Biology subjects

Zhang, C.

Publications and source records attributed to Zhang, C..

At least 163 records · Page 9Linked to original sources

bioRxiv2025

Nuclear N-Glycosylation Redefines the Glycoscape and Directs Cell Identity

Glycans modify nucleic acids, proteins and lipids in fundamental biological processes, but have long been considered confined to the secretory pathway and cell surface. Here, we challenge this view with evidence that N-glycosylation, a ubiquitous glycan modification, occurs within cell nuclei across tissues and species, exhibiting cell type-dependent abundance and localization patterns. Using a multi-faceted approach, we show that numerous classical membrane and secreted glycoproteins localize to the nucleus with N-glycan modifications, exemplified by the cell adhesion molecule L1CAM. Mechanistically, we show that the N-glycosylated L1CAM is transported to the nucleus by KPNB1, where it transcriptionally modulates neuronal differentiation. Moreover, this nuclear N-glycosylation event contributes to neurodevelopmental disorders and is pharmacologically reversible. These findings revise the cellular geography of N-glycosylation and expand the known routes of protein trafficking, and highlight nuclear N-glycosylation pathways as potential therapeutic entry points for developmental disorders.

cell biology↗

bioRxiv2025

Mitochondrial membrane junction-mediated ATP channeling drives activity-dependent glucose metabolism

Neurons and brown adipocytes rely on rapid ATP production from accelerated glucose metabolism to sustain bursts of activity upon stimulation, a process known as activity-dependent glucose metabolism. The first committed step in this pathway, the hexokinase I (HK1)-catalyzed phosphorylation of glucose, consumes ATP, raising the question of how this reaction can be accelerated when cytosolic ATP becomes limiting during stimulation. We identify Cell Cycle Exit and Neuronal Differentiation protein 1 (CEND1), expressed in both cell types, as a critical regulator of this process. Loss of CEND1 impairs activity-dependent glucose utilization, ATP generation, and stimulation-evoked activity both in vitro and in vivo. Mechanistically, CEND1 assembles a complex with HK1, voltage-dependent anion channel 1 (VDAC1), and adenine nucleotide translocase 1 (ANT1) at hemifusion-like membrane junction between the outer/inner mitochondrial membrane, channeling mitochondrially derived ATP directly to HK1. These findings uncover a previously unrecognized mechanism that sustains activity-dependent glucose metabolism, with broad implications for energy homeostasis in specialized cell types.

physiology↗

bioRxiv2025

Primate-specific adaptation of Ku protects transcriptomic integrity by suppressing Alu-mediated alternative splicing

Accurate pre-mRNA splicing is essential for the transfer of genetic information but faces unique challenge in higher primates due to the massive expansion of intronic Alu elements1-3. While studying Ku, the Ku70/Ku80 heterodimer best known for initiating non-homologous end-joining (NHEJ) by encircling DNA ends4, we discovered that Ku expression increased markedly during primate evolution in parallel with Alu expansion5. Ku binds double-stranded RNA (dsRNA) stem-loops, including those at the antisense Alu (asAlu) elements within introns5. Here, we show that Ku-depletion in human cells has a broad impact on splicing largely independent of cell-cycle states, NHEJ, or innate immune signaling, significantly affecting ~8-10% of quantifiable alternative splicing events. Mechanistically, Ku directly binds exonic asAlu to prevent their aberrant inclusion and binds asAlu within inverted-repeat Alu (irAlu) pairs flanking canonical exons to prevent exon skipping6. Among human tissues, Ku expression in the brain is consistently ~50% lower, correlating with more permissive expression of Alu-derived splice variants, particularly those encoding mitochondrial proteins and RNA-binding factors. Correspondingly, heterozygous Ku loss in patient causes developmental delay, neurological dysfunction, and acidosis. Together our findings identified Ku as a critical suppressor of Alu-associated alternative splicing co-opted during evolution with implications for primate brain function and human disease.

molecular biology↗

bioRxiv2025

A Combinatorial mRNA Therapy for Treating Rheumatoid Arthritis and Osteoarthritis by Inhibiting Inflammation and Promoting Cartilage Regeneration

Rheumatoid arthritis (RA) and osteoarthritis (OA) are debilitating joint disorders with distinct etiologies but share common pathological features of chronic inflammation and progressive cartilage damage. Current therapeutic options are largely palliative and yet fail to achieve desired effect. In this study, we developed a combinatorial mRNA therapy using a rationally engineered lipid nanoparticle (LNP) containing a novel ionizable lipid for enhanced cartilage penetration upon intra-articular administration. This LNP co-delivers mRNAs encoding two complementary therapeutic proteins: interleukin-1 receptor antagonist (IL-1Ra) for inflammation attenuation and a C-terminal truncated derivative of angiopoietin-like 3 (ANL3) for cartilage regeneration. In pre-clinical models including collagen-induced arthritis mice, TNF-transgenic mice with spontaneous RA and destabilization of medial meniscus (DMM) surgical OA model, this "one-shot" combinatorial therapy significantly ameliorated disease severity, reduced synovitis and bone erosion, and potently promoted the regeneration of hyaline-like cartilage. Mechanistically, transcriptomic profiling of joint specimens revealed that the combinatorial therapy concurrently suppressed inflammatory pathways and extracellular matrix degradation pathways, meanwhile upregulating anabolic genes facilitating chondrogenesis. Collectively, our study establishes a versatile and synergistic mRNA therapy that offers a potent disease-modifying immunomodulatory strategy capable of addressing the long-standing unmet needs in arthritis treatment.

immunology↗

bioRxiv2025

Quantification and Localisation of New Brain Lipid Synthesis Using Deuterium Oxide and High Resolution Mass Spectrometry

Myelin is the lipid-rich membrane that surrounds neuronal axons and is essential for neurological function in vertebrates. The development of therapeutics that stimulate myelin repair to treat demyelinating disorders such as multiple sclerosis is hampered by the inability to distinguish newly-synthesised from pre-existing myelin. This study aimed to develop a method to quantify and localise new myelin lipid synthesis in vivo. Deuterium oxide was administered for two weeks in the drinking water of mice fed normal chow, chow containing the demyelinating toxin cuprizone, or during spontaneous remyelination following cuprizone withdrawal. Liquid chromatography-tandem mass spectrometry and mass spectrometry imaging were used to quantify and localise the newly synthesised, deuterated lipids. While most glycerophospholipids were constitutively deuterated, deuteration of myelin-enriched sulfatides, hexosylceramides, and phosphatidylethanolamine plasmalogens was only apparent during remyelination. Deuterated hexosylceramide and phosphatidylethanolamine plasmalogen species were localised primarily to the corpus callosum, the white matter tract that is most heavily affected by cuprizone. Most deuterium atoms were found in the fatty acyl chains, indicative of de novo lipid synthesis. These methods provide the means to quantify and spatially profile dynamic lipid synthesis across diverse biological contexts, including understanding myelin homeostasis and preclinical evaluation of remyelinating therapeutics.

biochemistry↗

bioRxiv2025

Analyses of Genome-Wide Recombination in Sulfolobus islandicus

Recombination is a fundamental process that both maintains genome integrity and dramatically shapes evolutionary dynamics in populations from all domains of life. Here we cross two genetically distinct but highly syntenic sympatric strains of Sulfolobus (Saccharolobus) islandicus and identify the pattern of recombination across the archaeal chromosome. We quantify recombination breakpoints occurring at neutral chromosomal markers through high-throughput short read Illumina sequencing of selected recombinant strains. We focus on three datasets that provide different, but corroborating information: i) individual colony sequences, ii) large colony pools of strains sorted by background genotype, and iii) a large colony pool of combined background genotypes. For each dataset we apply a newly designed and optimized computational tool called MARBL (Multiply Anchored Recombination Breakpoint Locator) for quantifying breakpoints and testing their statistical significance relative to sources of error introduced by sequencing technology. By combining the results of each dataset, we determine the short tract recombination preference in this organism and discuss its implications for mechanisms of recombination in archaea. We demonstrate uniform rates of recombination throughout the chromosome, which differ from previous studies of natural variation and discuss this in light of previous data suggesting speciation occurs through selection against inter-species hybrids. Importance StatementRecombination is a critical process in all domains of life that facilitates the essential repair of chromosomes and exchanges variation between organisms. Here we investigate genome-wide patterns of recombination in the model archaeaon Saccharolobus islandicus to better understand mechanistic aspects of their DNA exchange and recombination processes. Our observations of recombination in a controlled laboratory setting identified patterns of recombination that support the ability to incorporate short pieces of DNA in a piecemeal fashion from larger transferred segments. By comparing these observations to inferred recombination from natural populations, we can gain insight into evolutionary parameters including natural selection that would be otherwise difficult to determine from genomic evidence alone.

microbiology↗

bioRxiv2025

Higher levels of antibiotic resistance are less competitive: the hidden ecological cost of no-metabolic cost resistance

Antibiotic resistance is often assumed to be constrained by fitness costs that limit the spread of highly resistant strains. Yet, many resistance mechanisms - including enzymatic antibiotic degradation - can arise with little or no metabolic cost, raising an important question: why is extreme resistance not more widespread? Here, we show that community-level interactions impose a hidden ecological cost on high resistance. By performing experiments with simple communities comprised of antibiotic resistant clinical isolates and an antibiotic susceptible strain, we find that when exposed to betalactam antibiotics, strains with a higher degree of antibiotic resistance can promote the survival of cohabiting susceptible strains. Guided by mean-field modeling, we find that highly resistant bacteria accelerate detoxification of the shared environment, shortening the period during which resistance confers a competitive advantage. Experiments with an engineered strain with tunable resistance level confirm that susceptible cells grow best in the presence of highly resistant strains. Importantly, this effect does not require evolved cooperation or active enzyme secretion; experimental and modeling results show that unavoidable processes, such as cell death or passive leakage, prevent complete privatization of resistance, giving rise to "accidental co-operation". These findings suggest that resistance evolution is not only shaped by intrinsic cellular costs but also by ecological feedback that limits the benefits of incremental increases in resistance. This result may be reflected in the phenotypic responses of the clinical strains tested in this work, which fell into distinct low- and high-resistance classes with no intermediate phenotypes. Thus, this work demonstrates the important role of community dynamics in understanding the evolution of antibiotic resistance and treatment outcomes.

microbiology↗

bioRxiv2025

A neuron type-specific microexon in Ank3/ankyrin-G modulates calcium activity and neuronal excitability

Recent studies have revealed many alternative exons differentially spliced across diverse neuron types in the mammalian brain, but their links to neuronal physiology remain unclear. Here we characterize a deeply conserved microexon E35a in Ank3 encoding ankyrin-G (AnkG), a multifaceted adaptor protein best known as a master organizer of the axon initial segment (AIS) and as a leading genetic risk factor for bipolar disorder. E35a is predominantly skipped in cortical glutamatergic neurons but included in cortical GABAergic neurons and cerebellar neurons, which is dictated by multiple neuronal splicing factors. In E35a-deletion mice we generated, interneurons show increased excitability and somatic Ca2+ activity, without disruption in AIS. Biochemical analyses suggest that E35a inclusion facilitates AnkG interaction with a protein complex involving inositol trisphosphate receptors (InsP3Rs) important for intracellular Ca2+ signaling. Alternative splicing therefore allows AnkG to modulate neuron type-specific excitability in addition to its ubiquitous pan-neuronal role in organizing the AIS.

neuroscience↗

bioRxiv2025

CryoDDM: CryoEM denoising diffusion model for heterogeneous conformational reconstruction

Heterogeneous protein reconstruction can reveal the relationship between protein dynamics and function, which represents one of the foremost challenges in cryogenic electron microscopy (cryo-EM) single-particle analysis (SPA). However, high-intensity noise results in inaccurate parameter estimation during classification and reconstruction, making it difficult to capture the subtle motions of proteins. Here, we present the Cryo-EM denoising diffusion model (CryoDDM), which denoises images while preserving high-frequency structural information, benefiting conformational heterogeneity classification and reconstruction. The image denoised by CryoDDM can be used to improve downstream analysis, enhance reconstruction resolution, discover new protein conformations, and map out conformational variations. Validated against tree diverse experimental datasets, spanning a proteasome, a membrane protein and a spike protein, our results consistently demonstrate the accuracy and superiority of CryoDDM. By guiding reconstruction with denoised images, CryoDDM enables high-resolution reconstruction of heterogeneous conformations, paving the way to illuminate fundamental questions in structural biology. The CryoDDM project are published at https://github.com/BIT-FuweiLi/CryoDDM.

bioinformatics↗

bioRxiv2025

Structural Basis of Asymmetric Class C GPCR activation and distinct Gq coupling induced by force stimulation

Mechanosensation is essential for diverse physiological processes. While many G protein-coupled receptors (GPCRs) are known to be mechanosensitive, the underlying mechanism remains largely unknown. Here, we reveal that mechanical force induces Gq activation of mGlu2, a balance modulator residing in kinocilia, but not other 7 mGlu members. Force activates mGlu2 through a conserved N-terminal force transduction motif (FTM) via a unique cis mechanism. We engineered a potent FTM-derived peptide agonist that recapitulates force-induced activation and resolved cryo-EM structures of apo-mGlu2, FTM-mGlu2, LFTM-{Psi}EK-mGlu2 and LFTM-{Psi}EK-mGlu2-Gq. The structures reveal that an atypical FTM binding to a previously uncharacterized pocket induces asymmetric 7TM domain rearrangement, enabling Gq coupling via an ICL1-TM3/6/7 interface, fundamentally distinct from the glutamate-induced Gi coupling mode of mGlu2. Compared with Gi-coupled mGlu2, the 5 helix of Gq rotated by 180{degrees}, penetrating deeper (8 [A]) into a hydrophobic pocket. Disruption of the M7947.32-F7806.57-F7766.53 hydrophobic triad core and a conformational propagation path predominantly comprising TM6-7 residues are identified as key elements mediating force induced mGlu2 activation. Further In vivo rescue experiments support that mGlu2s mechanosensitivity is dependent on FTM and is required for vestibular function. This work establishes a paradigm for class C GPCR mechanotransduction, revealing unprecedented structural mechanisms underlying force-induced Gq coupling and offering a chemical toolset to modulate mechanical signaling of GPCR.

cell biology↗

bioRxiv2025

The fitness costs of reproductive specialization scale inversely with organismal size

The evolution of reproductive specialization represents a fundamental innovation in multicellular life, yet the conditions favoring its evolution remain poorly understood. Here, we develop a population genetic framework that examines the fitness cost of reproductive specialization as a function of organism size. We show analytically that the costs of specialization decrease dramatically with organism size. For example, while a 4-cell organism with 50% somatic cells experiences a 50% reduction in population-level exponential growth rate (the same as the two-fold cost of sex), a hundred-cell organism faces only a 15% reduction, a thousand-cell organism 10%, a million-cell organism 5%, and a billion-cell organism merely 3.3%. This scaling relationship arises from the fact that proportionally more cellular growth in larger organisms is required for development, reducing the rate at which the fitness costs of specialization are compounded over multicellular generations. We contextualize our mathematical model with data from the volvocine green algae, showing that simple theoretical predictions closely match empirical measurements. While cellular differentiation demands that somatic advantages compensate for lost reproductive potential, we demonstrate that these compensatory requirements diminish with the logarithm of organism size, fundamentally altering the cost-benefit landscape for large organisms and potentially driving the evolution of a size-differentiation ratchet. This size-scaling relationship helps explain the broad association between large organismal size and multicellular complexity.

evolutionary biology↗

bioRxiv2025

Elucidation and engineering of arabinofuranosyltransferase to enable total de novo biosynthesis of paris saponins in yeast

Paris saponins (PSs) are structurally complex steroidal saponins that, due to their diverse glycosylation patterns, exhibit a range of significant pharmacological activities, including anti-tumor and antibacterial effects. However, incomplete characterization of the key enzymes responsible for glycosylation modifications has hindered their efficient heterologous biosynthesis. In this study, we reprogrammed the sugar donor specificity of a steroidal rhamnosyltransferase (UGT93M3) to enable the transfer of arabinofuranose (Araf). Through structural analysis, we identified key amino acid residues (368H/Q) that play an important role in determining Araf donor specificity. Guided by this insight, we successfully reconstructed the paris saponin I (PSI) biosynthetic pathway in Saccharomyces cerevisiae using engineered enzymes. To address challenges related to donor availability, we introduced UDP-sugar biosynthetic modules (UDP-Rha and UDP-Araf) into yeast. With this integrated platform, we were able to de novo produce a range of paris saponins, including diosgenin-3-O-glucosyl-(1[->]6)-glucoside (DGG), diosgenin-3-O-rhamnosyl(1[->]2) [glucosyl(1[->]6)]glucoside (DRGG) and paris saponin II. This work establishes a novel microbial platform for the sustainable production of paris saponins, particularly PSI, advancing the biosynthesis of steroidal glycosides and providing a potential strategy for the industrial-scale production of bioactive saponins. TeaserIn this study, we report the first complete de novo biosynthesis of four bioactive PSs, PSI, PSII, DGG and DRGG, in Saccharomyces cerevisiae from simple carbon source. Key advances include: (i) A single amino-acid switch (N368H) in the rhamnosyltransferase UGT93M3 endowed high-efficiency transfer of the rare five-membered arabinofuranose, solving a bottleneck in PS I biosynthesis; (ii) Elucidation of the molecular basis for sugar-donor specificity through AlphaFold3 docking and 300-ns molecular-dynamics simulations, revealing a histidine "latch" that stabilizes UDP-Araf in the catalytic pose; (iii) Construction of a 16-gene yeast chassis that integrates plant P450s, optimized glycosyltransferases, and de novo modules for UDP-rhamnose and UDP-arabinofuranose supply, achieving de novo microbial production of PS II, DGG, DRGG and PS I from glucose alone.

synthetic biology↗

bioRxiv2025

Trichome formation in Nicotiana benthamiana is induced by Agrobacterium

Plant trichomes are specialised epidermal appendages found on various organs of many plant species. They play important roles in plant protection against pests, pathogens, herbivores, resistance to abiotic stress, light perception, mechanosensitivity etc. They are of direct agricultural importance, providing fibre (e.g. cotton) and producing secondary metabolites essential for agriculture, medicine, and industry. Trichomes can be relatively sparsely distributed, making their biological investigation and commercial exploitation more challenging. Here we show that infiltration of Nicotiana benthamiana leaf tissue with certain nopaline-type Agrobacterium tumefacians strains induces extremely dense, localised formation of glandular trichomes 15 days post infiltration (dpi). This Agrobacterium-strain specific effect was shown to be due to the presence of a trans-zeatin synthase (tzs) gene located on some Ti plasmids, which results in the bacterial production of the cytokinin trans-zeatin. This simple procedure enables dramatically increased trichome density to be compared biologically with adjacent isogenic plant tissues on the same leaf.

plant biology↗

bioRxiv2025

Dynamic alterations of DNA methylation and transcriptome in adaptation to and recovery from the space environment

Space exploration presents tremendous health challenges. Here, we report time series of multi-omic and phenotypic profiles of seventeen astronauts from six China Manned Space missions with continuous spaceflight durations ranging from 13 to 180 days. We revealed a key role of DNA methylation regulation in reshaping gene expression patterns to adapt to the space environment. Long-duration spaceflight showed more alterations in epigenetic modifications correlated with alternative splicing and protein acetylation. During recovery, an "overrange rebound" phenomenon was observed, furthermore, a mathematical model was established to describe this implying important phenomenon. Moreover, we revealed the correlations between molecular alterations and phenotypic changes such as coagulation activation and bone intensity loss. Additionally, we performed ground-based simulation experiments to estimate the impacts of individual stressors in the space environment on DNA methylation. In summary, our study highlights the importance and complexity of epigenetic regulation in adaptation to and recovery from the space environment.

genomics↗

bioRxiv2025

Benchmarking TCR-pMHC structure prediction: a unified evaluation and CDR3-based functional insights

Interactions between T cell receptors (TCRs) and peptide-major histocompatibility complexes (pMHCs) are central to adaptive immunity. Recent advances in structure prediction tools have enabled atomic-level modeling of TCR-pMHC interactions. However, the lack of systematic evaluation forces practitioners to invest substantial resources in selecting appropriate tools. Here, we present a comprehensive benchmark of TCR-pMHC structure prediction with 70 previously unseen complexes and ten models spanning MSA-based, PLM-based, and docking-based approaches, revealing the superior modeling accuracy and docking quality of MSA-based methods, especially AlphaFold3. To further enhance the utility of AlphaFold3 predictions, we identify the pLDDT score of the TCR CDR3 region as an informative indicator of both structural correctness and functional relevance. Specifically, it enables up to 4.3% Top-1 success gain through reranking and captures mutation-induced affinity changes in over 80% of cases. Overall, our analysis would facilitate the practical usage of immune structure prediction models and guide the advancement of these models.

bioinformatics↗

bioRxiv2025

Engineering an Anaerobic Microenvironment to Empower Hydrogenase-Catalyzed Hydrogen Therapy for Diabetic Wound Healing

The inherent oxygen sensitivity of hydrogenases has long restricted their applications to anaerobic systems. Here, we present a hybrid peptide-nanocluster hydrogel that establishes a self-sustaining anaerobic microenvironment to enable hydrogenase-catalyzed hydrogen therapy under aerobic conditions. This system integrates a custom-designed Fmoc-KYF peptide hydrogel with integrated silver nanoclusters (AgNCs), which host an engineered [NiFe]-hydrogenase (3S1C-Hyd-2). Mechanistic studies combining molecular dynamics simulations and spectroscopic analysis revealed that hydrophobic pockets within the peptide network trap oxygen molecules, while photoexcited AgNCs rapidly deplete invasive O2. Our design ensures stable hydrogen evolution, effectively overcoming the oxygen sensitivity that has long restricted hydrogenase applications in biomedical contexts. In vitro, the in-situ-generated hydrogen effectively scavenges reactive oxygen species, suppresses pro-inflammatory cytokine production. In a diabetic mouse model, this light-activated system markedly accelerates wound closure (87% by day 11), reduces oxidative stress, promotes macrophage polarization toward a reparative phenotype, enhances angiogenesis, and upregulates tissue repair pathways, as confirmed by transcriptomic profiling. Our work establishes a new bioengineering paradigm for constructing anaerobic microenvironments through synergistic material-enzyme-light interactions, offering a versatile platform for hydrogenase-enabled therapeutic interventions in oxidative stress-related diseases.

biochemistry↗

bioRxiv2025

MAAD: Multidimensional Antiviral Antibody Database

Antibodies have emerged as central components of therapeutic strategies against viral infectious diseases, functioning as key effectors in both prevention and treatment. While traditional antibody discovery has relied heavily on high-throughput screening, the field is now shifting toward rational antibody design, which requires integrative insights into sequence-structure-function relationships. However, the absence of a standardized and well-annotated antibody database integrating these multidimensional features hampers systematic exploration, cross-pathogen comparison, and rational antibody design. Here, we introduce a "Multidimensional Antiviral Antibody Database" (MAAD; http://www.xxx), a curated platform dedicated to antibody nanobody and single-chain variable fragment targeting three high-impact RNA virus families, Coronaviridae (SARS-CoV-1, SARS-CoV-2, MERS-CoV), Orthomyxoviridae (influenza virus), and Pneumoviridae (respiratory syncytial virus, human metapneumovirus), due to the large, high-quality datasets accumulated in recent years. MAAD further incorporates a suite of interactive analysis modules, including CDR annotation, similarity-based CDR3 sequence analysis, V/J gene usage profiling, sequence-based clustering and structure-based antigen-antibody interfaces residues with per-site entropy and mutation rate profiling. These features enable in-depth exploration of antibody sequence characteristics, thereby facilitating functional and structural insights for rational antibody design. Together, by bridging antibody sequence, structure and function, MAAD offers an open and standardized platform that advances comparative antiviral research and supports therapeutic antibody discovery.

bioinformatics↗

bioRxiv2025

Bark-associated diazotroph communities are a cryptic source of nitrogen in forests

Nitrogen is an essential nutrient limiting forest productivity. Plants cannot access atmospheric N2 directly and rely on diazotrophic bacteria to fix nitrogen into bioavailable forms such as ammonium. Within forests, biological nitrogen fixation (BNF) occurs primarily in soils and root nodules. However it is unclear whether the extensive microbial communities recently discovered in tree bark can also fix nitrogen. Here we combine field measurements, metagenomic profiling, and biogeochemical assays to show that bark-dwelling diazotroph communities are abundant and active across diverse tree species. Bark from eight Australian tree species showed exceptionally high C:N ratios and depleted nitrogen stable isotope signatures ({delta}15N) consistent with locally fixed nitrogen, suggesting strong selection for diazotrophs. Consistently, bark microbial communities harbour phylogenetically and physiologically diverse diazotrophs, averaging [~]1012 cells m-2 and in higher relative abundance than underlying soils. Canonical and alternative nitrogenases were detected across eight bacterial phyla, primarily bark-adapted Alphaproteobacteria, Acidobacteriota, and Verrucomicrobiota, with strong signatures of purifying selection. Stable isotope labelling experiments demonstrated that bark-dwelling diazotrophs fix nitrogen at rates varying with tree species and habitat. In line with the presence of methane-oxidising diazotrophs, BNF was strongly stimulated by methane addition and suppressed by methanotroph inhibitors. Initial upscaling suggests bark-associated nitrogen fixation contributes up to 3.8 Tg N yr-1 globally ([~]6% of natural terrestrial BNF), with further studies required to constrain this budget and its contribution to tree nitrogen demands. Altogether, this discovery of substantial above-ground nitrogen inputs revises our understanding of forest nutrient cycles and redefines the functional scope of the caulosphere.

microbiology↗