Anaerobic methane oxidation is quantitatively important in deeper peat layers of boreal peatlands: Evidence from anaerobic incubations, in situ stable isotopes depth profiles, and microbial communities.

Boreal peatlands store most of their carbon in layers deeper than 0.5 m under anaerobic conditions, where carbon dioxide and methane are produced as terminal products of organic matter degradation. Since the global warming potential of methane is much greater than that of carbon dioxide, the balance between the production rates of these gases is important for future climate predictions. Herein, we aimed to understand whether anaerobic methane oxidation (AMO) could explain the high CO2/CH4 anaerobic production ratios that are widely observed for the deeper peat layers of boreal peatlands. Furthermore, we quantified the metabolic pathways of methanogenesis to examine whether hydrogenotrophic methanogenesis is a dominant methane production pathway for the presumably recalcitrant deeper peat. To assess the CH4 cycling in deeper peat, we combined laboratory anaerobic incubations with a pathway-specific inhibitor, in situ depth patterns of stable isotopes in CH4, and 16S rRNA gene amplicon sequencing for three representative boreal peatlands in Western Siberia. We found up to a 69 % reduction in CH4 production due to AMO, which largely explained the high CO2/CH4 anaerobic production ratios and the in situ depth-related patterns of δ13C and δD in methane. The absence of acetate accumulation after inhibiting acetotrophic methanogenesis and the presence of sulfate- and nitrate-reducing anaerobic acetate oxidizers in the deeper peat indicated that these microorganisms use SO42- and NO3- as electron acceptors. Acetotrophic methanogenesis dominated net CH4 production in the deeper peat, accounting for 81 ± 13 %. Overall, anaerobic oxidation is quantitatively important for the methane cycle in the deeper layers of boreal peatlands, affecting both methane and its main precursor concentrations.

Triazoles and Strobilurin Mixture Affects Soil Microbial Community and Incidences of Wheat Diseases.

Pesticides are widely used in agriculture as a pest control strategy. Despite the benefits of pesticides on crop yields, the persistence of chemical residues in soil has an unintended impact on non-targeted microorganisms. In the present study, we evaluated the potential adverse effects of a mixture of fungicides (difenoconazole, epoxiconazole, and kresoxim-methyl) on soil fungal and bacterial communities, as well as the manifestation of wheat diseases. In the fungicide-treated soil, the Shannon indices of both fungal and bacterial communities decreased, whereas the Chao1 indices did not differ compared to the control soil. Among bacterial taxa, the relative abundances of Arthrobacter and Sphingomonas increased in fungicide-treated soil due to their ability to utilize fungicides and other toxic compounds. Rhizopus and plant-beneficial Chaetomium were the dominant fungal genera, with their prevalence increasing by 2-4 times in the fungicide-treated soil. The genus Fusarium, which includes phytopathogenic species, which are notably responsible for root rot, was the most abundant taxon in each of the two conditions but its relative abundance was two times lower in fungicide-treated soils, consistent with a lower level of disease incidence in plants. The prediction of metabolic pathways revealed that the soil bacterial community had a high potential for degrading various pollutants, and the soil fungal community was in a state of recovery after the application of quinone outside inhibitor (QoI) fungicides. Fungicide-treated soil was characterized by an increase in soil microbial carbon, compared with the control soil. Collectively, the obtained results suggest that the application of difenoconazole, epoxiconazole, and kresoxim-methyl is an effective approach for pest control that does not pose a hazard for the soil ecosystem in the short term. However, it is necessary to carry out additional sampling to take into account the spatio-temporal impact of this fungicide mixture on the functional properties of the soil.

Mineral and Organic Fertilizers Distinctly Affect Fungal Communities in the Crop Rhizosphere.

Fungi represent a diverse group of organisms that play an essential role in maintaining soil health and ecosystem functioning. Plant root exudates form nutrient-rich niches that harbor specific fungal communities, or so-called rhizosphere mycobiomes. The long-term application of fertilizers supplies the soil with nutrients that may override the plant-related effects on rhizosphere fungal communities. Here, we assessed the effect of contrasting fertilization regimes on the composition, diversity, and abundance of bulk soil and rhizosphere mycobiomes of potato, white mustard, and maize under NPK (mineral fertilizers) or fresh cattle manure (organic fertilizers). Mineral and organic fertilizers led to distinct fungal communities in the rhizospheres of all studied crops, and the plant-related effects on the mycobiome were overridden by the effect of fertilization. The abundances of Ascomycota and Olpidiomycota were higher under manure, while the abundances of Basidiomycota and Monoblepharomycota increased under NPK. Manure input strongly increased fungal abundance but decreased fungal diversity and the total number of species. NPK had a slight effect on fungal diversity, but significantly increased the relative abundances of fungal phytopathogens, such as Alternaria and Fusarium. Our study shows that that potential plant species effects on the abundance and diversity of the rhizosphere mycobiomes are governed by long-term fertilization. Fertilization management could therefore be used to manipulate rhizosphere fungal communities and soilborne pathogen suppressiveness.

Does fresh farmyard manure introduce surviving microbes into soil or activate soil-borne microbiota?

Manure inputs into soil strongly affect soil microbial communities leading to shifts in microbial diversity and activity. It is still not clear whether these effects are caused mainly by the survival of microbes introduced with manure or by activation of the soil-borne microbiome. Here, we investigated how the soil microbiome was changed after the introduction of fresh farmyard cattle manure, and which microorganisms originating from manure survived in soil. Manure addition led to a strong increase in soil microbial biomass, gene copies abundances, respiration activity, and diversity. High-throughput sequencing analysis showed that higher microbial diversity in manured soil was caused mainly by activation of 113 soil-borne microbial genera which were mostly minor taxa in not-fertilized soil. Two weeks after manure input, 78% of the manure-associated genera were not detected anymore. Only 15 of 237 prokaryotic genera that originated from manure survived for 144 days in soil, and only 8 of them (primarily representatives of Clostridia class) were found in manured soil after winter. Thus, an increase in microbial biomass and diversity after manure input is caused mainly by activation of soil-borne microbial communities, while most exogenous microbes from manure do not survive in soil conditions after few months.

Temperature sensitivity of SOM decomposition is linked with a K-selected microbial community.

Temperature sensitivity (Q10 ) of soil organic matter (SOM) decomposition is a crucial parameter to predict the fate of soil carbon (C) under global warming. Nonetheless, the response pattern of Q10 to continuous warming and the underlying mechanisms are still under debate, especially considering the complex interactions between Q10 , SOM quality, and soil microorganisms. We examined the Q10 of SOM decomposition across a mean annual temperature (MAT) gradient from -1.9 to 5.1°C in temperate mixed forest ecosystems in parallel with SOM quality and bioavailability, microbial taxonomic composition, and functional genes responsible for organic carbon decomposition. Within this temperature gradient of 7.0°C, the Q10 values increased with MAT, but decreased with SOM bioavailability. The Q10 values increased with the prevalence of K-strategy of soil microbial community, which was characterized by: (i) high ratios of oligotrophic to copiotrophic taxa, (ii) ectomycorrhizal to saprotrophic fungi, (iii) functional genes responsible for degradation of recalcitrant to that of labile C, and (iv) low average 16S rRNA operon copy number. Because the recalcitrant organic matter was mainly utilized by the K-strategists, these findings independently support the carbon quality-temperature theory from the perspective of microbial taxonomic composition and functions. A year-long incubation experiment was performed to determine the response of labile and recalcitrant C pools to warming based on the two-pool model. The decomposition of recalcitrant SOM was more sensitive to increased temperature in southern warm regions, which might attribute to the dominance of K-selected microbial communities. It implies that climate warming would mobilize the larger recalcitrant pools in warm regions, exacerbating the positive feedback between increased MAT and CO2 efflux. This is the first attempt to link temperature sensitivity of SOM decomposition with microbial eco-strategies by incorporating the genetic information and disentangling the complex relationship between Q10 and soil microorganisms.

Spatial Changes in Microbial Communities along Different Functional Zones of a Free-Water Surface Wetland.

Constructed wetlands (CWs) are complicated ecosystems that include vegetation, sediments, and the associated microbiome mediating numerous processes in wastewater treatment. CWs have various functional zones where contrasting biochemical processes occur. Since these zones are characterized by different particle-size composition, physicochemical conditions, and vegetation, one can expect the presence of distinct microbiomes across different CW zones. Here, we investigated spatial changes in microbiomes along different functional zones of a free-water surface wetland located in Moscow, Russia. The microbiome structure was analyzed using Illumina MiSeq amplicon sequencing. We also determined particle diameter and surface area of sediments, as well as chemical composition of organic pollutants in different CW zones. Specific organic particle aggregates similar to activated sludge flocs were identified in the sediments. The highest accumulation of hydrocarbons was found in the zones with predominant sedimentation of fine fractions. Phytofilters had the highest rate of organic pollutants decomposition and predominance of Smithella, Ignavibacterium, and Methanothrix. The sedimentation tank had lower microbial diversity, and higher relative abundances of Parcubacteria, Proteiniclasticum, and Macellibacteroides, as well as higher predicted abundances of genes related to methanogenesis and methanotrophy. Thus, spatial changes in microbiomes of constructed wetlands can be associated with different types of wastewater treatment processes.

The impact of age on number and distribution of proliferating cells in subgranular zone in adult mouse brain.

The mouse brain retains an ability to produce hippocampal granule neurons during the mouse's entire lifespan. The neurons are produced in the subgranular zone (SGZ) located on the inner surface of the granule cell layer in the dentate gyrus (DG). In our study, we used a point cloud approach to characterize how the production and distribution of neural precursors for new hippocampal neurons change in the mouse brain relative to age. We found that the production of neural precursors decreases 64 fold from the age of 30 days to the age of 2.5 years. Within the SGZ the decline of cell proliferation continues during entire mouse life. We reconstructed the distribution of proliferating cells along the longitudinal axis of the SGZ and found that the highest number of proliferating cells are located approximately 0.75 mm from the dorsomedial end of the SGZ that corresponds to the most dorsal part of the DG in the mouse brain. The distribution of proliferating cells in the SGZ showed no apparent aggregations, periodicity or any other readily identifiable spatial characteristics. Proliferating cells in the SGZ tended to be located separately from other proliferating cells. About two thirds of them have no closely located other proliferating cells, and the remaining third is located in small clusters comprised of 2 or 3 proliferating cells. Based on our measurements, we calculated that from the age of 30 days to the age of 2.5 years 1.5 million neural precursors are produced in the SGZ.

Cell proliferation and neurogenesis in adult mouse brain.

Neurogenesis, the formation of new neurons, can be observed in the adult brain of many mammalian species, including humans. Despite significant progress in our understanding of adult neurogenesis, we are still missing data about the extent and location of production of neural precursors in the adult mammalian brain. We used 5-ethynyl-2'-deoxyuridine (EdU) to map the location of proliferating cells throughout the entire adult mouse brain and found that neurogenesis occurs at two locations in the mouse brain. The larger one we define as the main proliferative zone (MPZ), and the smaller one corresponds to the subgranular zone of the hippocampus. The MPZ can be divided into three parts. The caudate migratory stream (CMS) occupies the middle part of the MPZ. The cable of proliferating cells emanating from the most anterior part of the CMS toward the olfactory bulbs forms the rostral migratory stream. The thin layer of proliferating cells extending posteriorly from the CMS forms the midlayer. We have not found any additional aggregations of proliferating cells in the adult mouse brain that could suggest the existence of other major neurogenic zones in the adult mouse brain.

Dissecting molecular differences between Wnt coreceptors LRP5 and LRP6.

Low-density lipoprotein receptor-related proteins 5 and 6 (LRP5 and LRP6) serve as Wnt co-receptors for the canonical ß-catenin pathway. While LRP6 is essential for embryogenesis, both LRP5 and LRP6 play critical roles for skeletal remodeling, osteoporosis pathogenesis and cancer formation, making LRP5 and LRP6 key therapeutic targets for cancer and disease treatment. LRP5 and LRP6 each contain in the cytoplasmic domain five conserved PPPSPxS motifs that are pivotal for signaling and serve collectively as phosphorylation-dependent docking sites for the scaffolding protein Axin. However existing data suggest that LRP6 is more effective than LRP5 in transducing the Wnt signal. To understand the molecular basis that accounts for the different signaling activity of LRP5 and LRP6, we generated a series of chimeric receptors via swapping LRP5 and LRP6 cytoplasmic domains, LRP5C and LRP6C, and studied their Wnt signaling activity using biochemical and functional assays. We demonstrate that LRP6C exhibits strong signaling activity while LRP5C is much less active in cells. Recombinant LRP5C and LRP6C upon in vitro phosphorylation exhibit similar Axin-binding capability, suggesting that LRP5 and LRP6 differ in vivo at a step prior to Axin-binding, likely at receiving phosphorylation. We identified between the two most carboxyl PPPSPxS motifs an intervening "gap4" region that appears to account for much of the difference between LRP5C and LRP6C, and showed that alterations in this region are sufficient to enhance LRP5 PPPSPxS phosphorylation and signaling to levels comparable to LRP6 in cells. In addition we provide evidence that binding of phosphorylated LRP5 or LRP6 to Axin is likely direct and does not require the GSK3 kinase as a bridging intermediate as has been proposed. Our studies therefore uncover a new and important molecular tuning mechanism for differential regulation of LRP5 and LRP6 phosphorylation and signaling activity.

Characterization and development of novel small-molecules inhibiting GSK3 and activating Wnt signaling.

Glycogen synthase kinase 3 (GSK3) is an essential component of the Wnt signaling pathway and plays important roles in regulating cell proliferation, differentiation, and apoptosis. As GSK3 is abnormally upregulated in several diseases including type II diabetes, Alzheimer's disease and cancer, it has been regarded as a potential drug target. During zebrafish development, inhibition of GSK3 leads to ectopic activation of the Wnt pathway, resulting in a headless embryo. Using this phenotype as an assay we screened a chemical library of 4000 compounds and identified one novel compound, 3F8, which specifically inhibits eye and forebrain formation in zebrafish embryos, resembling a typical Wnt overexpression phenotype. Cell reporter assays, chemical informatics analysis and in vitro kinase experiments revealed that 3F8 is a selective GSK3 inhibitor, which is more potent than SB216763, a commonly used GSK3 inhibitor. Based on the structure of 3F8, a new generation of compounds inhibiting GSK3 was synthesized and validated by biological assays. Together, 3F8 and its derivatives could be useful as new reagents and potential therapeutic candidates for GSK3 related diseases.

DKK1 antagonizes Wnt signaling without promotion of LRP6 internalization and degradation.

DKK1 is a secreted protein that antagonizes Wnt signaling and plays essential roles in vertebrate embryogenesis including head induction, skeletal development, and limb patterning. DKK1 is also implicated in osteoporosis, arthritis, and cancer and represents a potential therapeutic target for the treatment of these diseases. DKK1 is a high affinity antagonistic ligand for LRP6, which is a Wnt coreceptor that acts together with the Frizzled serpentine receptor to initiate Wnt signal transduction. Two different models have been proposed to account for the mechanism by which DKK1 antagonizes LRP6 function. One model suggests that DKK1 binding to LRP6 disrupts Wnt-induced Frizzled-LRP6 complex formation, whereas the other model proposes that DKK1 interaction with LRP6 promotes LRP6 internalization and degradation, thereby reducing the cell surface LRP6 level. To clarify the molecular basis of DKK1 action, we examined how DKK1 affects the endogenous LRP6 in several mammalian cell lines including mouse embryonic fibroblasts. Here we show that DKK1 inhibits Wnt signaling but induces neither LRP6 down-regulation from the cell surface nor reduction of total LRP6 protein level and that DKK1 has no effect on the rate of continuous internalization of LRP6 and the half-life (about 4.7 h) of LRP6. We conclude that DKK1 inhibition of LRP6 is independent of LRP6 internalization and degradation.

R-spondin1 is a high affinity ligand for LRP6 and induces LRP6 phosphorylation and beta-catenin signaling.

R-spondin proteins are newly identified secreted molecules that activate beta-catenin signaling. However, the mechanism of R-spondin action and its relationship with Wnt signaling remain unclear. Here we show that human R-spondin1 (hRspo1) is a high affinity ligand for the Wnt co-receptor LRP6 (K(d) = 1.2 nm). hRspo1 induces glycogen synthase kinase 3-dependent phosphorylation and activation of LRP6. DKK1, an LRP6 antagonist, inhibits hRspo1-induced LRP6 phosphorylation. We further demonstrate that hRspo1 synergizes with Frizzled5 in Xenopus axis induction assays and induces the phosphorylation of Dishevelled, a cytoplasmic component downstream of Frizzled function. Our study reveals interesting similarity and distinction between Wnt and R-spondin signaling.

LRP5 mutations linked to high bone mass diseases cause reduced LRP5 binding and inhibition by SOST.

The low density lipoprotein (LDL) receptor-related protein 5 (LRP5) is a co-receptor for Wnt proteins and a major regulator in bone homeostasis. Human genetic studies have shown that recessive loss-of-function mutations in LRP5 are linked to osteoporosis, while on the contrary, dominant missense LRP5 mutations are associated with high bone mass (HBM) diseases. All LRP5 HBM mutations are clustered in a single region in the LRP5 extracellular domain and presumably result in elevated Wnt signaling in bone forming cells. Here we show that LRP5 HBM mutant proteins exhibit reduced binding to a secreted bone-specific LRP5 antagonist, SOST, and consequently are more refractory to inhibition by SOST. As loss-of-function mutations in the SOST gene are associated with Sclerosteosis, another disorder of excessive bone growth, our study suggests that the SOST-LRP5 antagonistic interaction plays a central role in bone mass regulation and may represent a nodal point for therapeutic intervention for osteoporosis and other bone diseases.