrane domain, while the bulk of the approximately 42 kDa protein is located in the periplasm. Using an N-terminal cleavable signal peptide, the major portion of G3P is translocated into the periplasm via SecYEG with the help of SecA and the membrane potential. Targeting, on the other hand, could not be clearly assigned to one of the known post- or co-translational pathways. Although contact via disulfide crosslink studies to Ffh, the protein component of the ribonucleoprotein SRP, was observed via stalled ribosome nascent chains (RNCs), insertion into the membrane in vivo was independent of Ffh. Even when the interaction between SecY and FtsY, the receptor for SRP at the membrane, was impaired, G3P was inserted via SecYEG. Although the chaperone SecB was able to bind to G3P in vitro, G3P inserted independently of SecB in vivo. For membrane incorporation of G3P, it was shown that YidC is required in vivo in addition to SecYEG. Disulphide crosslink studies demonstrated that G3P first contacts the plug domain TM2b and lateral gate (TM2a and TM7) via the signal peptide of G3P, and finally the C-terminal transmembrane domain of G3P contacts YidC via TM3 and TM5 of the hydrophilic slide. Based on these contact sites, a possible insertion model was confirmed, with SecY and YidC mediating defined steps in the insertion process, providing new insights into this largely unknown process.
Prediction of protein-protein complexes by combining size exclusion chromatography and mass spectrometric analysis
(2021) Gilbert, Max; Schulze, Waltraud
Two major objectives were pursued and met in this study. First, the goal was to add to the scientific toolbox a diligent method for uncovering PPi dynamics on a proteomic scale, with a focus on plant membranes. There are large-scale or high-throughput approaches, but they rely on genetically modified proteins or heterologous expression systems to describe PPi outside of their natural context. Similarly, those methods are incapable of describing the dynamics of protein interactions. In course of this study, a co-elution based approach was combined with modern mass spectrometric label free quantification in order to investigate PPi and interaction dynamics on a proteomic scale. A rigorous data processing pipeline was developed to not only address known fallacies of using co-elution based methods (such as for example random co elution), but also to access and utilize meta-information in form of protein abundance and protein network connectivity to draw conclusions not only on proteomic scale, but also for individual proteins. In total, 6.928 individual proteins extracted from Arabidopsis thaliana root membranes were detected under different nutritional conditions (full nutrition, nitrogen starvation and nitrogen resupply). The data processing pipeline described in this study was used to predict and discover connectivity information for at least 2.058 of these proteins. Each step in data processing was validated by comparison to database confirmed interactions to improve filtering criteria. Protein abundance was evaluated through a unique ranking system, allowing a seamless integration as network attributes for each condition. From the suggested interaction data, an interactome network of the various nutritional conditions was reconstructed. Using different network parameters from graph theory, protein significance and dynamic conditional changes were described. Second, this study applied the aforementioned approach to identify relevant proteins involved in nitrogen signaling in Arabidopsis thaliana root membranes. Through correlation analysis and network reconstruction, receptor kinase AT5G49770 was identified as a component of the nitrogen signaling network that collaborates with co-receptor QSK1, BAK1, the nitrogen transporter NRT2.1 and proton pump AHA2. In response to nitrogen deficiency, the network parameters of AT5G49770 reacted strongly and its involvement was demonstrated by a phenotypic similarity to knock-out lines of NRT2.1, NRT1.1 and AHA2 during a root growth assay of Arabidopsis seedlings. The interaction between QSK1 and BAK1 was further confirmed using FRET/FLIM microscopy and pulldown assays. These findings show that combining a co-elution based approach with a rigorous data processing pipeline and network analysis is suitable to study the protein interaction environment and signal response dynamics in plant root membranes. The modular experimental design allows for a simple adaptation to study different stimuli and the unbiased proteomic approach yields results for proteins regardless of the individual scientific focus. Meta-information such as protein abundance and network connectivity parameters can be used to prospect and identify important proteins involved in stress response dynamics. The author of this study is confident that the proteomic data produced can be utilized in further research and contributes to the understanding of nitrogen signaling in plant root membranes. Through integration of the data processing pipeline and adaptation to different scientific scenarios, valuable information beyond protein interaction is gained. Thus, this work makes an important contribution to the advancement of proteomic analysis and data interpretation methodology.

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