Massive C loss from subalpine grassland soil with seasonal warming larger than 1.5 °C in an altitudinal transplantation experiment

Climate change is associated with a change in soil organic carbon (SOC) stocks, implying a feedback mechanism on global warming. Grassland soils represent 28 % of the global soil C sink and are therefore important for the atmospheric greenhouse gas concentration. In a field experiment in the Swiss Alps we recorded changes in the ecosystem organic carbon stock under climate change conditions, while quantifying the ecosystem C fluxes at the same time (ecosystem respiration, gross primary productivity, C export in plant material and leachate water). We exposed 216 grassland monoliths to six different climate scenarios (CS) in an altitudinal transplantation experiment. In addition, we applied an irrigation treatment (+12–21 % annual precipitation) and an N deposition treatment (+3 and +15 kg N ha−1 a−1) in a factorial design, simulating summer-drought mitigation and atmospheric N pollution. In five years the ecosystem C stock, consisting of plant C and SOC, dropped dramatically by about −14 % (−1034 ± 610 g C m−2) with the CS treatment representing a +3.0 °C seasonal (Apr.–Oct.) warming. N deposition and the irrigation treatment caused no significant effects. Measurements of C fluxes revealed that ecosystem respiration increased by 10 % at the +1.5 °C warmer CS site and by 38 % at the +3 °C warmer CS site (P ≤ 0.001 each), compared to the CS reference site with no warming. However, gross primary productivity was unaffected by warming, as were the amounts of exported C in harvested plant material and leachate water (dissolved organic C). As a result, the five year C flux balance resulted in a climate scenario effect of −936 ± 138 g C m−2 at the +3.0 °C CS, similar to the C stock climate scenario effect. It is likely that this dramatic C loss of the grassland is a transient effect before a new, climate adjusted steady state is reached.

Txnip enhances Fitness of Dnmt3a-Mutant Hematopoietic Stem Cells Via p21

Clonal hematopoiesis (CH) refers to the age-related expansion of specific clones in the blood system and manifests from somatic mutations acquired in hematopoietic stem cells (HSCs). Approximately 50% of CH variants occur in the gene DNMT3A. While DNMT3A-mutant CH becomes almost ubiquitous in aging humans, a unifying molecular mechanism to illuminate how DNMT3A-mutant HSCs outcompete their counterparts is still lacking. Here, we used interferon-gamma (IFNg) as a model to study the mechanisms by which Dnmt3a mutations increase HSC fitness under recurrent hematopoietic stress. To represent the spectrum of DNMT3A variants found in humans, mouse genetic models were generated; Dnmt3a heterozygous (Vav-Cre; Dnmt3afl/+ = Dnmt3aHET) and homozygous (Vav-Cre; Dnmt3afl/fl = Dnmt3aKO) hematopoietic loss-of-function, and a knock-in model analogous to the hotspot point mutation most prevalent in AML (Vav-Cre; Dnmt3aR878H/+ = Dnmt3aR878). When Dnmt3a-mutant cells were competitively transplanted with wild-type (WT) competitor bone marrow (BM) cells and challenged with different inflammatory and proliferative stressors, Dnmt3aKO and Dnmt3aR878 HSCs were specifically resistant to the deleterious effects of IFNg on HSC self-renewal and clonal expansion. This insensitivity was also confirmed in a humanized mouse model where human CD34 + cord blood cells edited with DNMT3A-targeting gRNAs were xenografted into recipient mice and episodically exposed to human recombinant IFNg. DNMT3A mutant cells maintained their clone size, whereas AAVS1-targeted cells (control) were depleted over serial transplantation. These data suggest that Dnmt3a-mutant HSCs, mimicked DNMT3A-mutated human HSCs and are specifically resistant to IFNg-mediated depletion. One explanation for the observed resistance is that Dnmt3a-mutant HSCs have a fitness advantage under IFNg challenge. Therefore, we generated a novel mouse model to directly quantify the competition between Dnmt3a-mutant and WT HSCs. 10% donor BM cells (CD45.2; WT or Dnmt3a-mutant), 10% WT competitor BM cells (CD45.1/2; Ubc-GFP+) and 80% BM cells (CD45.1; IFNgr1KO; Rosa-M2-rtTA-IFNg) that express IFNg by doxycycline were transplanted into CD45.1 recipient mice. To normalize the effect of doxycycline, chimera made with 80% BM cells (CD45.1; IFNgr1KO; Rosa-M2-rtTA) were also transplanted into recipients. Our result from this transplantation experiment showed Dnmt3a-mutant HSCs resisted IFNg-mediated depletion due to an enhanced fitness advantage. Genetic ablation of IFNgr1 from Dnmt3a-mutant mice revealed that IFNg signaling is cell-intrinsically required by clonal expansion of Dnmt3a-mutant HSCs. In parallel, when HSCs were transplanted into IFNg-deficient recipient mice, clonal expansion of Dnmt3aKO HSCs but not WT HSCs was significantly compromised, suggesting IFNg signaling is also cell-extrinsically crucial for the clonal expansion of Dnmt3a-mutant HSCs in vivo. Mechanistically, DNA hypomethylation-associated over-expression of Thioredoxin-interacting protein (Txnip) in Dnmt3a-mutant HSCs was identified by coupling single-cell RNA-sequencing and Whole-Genome Bisulfite sequencing. The sustained Txnip levels in Dnmt3aKO HSCs led to p53 stabilization and upregulation of p21 under IFNg challenge, further correlated with a retained quiescence and resistance to apoptosis in response to IFNg exposure. Implementing biochemical studies, we observed Txnip mediated an enrichment of p53 at p21 promoter under IFNg exposure in Dnmt3aKO but not WT 32D murine myeloid cell line. Knocking down Txnip by shRNA normalized p53 occupancy at p21 promoter and rescued IFNg-associated p21 upregulation in Dnmt3aKO 32D cells. Functionally, knocking down Txnip and p21 re-sensitized Dnmt3aKO HSCs to IFNg-induced cell cycle activation and apoptosis. In vivo, down-regulation of p21 had no effect on WT HSCs in response to IFNg exposure, but it completely primed Dnmt3aKO and Dnmt3aR878 HSCs to IFNg-induced exhaustion in a transplantation experiment. Taken together, our data highlighted a Txnip-p53-p21 pathway that preserves the functional potential of Dnmt3a-mutant HSCs under conditions of inflammatory stress, which suggests a novel mechanism to explain the increased fitness of Dnmt3a-mutant HSCs and supports rationale for developing interventions to mitigate expansion of pre-malignant clones as a method of blood cancer prevention. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Targeting M2-Tumor Associated Macrophages By Arginase-1 and PD-L1 in Regulating Juvenile Myelomonocytic Leukemia (JMML) Development and Relapse

Tumor-associated macrophages (TAMs) are a key component of tumor-infiltrating immune cells. Macrophages are largely characterized as M1 or M2 types, and TAMs have been shown to express an M2-like phenotype. TAMs endorse tumor progression and contribute to resistance to chemotherapies. However, it is unclear what the composition of M2 macrophages is in patients with Juvenile myelomonocytic leukemia (JMML) and how do these cells mechanistically contribute to JMML and/or relapse after bone marrow transplantation. To study the role of M2- TAMs in JMML development, we first examined the bulk RNA-sequence data in 90 JMML patients. These data demonstrated a significant increase in the expression of arginase-1 (Arg-1) and programmed cell death-1 (PD-1). Furthermore, single cell RNA-sequencing analysis of monocytes/macrophages from 4 JMML patients revealed higher expression of M2- macrophage markers/genes such as IL-10, CD163, MRC1/CD206, TGF-β1 and IL-1R1 compared to M1 macrophage (CD80, CCR7, IL-6, CXCL10, CXCL11 and TNF) expression. We hypothesized that in JMML, inflammatory myeloid cells including neutrophils and M2-macrophages express higher levels of arginase and PD-1, which may contribute to the local suppression of immune responses and damage the bone marrow microenvironment (BME) leading to poor engraftment of normal donor cells, resulting in relapse. To study how alterations in bone marrow (BM) macrophages (M1/M2) contribute to JMML development and relapse, we utilized a mouse model bearing Shp2 E76K mutation (Ptpn11 E76K/+) driven by lysosome-cre (Ptpn11 E76K/+; LysM-Cre+, indicated as Shp2* mice hereafter). This model is frequently used to study JMML as it manifests cardinal features of human JMML. In a competitive transplantation experiment using, Shp2* + Boy/J BM cells (1:1 ratio) transplanted into lethally irradiated Shp2* recipient mice, we show that Shp2* mutant cells out compete WT BoyJ cells and result in rapid growth of CD45.2+ Shp2* mutant mature myeloid cells, hematopoietic stem and progenitors (HSC/Ps) and M2- macrophages (F4/80+/CD206+) in the BM and spleen leading to leukemia relapse. To determine if modulating Arg-1 and PD-1/PD-L1 levels in the background of Shp2* mutant leukemic stem cells in Shp2* recipients would alter the overall engraftment and JMML development and relapse, we again performed a competitive transplantation experiment using, Shp2* + Boy/J (BM cells, 1:1 ratio) into Shp2* and WT recipient mice. After 8 weeks post transplantation, we investigated the role of Arg-1 and PD-L1 in Shp2* recipients using pharmacological inhibitors, CB-1158 (Arg-1 inhibitor; 100 mg/kg, orally) + anti-PD-L1 antibody (10 mg/kg, i.p) for 30 days. The Arg-1 + PD-L1 treatment significantly reduced the number of white blood cells, neutrophils, monocytes and improved RBC and platelet counts. The spleen and liver weights were significantly rescued as well. Interestingly, CD45.1 WT donor cells in the PB, BM, and spleen were significantly increased and a significant reduction of Shp2* mutant CD45.2+ mature myeloid cells in the PB, BM, and spleen was observed. Importantly, the frequency and absolute number of leukemic blasts, LSK (Lin-/Sca1+/c-KIT+) cells, short term hematopoietic cells (ST-HSCs), common myeloid progenitors (CMP), granulocyte macrophage progenitors (GMP) and megakaryocyte erythroid progenitors (MEP) were significantly reduced. Furthermore, the M2- TAMs were significantly reduced in the BM and spleen of Arg-1 + PD-L1 drug treated group compared to vehicle treated mice. Notably the CD8+ T-cells (IFN-γ+ and TNF-α+) were significantly improved in the drug treated mice. These data suggest that the suppression of arginase-1 allows for the arginine levels to increase, which promotes the proliferation of T-cells. Increasing arginine levels also promotes an anti-tumor immune response resulting in the emergence of CD45.1 WT HSCs as opposed to mutant CD45.2 HSCs, suggesting that Arg-1 + PD-L1 treatment is a novel therapeutic approach to treat patients with JMML and for preventing leukemia relapse after BM transplantation. Disclosures No relevant conflicts of interest to declare.

A transplantation experiment yields no evidence for phenotypic plasticity in shell band width in Cepaea nemoralis

The common European grove snail, Cepaea nemoralis (Helicidae), has been a model species in genetics and evolutionary biology for over a century thanks largely to its genetic shell colour polymorphism. Although most aspects of its shell colour variation are known to be purely genetic, with little or no phenotypic plasticity involved, the width of the spiral bands has been suspected to display a certain amount of plasticity. To test this, we conducted a transplantation experiment, in which 548 growing banded snails were marked and transplanted, either within the same habitat (open or closed vegetation, displaying 19% and 61% band fusion, respectively) or between habitat types. The numbers recaptured were low: 8%, 5% after removal of individuals that had not grown. Based on these samples, we did not find any substantial influence of transplantation on band width.

Site-Specific Microbial Decomposer Communities Do Not Imply Faster Decomposition: Results from a Litter Transplantation Experiment

Microbes drive leaf litter decomposition, and their communities are adapted to the local vegetation providing that litter. However, whether these local microbial communities confer a significant home-field advantage in litter decomposition remains unclear, with contrasting results being published. Here, we focus on a litter transplantation experiment from oak forests (home site) to two away sites without oak in South Tyrol (Italy). We aimed to produce an in-depth analysis of the fungal and bacterial decomposer communities using Illumina sequencing and qPCR, to understand whether local adaptation occurs and whether this was associated with litter mass loss dynamics. Temporal shifts in the decomposer community occurred, reflecting changes in litter chemistry over time. Fungal community composition was site dependent, while bacterial composition did not differ across sites. Total litter mass loss and rates of litter decomposition did not change across sites. Litter quality influenced the microbial community through the availability of different carbon sources. Additively, our results do not support the hypothesis that locally adapted microbial decomposers lead to a greater or faster mass loss. It is likely that high functional redundancy within decomposer communities regulated the decomposition, and thus greater future research attention should be given to trophic guilds rather than taxonomic composition.

Seed provenance determines germination responses of Rumex crispus (L.) under water stress and nutrient availability

Aims Seeds of Rumex crispus from six provenances were studied in relation to their germination under drought and presence of nitrogen in the germination and emergence media. We also investigated whether adaptation to soil increases the ability of the species to colonize and establish in contrasting environments along a longitudinal gradient in western Spain by means of a reciprocal transplantation experiment. Methods We conducted a germination trial in the lab to test for the germination responses to water scarcity along a polyethylene glycol gradient and to varying concentrations of nitrogen compounds. Simultaneously reciprocal transplantations experiment was conducted, where seeds from six provenances were grown in the soils from the very same provenances. Seedling emergence, survivorship and fitness-related variables were measured in all plots. Important Findings We found that R. crispus has a cold-stratification requirement that enhances its germination. Significant differences between the six provenances were detected for time-to-germination, total seedling emergence, plant mortality and reproductive effort in all the experiments. The differences between provenances with respect to germination were confirmed by the significant statistical analyses of the variance, thus providing evidence that seeds from parent plants grown in different environmental conditions have an intrinsically different abilities to germinate and establish. Soil nitrogen content where seed germination and seedlings establish also play an important role in their performance in terms of survivorship and reproduction, being the higher levels of inorganic nitrogen and of microbial biomass those that increased biomass production, enhanced inflorescence formation and reduced plant mortality. We conclude that one of the main reasons for the spread and maintenance of R. crispus would be the increased levels of nitrogen in agricultural soils.