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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.
Incorporation of noncanonical amino acids (ncAAs) with bioorthogonal reactive groups by amber suppression allows the generation of synthetic proteins with desired novel properties. Such modified molecules are in high demand for basic research and therapeutic applications such as cancer treatment and in vivo imaging. The positioning of the ncAA-responsive codon within the protein's coding sequence is critical in order to maintain protein function, achieve high yields of ncAA-containing protein, and allow effective conjugation. Cell-free ncAA incorporation is of particular interest due to the open nature of cell-free systems and their concurrent ease of manipulation. In this study, we report a straightforward workflow to inquire ncAA positions in regard to incorporation efficiency and protein functionality in a Chinese hamster ovary (CHO) cell-free system. As a model, the well-established orthogonal translation components Escherichia coli tyrosyl-tRNA synthetase (TyrRS) and tRNATyr(CUA) were used to site-specifically incorporate the ncAA p-azido-l-phenylalanine (AzF) in response to UAG codons. A total of seven ncAA sites within an anti-epidermal growth factor receptor (EGFR) single-chain variable fragment (scFv) N-terminally fused to the red fluorescent protein mRFP1 and C-terminally fused to the green fluorescent protein sfGFP were investigated for ncAA incorporation efficiency and impact on antigen binding. The characterized cell-free dual fluorescence reporter system allows screening for ncAA incorporation sites with high incorporation efficiency that maintain protein activity. It is parallelizable, scalable, and easy to operate. We propose that the established CHO-based cell-free dual fluorescence reporter system can be of particular interest for the development of antibody-drug conjugates (ADCs).
The benefits of counting butterflies: recommendations for a successful citizen science project
(2022)
Citizen science (CS) projects, being popular across many fields of science, have recently also become a popular tool to collect biodiversity data. Although the benefits of such projects for science and policy making are well understood, relatively little is known about the benefits participants get from these projects as well as their personal backgrounds and motivations. Furthermore, very little is known about their expectations. We here examine these aspects, with the citizen science project "German Butterfly Monitoring" as an example. A questionnaire was sent to all participants of the project and the responses to the questionnaire indicated the following: center dot Most transect walkers do not have a professional background in this field, though they do have a high educational level, and are close to retirement, with a high number of females; center dot An important motivation to join the project is to preserve the natural environment and to contribute to scientific knowledge; center dot Participants benefit by enhancing their knowledge about butterflies and especially their ability to identify different species (taxonomic knowledge); center dot Participants do not have specific expectations regarding the project beyond proper management and coordination, but have an intrinsic sense of working for a greater good. The willingness to join a project is higher if the project contributes to the solution of a problem discussed in the media (here, insect decline). Based on our findings from the analysis of the questionnaire we can derive a set of recommendations for establishing a successful CS project. These include the importance of good communication, e.g., by explaining what the (scientific) purpose of the project is and what problems are to be solved with the help of the data collected in the project. The motivation to join a CS project is mostly intrinsic and CS is a good tool to engage people during difficult times such as the COVID-19 pandemic, giving participants the feeling of doing something useful.
Human induced pluripotent stem cells (hiPSCs) are highly sensitive to extrinsic physical and biochemical signals from their extracellular microenvironments. In this study, we analyzed the effect of cyclic temperature changes on hiPSCs behaviors, especially by means of scanning force microscopy (BIO-AFM). The alternation in cellular mechanics, as well as the secretion and pattern of deposition of extracellular matrix (ECM) protein in hiPSCs were evaluated. The arrangement of the actin cytoskeleton changed with the variation of the temperature. The rearranged cytoskeleton architecture led to the subsequent changes in cell mechanics (Young's modulus of hiPSCs). With the exposure to the cyclic cold stimuli, an increase in the average surface roughness (Ra) and roughness mean square (RMS) was detected. This observation might be at least in part due to the upregulated secretion of Laminin alpha 5 during repeated temporary cooling. The expression of pluripotent markers, NANOG and SOX2, was not impaired in hiPSCs, when exposed to the cyclic cold stimuli for 24 h. Our findings provide an insight into the effect of temperature on the hiPSC behaviors, which may contribute to a better understanding of the application of locally controlled therapeutic hypothermia.
1. Microplastics in soils have become an important threat for terrestrial systems as they may potentially alter the geochemical/biophysical soil environment and can interact with drought. As microplastics may affect soil water content, this could exacerbate the well-known negative effects of drought on ecosystem functionality. Thus, functions including litter decomposition, soil aggregation or those related with nutrient cycling can be altered. Despite this potential interaction, we know relatively little about how microplastics, under different soil water conditions, affect ecosystem functions and multifunctionality.
2. To address this gap, we performed an experiment using grassland plant communities growing in microcosms. Microplastic fibres (absent, present) and soil water conditions (well-watered, drought) were applied in a fully factorial design. At harvest, we measured soil ecosystem functions related to nutrient cycling (beta-glucosaminidase, beta-D-cellobiosidase, phosphatase, beta-glucosidase enzymes), respiration, nutrient retention, pH, litter decomposition and soil aggregation (water stable aggregates). As terrestrial systems provide these functions simultaneously, we also assessed ecosystem multifunctionality, an index that encompasses the array of ecosystem functions measured here.
3. We found that the interaction between microplastic fibres and drought affected ecosystem functions and multifunctionality. Drought had negatively affected nutrient cycling by decreasing enzymatic activities by up to similar to 39%, while microplastics increased soil aggregation by similar to 18%, soil pH by similar to 4% and nutrient retention by up to similar to 70% by diminishing nutrient leaching. Microplastic fibres also impacted soil enzymes, respiration and ecosystem multifunctionality, but importantly, the direction of these effects depended on soil water status. That is, under well-watered conditions, these functions decreased with microplastic fibres by up to similar to 34% while under drought they had similar values irrespective of the microplastic presence, or tended to increase with microplastics. Litter decomposition had a contrary pattern increasing with microplastics by similar to 6% under well-watered conditions while decreasing to a similar percentage under drought.
4. Synthesis and applications. Single ecosystem functions can be positively or negatively affected by microplastics fibres depending on soil water status. However, our results suggest that microplastic fibres may cause negative effects on ecosystem soil multifunctionality of a similar magnitude as drought. Thus, strategies to counteract this new global change factor are necessary.
Full-length transcriptome
(2021)
Fish is considered as a supreme model for clarifying the evolution and regulatory mechanism of vertebrate immunity. However, the knowledge of distinct immune cell populations in fish is still limited, and further development of techniques advancing the identification of fish immune cell populations and their functions are required. Single cell RNA-seq (scRNA-seq) has provided a new approach for effective in-depth identification and characterization of cell subpopulations. Current approaches for scRNA-seq data analysis usually rely on comparison with a reference genome and hence are not suited for samples without any reference genome, which is currently very common in fish research. Here, we present an alternative, i.e. scRNA-seq data analysis with a full-length transcriptome as a reference, and evaluate this approach on samples from Epinephelus coioides-a teleost without any published genome. We show that it reconstructs well most of the present transcripts in the scRNA-seq data achieving a sensitivity equivalent to approaches relying on genome alignments of related species. Based on cell heterogeneity and known markers, we characterized four cell types: T cells, B cells, monocytes/macrophages (Mo/M phi) and NCC (non-specific cytotoxic cells). Further analysis indicated the presence of two subsets of Mo/M phi including M1 and M2 type, as well as four subsets in B cells, i.e. mature B cells, immature B cells, pre B cells and early-pre B cells. Our research will provide new clues for understanding biological characteristics, development and function of immune cell populations of teleost. Furthermore, our approach provides a reliable alternative for scRNA-seq data analysis in teleost for which no reference genome is currently available.
Species community structures of Afrotropical butterflies differ depending on the monitoring method
(2022)
Standardised biodiversity assessment is crucial to understand community structures and population dynamics of animals.
There exist various methods to monitor biodiversity. Approaches differ depending on the target species group and the aim of study, and show advantages and disadvantages.
The obtained data and results are influenced by local environmental conditions and seasonal variability.
In a comparative approach, we studied butterfly diversity and community structure in the dryland savannah biome of south-eastern Kenya with two different methods, transect counts and bait trapping.
We repeatedly collected data throughout the dry and rainy seasons, in both near natural and anthropogenically influenced landscapes. Significantly more species and individuals were recorded by transect counts than by bait trapping, though the larger and more mobile Nymphalid species (and in particular representatives of the genus Charaxes) were comparatively overrepresented in traps.
The transect data revealed much more pronounced effects of land-use and seasonality than the trap data. These results show that the choice of data collection methods must depend on the general research question, habitat conditions and season. To study the relative variation of species diversity and abundance, the collection of a fraction of the total species diversity might be sufficient.
However, if the focus is on a largely complete recording of species diversity, the use of various collection methods is essential. More specifically, our data clearly demonstrate that transect counts represent a reasonable method for assessing butterfly diversity for the African dryland savannah region, but fails to fully capture occurrences of all species. Bait trapping can be used only as a supplementary method for assessing some few highly mobile low-density species.
Genomic prediction has revolutionized crop breeding despite remaining issues of transferability of models to unseen environmental conditions and environments. Usage of endophenotypes rather than genomic markers leads to the possibility of building phenomic prediction models that can account, in part, for this challenge. Here, we compare and contrast genomic prediction and phenomic prediction models for 3 growth-related traits, namely, leaf count, tree height, and trunk diameter, from 2 coffee 3-way hybrid populations exposed to a series of treatment-inducing environmental conditions. The models are based on 7 different statistical methods built with genomic markers and ChlF data used as predictors. This comparative analysis demonstrates that the best-performing phenomic prediction models show higher predictability than the best genomic prediction models for the considered traits and environments in the vast majority of comparisons within 3-way hybrid populations. In addition, we show that phenomic prediction models are transferrable between conditions but to a lower extent between populations and we conclude that chlorophyll a fluorescence data can serve as alternative predictors in statistical models of coffee hybrid performance. Future directions will explore their combination with other endophenotypes to further improve the prediction of growth-related traits for crops.
The Lena Delta in Siberia is the largest delta in the Arctic and as a snow-dominated ecosystem particularly vulnerable to climate change.
Using the two decades of MODerate resolution Imaging Spectroradiometer satellite acquisitions, this study investigates interannual and spatial variability of snow-cover duration and summer vegetation vitality in the Lena Delta.
We approximated snow by the application of the normalized difference snow index and vegetation greenness by the normalized difference vegetation index (NDVI). We consolidated the analyses by integrating reanalysis products on air temperature from 2001 to 2021, and air temperature, ground temperature, and the date of snow-melt from time-lapse camera (TLC) observations from the Samoylov observatory located in the central delta.
We extracted spring snow-cover duration determined by a latitudinal gradient. The 'regular year' snow-melt is transgressing from mid-May to late May within a time window of 10 days across the delta.
We calculated yearly deviations per grid cell for two defined regions, one for the delta, and one focusing on the central delta. We identified an ensemble of early snow-melt years from 2012 to 2014, with snow-melt already starting in early May, and two late snow-melt years in 2004 and 2017, with snow-melt starting in June. In the times of TLC recording, the years of early and late snow-melt were confirmed.
In the three summers after early snow-melt, summer vegetation greenness showed neither positive nor negative deviations. Whereas, vegetation greenness was reduced in 2004 after late snow-melt together with the lowest June monthly air temperature of the time series record. Since 2005, vegetation greenness is rising, with maxima in 2018 and 2021.
The NDVI rise since 2018 is preceded by up to 4 degrees C warmer than average June air temperature. The ongoing operation of satellite missions allows to monitor a wide range of land surface properties and processes that will provide urgently needed data in times when logistical challenges lead to data gaps in land-based observations in the rapidly changing Arctic.
The production of volatile organic compounds (VOCs) represents a promising strategy of plant-beneficial bacteria to control soil-borne phytopathogens.
Pseudomonas sp. PICF6 and Pseudomonas simiae PICF7 are two indigenous inhabitants of olive roots displaying effective biological control against Verticillium dahliae. Additionally, strain PICF7 is able to promote the growth of barley and Arabidopsis thaliana, VOCs being involved in the growth of the latter species.
In this study, the antagonistic capacity of these endophytic bacteria against relevant phytopathogens (Verticillium spp., Rhizoctonia solani, Sclerotinia sclerotiorum and Fusarium oxysporum f.sp. lycopersici) was assessed. Under in vitro conditions, PICF6 and PICF7 were only able to antagonize representative isolates of V. dahliae and V. longisporum. Remarkably, both strains produced an impressive portfolio of up to twenty VOCs, that included compounds with reported antifungal (e.g., 1-undecene, (methyldisulfanyl) methane and 1-decene) or plant growth promoting (e.g., tridecane, 1-decene) activities. Moreover, their volatilomes differed strongly in the absence and presence of V. dahliae.
For example, when co incubated with the defoliating pathotype of V. dahliae, the antifungal compound 4-methyl-2,6-bis(2-methyl-2-propanyl)phenol was produced. Results suggest that volatiles emitted by these endophytes may differ in their modes of action, and that potential benefits for the host needs further investigation in planta.
Since the beginning of the Anthropocene, lacustrine biodiversity has been influenced by climate change and human activities. These factors advance the spread of harmful cyanobacteria in lakes around the world, which affects water quality and impairs the aquatic food chain. In this study, we assessed changes in cyanobacterial community dynamics via sedimentary DNA (sedaDNA) from well-dated lake sediments of Lake Tiefer See, which is part of the Klocksin Lake Chain spanning the last 350 years. Our diversity and community analysis revealed that cyanobacterial communities form clusters according to the presence or absence of varves. Based on distance-based redundancy and variation partitioning analyses (dbRDA and VPA) we identified that intensified lake circulation inferred from vegetation openness reconstructions, delta C-13 data (a proxy for varve preservation) and total nitrogen content were abiotic factors that significantly explained the variation in the reconstructed cyanobacterial community from Lake Tiefer See sediments. Operational taxonomic units (OTUs) assigned to Microcystis sp. and Aphanizomenon sp. were identified as potential eutrophication-driven taxa of growing importance since circa common era (ca. CE) 1920 till present. This result is corroborated by a cyanobacteria lipid biomarker analysis. Furthermore, we suggest that stronger lake circulation as indicated by non-varved sediments favoured the deposition of the non-photosynthetic cyanobacteria sister clade Sericytochromatia, whereas lake bottom anoxia as indicated by subrecent- and recent varves favoured the Melainabacteria in sediments. Our findings highlight the potential of high-resolution amplicon sequencing in investigating the dynamics of past cyanobacterial communities in lake sediments and show that lake circulation, anoxic conditions, and human-induced eutrophication are main factors explaining variations in the cyanobacteria community in Lake Tiefer See during the last 350 years.
Microbe-mediated enzymatic hydrolysis of organic matter entails the production of hydrolysate, the recovery of which may be more or less efficient. The selfish uptake mechanism, recently discovered, allows microbes to hydrolyze polysaccharides and take up large oligomers, which are then degraded in the periplasmic space. By minimizing the hydrolysate loss, selfish behaviour may be profitable for free-living cells dwelling in a patchy substrate landscape. However, selfish uptake seems to be tailored to algal-derived polysaccharides, abundant in organic particles, suggesting that particle-attached microbes may use this strategy. We tracked selfish polysaccharides uptake in surface microbial communities of the northeastern Mediterranean Sea, linking the occurrence of this processing mode with microbial lifestyle. Additionally, we set up fluorescently labelled polysaccharides incubations supplying phytodetritus to investigate a 'pioneer' scenario for particle-attached microbes. Under both conditions, selfish behaviour was almost exclusively carried out by particle-attached microbes, suggesting that this mechanism may represent an advantage in the race for particle exploitation. Our findings shed light on the selfish potential of particle-attached microbes, suggesting multifaceted foraging strategies exerted by particle colonizers.
Consumers are increasingly demanding higher quality and safety standards for the products they consume, and one of this is wheat flour, the basis of a wide variety of processed products. This major component in the diet of many communities can be contaminated by microorganisms before the grain harvest, or during the grain storage right before processing. These microorganisms include several fungal species, many of which produce mycotoxins, secondary metabolites that can cause severe acute and chronic disorders. Yet, we still know little about the overall composition of fungal communities associated with wheat flour. In this study, we contribute to fill this gap by characterizing the fungal microbiome of different types of wheat flour using culture-dependent and -independent techniques. Qualitatively, these approaches suggested similar results, highlighting the presence of several fungal taxa able to produce mycotoxins. In-vitro isolation of fungal species suggest a higher frequency of Penicillium, while metabarcoding suggest a higher abundance of Alternaria. This discrepancy might reside on the targeted portion of the community (alive vs. overall) or in the specific features of each technique. Thus, this study shows that commercial wheat flour hosts a wide fungal diversity with several taxa potentially representing concerns for consumers, aspects that need more attention throughout the food production chain.
Wildfires, as a key disturbance in forest ecosystems, are shaping the world's boreal landscapes. Changes in fire regimes are closely linked to a wide array of environmental factors, such as vegetation composition, climate change, and human activity. Arctic and boreal regions and, in particular, Siberian boreal forests are experiencing rising air and ground temperatures with the subsequent degradation of permafrost soils leading to shifts in tree cover and species composition. Compared to the boreal zones of North America or Europe, little is known about how such environmental changes might influence long-term fire regimes in Russia. The larch-dominated eastern Siberian deciduous boreal forests differ markedly from the composition of other boreal forests, yet data about past fire regimes remain sparse. Here, we present a high-resolution macroscopic charcoal record from lacustrine sediments of Lake Khamra (southwest Yakutia, Siberia) spanning the last ca. 2200 years, including information about charcoal particle sizes and morphotypes. Our results reveal a phase of increased charcoal accumulation between 600 and 900 CE, indicative of relatively high amounts of burnt biomass and high fire frequencies. This is followed by an almost 900-year-long period of low charcoal accumulation without significant peaks likely corresponding to cooler climate conditions. After 1750 CE fire frequencies and the relative amount of biomass burnt start to increase again, coinciding with a warming climate and increased anthropogenic land development after Russian colonization. In the 20th century, total charcoal accumulation decreases again to very low levels despite higher fire frequency, potentially reflecting a change in fire management strategies and/or a shift of the fire regime towards more frequent but smaller fires. A similar pattern for different charcoal morphotypes and comparison to a pollen and non-pollen palynomorph (NPP) record from the same sediment core indicate that broad-scale changes in vegetation composition were probably not a major driver of recorded fire regime changes. Instead, the fire regime of the last two millennia at Lake Khamra seems to be controlled mainly by a combination of short-term climate variability and anthropogenic fire ignition and suppression.
Relationships between climate, species composition, and species richness are of particular importance for understanding how boreal ecosystems will respond to ongoing climate change. This study aims to reconstruct changes in terrestrial vegetation composition and taxa richness during the glacial Late Pleistocene and the interglacial Holocene in the sparsely studied southeastern Yakutia (Siberia) by using pollen and sedimentary ancient DNA (sedaDNA) records. Pollen and sedaDNA metabarcoding data using the trnL g and h markers were obtained from a sediment core from Lake Bolshoe Toko. Both proxies were used to reconstruct the vegetation composition, while metabarcoding data were also used to investigate changes in plant taxa richness. The combination of pollen and sedaDNA approaches allows a robust estimation of regional and local past terrestrial vegetation composition around Bolshoe Toko during the last similar to 35,000 years. Both proxies suggest that during the Late Pleistocene, southeastern Siberia was covered by open steppe-tundra dominated by graminoids and forbs with patches of shrubs, confirming that steppe-tundra extended far south in Siberia. Both proxies show disturbance at the transition between the Late Pleistocene and the Holocene suggesting a period with scarce vegetation, changes in the hydrochemical conditions in the lake, and in sedimentation rates. Both proxies document drastic changes in vegetation composition in the early Holocene with an increased number of trees and shrubs and the appearance of new tree taxa in the lake's vicinity. The sedaDNA method suggests that the Late Pleistocene steppe-tundra vegetation supported a higher number of terrestrial plant taxa than the forested Holocene. This could be explained, for example, by the "keystone herbivore" hypothesis, which suggests that Late Pleistocene megaherbivores were able to maintain a high plant diversity. This is discussed in the light of the data with the broadly accepted species-area hypothesis as steppe-tundra covered such an extensive area during the Late Pleistocene.
The Brassica napus seed microbiota is cultivar-specific and transmitted via paternal breeding lines
(2022)
Seed microbiota influence germination and plant health and have the potential to improve crop performance, but the factors that determine their structure and functions are still not fully understood.
Here, we analysed the impact of plant-related and external factors on seed endophyte communities of 10 different oilseed rape (Brassica napus L.) cultivars from 26 field sites across Europe.
All seed lots harboured a high abundance and diversity of endophytes, which were dominated by six genera: Ralstonia, Serratia, Enterobacter, Pseudomonas, Pantoea, and Sphingomonas.
The cultivar was the main factor explaining the variations in bacterial diversity, abundance and composition. In addition, the latter was significantly influenced by diverse biotic and abiotic factors, for example host germination rates and disease resistance against Plasmodiophora brassicae.
A set of bacterial biomarkers was identified to discriminate between characteristics of the seeds, for example Sphingomonas for improved germination and Brevundimonas for disease resistance.
Application of a Bayesian community approach suggested vertical transmission of seed endophytes, where the paternal parent plays a major role and might even determine the germination performance of the offspring.
This study contributes to the understanding of seed microbiome assembly and underlines the potential of the microbiome to be implemented in crop breeding and biocontrol programmes.
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.
Urokinase-type plasminogen activator is widely discussed as a marker for cancer prognosis and diagnosis and as a target for cancer therapies. Together with its receptor, uPA plays an important role in tumorigenesis, tumor progression and metastasis. In the present study, systematic evolution of ligands by exponential enrichment (SELEX) was used to select single-stranded DNA aptamers targeting different forms of human uPA. Selected aptamers allowed the distinction between HMW-uPA and LMW-uPA, and therefore, presumably, have different binding regions. Here, uPAapt-02-FR showed highly affine binding with a K-D of 0.7 nM for HMW-uPA and 21 nM for LMW-uPA and was also able to bind to pro-uPA with a K-D of 14 nM. Furthermore, no cross-reactivity to mouse uPA or tissue-type plasminogen activator (tPA) was measured, demonstrating high specificity. Suppression of the catalytic activity of uPA and inhibition of uPAR-binding could be demonstrated through binding with different aptamers and several of their truncated variants. Since RNA aptamers are already known to inhibit uPA-uPAR binding and other pathological functions of the uPA system, these aptamers represent a novel, promising tool not only for detection of uPA but also for interfering with the pathological functions of the uPA system by additionally inhibiting uPA activity.
Biodegradation of polyester polyurethane by the marine fungus Cladosporium halotolerans 6UPA1
(2022)
Lack of degradability and the accumulation of polymeric wastes increase the risk for the health of the environment. Recently, recycling of polymeric waste materials becomes increasingly important as raw materials for polymer synthesis are in short supply due to the rise in price and supply chain disruptions. As an important polymer, polyurethane (PU) is widely used in modern life, therefore, PU biodegradation is desirable to avoid its accumulation in the environment. In this study, we isolated a fungal strain Cladosporium halotolerans from the deep sea which can grow in mineral medium with a polyester PU (Impranil DLN) as a sole carbon source. Further, we demonstrate that it can degrade up to 80% of Impranil PU after 3 days of incubation at 28 celcius by breaking the carbonyl groups (1732 cm(-1)) and C-N-H bonds (1532 cm(-1) and 1247 cm(-1)) as confirmed by Fourier-transform infrared (FTIR) spectroscopy analysis. Gas chromatography-mass spectrometry (GC-MS) analysis revealed polyols and alkanes as PU degradation intermediates, indicating the hydrolysis of ester and urethane bonds. Esterase and urease activities were detected in 7 days-old cultures with PU as a carbon source. Transcriptome analysis showed a number of extracellular protein genes coding for enzymes such as cutinase, lipase, peroxidase and hydrophobic surface binding proteins A (HsbA) were expressed when cultivated on Impranil PU. The yeast two-hybrid assay revealed that the hydrophobic surface binding protein ChHsbA1 directly interacts with inducible esterases, ChLip1 (lipase) and ChCut1 (cutinase). Further, the KEGG pathway for "fatty acid degradation " was significantly enriched in Impranil PU inducible genes, indicating that the fungus may use the degradation intermediates to generate energy via this pathway. Taken together, our data indicates secretion of both esterase and hydrophobic surface binding proteins by C. halotolerans plays an important role in Impranil PU absorption and subsequent degradation. Our study provides a mechanistic insight into Impranil PU biodegradation by deep sea fungi and provides the basis for future development of biotechnological PU recycling.
As a critical part of plant immunity, cells that are attacked by pathogens undergo rapid transcriptional reprogramming to minimize virulence. Many bacterial phytopathogens use type III effector (T3E) proteins to interfere with plant defense responses, including this transcriptional reprogramming. Here, we show that Xanthomonas outer protein S (XopS), a T3E of Xanthomonas campestris pv. vesicatoria (Xcv), interacts with and inhibits proteasomal degradation of WRKY40, a transcriptional regulator of defense gene expression. Virus-induced gene silencing of WRKY40 in pepper (Capsicum annuum) enhanced plant tolerance to Xcv infection, indicating that WRKY40 represses immunity. Stabilization of WRKY40 by XopS reduces the expression of its targets, which include salicylic acid-responsive genes and the jasmonic acid signaling repressor JAZ8. Xcv bacteria lacking XopS display significantly reduced virulence when surface inoculated onto susceptible pepper leaves. XopS delivery by Xcv, as well as ectopic expression of XopS in Arabidopsis thaliana or Nicotiana benthamiana, prevented stomatal closure in response to bacteria and biotic elicitors. Silencing WRKY40 in pepper or N. benthamiana abolished XopS's ability to prevent stomatal closure. This suggests that XopS interferes with both preinvasion and apoplastic defense by manipulating WRKY40 stability and downstream gene expression, eventually altering phytohormone crosstalk to promote pathogen proliferation.