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Genetic and biochemical characterization of a radical SAM enzyme required for post-translational glutamine methylation of methyl-coenzyme M reductase

ABSTRACT Methyl-coenzyme M reductase (MCR), the key catalyst in the anoxic production and consumption of methane, contains an unusual 2-methylglutamine residue within its active site. In vitro data show that a B12-dependent radical SAM (rSAM) enzyme, designated MgmA, is responsible for this post-translational modification (PTM). Here, we show that two different MgmA homologs are able to methylate MCR in vivo when expressed in Methanosarcina acetivorans , an organism that does not normally possess this PTM. M. acetivorans strains expressing MgmA showed small, but significant, reductions in growth rates and yields on methylotrophic substrates. Structural characterization of the Ni(II) form of Gln-methylated M. acetivorans MCR revealed no significant differences in the protein fold between the modified and unmodified enzyme; however, the purified enzyme contained the heterodisulfide reaction product, as opposed to the free cofactors found in eight prior M. acetivorans MCR structures, suggesting that substrate/product binding is altered in the modified enzyme. Structural characterization of MgmA revealed a fold similar to other B12-dependent rSAMs, with a wide active site cleft capable of binding an McrA peptide in an extended, linear conformation. IMPORTANCE Methane plays a key role in the global carbon cycle and is an important driver of climate change. Because MCR is responsible for nearly all biological methane production and most anoxic methane consumption, it plays a major role in setting the atmospheric levels of this important greenhouse gas. Thus, a detailed understanding of this enzyme is critical for the development of methane mitigation strategies.

Rodriguez Carrero, Roy J. (ORCID:0000000184475641)↗

Energy Conservation and Hydrogenase Function in Methanogenic Archaea, in Particular the Genus Methanosarcina

The biological production of methane is vital to the global carbon cycle and accounts for ca. 74% of total methane emissions. The organisms that facilitate this process, methanogenic archaea, belong to a large and phylogenetically diverse group that thrives in a wide range of anaerobic environments. Two main subgroups exist within methanogenic archaea: those with and those without cytochromes. Although a variety of metabolisms exist within this group, the reduction of growth substrates to methane using electrons from molecular hydrogen is, in a phylogenetic sense, the most widespread methanogenic pathway. Methanogens without cytochromes typically generate methane by the reduction of CO 2 with electrons derived from H 2 , formate, or secondary alcohols, generating a transmembrane ion gradient for ATP production via an Na +- translocating methyltransferase (Mtr). These organisms also conserve energy with a novel flavin-based electron bifurcation mechanism, wherein the endergonic reduction of ferredoxin is facilitated by the exergonic reduction of a disulfide terminal electron acceptor coupled to either H 2 or formate oxidation. Methanogens that utilize cytochromes have a broader substrate range, and can convert acetate and methylated compounds to methane, in addition to the ability to reduce CO 2 . Cytochrome-containing methanogens are able to supplement the ion motive force generated by Mtr with an H + -translocating electron transport system. In both groups, enzymes known as hydrogenases, which reversibly interconvert protons and electrons to molecular hydrogen, play a central role in the methanogenic process. In this review, we discuss recent insight into methanogen metabolism and energy conservation mechanisms with a particular focus on the genus Methanosarcina.

59 BASIC BIOLOGICAL SCIENCES↗

Predation by a ciliate community mediates temperature and nutrient effects on a peatland prey prokaryotic community

Temperature significantly impacts microbial communities’ composition and function, which plays a vital role in the global carbon cycle that determines climate change. Nutrient influxes often accompany rising temperatures due to human activity. While ecological interactions between different microorganisms could shape their response to environmental change, we do not understand how predation may influence these responses in a warmer and increasingly nutrient-rich world. Here, we assess whether predation by a ciliate community of bacterial consumers influences changes in the diversity, biomass, and function of a freshwater prokaryotic community under different temperature and nutrient conditions. We found that predator presence mediates the effects of temperature and nutrients on the total prokaryotic community biomass and composition through various mechanisms, including direct and indirect effects. However, the total community function was resilient. Our study supports previous findings that temperature and nutrients are essential drivers of microbial community composition and function but also demonstrates how predation can mediate these effects, indicating that the biotic context is as important as the abiotic context to understanding microbial responses to novel climates.

ciliates↗

Methylotrophy in the Mire: direct and indirect routes for methane production in thawing permafrost

While wetlands are major sources of biogenic methane (CH 4 ), our understanding of resident microbial metabolism is incomplete, which compromises the prediction of CH 4 emissions under ongoing climate change. Here, we employed genome-resolved multi-omics to expand our understanding of methanogenesis in the thawing permafrost peatland of Stordalen Mire in Arctic Sweden. In quadrupling the genomic representation of the site’s methanogens and examining their encoded metabolism, we revealed that nearly 20% of the metagenome-assembled genomes (MAGs) encoded the potential for methylotrophic methanogenesis. Further, 27% of the transcriptionally active methanogens expressed methylotrophic genes; for Methanosarcinales and Methanobacteriales MAGs, these data indicated the use of methylated oxygen compounds (e.g., methanol), while for Methanomassiliicoccales, they primarily implicated methyl sulfides and methylamines. In addition to methanogenic methylotrophy, >1,700 bacterial MAGs across 19 phyla encoded anaerobic methylotrophic potential, with expression across 12 phyla. Metabolomic analyses revealed the presence of diverse methylated compounds in the Mire, including some known methylotrophic substrates. Active methylotrophy was observed across all stages of a permafrost thaw gradient in Stordalen, with the most frozen non-methanogenic palsa found to host bacterial methylotrophy and the partially thawed bog and fully thawed fen seen to house both methanogenic and bacterial methylotrophic activities. Methanogenesis across increasing permafrost thaw is thus revised from the sole dominance of hydrogenotrophic production and the appearance of acetoclastic at full thaw to consider the co-occurrence of methylotrophy throughout. Collectively, these findings indicate that methanogenic and bacterial methylotrophy may be an important and previously underappreciated component of carbon cycling and emissions in these rapidly changing wetland habitats.

59 BASIC BIOLOGICAL SCIENCES↗

Giant Virus Infection Signatures Are Modulated by Euphotic Zone Depth Strata and Iron Regimes of the Subantarctic Southern Ocean

Viruses can alter the abundance, evolution, and metabolism of microorganisms in the ocean, playing a key role in water column biogeochemistry and global carbon cycles. Large efforts to measure the contribution of eukaryotic microorganisms (e.g., protists) to the marine food web have been made, yet the in situ activities of the ecologically relevant viruses that infect these organisms are not well characterized. Viruses within the phylum Nucleocytoviricota (“giant viruses”) are known to infect a diverse range of ecologically relevant marine protists, yet how these viruses are influenced by environmental conditions remains under-characterized. By employing metatranscriptomic analyses of in situ microbial communities along a temporal and depthresolved gradient, we describe the diversity of giant viruses at the Southern Ocean Time Series (SOTS), a site within the subpolar Southern Ocean. Using a phylogeny-guided taxonomic assessment of detected giant virus genomes and metagenome-assembled genomes, we observed depth-dependent structuring of divergent giant virus families mirroring dynamic physicochemical gradients in the stratified euphotic zone. Analyses of transcribed metabolic genes from giant viruses suggest viral metabolic reprogramming of hosts from the surface to a 200-m depth. Lastly, using on-deck incubations reflecting a gradient of iron availability, we show that modulating iron regimes influences the activity of giant viruses in the field. Specifically, we show enhanced infection signatures of giant viruses under both iron-replete and iron-limited conditions. Collectively, these results expand our understanding of how the water column’s vertical biogeography and chemical surroundings affect an important group of viruses within the Southern Ocean

59 BASIC BIOLOGICAL SCIENCES↗

Long-Term Cellulose Enrichment Selects for Highly Cellulolytic Consortia and Competition for Public Goods

Microbial communities are a key driver of the carbon cycle through the breakdown of complex polysaccharides in diverse environments including soil, marine systems, and the mammalian gut. However, due to the complexity of these communities, the species-species interactions that impact community structure and ultimately shape the rate of decomposition are difficult to define.

microbial interactions↗

Functional insights of novel Bathyarchaeia reveal metabolic versatility in their role in peatlands of the Peruvian Amazon

ABSTRACT The decomposition of soil organic carbon within tropical peatlands is influenced by the functional composition of the microbial community. In this study, building upon our previous work, we recovered a total of 28 metagenome-assembled genomes (MAGs) classified as Bathyarchaeia from the tropical peatlands of the Pastaza-Marañón Foreland Basin (PMFB) in the Amazon. Using phylogenomic analyses, we identified nine genus-level clades to have representatives from the PMFB, with four forming a putative novel family (“CandidatusPaludivitaceae”) endemic to peatlands. We focus on theCa. Paludivitaceae MAGs due to the novelty of this group and the limited understanding of their role within tropical peatlands. Functional analysis of these MAGs reveals that this putative family comprises facultative anaerobes, possessing the genetic potential for oxygen, sulfide, or nitrogen oxidation. This metabolic versatility can be coupled to the fermentation of acetoin, propanol, or proline. The other clades outsideCa. Paludivitaceae are putatively capable of acetogenesis andde novoamino acid biosynthesis and encode a high amount of Fe 3+ transporters. Crucially, theCa. Paludivitaceae are predicted to be carboxydotrophic, capable of utilizing CO for energy generation or biomass production. Through this metabolism, they could detoxify the environment from CO, a byproduct of methanogenesis, or produce methanogenic substrates like CO 2 and H 2 . Overall, our results show the complex metabolism and various lineages of Bathyarchaeia within tropical peatlands pointing to the need to further evaluate their role in these ecosystems. IMPORTANCE With the expansion of theCandidatusPaludivitaceae family by the assembly of 28 new metagenome assembled genomes, this study provides novel insights into their metabolic diversity and ecological significance in peatland ecosystems. From a comprehensive phylogenic and functional analysis, we have elucidated their putative unique facultative anaerobic capabilities and CO detoxification potential. This research highlights their crucial role in carbon cycling and greenhouse gas regulation. These findings are essential for resolving the microbial processes affecting peat soil stability, offering new perspectives on the ecological roles of previously underexplored and underrepresented archaeal populations.

Microbiology↗

Transcriptional response of Methanosarcina acetivorans to repression of the energy-conserving methanophenazine: CoM-CoB heterodisulfide reductase enzyme HdrED

ABSTRACT Methane-producing archaea are key organisms in the anaerobic carbon cycle. These organisms, also called methanogens, grow by converting substrate to methane gas in a process called methanogenesis. Previous research showed that the reduction of the terminal electron acceptor is the rate-limiting step in methanogenesis by Methanosarcina acetivorans . In order to gain insight into how the cells sense and respond to the availability of the terminal electron acceptor, we designed an experiment to deplete cells of the essential terminal oxidase enzyme, HdrED. We found that the depletion of HdrED in vivo results in a higher abundance of transcripts for methyltransferases ( mtaC2, mtaB3, mtaC3 ), coenzyme B biosynthesis, C1 metabolism, and pyrimidine compounds. In most cases, these changes were distinct from transcript abundance changes observed during the transition from exponential growth to stationary phase cultures. These data implicate the methylotrophic methanogenesis regulator MsrC (MA4383) in CoM-S-S-CoB heterodisulfide sensing and indicate cells have a specific mechanism to sense intracellular ratio of CoM-S-S-CoB, coenzyme M, and coenzyme B thiols and further suggest transcripts encoding translation and methanogenesis functions are controlled by feed-forward regulation depending on substrate availability. IMPORTANCE Methanosarcina is an emerging model archaeon and synthetic biology platform for the production of renewable energy and sustainable chemicals to reduce dependence on petroleum. Research into metabolic networks and gene regulation in this organism and other methanogens will inform genome-scale metabolic modeling and microbial function prediction in uncultured or non-model anaerobes and archaea. This study suggests methanogens use unknown mechanisms to efficiently couple methanogenesis to gene regulation via CoM-S-S-CoB and ATP availability.

Buan, Nicole R. (ORCID:000000027560973X)↗

Plant Physiology Increases the Magnitude and Spread of the Transient Climate Response to CO 2 in CMIP6 Earth System Models

Increasing concentrations of CO 2 in the atmosphere influence climate both through CO 2 ’s role as a greenhouse gas and through its impact on plants. Plants respond to atmospheric CO 2 concentrations in several ways that can alter surface energy and water fluxes and thus surface climate, including changes in stomatal conductance, water use, and canopy leaf area. These plant physiological responses are already embedded in most Earth system models, and a robust literature demonstrates that they can affect global-scale temperature. However, the physiological contribution to transient warming has yet to be assessed systematically in Earth system models. Here this gap is addressed using carbon cycle simulations from phases 5 and 6 of the Coupled Model Intercomparison Project (CMIP) to isolate the radiative and physiological contributions to the transient climate response (TCR), which is defined as the change in globally averaged near-surface air temperature during the 20-yr window centered on the time of CO 2 doubling relative to preindustrial CO 2 concentrations. In CMIP6 models, the physiological effect contributes 0.12°C ( σ : 0.09°C; range: 0.02°–0.29°C) of warming to the TCR, corresponding to 6.1% of the full TCR ( σ : 3.8%; range: 1.4%–13.9%). Moreover, variation in the physiological contribution to the TCR across models contributes disproportionately more to the intermodel spread of TCR estimates than it does to the mean. The largest contribution of plant physiology to CO 2 -forced warming—and the intermodel spread in warming—occurs over land, especially in forested regions.

54 ENVIRONMENTAL SCIENCES↗

Variation in forest root image annotation by experts, novices, and AI

Abstract Background The manual study of root dynamics using images requires huge investments of time and resources and is prone to previously poorly quantified annotator bias. Artificial intelligence (AI) image-processing tools have been successful in overcoming limitations of manual annotation in homogeneous soils, but their efficiency and accuracy is yet to be widely tested on less homogenous, non-agricultural soil profiles, e.g., that of forests, from which data on root dynamics are key to understanding the carbon cycle. Here, we quantify variance in root length measured by human annotators with varying experience levels. We evaluate the application of a convolutional neural network (CNN) model, trained on a software accessible to researchers without a machine learning background, on a heterogeneous minirhizotron image dataset taken in a multispecies, mature, deciduous temperate forest. Results Less experienced annotators consistently identified more root length than experienced annotators. Root length annotation also varied between experienced annotators. The CNN root length results were neither precise nor accurate, taking ~ 10% of the time but significantly overestimating root length compared to expert manual annotation ( p = 0.01). The CNN net root length change results were closer to manual ( p = 0.08) but there remained substantial variation. Conclusions Manual root length annotation is contingent on the individual annotator. The only accessible CNN model cannot yet produce root data of sufficient accuracy and precision for ecological applications when applied to a complex, heterogeneous forest image dataset. A continuing evaluation and development of accessible CNNs for natural ecosystems is required.

Handy, Grace↗

Warming-induced permafrost thaw exacerbates tundra soil carbon decomposition mediated by microbial community

BACKGROUND:It is well-known that global warming has effects on high-latitude tundra underlain with permafrost. This leads to a severe concern that decomposition of soil organic carbon (SOC) previously stored in this region, which accounts for about 50% of the world's SOC storage, will cause positive feedback that accelerates climate warming. We have previously shown that short-term warming (1.5 years) stimulates rapid, microbe-mediated decomposition of tundra soil carbon without affecting the composition of the soil microbial community (based on the depth of 42684 sequence reads of 16S rRNA gene amplicons per 3 g of soil sample). RESULTS:We show that longer-term (5 years) experimental winter warming at the same site altered microbial communities (p < 0.040). Thaw depth correlated the strongest with community assembly and interaction networks, implying that warming-accelerated tundra thaw fundamentally restructured the microbial communities. Both carbon decomposition and methanogenesis genes increased in relative abundance under warming, and their functional structures strongly correlated (R 2 > 0.725, p < 0.001) with ecosystem respiration or CH4 flux. CONCLUSIONS:Our results demonstrate that microbial responses associated with carbon cycling could lead to positive feedbacks that accelerate SOC decomposition in tundra regions, which is alarming because SOC loss is unlikely to subside owing to changes in microbial community composition. Video Abstract.

59 BASIC BIOLOGICAL SCIENCES↗

Increased replication of dissimilatory nitrate-reducing bacteria leads to decreased anammox bioreactor performance

Background: Anaerobic ammonium oxidation (anammox) is a biological process employed to remove reactive nitrogen from wastewater. While a substantial body of literature describes the performance of anammox bioreactors under various operational conditions and perturbations, few studies have resolved the metabolic roles of their core microbial community members. Results: Here, we used metagenomics to study the microbial community of a laboratory-scale anammox bioreactor from inoculation, through a performance destabilization event, to robust steady-state performance. Metabolic analyses revealed that nutrient acquisition from the environment is selected for in the anammox community. Dissimilatory nitrate reduction to ammonium (DNRA) was the primary nitrogen removal pathway that competed with anammox. Increased replication of bacteria capable of DNRA led to the out-competition of anammox bacteria, and the loss of the bioreactor's nitrogen removal capacity. These bacteria were highly associated with the anammox bacterium and considered part of the core microbial community. Conclusions: Our findings highlight the importance of metabolic interdependencies related to nitrogen- and carbon-cycling within anammox bioreactors and the potentially detrimental effects of bacteria that are otherwise considered core microbial community members.

59 BASIC BIOLOGICAL SCIENCES↗

HT-SIP: a semi-automated stable isotope probing pipeline identifies cross-kingdom interactions in the hyphosphere of arbuscular mycorrhizal fungi

Abstract Background Linking the identity of wild microbes with their ecophysiological traits and environmental functions is a key ambition for microbial ecologists. Of many techniques that strive for this goal, Stable-isotope probing—SIP—remains among the most comprehensive for studying whole microbial communities in situ. In DNA-SIP, actively growing microorganisms that take up an isotopically heavy substrate build heavier DNA, which can be partitioned by density into multiple fractions and sequenced. However, SIP is relatively low throughput and requires significant hands-on labor. We designed and tested a semi-automated, high-throughput SIP (HT-SIP) pipeline to support well-replicated, temporally resolved amplicon and metagenomics experiments. We applied this pipeline to a soil microhabitat with significant ecological importance—the hyphosphere zone surrounding arbuscular mycorrhizal fungal (AMF) hyphae. AMF form symbiotic relationships with most plant species and play key roles in terrestrial nutrient and carbon cycling. Results Our HT-SIP pipeline for fractionation, cleanup, and nucleic acid quantification of density gradients requires one-sixth of the hands-on labor compared to manual SIP and allows 16 samples to be processed simultaneously. Automated density fractionation increased the reproducibility of SIP gradients compared to manual fractionation, and we show adding a non-ionic detergent to the gradient buffer improved SIP DNA recovery. We applied HT-SIP to 13 C-AMF hyphosphere DNA from a 13 CO 2 plant labeling study and created metagenome-assembled genomes (MAGs) using high-resolution SIP metagenomics (14 metagenomes per gradient). SIP confirmed the AMF Rhizophagus intraradices and associated MAGs were highly enriched (10–33 atom% 13 C), even though the soils’ overall enrichment was low (1.8 atom% 13 C). We assembled 212 13 C-hyphosphere MAGs; the hyphosphere taxa that assimilated the most AMF-derived 13 C were from the phyla Myxococcota, Fibrobacterota, Verrucomicrobiota, and the ammonia-oxidizing archaeon genus Nitrososphaera . Conclusions Our semi-automated HT-SIP approach decreases operator time and improves reproducibility by targeting the most labor-intensive steps of SIP—fraction collection and cleanup. We illustrate this approach in a unique and understudied soil microhabitat—generating MAGs of actively growing microbes living in the AMF hyphosphere (without plant roots). The MAGs’ phylogenetic composition and gene content suggest predation, decomposition, and ammonia oxidation may be key processes in hyphosphere nutrient cycling.

59 BASIC BIOLOGICAL SCIENCES↗

How to engineer the unknown: Advancing a quantitative and predictive understanding of plant and soil biology to address climate change

Our basic understanding of carbon cycling in the biosphere remains qualitative and incomplete, precluding our ability to effectively engineer novel solutions to climate change. How can we attempt to engineer the unknown? This challenge has been faced before in plant biology, providing a roadmap to guide future efforts. We use examples from over a century of photosynthesis research to illustrate the key principles that will set future plant engineering on a solid footing, namely, an effort to identify the key control variables, quantify the effects of systematically tuning these variables, and use theory to account for these observations. The main contributions of plant synthetic biology will stem not from delivering desired genotypes but from enabling the kind of predictive understanding necessary to rationally design these genotypes in the first place. Only then will synthetic plant biology be able to live up to its promise.

59 BASIC BIOLOGICAL SCIENCES↗

Active layer depth and soil properties impact specific leaf area variation and ecosystem productivity in a boreal forest

Specific leaf area (SLA, leaf area per unit dry mass) is a key canopy structural characteristic, a measure of photosynthetic capacity, and an important input into many terrestrial process models. Although many studies have examined SLA variation, relatively few data exist from high latitude, climate-sensitive permafrost regions. We measured SLA and soil and topographic properties across a boreal forest permafrost transition, in which dominant tree species changed as permafrost deepened from 54 to >150 cm over 75 m hillslope transects in Caribou-Poker Creeks Research Watershed, Alaska. We characterized both linear and threshold relationships between topographic and edaphic variables and SLA and developed a conceptual model of these relationships. We found that the depth of the soil active layer above permafrost was significantly and positively correlated with SLA for both coniferous and deciduous boreal tree species. Intraspecific SLA variation was associated with a fivefold increase in net primary production, suggesting that changes in active layer depth due to permafrost thaw could strongly influence ecosystem productivity. While this is an exploratory study to begin understanding SLA variation in a non-contiguous permafrost system, our results indicate the need for more extensive evaluation across larger spatial domains. These empirical relationships and associated uncertainty can be incorporated into ecosystem models that use dynamic traits, improving our ability to predict ecosystem-level carbon cycling responses to ongoing climate change.

59 BASIC BIOLOGICAL SCIENCES↗

Carbonyl sulfide fluxes and concentrations from midwestern US (Bondville, FermiLab and Chicago)

This data package includes measurements of carbonyl sulfide (OCS) fluxes and concentrations from flux towers in the midwestern US. The measurements were made at AmeriFlux towers and thus the data package also includes a subset of the standard eddy covariance and meteorological data (e.g. VPD and NEE) that are also available from the AmeriFlux portal. The measurements presented here were made to provide an independent constraint on whole ecosystem stomatal conductance from these ecosystems with the goal of testing how the relationship between gross primary production and stomatal conductance varies through the growing season. Furthermore, we derived whole canopy nighttime stomatal conductance estimates to test how stomatal conductance varies between native prairie and maize ecosystems. All data presented here were discussed in the publication "Seasonal evolution of canopy stomatal conductance for a prairie and maize field in the midwestern US from continuous carbonyl sulfide fluxes" in Geophysical Research Letters (doi: 10.1029/2019GL085652).

54 ENVIRONMENTAL SCIENCES↗

ESS-DIVE reporting format for field measurements of soil respiration

This is a new data and metadata reporting format for soil respiration data, based on engagement with a wide range of researchers in the earth and ecological sciences as well as expert advisory panels. Reporting formats are intended to be ‘bottom-up’, community-developed documents and templates that can enable consistent, findable, accessible, interoperable, and reproducible (FAIR) data; our goal was a reporting format that would be relevant and useful for synthesis activities, optimizing data discoverability and usability while not placing an undue burden on data contributors. The format is split into two levels of information—metadata about chambers (used to measure soil respiration in the field), and the measured data themselves. The file data_reportingformat_template.xlsx contains information on how the data table should be structured, including field names, field descriptions, and units; the chammetadata_reportingformat_template.xlsx file contains similar information for the chamber-level metadata. Fundamentally, this format aims to enable FAIR data through an easy-to-use guide for data providers interested in formatting their data in a common way, and provide ‘future-proofing’ capabilities to support reanalyses of soil respiration data, a globally-important component of the carbon cycle.

54 ENVIRONMENTAL SCIENCES↗

Comparing the Recovery of Arbuscular and Ectomycorrhizal Stands from Long-Term Nitrogen Fertilization at the Fernow Experimental Forest, WV (2021 and 2022)

This data was generated to answer the research question: After the end of a 30-year nitrogen (N) fertilization experiment in an Eastern temperate forest, to what extent do plant microbial interactions and carbon cycling in arbuscular mycorrhizal (AM) and ectomycorrhizal (ECM) dominated stands follow different recovery trajectories?The soil and the roots used in this dataset were sampled from two watersheds (N-fertilized watershed 3 and Reference watershed 7) with 6 AM and 6 ECM dominated stands each, over the course of three months (June, July, August) in 2021 and 2022. The "All_Roots.csv" data includes fine root biomass (in g) scaled to sampled soil (m2) (Scaled_Rt_Bm_gm2), and AM and ECM root colonization (%) in two soil fractions: the organic horizon (O) and the mineral horizon (B). The "All_Enzymes.csv" data includes extracellular soil enzyme activity (N-acetyl-glucosaminidase (NAG), acid phosphatase (AP), β-glucosidase (BG), phenol oxidase and peroxidase), and the ratios of BG to AP (BG:AP) and BG to NAG (BG:NAG) in three soil fractions: the organic horizon (O), the bulk soil (B), and the rhizosphere (R).The "All_Nmin" data includes inorganic N (NO3- and NH4+) pools (unit: μg of N per g of dry soil) measured pre- (I_Nitrate_ugNgsoil and I_Ammonia_ugNgsoil) and post-incubation (F_Nitrate_ugNgsoil and F_Ammonia_ugNgsoil), as well as calculated nitrification (Nitrification_day) and nitrogen mineralization (Mineralization_day) rates per day, in three soil fractions: the organic horizon (O), the bulk soil (B), and the rhizosphere (R).

54 ENVIRONMENTAL SCIENCES↗