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Background
Teleost fishes comprise more than half of the vertebrate species. Within teleosts, most phylogenies consider the split between Osteoglossomorpha and Euteleosteomorpha/Otomorpha as basal, preceded only by the derivation of the most primitive group of teleosts, the Elopomorpha. While Osteoglossomorpha are generally species poor, the taxon contains the African weakly electric fish (Mormyroidei), which have radiated into numerous species. Within the mormyrids, the genus Campylomormyrus is mostly endemic to the Congo Basin. Campylomormyrus serves as a model to understand mechanisms of adaptive radiation and ecological speciation, especially with regard to its highly diverse species-specific electric organ discharges (EOD). Currently, there are few well-annotated genomes available for electric fish in general and mormyrids in particular. Our study aims at producing a high-quality genome assembly and to use this to examine genome evolution in relation to other teleosts. This will facilitate further understanding of the evolution of the osteoglossomorpha fish in general and of electric fish in particular.
Results
A high-quality weakly electric fish (C. compressirostris) genome was produced from a single individual with a genome size of 862 Mb, consisting of 1,497 contigs with an N50 of 1,399 kb and a GC-content of 43.69%. Gene predictions identified 34,492 protein-coding genes, which is a higher number than in the two other available Osteoglossomorpha genomes of Paramormyrops kingsleyae and Scleropages formosus. A Computational Analysis of gene Family Evolution (CAFE5) comparing 33 teleost fish genomes suggests an overall faster gene family turnover rate in Osteoglossomorpha than in Otomorpha and Euteleosteomorpha. Moreover, the ratios of expanded/contracted gene family numbers in Osteoglossomorpha are significantly higher than in the other two taxa, except for species that had undergone an additional genome duplication (Cyprinus carpio and Oncorhynchus mykiss). As potassium channel proteins are hypothesized to play a key role in EOD diversity among species, we put a special focus on them, and manually curated 16 Kv1 genes. We identified a tandem duplication in the KCNA7a gene in the genome of C. compressirostris.
Conclusions
We present the fourth genome of an electric fish and the third well-annotated genome for Osteoglossomorpha, enabling us to compare gene family evolution among major teleost lineages. Osteoglossomorpha appear to exhibit rapid gene family evolution, with more gene family expansions than contractions. The curated Kv1 gene family showed seven gene clusters, which is more than in other analyzed fish genomes outside Osteoglossomorpha. The KCNA7a, encoding for a potassium channel central for EOD production and modulation, is tandemly duplicated which may related to the diverse EOD observed among Campylomormyrus species.
The use of automated tools to reconstruct lipid metabolic pathways is not warranted in plants. Here, the authors construct Plant Lipid Module for Arabidopsis rosette using constraint-based modeling, demonstrate its integration in other plant metabolic models, and use it to dissect the genetic architecture of lipid metabolism.
Lipids play fundamental roles in regulating agronomically important traits. Advances in plant lipid metabolism have until recently largely been based on reductionist approaches, although modulation of its components can have system-wide effects. However, existing models of plant lipid metabolism provide lumped representations, hindering detailed study of component modulation. Here, we present the Plant Lipid Module (PLM) which provides a mechanistic description of lipid metabolism in the Arabidopsis thaliana rosette. We demonstrate that the PLM can be readily integrated in models of A. thaliana Col-0 metabolism, yielding accurate predictions (83%) of single lethal knock-outs and 75% concordance between measured transcript and predicted flux changes under extended darkness. Genome-wide associations with fluxes obtained by integrating the PLM in diel condition- and accession-specific models identify up to 65 candidate genes modulating A. thaliana lipid metabolism. Using mutant lines, we validate up to 40% of the candidates, paving the way for identification of metabolic gene function based on models capturing natural variability in metabolism.
Progressive habitat fragmentation threatens plant species with narrow habitat requirements. While local environmental conditions define population growth rates and recruitment success at the patch level, dispersal is critical for population viability at the landscape scale. Identifying the dynamics of plant meta-populations is often confounded by the uncertainty about soil-stored population compartments. We combined a landscape-scale assessment of an amphibious plant's population structure with measurements of dispersal complexity in time to track dispersal and putative shifts in functional connectivity. Using 13 microsatellite markers, we analyzed the genetic structure of extant Oenanthe aquatica populations and their soil seed banks in a kettle hole system to uncover hidden connectivity among populations in time and space. Considerable spatial genetic structure and isolation-by-distance suggest limited gene flow between sites. Spatial isolation and patch size showed minor effects on genetic diversity. Genetic similarity found among extant populations and their seed banks suggests increased local recruitment, despite some evidence of migration and recent colonization. Results indicate stepping-stone dispersal across adjacent populations. Among permanent and ephemeral demes the resulting meta-population demography could be determined by source-sink dynamics. Overall, these spatiotemporal connectivity patterns support mainland-island dynamics in our system, highlighting the importance of persistent seed banks as enduring sources of genetic diversity.
Metabolic engineering of microalgae offers a promising solution for sustainable biofuel production, and rational design of engineering strategies can be improved by employing metabolic models that integrate enzyme turnover numbers. However, the coverage of turnover numbers for Chlamydomonas reinhardtii, a model eukaryotic microalga accessible to metabolic engineering, is 17-fold smaller compared to the heterotrophic cell factory Saccharomyces cerevisiae. Here we generate quantitative protein abundance data of Chlamydomonas covering 2337 to 3708 proteins in various growth conditions to estimate in vivo maximum apparent turnover numbers. Using constrained-based modeling we provide proxies for in vivo turnover numbers of 568 reactions, representing a 10-fold increase over the in vitro data for Chlamydomonas. Integration of the in vivo estimates instead of in vitro values in a metabolic model of Chlamydomonas improved the accuracy of enzyme usage predictions. Our results help in extending the knowledge on uncharacterized enzymes and improve biotechnological applications of Chlamydomonas.
Aquatic ecosystems are frequently overlooked as fungal habitats, although there is increasing evidence that their diversity and ecological importance are greater than previously considered. Aquatic fungi are critical and abundant components of nutrient cycling and food web dynamics, e.g., exerting top-down control on phytoplankton communities and forming symbioses with many marine microorganisms. However, their relevance for microphytobenthic communities is almost unexplored. In the light of global warming, polar regions face extreme changes in abiotic factors with a severe impact on biodiversity and ecosystem functioning. Therefore, this study aimed to describe, for the first time, fungal diversity in Antarctic benthic habitats along the salinity gradient and to determine the co-occurrence of fungal parasites with their algal hosts, which were dominated by benthic diatoms. Our results reveal that Ascomycota and Chytridiomycota are the most abundant fungal taxa in these habitats. We show that also in Antarctic waters, salinity has a major impact on shaping not just fungal but rather the whole eukaryotic community composition, with a diversity of aquatic fungi increasing as salinity decreases. Moreover, we determined correlations between putative fungal parasites and potential benthic diatom hosts, highlighting the need for further systematic analysis of fungal diversity along with studies on taxonomy and ecological roles of Chytridiomycota.
This study focuses on three key aspects: (a) crude throat swab samples in a viral transport medium (VTM) as templates for RT-LAMP reactions; (b) a biotinylated DNA probe with enhanced specificity for LFA readouts; and (c) a digital semi-quantification of LFA readouts. Throat swab samples from SARS-CoV-2 positive and negative patients were used in their crude (no cleaning or pre-treatment) forms for the RT-LAMP reaction. The samples were heat-inactivated but not treated for any kind of nucleic acid extraction or purification. The RT-LAMP (20 min processing time) product was read out by an LFA approach using two labels: FITC and biotin. FITC was enzymatically incorporated into the RT-LAMP amplicon with the LF-LAMP primer, and biotin was introduced using biotinylated DNA probes, specifically for the amplicon region after RT-LAMP amplification. This assay setup with biotinylated DNA probe-based LFA readouts of the RT-LAMP amplicon was 98.11% sensitive and 96.15% specific. The LFA result was further analysed by a smartphone-based IVD device, wherein the T-line intensity was recorded. The LFA T-line intensity was then correlated with the qRT-PCR Ct value of the positive swab samples. A digital semi-quantification of RT-LAMP-LFA was reported with a correlation coefficient of R2 = 0.702. The overall RT-LAMP-LFA assay time was recorded to be 35 min with a LoD of three RNA copies/µL (Ct-33). With these three advancements, the nucleic acid testing-point of care technique (NAT-POCT) is exemplified as a versatile biosensor platform with great potential and applicability for the detection of pathogens without the need for sample storage, transportation, or pre-processing.
The physiological dependence of animals on dietary intake of vitamins, amino acids, and fatty acids is ubiquitous. Sharp differences in the availability of these vital dietary biomolecules among different resources mean that consumers must adopt a range of strategies to meet their physiological needs. We review the emerging work on omega-3 long-chain polyunsaturated fatty acids, focusing predominantly on predator-prey interactions, to illustrate that trade-off between capacities to consume resources rich in vital biomolecules and internal synthesis capacity drives differences in phenotype and fitness of consumers. This can then feedback to impact ecosystem functioning. We outline how focus on vital dietary biomolecules in eco-eco-devo dynamics can improve our understanding of anthropogenic changes across multiple levels of biological organization.
Starch is a biopolymer for which, despite its simple composition, understanding the precise mechanism behind its formation and regulation has been challenging. Several approaches and bioanalytical tools can be used to expand the knowledge on the different parts involved in the starch metabolism. In this sense, a comprehensive analysis targeting two of the main groups of molecules involved in this process: proteins, as effectors/regulators of the starch metabolism, and maltodextrins as starch components and degradation products, was conducted in this research work using potato plants (Solanum tuberosum L. cv. Desiree) as model of study. On one side, proteins physically interacting to potato starch were isolated and analyzed through mass spectrometry and western blot for their identification. Alternatively, starch interacting proteins were explored in potato tubers from transgenic plants having antisense inhibition of starch-related enzymes and on tubers stored under variable environmental conditions. Most of the proteins recovered from the starch granules corresponded to previously described proteins having a specific role in the starch metabolic pathway. Another set of proteins could be grouped as protease inhibitors, which were found weakly interacting to starch. Variations in the protein profile obtained after electrophoresis separation became clear when tubers were stored under different temperatures, indicating a differential expression of proteins in response to changing environmental conditions.
On the other side, since maltodextrin metabolism is thought to be involved in both starch initiation and degradation, soluble maltooligosaccharide content in potato tubers was analyzed in this work under diverse experimental variables. For this, tuber disc samples from wild type and transgenic lines strongly repressing either the plastidial or cytosolic form of the -glucan phosphorylase and phosphoglucomutase were incubated with glucose, glucose-6-phosphate, and glucose-1-phosphate solutions to evaluate the influence of such enzymes on the conversion of the carbon sources into soluble maltodextrins, in comparison to wild-type samples. Relative maltodextrin amounts analyzed through capillary electrophoresis equipped with laser-induced fluorescence (CE-LIF) revealed that tuber discs could immediately uptake glucose-1-phosphate and use it to produce maltooligosaccharides with a degree of polymerization of up to 30 (DP30), in contrast to transgenic tubers with strong repression of the plastidial glucan phosphorylase. The results obtained from the maltodextrin analysis support previous indications that a specific transporter for glucose-1-phosphate may exist in both the plant cells and the plastidial membranes, thereby allowing a glucose-6-phosphate independent transport. Furthermore, it confirms that the plastidial glucan phosphorylase is responsible for producing longer maltooligosaccharides in the plastids by catalyzing a glucan polymerization reaction when glucose-1-phosphate is available. All these findings contribute to a better understanding of the role of the plastidial glucan phosphorylase as a key enzyme directly involved in the synthesis and degradation of glucans and their implication on starch metabolism.
Cells are built from a variety of macromolecules and metabolites. Both, the proteome and the metabolome are highly dynamic and responsive to environmental cues and developmental processes. But it is not their bare numbers, but their interactions that enable life. The protein-protein (PPI) and protein-metabolite interactions (PMI) facilitate and regulate all aspects of cell biology, from metabolism to mitosis. Therefore, the study of PPIs and PMIs and their dynamics in a cell-wide context is of great scientific interest. In this dissertation, I aim to chart a map of the dynamic PPIs and PMIs across metabolic and cellular transitions. As a model system, I study the shift from the fermentative to the respiratory growth, known as the diauxic shift, in the budding yeast Saccharomyces cerevisiae. To do so, I am applying a co-fractionation mass spectrometry (CF-MS) based method, dubbed protein metabolite interactions using size separation (PROMIS). PROMIS, as well as comparable methods, will be discussed in detail in chapter 1.
Since PROMIS was developed originally for Arabidopsis thaliana, in chapter 2, I will describe the adaptation of PROMIS to S. cerevisiae. Here, the obtained results demonstrated a wealth of protein-metabolite interactions, and experimentally validated 225 previously predicted PMIs. Applying orthogonal, targeted approaches to validate the interactions of a proteogenic dipeptide, Ser-Leu, five novel protein-interactors were found. One of those proteins, phosphoglycerate kinase, is inhibited by Ser-Leu, placing the dipeptide at the regulation of glycolysis.
In chapter 3, I am presenting PROMISed, a novel web-tool designed for the analysis of PROMIS- and other CF-MS-datasets. Starting with raw fractionation profiles, PROMISed enables data pre-processing, profile deconvolution, scores differences in fractionation profiles between experimental conditions, and ultimately charts interaction networks. PROMISed comes with a user-friendly graphic interface, and thus enables the routine analysis of CF-MS data by non-computational biologists.
Finally, in chapter 4, I applied PROMIS in combination with the isothermal shift assay to the diauxic shift in S. cerevisiae to study changes in the PPI and PMI landscape across this metabolic transition. I found a major rewiring of protein-protein-metabolite complexes, exemplified by the disassembly of the proteasome in the respiratory phase, the loss of interaction of an enzyme involved in amino acid biosynthesis and its cofactor, as well as phase and structure specific interactions between dipeptides and enzymes of central carbon metabolism.
In chapter 5, I am summarizing the presented results, and discuss a strategy to unravel the potential patterns of dipeptide accumulation and binding specificities. Lastly, I recapitulate recently postulated guidelines for CF-MS experiments, and give an outlook of protein interaction studies in the near future.
Plant metabolism serves as the primary mechanism for converting assimilated carbon into essential compounds crucial for plant growth and ultimately, crop yield. This renders it a focal point of research with significant implications. Despite notable strides in comprehending the genetic principles underpinning metabolism and yield, there remains a dearth of knowledge regarding the genetic factors responsible for trait variation under varying environmental conditions. Given the burgeoning global population and the advancing challenges posed by climate change, unraveling the intricacies of metabolic and yield responses to water scarcity became increasingly important in safeguarding food security.
Our research group has recently started to work on the genetic resources of legume species. To this end, the study presented here investigates the metabolic diversity across five different legume species at a tissue level, identifying species-specific biosynthesis of alkaloids as well as iso-/flavonoids with diverse functional groups, namely prenylation, phenylacylation as well as methoxylation, to create a resource for follow up studies investigation the metabolic diversity in natural diverse populations of legume species.
Following this, the second study investigates the genetic architecture of drought-induced changes in a global common bean population. Here, a plethora of quantitative trait loci (QTL) associated with various traits are identified by performing genome-wide association studies (GWAS), including for lipid signaling. On this site, overexpression of candidates highlighted the induction of several oxylipins reported to be pivotal in coping with harsh environmental conditions such as water scarcity.
Diverging from the common bean and GWAS, the following study focuses on identifying drought-related QTL in tomato using a bi-parental breeding population. This descriptive study highlights novel multi-omic QTL, including metabolism, photosynthesis as well as fruit setting, some of which are uniquely assigned under drought. Compared to conventional approaches using the bi-parental IL population, the study presented improves the resolution by assessing further backcrossed ILs, named sub-ILs.
In the final study, a photosynthetic gene, namely a PetM subunit of the cytochrome b6f complex encoding gene, involved in electron flow is characterized in an horticultural important crop. While several advances have been made in model organisms, this study highlights the transition of this fundamental knowledge to horticultural important crops, such as tomato, and investigates its function under differing light conditions. Overall, the presented thesis combines different strategies in unveiling the genetic components in multi-omic traits under drought using conventional breeding populations as well as a diverse global population. To this end, it allows a comparison of either approach and highlights their strengths and weaknesses.
The capillary-venous pathology cerebral cavernous malformation (CCM) is caused by loss of CCM1/Krev interaction trapped protein 1 (KRIT1), CCM2/MGC4607, or CCM3/PDCD10 in some endothelial cells. Mutations of CCM genes within the brain vasculature can lead to recurrent cerebral hemorrhages. Pharmacological treatment options are urgently needed when lesions are located in deeply-seated and in-operable regions of the central nervous system. Previous pharmacological suppression screens in disease models of CCM led to the discovery that treatment with retinoic acid improved CCM phenotypes. This finding raised a need to investigate the involvement of retinoic acid in CCM and test whether it has a curative effect in preclinical mouse models. Here, we show that components of the retinoic acid synthesis and degradation pathway are transcriptionally misregulated across disease models of CCM. We complemented this analysis by pharmacologically modifying retinoic acid levels in zebrafish and human endothelial cell models of CCM, and in acute and chronic mouse models of CCM. Our pharmacological intervention studies in CCM2-depleted human umbilical vein endothelial cells (HUVECs) and krit1 mutant zebrafish showed positive effects when retinoic acid levels were increased. However, therapeutic approaches to prevent the development of vascular lesions in adult chronic murine models of CCM were drug regiment-sensitive, possibly due to adverse developmental effects of this hormone. A treatment with high doses of retinoic acid even worsened CCM lesions in an adult chronic murine model of CCM. This study provides evidence that retinoic acid signaling is impaired in the CCM pathophysiology and suggests that modification of retinoic acid levels can alleviate CCM phenotypes.
Cell-level systems biology model to study inflammatory bowel diseases and their treatment options
(2023)
To help understand the complex and therapeutically challenging inflammatory bowel diseases (IBDs), we developed a systems biology model of the intestinal immune system that is able to describe main aspects of IBD and different treatment modalities thereof. The model, including key cell types and processes of the mucosal immune response, compiles a large amount of isolated experimental findings from literature into a larger context and allows for simulations of different inflammation scenarios based on the underlying data and assumptions. In the context of a large and diverse virtual IBD population, we characterized the patients based on their phenotype (in contrast to healthy individuals, they developed persistent inflammation after a trigger event) rather than on a priori assumptions on parameter differences to a healthy individual. This allowed to reproduce the enormous diversity of predispositions known to lead to IBD. Analyzing different treatment effects, the model provides insight into characteristics of individual drug therapy. We illustrate for anti-TNF-alpha therapy, how the model can be used (i) to decide for alternative treatments with best prospects in the case of nonresponse, and (ii) to identify promising combination therapies with other available treatment options.
Dielektrophorese ist die Manipulation polarisierbarer Partikel durch inhomogene elektrische Wechselfelder. In dieser Arbeit wurden drei verschiedene Enzyme durch Dielektrophorese immobilisiert und anschließend hinsichtlich ihrer katalytischen Aktivität untersucht: Meerrettichperoxidase, Cholinoxidase aus Alcaligenes sp. und Glucoseoxidase aus Aspergillus niger. Die Immobilisierung erfolgte durch Dielektrophorese auf nano-Elektrodenarrays aus Wolfram-Zylindern mit 500 nm Durchmesser oder aus Titannitrid-Ringen mit 20 nm Breite. Die Immobilisierung der Enzyme konnte fluoreszenzmikroskopisch entweder anhand der intrinsischen Fluoreszenz oder aufgrund einer Fluoreszenzmarkierung vor oder nach der Immobilisierung für alle getesteten Enzyme nachgewiesen werden. Die Messung der Enzymaktivität erfolgte quantitativ durch den direkten oder indirekten Nachweis des gebildeten Produktes oder, im Falle der Cholinoxidase, durch Beobachtung der intrinsischen Fluoreszenz des Cofaktors FAD, die vom Oxidationszustand dieses Enzyms abhängt. Für die Meerrettichperoxidase konnte so eine hohe erhaltene Enzymaktivität nach der Immobilisierung nachgewiesen werden. Die Aktivität der permanent immobilisierten Fraktion der Meerrettichperoxidase entsprach bis zu 47 % der höchstmöglichen Aktivität einer Monolage dieses Enzyms auf den Elektroden des Chips. Diese Aktivität kann als aktive, aber zufällig gegenüber der Oberfläche ausgerichtete Enzymschicht interpretiert werden. Für die permanent immobilisierte Glucoseoxidase wurde nur eine Aktivität entsprechend <1,3 % der Aktivität einer solchen Enzymschicht detektiert, während für die immobilisierte Cholinoxidase gar keine Aktivität nachgewiesen werden konnte. Die Aktivität der durch DEP immobilisierten Enzyme konnte somit quantitativ bestimmt werden. Der Anteil an erhaltener Aktivität hängt dabei stark vom verwendeten Enzym ab.
Leaves exhibit cells with varying degrees of shape complexity along the proximodistal axis. Heterogeneities in growth directions within individual cells bring about such complexity in cell shape. Highly complex and interconnected gene regulatory networks and signaling pathways have been identified to govern these processes. In addition, the organization of cytoskeletal networks and cell wall mechanical properties greatly influences the regulation of cell shape. Research has shown that microtubules are involved in regulating cellulose deposition and direc-tion of cell growth. However, comprehensive analysis of the regulation of the actin cytoskele-ton in cell shape regulation has not been well studied.
This thesis provides evidence that actin regulates aspects of cell growth, division, and direction-al expansion that impacts morphogenesis of developing leaves. The jigsaw puzzle piece mor-phology of epidermal pavement cells further serves as an ideal system to investigate the com-plex process of morphogenetic processes occurring at the cellular level. Here we have em-ployed live cell based imaging studies to track the development of pavement cells in actin com-promised conditions. Genetic perturbation of two predominantly expressed vegetative actin genes ACTIN2 and ACTIN7 results in delayed emergence of the cellular protrusions in pave-ment cells. Perturbation of actin also impacted the organization of microtubule in these cells that is known to promote emergence of cellular protrusions. Further, live-cell imaging of actin or-ganization revealed a correlation with cell shape, suggesting that actin plays a role in influencing pavement cell morphogenesis.
In addition, disruption of actin leads to an increase in cell size along the leaf midrib, with cells being highly anisotropic due to reduced cell division. The reduction of cell division further im-pacted the morphology of the entire leaf, with the mutant leaves being more curved. These re-sults suggests that actin plays a pivotal role in regulating morphogenesis at the cellular and tis-sue scales thereby providing valuable insights into the role of the actin cytoskeleton in plant morphogenesis.
Development of a CRISPR/Cas gene editing technique for the coccolithophore Chrysotila carterae
(2024)
Actin is one of the most highly conserved proteins in eukaryotes and distinct actin-related proteins with filament-forming properties are even found in prokaryotes. Due to these commonalities, actin-modulating proteins of many species share similar structural properties and proposed functions. The polymerization and depolymerization of actin are critical processes for a cell as they can contribute to shape changes to adapt to its environment and to move and distribute nutrients and cellular components within the cell. However, to what extent functions of actin-binding proteins are conserved between distantly related species, has only been addressed in a few cases. In this work, functions of Coronin-A (CorA) and Actin-interacting protein 1 (Aip1), two proteins involved in actin dynamics, were characterized. In addition, the interchangeability and function of Aip1 were investigated in two phylogenetically distant model organisms. The flowering plant Arabidopsis thaliana (encoding two homologs, AIP1-1 and AIP1-2) and in the amoeba Dictyostelium discoideum (encoding one homolog, DdAip1) were chosen because the functions of their actin cytoskeletons may differ in many aspects. Functional analyses between species were conducted for AIP1 homologs as flowering plants do not harbor a CorA gene.
In the first part of the study, the effect of four different mutation methods on the function of Coronin-A protein and the resulting phenotype in D. discoideum was revealed in two genetic knockouts, one RNAi knockdown and a sudden loss-of-function mutant created by chemical-induced dislocation (CID). The advantages and disadvantages of the different mutation methods on the motility, appearance and development of the amoebae were investigated, and the results showed that not all observed properties were affected with the same intensity. Remarkably, a new combination of Selection-Linked Integration and CID could be established.
In the second and third parts of the thesis, the exchange of Aip1 between plant and amoeba was carried out. For A. thaliana, the two homologs (AIP1-1 and AIP1-2) were analyzed for functionality as well as in D. discoideum. In the Aip1-deficient amoeba, rescue with AIP1-1 was more effective than with AIP1-2. The main results in the plant showed that in the aip1-2 mutant background, reintroduced AIP1-2 displayed the most efficient rescue and A. thaliana AIP1-1 rescued better than DdAip1. The choice of the tagging site was important for the function of Aip1 as steric hindrance is a problem. The DdAip1 was less effective when tagged at the C-terminus, while the plant AIP1s showed mixed results depending on the tag position. In conclusion, the foreign proteins partially rescued phenotypes of mutant plants and mutant amoebae, despite the organisms only being very distantly related in evolutionary terms.
Arachidonsäurelipoxygenasen (ALOX-Isoformen) sind Lipid-peroxidierenden Enzyme, die bei der Zelldifferenzierung und bei der Pathogenese verschiedener Erkrankungen bedeutsam sind. Im menschlichen Genom gibt es sechs funktionelle ALOX-Gene, die als Einzelkopiegene vorliegen. Für jedes humane ALOX-Gen gibt es ein orthologes Mausgen. Obwohl sich die sechs humanen ALOX-Isoformen strukturell sehr ähnlich sind, unterscheiden sich ihre funktionellen Eigenschaften deutlich voneinander. In der vorliegenden Arbeit wurden vier unterschiedliche Fragestellungen zum Vorkommen, zur biologischen Rolle und zur Evolutionsabhängigkeit der enzymatischen Eigenschaften von Säugetier-ALOX-Isoformen untersucht:
1) Spitzhörnchen (Tupaiidae) sind evolutionär näher mit dem Menschen verwandt als Nagetiere und wurden deshalb als Alternativmodelle für die Untersuchung menschlicher Erkrankungen vorgeschlagen. In dieser Arbeit wurde erstmals der Arachidonsäurestoffwechsel von Spitzhörnchen untersucht. Dabei wurde festgestellt, dass im Genom von Tupaia belangeri vier unterschiedliche ALOX15-Gene vorkommen und die Enzyme sich hinsichtlich ihrer katalytischen Eigenschaften ähneln. Diese genomische Vielfalt, die weder beim Menschen noch bei Mäusen vorhanden ist, erschwert die funktionellen Untersuchungen zur biologischen Rolle des ALOX15-Weges. Damit scheint Tupaia belangeri kein geeigneteres Tiermodel für die Untersuchung des ALOX15-Weges des Menschen zu sein.
2) Entsprechend der Evolutionshypothese können Säugetier-ALOX15-Orthologe in Arachidonsäure-12-lipoxygenierende- und Arachidonsäure-15-lipoxygenierende Enzyme eingeteilt werden. Dabei exprimieren Säugetierspezies, die einen höheren Evolutionsgrad als Gibbons aufweisen, Arachidonsäure-15-lipoxygenierende ALOX15-Orthologe, während evolutionär weniger weit entwickelte Säugetiere Arachidonsäure-12 lipoxygenierende Enzyme besitzen. In dieser Arbeit wurden elf neue ALOX15-Orthologe als rekombinante Proteine exprimiert und funktionell charakterisiert. Die erhaltenen Ergebnisse fügen sich widerspruchsfrei in die Evolutionshypothese ein und verbreitern deren experimentelle Basis. Die experimentellen Daten bestätigen auch das Triadenkonzept.
3) Da humane und murine ALOX15B-Orthologe unterschiedliche funktionelle Eigenschaften aufweisen, können Ergebnisse aus murinen Krankheitsmodellen zur biologischen Rolle der ALOX15B nicht direkt auf den Menschen übertragen werden. Um die ALOX15B-Orthologen von Maus und Mensch funktionell einander anzugleichen, wurden im Rahmen der vorliegenden Arbeit Knock-in Mäuse durch die In vivo Mutagenese mittels CRISPR/Cas9-Technik hergestellt. Diese exprimieren eine humanisierte Mutante (Doppelmutation von Tyrosin603Asparaginsäure+Histidin604Valin) der murinen Alox15b. Diese Mäuse waren lebens- und fortpflanzungsfähig, zeigten aber geschlechtsspezifische Unterschiede zu ausgekreuzten Wildtyp-Kontrolltieren im Rahmen ihre Individualentwicklung.
4) In vorhergehenden Untersuchungen zur Rolle der ALOX15B in Rahmen der Entzündungsreaktion wurde eine antiinflammatorische Wirkung des Enzyms postuliert. In der vorliegenden Arbeit wurde untersucht, ob eine Humanisierung der murinen Alox15b die Entzündungsreaktion in zwei verschiedenen murinen Entzündungsmodellen beeinflusst. Eine Humanisierung der murinen Alox15b führte zu einer verstärkten Ausbildung von Entzündungssymptomen im induzierten Dextran-Natrium-Sulfat-Kolitismodell. Im Gegensatz dazu bewirkte die Humanisierung der Alox15b eine Abschwächung der Entzündungssymptome im Freund‘schen Adjuvans Pfotenödemmodell. Diese Daten deuten darauf hin, dass sich die Rolle der ALOX15B in verschiedenen Entzündungsmodellen unterscheidet.
Microalgae have been recognized as a promising green production platform for recombinant proteins. The majority of studies on recombinant protein expression have been conducted in the green microalga C. reinhardtii. While promising improvement regarding nuclear transgene expression in this alga has been made, it is still inefficient due to epigenetic silencing, often resulting in low yields that are not competitive with other expressor organisms. Other microalgal species might be better suited for high-level protein expression, but are limited in their availability of molecular tools.
The red microalga Porphyridium purpureum recently emerged as candidate for the production of recombinant proteins. It is promising in that transformation vectors are episomally maintained as autonomously replicating plasmids in the nucleus at a high copy number, thus leading to high expression values in this red alga.
In this work, we expand the genetic tools for P. purpureum and investigate parameters that govern efficient transgene expression. We provide an improved transformation protocol to streamline the generation of transgenic lines in this organism. After being able to efficiently generate transgenic lines, we showed that codon usage is a main determinant of high-level transgene expression, not only at the protein level but also at the level of mRNA accumulation. The optimized expression constructs resulted in YFP accumulation up to an unprecedented 5% of the total soluble protein. Furthermore, we designed new constructs conferring efficient transgene expression into the culture medium, simplifying purification and harvests of recombinant proteins. To further improve transgene expression, we tested endogenous promoters driving the most highly transcribed genes in P. purpureum and found minor increase of YFP accumulation.
We employed the previous findings to express complex viral antigens from the hepatitis B virus and the hepatitis C virus in P. purpureum to demonstrate its feasibility as producer of biopharmaceuticals. The viral glycoproteins were successfully produced to high levels and could reach their native confirmation, indicating a functional glycosylation machinery and an appropriate folding environment in this red alga. We could successfully upscale the biomass production of transgenic lines and with that provide enough material for immunization trials in mice that were performed in collaboration. These trials showed no toxicity of neither the biomass nor the purified antigens, and, additionally, the algal-produced antigens were able to elicit a strong and specific immune response.
The results presented in this work pave the way for P. purpureum as a new promising producer organism for biopharmaceuticals in the microalgal field.
Heat stress (HS) is a major abiotic stress that negatively affects plant growth and productivity. However, plants have developed various adaptive mechanisms to cope with HS, including the acquisition and maintenance of thermotolerance, which allows them to respond more effectively to subsequent stress episodes. HS memory includes type II transcriptional memory which is characterized by enhanced re-induction of a subset of HS memory genes upon recurrent HS. In this study, new regulators of HS memory in A. thaliana were identified through the characterization of rein mutants.
The rein1 mutant carries a premature stop in CYCLIN-DEPENDENT-KINASE 8 (CDK8) which is part of the cyclin kinase module of the Mediator complex. Rein1 seedlings show impaired type II transcriptional memory in multiple heat-responsive genes upon re-exposure to HS. Additionally, the mutants exhibit a significant deficiency in HS memory at the physiological level. Interaction studies conducted in this work indicate that CDK8 associates with the memory HEAT SHOCK FACTORs HSAF2 and HSFA3. The results suggest that CDK8 plays a crucial role in HS memory in plants together with other memory HSFs, which may be potential targets of the CDK8 kinase function. Understanding the role and interaction network of the Mediator complex during HS-induced transcriptional memory will be an exciting aspect of future HS memory research.
The second characterized mutant, rein2, was selected based on its strongly impaired pAPX2::LUC re-induction phenotype. In gene expression analysis, the mutant revealed additional defects in the initial induction of HS memory genes. Along with this observation, basal thermotolerance was impaired similarly as HS memory at the physiological level in rein2. Sequencing of backcrossed bulk segregants with subsequent fine mapping narrowed the location of REIN2 to a 1 Mb region on chromosome 1. This interval contains the At1g65440 gene, which encodes the histone chaperone SPT6L. SPT6L interacts with chromatin remodelers and bridges them to the transcription machinery to regulate nucleosome and Pol II occupancy around the transcriptional start site. The EMS-induced missense mutation in SPT6L may cause altered HS-induced gene expression in rein2, possibly triggered by changes in the chromatin environment resulting from altered histone chaperone function.
Expanding research on screen-derived factors that modify type II transcriptional memory has the potential to enhance our understanding of HS memory in plants. Discovering connections between previously identified memory factors will help to elucidate the underlying network of HS memory. This knowledge can initiate new approaches to improve heat resilience in crops.
This work analyzed functional and regulatory aspects of the so far little characterized EPSIN N-terminal Homology (ENTH) domain-containing protein EPSINOID2 in Arabidopsis thaliana. ENTH domain proteins play accessory roles in the formation of clathrin-coated vesicles (CCVs) (Zouhar and Sauer 2014). Their ENTH domain interacts with membranes and their typically long, unstructured C-terminus contains binding motifs for adaptor protein complexes and clathrin itself. There are seven ENTH domain proteins in Arabidopsis. Four of them possess the canonical long C-terminus and participate in various, presumably CCV-related intracellular transport processes (Song et al. 2006; Lee et al. 2007; Sauer et al. 2013; Collins et al. 2020; Heinze et al. 2020; Mason et al. 2023). The remaining three ENTH domain proteins, however, have severely truncated C-termini and were termed EPSINOIDs (Zouhar and Sauer 2014; Freimuth 2015). Their functions are currently unclear. Preceding studies focusing on EPSINOID2 indicated a role in root hair formation: epsinoid2 T DNA mutants exhibited an increased root hair density and EPSINOID2-GFP was specifically located in non-hair cell files in the Arabidopsis root epidermis (Freimuth 2015, 2019).
In this work, it was clearly shown that loss of EPSINOID2 leads to an increase in root hair density through analyses of three independent mutant alleles, including a newly generated CRISPR/Cas9 full deletion mutant. The ectopic root hairs emerging from non-hair positions in all epsinoid2 mutant alleles are most likely not a consequence of altered cell fate, because extensive genetic analyses placed EPSINOID2 downstream of the established epidermal patterning network. Thus, EPSINOID2 seems to act as a cell autonomous inhibitor of root hair formation. Attempts to confirm this hypothesis by ectopically overexpressing EPSINOID2 led to the discovery of post-transcriptional and -translational regulation through different mechanisms. One involves the little characterized miRNA844-3p. Interference with this pathway resulted in ectopic EPSINOID2 overexpression and decreased root hair density, confirming it as negative factor in root hair formation. A second mechanism likely involves proteasomal degradation. Treatment with proteasomal inhibitor MG132 led to EPSINOID2-GFP accumulation, and a KEN box degron motif was identified in the EPSINOID2 sequence associated with degradation through a ubiquitin/proteasome-dependent pathway. In line with a tight dose regulation, genetic analyses of all three mutant alleles indicate that EPSINOID2 is haploinsufficient. Lastly, it was revealed that, although EPSINOID2 promoter activity was found in all epidermal cells, protein accumulation was observed in N-cells only, hinting at yet another layer of regulation.
Moss-microbe associations are often characterised by syntrophic interactions between the microorganisms and their hosts, but the structure of the microbial consortia and their role in peatland development remain unknown.
In order to study microbial communities of dominant peatland mosses, Sphagnum and brown mosses, and the respective environmental drivers, four study sites representing different successional stages of natural northern peatlands were chosen on a large geographical scale: two brown moss-dominated, circumneutral peatlands from the Arctic and two Sphagnum-dominated, acidic peat bogs from subarctic and temperate zones.
The family Acetobacteraceae represented the dominant bacterial taxon of Sphagnum mosses from various geographical origins and displayed an integral part of the moss core community. This core community was shared among all investigated bryophytes and consisted of few but highly abundant prokaryotes, of which many appear as endophytes of Sphagnum mosses. Moreover, brown mosses and Sphagnum mosses represent habitats for archaea which were not studied in association with peatland mosses so far. Euryarchaeota that are capable of methane production (methanogens) displayed the majority of the moss-associated archaeal communities. Moss-associated methanogenesis was detected for the first time, but it was mostly negligible under laboratory conditions. Contrarily, substantial moss-associated methane oxidation was measured on both, brown mosses and Sphagnum mosses, supporting that methanotrophic bacteria as part of the moss microbiome may contribute to the reduction of methane emissions from pristine and rewetted peatlands of the northern hemisphere.
Among the investigated abiotic and biotic environmental parameters, the peatland type and the host moss taxon were identified to have a major impact on the structure of moss-associated bacterial communities, contrarily to archaeal communities whose structures were similar among the investigated bryophytes. For the first time it was shown that different bog development stages harbour distinct bacterial communities, while at the same time a small core community is shared among all investigated bryophytes independent of geography and peatland type.
The present thesis displays the first large-scale, systematic assessment of bacterial and archaeal communities associated both with brown mosses and Sphagnum mosses. It suggests that some host-specific moss taxa have the potential to play a key role in host moss establishment and peatland development.
Long-term bacteria-fungi-plant associations in permafrost soils inferred from palaeometagenomics
(2024)
The arctic is warming 2 – 4 times faster than the global average, resulting in a strong feedback on northern ecosystems such as boreal forests, which cover a vast area of the high northern latitudes. With ongoing global warming, the treeline subsequently migrates northwards into tundra areas. The consequences of turning ecosystems are complex: on the one hand, boreal forests are storing large amounts of global terrestrial carbon and act as a carbon sink, dragging carbon dioxide out of the global carbon cycle, suggesting an enhanced carbon uptake with increased tree cover. On the other hand, with the establishment of trees, the albedo effect of tundra decreases, leading to enhanced soil warming. Meanwhile, permafrost thaws, releasing large amounts of previously stored carbon into the atmosphere. So far, mainly vegetation dynamics have been assessed when studying the impact of warming onto ecosystems. Most land plants are living in close symbiosis with bacterial and fungal communities, sustaining their growth in nutrient poor habitats. However, the impact of climate change on these subsoil communities alongside changing vegetation cover remains poorly understood. Therefore, a better understanding of soil community dynamics on multi millennial timescales is inevitable when addressing the development of entire ecosystems. Unravelling long-term cross-kingdom dependencies between plant, fungi, and bacteria is not only a milestone for the assessment of warming on boreal ecosystems. On top, it also is the basis for agriculture strategies to sustain society with sufficient food in a future warming world.
The first objective of this thesis was to assess ancient DNA as a proxy for reconstructing the soil microbiome (Manuscripts I, II, III, IV). Research findings across these projects enable a comprehensive new insight into the relationships of soil microorganisms to the surrounding vegetation. First, this was achieved by establishing (Manuscript I) and applying (Manuscript II) a primer pair for the selective amplification of ancient fungal DNA from lake sediment samples with the metabarcoding approach. To assess fungal and plant co-variation, the selected primer combination (ITS67, 5.8S) amplifying the ITS1 region was applied on samples from five boreal and arctic lakes. The obtained data showed that the establishment of fungal communities is impacted by warming as the functional ecological groups are shifting. Yeast and saprotroph dominance during the Late Glacial declined with warming, while the abundance of mycorrhizae and parasites increased with warming. The overall species richness was also alternating. The results were compared to shotgun sequencing data reconstructing fungi and bacteria (Manuscripts III, IV), yielding overall comparable results to the metabarcoding approach. Nonetheless, the comparison also pointed out a bias in the metabarcoding, potentially due to varying ITS lengths or copy numbers per genome.
The second objective was to trace fungus-plant interaction changes over time (Manuscripts II, III). To address this, metabarcoding targeting the ITS1 region for fungi and the chloroplast P6 loop for plants for the selective DNA amplification was applied (Manuscript II). Further, shotgun sequencing data was compared to the metabarcoding results (Manuscript III). Overall, the results between the metabarcoding and the shotgun approaches were comparable, though a bias in the metabarcoding was assumed. We demonstrated that fungal shifts were coinciding with changes in the vegetation. Yeast and lichen were mainly dominant during the Late Glacial with tundra vegetation, while warming in the Holocene lead to the expansion of boreal forests with increasing mycorrhizae and parasite abundance. Aside, we highlighted that Pinaceae establishment is dependent on mycorrhizal fungi such as Suillineae, Inocybaceae, or Hyaloscypha species also on long-term scales.
The third objective of the thesis was to assess soil community development on a temporal gradient (Manuscripts III, IV). Shotgun sequencing was applied on sediment samples from the northern Siberian lake Lama and the soil microbial community dynamics compared to ecosystem turnover. Alongside, podzolization processes from basaltic bedrock were recovered (Manuscript III). Additionally, the recovered soil microbiome was compared to shotgun data from granite and sandstone catchments (Manuscript IV, Appendix). We assessed if the establishment of the soil microbiome is dependent on the plant taxon and as such comparable between multiple geographic locations or if the community establishment is driven by abiotic soil properties and as such the bedrock area. We showed that the development of soil communities is to a great extent driven by the vegetation changes and temperature variation, while time only plays a minor role. The analyses showed general ecological similarities especially between the granite and basalt locations, while the microbiome on species-level was rather site-specific. A greater number of correlated soil taxa was detected for deep-rooting boreal taxa in comparison to grasses with shallower roots. Additionally, differences between herbaceous taxa of the late Glacial compared to taxa of the Holocene were revealed.
With this thesis, I demonstrate the necessity to investigate subsoil community dynamics on millennial time scales as it enables further understanding of long-term ecosystem as well as soil development processes and such plant establishment. Further, I trace long-term processes leading to podzolization which supports the development of applied carbon capture strategies under future global warming.
The African weakly electric fishes (Mormyridae) exhibit a remarkable adaptive radiation possibly due to their species-specific electric organ discharges (EODs). It is produced by a muscle-derived electric organ that is located in the caudal peduncle. Divergence in EODs acts as a pre-zygotic isolation mechanism to drive species radiations. However, the mechanism behind the EOD diversification are only partially understood.
The aim of this study is to explore the genetic basis of EOD diversification from the gene expression level across Campylomormyrus species/hybrids and ontogeny. I firstly produced a high quality genome of the species C. compressirostris as a valuable resource to understand the electric fish evolution.
The next study compared the gene expression pattern between electric organs and skeletal muscles in Campylomormyrus species/hybrids with different types of EOD duration. I identified several candidate genes with an electric organ-specific expression, e.g. KCNA7a, KLF5, KCNJ2, SCN4aa, NDRG3, MEF2. The overall genes expression pattern exhibited a significant association with EOD duration in all analyzed species/hybrids. The expression of several candidate genes, e.g. KCNJ2, KLF5, KCNK6 and KCNQ5, possibly contribute to the regulation of EOD duration in Campylomormyrus due to their increasing or decreasing expression. Several potassium channel genes showed differential expression during ontogeny in species and hybrid with EOD alteration, e.g. KCNJ2.
I next explored allele specific expression of intragenus hybrids by crossing the duration EOD species C. compressirostris with the medium duration EOD species C. tshokwe and the elongated duration EOD species C. rhynchophorus. The hybrids exhibited global expression dominance of the C. compressirostris allele in the adult skeletal muscle and electric organ, as well as in the juvenile electric organ. Only the gene KCNJ2 showed dominant expression of the allele from C. rhynchophorus, and this was increasingly dominant during ontogeny. It hence supported our hypothesis that KCNJ2 is a key gene of regulating EOD duration. Our results help us to understand, from a genetic perspective, how gene expression effect the EOD diversification in the African weakly electric fish.
This thesis focuses on the molecular evolution of Macroscelidea, commonly referred to as sengis. Sengis are a mammalian order belonging to the Afrotherians, one of the four major clades of placental mammals. Sengis currently consist of twenty extant species, all of which are endemic to the African continent. They can be separated in two families, the soft-furred sengis (Macroscelididae) and the giant sengis (Rhynchocyonidae). While giant sengis can be exclusively found in forest habitats, the different soft-furred sengi species dwell in a broad range of habitats, from tropical rain-forests to rocky deserts.
Our knowledge on the evolutionary history of sengis is largely incomplete. The high level of superficial morphological resemblance among different sengi species (especially the soft-furred sengis) has for example led to misinterpretations of phylogenetic relationships, based on morphological characters. With the rise of DNA based taxonomic inferences, multiple new genera were defined and new species described. Yet, no full taxon molecular phylogeny exists, hampering the answering of basic taxonomic questions. This lack of knowledge can be to some extent attributed to the limited availability of fresh-tissue samples for DNA extraction. The broad African distribution, partly in political unstable regions and low population densities complicate contemporary sampling approaches. Furthermore, the DNA information available usually covers only short stretches of the mitochondrial genome and thus a single genetic locus with limited informational content.
Developments in DNA extraction and library protocols nowadays offer the opportunity to access DNA from museum specimens, collected over the past centuries and stored in natural history museums throughout the world. Thus, the difficulties in fresh-sample acquisition for molecular biological studies can be overcome by the application of museomics, the research field which emerged from those laboratory developments.
This thesis uses fresh-tissue samples as well as a vast collection museum specimens to investigate multiple aspects about the macroscelidean evolutionary history. Chapter 4 of this thesis focuses on the phylogenetic relationships of all currently known sengi species. By accessing DNA information from museum specimens in combination of fresh tissue samples and publicly available genetic resources it produces the first full taxon molecular phylogeny of sengis. It confirms the monophyly of the genus Elephantulus and discovers multiple deeply divergent lineages within different species, highlighting the need for species specific approaches. The study furthermore focuses on the evolutionary time frame of sengis by evaluating the impact of commonly varied parameters on tree dating. The results of the study show, that the mitochondrial information used in previous studies to temporal calibrate the Macroscelidean phylogeny led to an overestimation of node ages within sengis. Especially soft-furred sengis are thus much younger than previously assumed. The refined knowledge of nodes ages within sengis offer the opportunity to link e.g. speciation events to environmental changes.
Chapter 5 focuses on the genus Petrodromus with its single representative Petrodromus tetradactylus. It again exploits the opportunities of museomics and gathers a comprehensive, multi-locus genetic dataset of P. tetradactylus individuals, distributed across most the known range of this species. It reveals multiple deeply divergent lineages within Petrodromus, whereby some could possibly be associated to previously described sub-species, at least one was formerly unknown. It underscores the necessity for a revision of the genus Petrodromus through the integration of both molecular and morphological evidence. The study, furthermore identifies changing forest distributions through climatic oscillations as main factor shaping the genetic structure of Petrodromus.
Chapter 6 uses fresh tissue samples to extent the genomic resources of sengis by thirteen new nuclear genomes, of which two were de-novo assembled. An extensive dataset of more than 8000 protein coding one-to-one orthologs allows to further refine and confirm the temporal time frame of sengi evolution found in Chapter 4. This study moreover investigates the role of gene-flow and incomplete lineage sorting (ILS) in sengi evolution. In addition it identifies clade specific genes of possible outstanding evolutionary importance and links them to potential phenotypic traits affected. A closer investigation of olfactory receptor proteins reveals clade specific differences. A comparison of the demographic past of sengis to other small African mammals does not reveal a sengi specific pattern.
The plant cell wall plays several crucial roles during plant development with its integrity acting as key signalling component for growth regulation during biotic and abiotic stresses. Cellulose microfibrils, the principal load-bearing components is the major component of the primary cell wall, whose synthesis is mediated by microtubule-associated CELLULOSE SYNTHASE (CESA) COMPLEXES (CSC). Previous studies have shown that CSC interacting proteins COMPANION OF CELLULOSE SYNTHASE (CC) facilitate sustained cellulose synthesis during salt stress by promoting repolymerization of cortical microtubules. However, our understanding of cellulose synthesis during salt stress remains incomplete.
In this study, a pull-down of CC1 protein led to the identification of a novel interactor, termed LEA-like. Phylogenetic analysis revealed that LEA-like belongs to the LATE EMBRYOGENESIS ABUNDANT (LEA) protein family, specifically to the LEA_2 subgroup, showing a close relationship with the CC proteins. Roots of the double mutants lea-like and its closest homolog emb3135 exhibited hypersensitivity when grown on cellulose synthesis inhibitors. Further analysis of higher-order mutants of lea-like, emb3135, and cesa6 demonstrated a genetic interaction between them indicating a significant role in cellulose synthesis.
Live-cell imaging revealed that both LEA-like and EMB3135 migrated with the CSC at the plasma membrane along microtubule tracks in control and oryzalin-treated conditions which destabilize microtubules, suggesting a tight interaction. Investigation of fluorescently labeled lines of different domains of the LEA-like protein revealed that the N-terminal cytosolic domain of LEA-like colocalizes with microtubules, suggesting a physical association between the two.
Considering the established role of LEA proteins in abiotic stress tolerance, we performed phenotypic analysis of the mutant under various stresses. Growth of double mutants of lea-like and emb3135 on NaCl containing media resulted in swelling of root cell indicating a putative role in salt stress tolerance. Supportive of this the quadruple mutant, lacking LEA-like, EMB3135, CC1, and CC2 proteins, exhibited a severe root growth defect on NaCl media compared to control conditions. Live-cell imaging revealed that under salt stress, the LEA-like protein forms aggregates in the plasma membrane.
In conclusion, this study has unveiled two novel interactors of the CSC that act with the CC proteins that regulate plant growth in response to salt stress providing new insights into the intricate regulation of cellulose synthesis, particularly under such conditions.
Recently crops encounter an increased number of individual and combined abiotic and biotic stress, which severely affect their growth and yield. Plants are associated with a large number of microorganisms including beneficial as well as pathogenic microorganisms. The interaction of plants with beneficial microorganisms can exert a substantial impact on plant growth and health and their potential can be utilized in sustainable plant production systems. Currently, climate change will increase the impact of stress on crops which will more likely be exposed to combined abiotic and biotic stress. At present, knowledge on how abiotic and biotic stress and the combination of both stresses affect the plant performance and the microbiome is limited. Soil-borne pathogens are responsible for relevant economic losses and are difficult to control. The root bacterial endophytes have shown potential in alleviating stress on plants and improving crop yield and quality. This raises the question how individual abiotic stress like salinity (ionic) and drought (osmotic) and the combination with biotic stress (Verticillium dahliae or Fusarium oxysporum) affects the root microbiota and thus the performance of the plant. Therefore, the goal of this thesis was to improve the understanding of the impact of individual and combined biotic and abiotic stress especially the endophytic root microbiota and thus plant performance. The work is focused on the economically important horticultural crop tomato. The bacterial rootendophytes of tomato plants exposed to individual and combined abiotic and biotic stress was studied with culture-independent and culture-dependent methods. Bacterial root endophytes obtained from tomato roots exposed to individual and combined stress were characterized for their traits that are beneficial to plant growth and health in in vitro and in vivo assays. Finally, the efficacy of selected endophytes in alleviating individual and combined abiotic and biotic stress in tomato plants was assessed. Furthermore, stress conditions can alter the composition of root exudates and volatiles, which may in turn affect the root microbiota assembly. Therefore, the volatile profiles of healthy and pathogen (F. oxysporum) infected tomato roots grown in soil was investigated. A soil olfactometer was established to study the impact of root volatiles of healthy and infected tomato on migration of applied beneficial bacteria. The results of tomato characteristics (plant growth, photosynthesis rate) confirmed the negative effect of individual abiotic and biotic stress reported in other studies. However, the response of combined abiotic stress with biotic stress on plant growth varied depending on the type of combined stress.. For instance, a significant higher negative impact on plant growth was observed when tomato plants were cultivated under ionic stress and infected with F. oxysporum. No additional negative effect on plant growth was observed when tomato was infected with V. dahliae. Both culture-dependent and cultureindependent analyses of the root microbiota revealed that individual and combined abiotic and biotic stress alter the root microbiota structure and diversity of tomato. A significantly lower number of cultivable root endophytes was obtained from roots exposed to ionic stress. 16S rRNA amplicon analysis revealed a stronger impact on the diversity of root-associated bacteria in comparison to biotic stress. The endophytes were characterized as member of the phyla Proteobacteria, Actinobacteria and Firmicutes, and members of Bacteriodetes were only detected by culture-independent approach. A total of 683 cultivable bacterial endophytes were characterized using various in vitro and in vivo plant growth-promoting (PGP) assays. As expected, the highest number of root endophytes with tolerance to ionic stress were obtained from tomato roots exposed to ionic stress. Comparably, a high percentage of root endophytes isolated from roots exposed to osmotic were tolerant to osmotic stress showing that the environment affects the selection of microorganisms by the plant. Interestingly, endopyhtes obtained from roots exposed to abiotic stress showed no traits related to plant growth promotion. Based on in vivo and in vitro traits, five selected endophytes were able to alleviate abiotic and biotic stress on plants. These endophytes were obtained from tomato roots infected with V. dahliae. The blend of root emitted volatiles also differed between healthy and F. oxysporum infected tomato plants. The olfactometer setup results highlighted that root volatiles were involved in attraction of bacteria to the plant roots and beneficial bacteria were observed to migrate towards both, diseased and healthy plants in comparable density. It is proposed that root volatiles from healthy and pathogen infected plants not only work as signals but are also used as an energy source for the rhizosphere bacteria. Concluding, the results of this study indicate that abiotic and biotic stress altered the bacterial rootendophytes and thus affects plant performance. The treatment of plants with beneficial microorganisms reduced the negative impact of stress conditions on plant performance. However, more studies using the selected isolates must be performed in the field for drawing inferences on the efficacy of the selected bacterial isolates in ameliorating the effect of abiotic and biotic stress in plants. The extensive isolate collection will serve as a basis for conducting investigations of root-associated bacteria on plant performance. This is important for the development of new plant protection strategies.
Bioelectrochemical investigation of E. coli TMAO reductase and R. capsulatus formate dehydrogenase
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The European fire-bellied toad (Bombina bombina) is regarded as one of the most threatened species of amphibians in central Europe and is particularly affected by environmental perturbations. During the last decades population numbers in Germany have declined drastically due to pollution, eutrophication and habitat fragmentation. Illegal translocations resulted in an introgression from southern genotypes (probably Austrian) into three local Bombina populations (Northern Germany and Southern Sweden) belonging to the northern lineage of the species. Interestingly, these populations show high frequencies of allochthonous (non-local) alleles at multiple loci and outperform the autochthonous populations in terms of their body condition. Over a time period of ten years, I could show that the Southern lineage haplo- and genotypes are still present in the North and that frequencies of introgressed haplotypes in allochthonous populations did not increase over time. However, the introgression itself expanded towards adjacent populations while the overall haplotype diversity has decreased. In contrast, southern lineage genotypes for two candidate genes under selection, the (immunity) MHC class II gene, as well as the (temperature) stress response HSP70 kDa gene, either do not occur at all or only at low frequencies in northern populations. Furthermore, these alleles do not seem to follow the introgression pattern, as they are also present in non-introgressed populations. This thesis tested two possible outcomes of introgressive hybridization in Northern B. bombina populations: (1) local populations (autochthonous) of Bombina bombina are highly adapted to their environments so that introgression of alien genes causes outbreeding depression or (2) local populations of Bombina bombina potentially lack adaptive variation so that introgression of alien genes causes genetic rescue and promotes adaptive change. I found that this unintentional experiment, as a result of illegal translocations imitating introgression of alien genes coming from a southern population (potentially adapted to warmer climate) into a northern lineage (potentially adapted to local pathogens), has increased the genetic diversity and improved fitness in introgressed northern populations, without disrupting local adaptation in the threatened amphibian species B. bombina, favouring the genetic rescue hypothesis. These results and conclusions represent relevant information for future conservation plans, including supportive breeding programmes for fire-bellied toads in Northern Germany and Southern Sweden.