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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.
The global drylands cover nearly half of the terrestrial surface and are home to more than two billion people. In many drylands, ongoing land-use change transforms near-natural savanna vegetation to agricultural land to increase food production. In Southern Africa, these heterogenous savanna ecosystems are also recognized as habitats of many protected animal species, such as elephant, lion and large herds of diverse herbivores, which are of great value for the tourism industry. Here, subsistence farmers and livestock herder communities often live in close proximity to nature conservation areas. Although these land-use transformations are different regarding the future they aspire to, both processes, nature conservation with large herbivores and agricultural intensification, have in common, that they change the vegetation structure of savanna ecosystems, usually leading to destruction of trees, shrubs and the woody biomass they consist of.
Such changes in woody vegetation cover and biomass are often regarded as forms of land degradation and forest loss. Global forest conservation approaches and international programs aim to stop degradation processes, also to conserve the carbon bound within wood from volatilization into earth’s atmosphere. In search for mitigation options against global climate change savannas are increasingly discussed as potential carbon sinks. Savannas, however, are not forests, in that they are naturally shaped by and adapted to disturbances, such as wildfires and herbivory. Unlike in forests, disturbances are necessary for stable, functioning savanna ecosystems and prevent these ecosystems from forming closed forest stands. Their consequently lower levels of carbon storage in woody vegetation have long been the reason for savannas to be overlooked as a potential carbon sink but recently the question was raised if carbon sequestration programs (such as REDD+) could also be applied to savanna ecosystems. However, heterogenous vegetation structure and chronic disturbances hamper the quantification of carbon stocks in savannas, and current procedures of carbon storage estimation entail high uncertainties due to methodological obstacles. It is therefore challenging to assess how future land-use changes such as agricultural intensification or increasing wildlife densities will impact the carbon storage balance of African drylands.
In this thesis, I address the research gap of accurately quantifying carbon storage in vegetation and soils of disturbance-prone savanna ecosystems. I further analyse relevant drivers for both ecosystem compartments and their implications for future carbon storage under land-use change. Moreover, I show that in savannas different carbon storage pools vary in their persistence to disturbance, causing carbon bound in shrub vegetation to be most likely to experience severe losses under land-use change while soil organic carbon stored in subsoils is least likely to be impacted by land-use change in the future.
I start with summarizing conventional approaches to carbon storage assessment and where and for which reasons they fail to accurately estimated savanna ecosystem carbon storage. Furthermore, I outline which future-making processes drive land-use change in Southern Africa along two pathways of land-use transformation and how these are likely to influence carbon storage. In the following chapters, I propose a new method of carbon storage estimation which is adapted to the specific conditions of disturbance-prone ecosystems and demonstrate the advantages of this approach in relation to existing forestry methods. Specifically, I highlight sources for previous over- and underestimation of savanna carbon stocks which the proposed methodology resolves. In the following chapters, I apply the new method to analyse impacts of land-use change on carbon storage in woody vegetation in conjunction with the soil compartment. With this interdisciplinary approach, I can demonstrate that indeed both, agricultural intensification and nature conservation with large herbivores, reduce woody carbon storage above- and belowground, but partly sequesters this carbon into the soil organic carbon stock. I then quantify whole-ecosystem carbon storage in different ecosystem compartments (above- and belowground woody carbon in shrubs and trees, respectively, as well as topsoil and subsoil organic carbon) of two savanna vegetation types (scrub savanna and savanna woodland). Moreover, in a space-for-time substitution I analyse how land-use changes impact carbon storage in each compartment and in the whole ecosystem. Carbon storage compartments are found to differ in their persistence to land-use change with carbon bound in shrub biomass being least persistent to future changes and subsoil organic carbon being most stable under changing land-use. I then explore which individual land-use change effects act as drivers of carbon storage through Generalized Additive Models (GAMs) and uncover non-linear effects, especially of elephant browsing, with implications for future carbon storage. In the last chapter, I discuss my findings in the larger context of this thesis and discuss relevant implications for land-use change and future-making decisions in rural Africa.
Resolving the evolutionary history of two hippotragin antelopes using archival and ancient DNA
(2024)
African antelopes are iconic but surprisingly understudied in terms of their genetics, especially when it comes to their evolutionary history and genetic diversity. The age of genomics provides an opportunity to investigate evolution using whole nuclear genomes. Decreasing sequencing costs enable the recovery of multiple loci per genome, giving more power to single specimen analyses and providing higher resolution insights into species and populations that can help guide conservation efforts. This age of genomics has only recently begun for African antelopes. Many African bovids have a declining population trend and hence, are often endangered. Consequently, contemporary samples from the wild are often hard to collect. In these cases, ex situ samples from contemporary captive populations or in the form of archival or ancient DNA (aDNA) from historical museum or archaeological/paleontological specimens present a great research opportunity with the latter two even offering a window to information about the past. However, the recovery of aDNA is still considered challenging from regions with prevailing climatic conditions that are deemed adverse for DNA preservation like the African continent. This raises the question if DNA recovery from fossils as old as the early Holocene from these regions is possible.
This thesis focuses on investigating the evolutionary history and genetic diversity of two species: the addax (Addax nasomaculatus) and the blue antelope (Hippotragus leucophaeus). The addax is critically endangered and might even already be extinct in the wild, while the blue antelope became extinct ~1800 AD, becoming the first extinct large African mammal species in historical times. Together, the addax and the blue antelope can inform us about current and past extinction events and the knowledge gained can help guide conservation efforts of threatened species. The three studies used ex situ samples and present the first nuclear whole genome data for both species. The addax study used historical museum specimens and a contemporary sample from a captive population. The two studies on the blue antelope used mainly historical museum specimens but also fossils, and resulted in the recovery of the oldest paleogenome from Africa at that time.
The aim of the first study was to assess the genetic diversity and the evolutionary history of the addax. It found that the historical wild addax population showed only limited phylogeographic structuring, indicating that the addax was a highly mobile and panmictic population and suggesting that the current European captive population might be missing the majority of the historical mitochondrial diversity. It also found the nuclear and mitochondrial diversity in the addax to be rather low compared to other wild ungulate species. Suggestions on how to best save the remaining genetic diversity are presented. The European zoo population was shown to exhibit no or only minor levels of inbreeding, indicating good prospects for the restoration of the species in the wild. The trajectory of the addax’s effective population size indicated a major bottleneck in the late Pleistocene and a low effective population size well before recent human impact led to the species being critically endangered today.
The second study set out to investigate the identities of historical blue antelope specimens using aDNA techniques. Results showed that six out of ten investigated specimens were misidentified, demonstrating the blue antelope to be one of the scarcest mammal species in historical natural history collections, with almost no bone reference material. The preliminary analysis of the mitochondrial genomes suggested a low diversity and hence low population size at the time of the European colonization of southern Africa.
Study three presents the results of the analyses of two blue antelope nuclear genomes, one ~200 years old and another dating to the early Holocene, 9,800–9,300 cal years BP. A fossil-calibrated phylogeny dated the divergence time of the three historically extant Hippotragus species to ~2.86 Ma and demonstrated the blue and the sable antelope (H. niger) to be sister species. In addition, ancient gene flow from the roan (H. equinus) into the blue antelope was detected. A comparison with the roan and the sable antelope indicated that the blue antelope had a much lower nuclear diversity, suggesting a low population size since at least the early Holocene. This concurs with findings from the fossil record that show a considerable decline in abundance after the Pleistocene–Holocene transition. Moreover, it suggests that the blue antelope persisted throughout the Holocene regardless of a low population size, indicating that human impact in the colonial era was a major factor in the blue antelope’s extinction.
This thesis uses aDNA analyses to provide deeper insights into the evolutionary history and genetic diversity of the addax and the blue antelope. Human impact likely was the main driver of extinction in the blue antelope, and is likely the main factor threatening the addax today. This thesis demonstrates the value of ex situ samples for science and conservation, and suggests to include genetic data for conservation assessments of species. It further demonstrates the beneficial use of aDNA for the taxonomic identification of historically important specimens in natural history collections. Finally, the successful retrieval of a paleogenome from the early Holocene of Africa using shotgun sequencing shows that DNA retrieval from samples of that age is possible from regions generally deemed unfavorable for DNA preservation, opening up new research opportunities. All three studies enhance our knowledge of African antelopes, contributing to the general understanding of African large mammal evolution and to the conservation of these and similarly threatened species.
Carbohydrates play a vital role in all living organisms; serving as a cornerstone in primary metabolism through the release of energy from their hydrolysis and subsequent re-utilization (Apriyanto et al., 2022). Starch is the principal carbohydrate reserve in plants, providing essential energy for plant growth. Furthermore, starch serves as a significant carbohydrate source in the human diet. Beyond its nutritional value, starch has extensive industrial application associated with many aspects of human society, such as feed, pharmacy, textiles, and the production of biodegradable plastics. Understanding the mechanisms underlying starch metabolism in plants carries multifaceted benefits. Not only does it contribute to increasing crop yield and refining grain quality, but also can improve the efficiency of industrial applications.
Starch in plants is categorized into two classes based on their location and function: transitory starch and storage starch. Transitory starch is produced in chloroplasts of autotrophic tissues/organs, such as leaves. It is synthesized during the day and degraded during the night. Storage starch is synthesized in heterotrophic tissues/organs, such as endosperm, roots and tubers, which is utilized for plant reproduction and industrial application in human life. Most studies aiming to comprehend starch metabolism of Arabidopsis thaliana primarily focus on transitory starch.
Starch is stored as granular form in chloroplast and amyloplast. The parameters of starch granules, including size, morphology, and quantity per chloroplast serve as indicators of starch metabolism status. However, the understanding of their regulatory mechanism is still incomplete. In this research, I initially employed a simple and adapted method based on laser confocal scanning microscopy (LCSM) to observe size, morphology and quantity of starch granules within chloroplasts in Arabidopsis thaliana in vivo. This method facilitated a rapid and versatile analysis of starch granule parameters across numerous samples. Utilizing this approach, I compared starch granule number per chloroplast between mesophyll cells and guard cells in both wild type plants (Col-0) and several starch related mutants. The results revealed that the granule number is distinct between mesophyll cells and guard cells, even within the same genetic background, suggesting that guard cells operate a unique regulatory mechanism of starch granule number.
Subsequently, I redirected my attention toward examining starch morphology. Through microscopy analyses, I observed a gradual alteration in starch granule morphology in certain mutants during leaf aging. Specifically, in mutants such as sex1-8 and dpe2phs1ss4, there was a progressive alteration in starch granule morphology over time. Conversely, in Col-0 and ss4 mutant, these morphological alterations were not evident. This discovery suggests a new perspective to understand the development of starch morphology.
Further investigation revealed that mutants lacking either Disproportionating enzyme 2 (DPE2) or MALTOSE-EXCESS 1 (MEX1) exhibited gradual alterations in starch morphology with leaf aging. Notably, the most severe effects on starch morphology occurred in double mutants lacking either DPE2 or MEX1 in conjunction with a lack of starch synthase 4 (SS4). In these mutations, a transformation of the starch granule morphology from the typical discoid morphology to oval and eventually to a spherical shape.
To investigate the changes in the internal structure of starch during this alteration, I analyzed the chain length distribution (CLD) of the amylopectin of young, intermediate and old leaves of the mutants. Throughout starch granule development, I found an increased presence of short glucan chains within the granules, particularly evident in dpe2ss4 and mex1ss4 mutants, as well as their parental single mutants. Notably, the single mutant ss4 also showed an affected granule morphology, albeit not influenced by leaf aging..
The CLD pattern of the amylopectin reflects an integrative regulation involving several participants in starch synthesis, including starch synthases (SSs), starch branching/debranching enzymes (SBEs/DBEs). Therefore, I further detected the expression of related genes on transcription level and the enzymatic activity of their respective proteins. Results indicated altered gene expression of several regulators in these mutants, particularly demonstrating dramatic alterations in dpe2 and dpe2ss4 with leaf aging. These changes corresponded with the observed alterations in starch granule morphology.
Taken together, I have identified and characterized a progressive alteration in starch granule morphology primarily resulting from the deficiencies in DPE2 and MEX1. Furthermore, I have associated the CLD pattern with the granule morphogenesis, as well as the gene expression and enzymatic activity of proteins involved in starch synthesis. Unlike SS4, which is implicated in starch initiation, MEX1 and DPE2 are involved into starch degradation. MEX1 is located in chloroplast envelope and DPE2 is situated in the cytosol. Considering the locations and known functions of DPE2/MEX1 and SS4, I infer that there might be two pathways influencing starch morphology: an initiation-affected pathway via SS4 and a degradation-affected pathway via DPE2/MEX1.
Overcoming natural biomass limitations in gram-negative bacteria through synthetic carbon fixation
(2024)
The carbon demands of an ever-increasing human population and the concomitant rise in net carbon emissions requires CO2 sequestering approaches for production of carbon-containing molecules. Microbial production of carbon-containing products from plant-based sugars could replace current fossil-based production. However, this form of sugar-based microbial production directly competes with human food supply and natural ecosystems. Instead, one-carbon feedstocks derived from CO2 and renewable energy were proposed as an alternative. The one carbon molecule formate is a stable, readily soluble and safe-to-store energetic mediator that can be electrochemically generated from CO2 and (excess off-peak) renewable electricity. Formate-based microbial production could represent a promising approach for a circular carbon economy. However, easy-to-engineer and efficient formate-utilizing microbes are lacking. Multiple synthetic metabolic pathways were designed for better-than-nature carbon fixation. Among them, the reductive glycine pathway was proposed as the most efficient pathway for aerobic formate assimilation. While some of these pathways have been successfully engineered in microbial hosts, these synthetic strains did so far not exceed the performance of natural strains. In this work, I engineered and optimized two different synthetic formate assimilation pathways in gram-negative bacteria to exceed the limits of a natural carbon fixation pathway, the Calvin cycle.
The first chapter solidified Cupriavidus necator as a promising formatotrophic host to produce value-added chemicals. The formate tolerance of C. necator was assessed and a production pathway for crotonate established in a modularized fashion. Last, bioprocess optimization was leveraged to produce crotonate from formate at a titer of 148 mg/L.
In the second chapter, I chromosomally integrated and optimized the synthetic reductive glycine pathway in C. necator using a transposon-mediated selection approach. The insertion methodology allowed selection for condition-specific tailored pathway expression as improved pathway performance led to better growth. I then showed my engineered strains to exceed the biomass yields of the Calvin cycle utilizing wildtype C. necator on formate. This demonstrated for the first time the superiority of a synthetic formate assimilation pathway and by extension of synthetic carbon fixation efforts as a whole.
In chapter 3, I engineered a segment of a synthetic carbon fixation cycle in Escherichia coli. The GED cycle was proposed as a Calvin cycle alternative that does not perform a wasteful oxygenation reaction and is more energy efficient. The pathways simple architecture and reasonable driving force made it a promising candidate for enhanced carbon fixation. I created a deletion strain that coupled growth to carboxylation via the GED pathway segment. The CO2 dependence of the engineered strain and 13C-tracer analysis confirmed operation of the pathway in vivo.
In the final chapter, I present my efforts of implementing the GED cycle also in C. necator, which might be a better-suited host, as it is accustomed to formatotrophic and hydrogenotrophic growth. To provide the carboxylation substrate in vivo, I engineered C. necator to utilize xylose as carbon source and created a selection strain for carboxylase activity. I verify activity of the key enzyme, the carboxylase, in the decarboxylative direction. Although CO2-dependent growth of the strain was not obtained, I showed that all enzymes required for operation of the GED cycle are active in vivo in C. necator.
I then evaluate my success with engineering a linear and cyclical one-carbon fixation pathway in two different microbial hosts. The linear reductive glycine pathway presents itself as a much simpler metabolic solution for formate dependent growth over the sophisticated establishment of hard-to-balance carbon fixation cycles. Last, I highlight advantages and disadvantages of C. necator as an upcoming microbial benchmark organism for synthetic metabolism efforts and give and outlook on its potential for the future of C1-based manufacturing.
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.
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.
Due to their sessile lifestyle, plants are constantly exposed to pathogens and possess a multi-layered immune system that prevents infection. The first layer of immunity called pattern-triggered immunity (PTI), enables plants to recognise highly conserved molecules that are present in pathogens, resulting in immunity from non-adaptive pathogens. Adapted pathogens interfere with PTI, however the second layer of plant immunity can recognise these virulence factors resulting in a constant evolutionary battle between plant and pathogen. Xanthomonas campestris pv. vesicatoria (Xcv) is the causal agent of bacterial leaf spot disease in tomato and pepper plants. Like many Gram-negative bacteria, Xcv possesses a type-III secretion system, which it uses to translocate type-III effectors (T3E) into plant cells. Xcv has over 30 T3Es that interfere with the immune response of the host and are important for successful infection. One such effector is the Xanthomonas outer protein M (XopM) that shows no similarity to any other known protein. Characterisation of XopM and its role in virulence was the focus of this work.
While screening a tobacco cDNA library for potential host target proteins, the vesicle-associated membrane protein (VAMP)-associated protein 1-2 like (VAP12) was identified. The interaction between XopM and VAP12 was confirmed in the model species Nicotiana benthamiana and Arabidopsis as well as in tomato, a Xcv host. As plants possess multiple VAP proteins, it was determined that the interaction of XopM and VAP is isoform specific.
It could be confirmed that the major sperm protein (MSP) domain of NtVAP12 is sufficient for binding XopM and that binding can be disrupted by substituting one amino acid (T47) within this domain. Most VAP interactors have at least one FFAT (two phenylalanines [FF] in an acidic tract) related motif, screening the amino acid sequence of XopM showed that XopM has two FFAT-related motifs. Substitution of the second residue of each FFAT motif (Y61/F91) disrupts NtVAP12 binding, suggesting that these motifs cooperatively mediate this interaction. Structural modelling using AlphaFold further confirmed that the unstructured N-terminus of XopM binds NtVAP12 at its MSP domain, which was further confirmed by the generation of truncated XopM variants.
Infection of pepper leaves, with a XopM deficient Xcv strain did not result in a reduction of virulence in comparison to the Xcv wildtype, showing that the function of XopM during infection is redundant. Virus-induced gene silencing of NbVAP12 in N. benthamiana plants also did not affect Xcv virulence, which further indicated that interaction with VAP12 is also non-essential for Xcv virulence. Despite such findings, ectopic expression of wildtype XopM and XopMY61A/F91A in transgenic Arabidopsis seedlings enhanced the growth of a non-pathogenic Pseudomonas syringae pv. tomato (Pst) DC3000 strain. XopM was found to interfere with the PTI response allowing Pst growth independent of its binding to VAP. Furthermore, transiently expressed XopM could suppress reactive oxygen species (ROS; one of the earliest PTI responses) production in N. benthamiana leaves. The FFAT double mutant XopMY61A/F91A as well as the C-terminal truncation variant XopM106-519 could still suppress the ROS response while the N-terminal variant XopM1-105 did not. Suppression of ROS production is therefore independent of VAP binding. In addition, tagging the C-terminal variant of XopM with a nuclear localisation signal (NLS; NLS-XopM106-519) resulted in significantly higher ROS production than the membrane localising XopM106-519 variant, indicating that XopM-induced ROS suppression is localisation dependent.
To further characterise XopM, mass spectrometry techniques were used to identify post-translational modifications (PTM) and potential interaction partners. PTM analysis revealed that XopM contains up to 21 phosphorylation sites, which could influence VAP binding. Furthermore, proteins of the Rab family were identified as potential plant protein interaction partners. Rab proteins serve a multitude of functions including vesicle trafficking and have been previously identified as T3E host targets. Taking this into account, a model of virulence of XopM was proposed, with XopM anchoring itself to VAP proteins to potentially access plasma membrane associated proteins. XopM possibly interferes with vesicle trafficking, which in turn suppresses ROS production through an unknown mechanism.
In this work it was shown that XopM targets VAP proteins. The data collected suggests that this T3E uses VAP12 to anchor itself into the right place to carry out its function. While more work is needed to determine how XopM contributes to virulence of Xcv, this study sheds light onto how adapted pathogens overcome the immune response of their hosts. It is hoped that such knowledge will contribute to the development of crops resistant to Xcv in the future.
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.