Subtropical agroforestry systems (AFSs) are increasingly challenged by combined abiotic stresses (e.g., drought + high temperature), which severely threaten ecosystem stability and productivity. The coevolution of rhizosphere microbiome and plant stress-resistant metabolism is hypothesized to be a key adaptive mechanism, but the underlying processes and driving factors remain unclear. Here, we conducted a long-term field survey (5 years) and controlled simulation experiments to explore the coevolutionary patterns of rhizosphere microbiome and plant stress-resistant metabolism in four typical subtropical AFSs (bamboo-tea intercropping, camphor-corn intercropping, mango-grass intercropping, and natural secondary forest-agricultural crop mixed system) under drought-high temperature (D-HT) combined stress. Integrating metabolomics, metagenomics, and transcriptomics, we characterized plant stress-resistant metabolite profiles, rhizosphere microbial community structure, and functional gene co-expression networks. Results showed that the natural secondary forest-agricultural crop mixed system (high plant diversity) exhibited the strongest adaptive capacity to D-HT stress, with the highest plant survival rate (82.3%) and yield stability (yield reduction rate: 12.7%). This was accompanied by distinct coevolutionary patterns: (1) Plant stress-resistant metabolites (e.g., abscisic acid, jasmonic acid, phenols) and rhizosphere beneficial microbes (e.g., Arthrobacter, Bacillus, mycorrhizal fungi) formed stable co-occurrence networks, with 32.5% of metabolites significantly correlating with microbial functional genes; (2) Transcriptomic analysis revealed that plants in high-diversity AFSs upregulated genes involved in metabolite synthesis (e.g., PAL, LOX, NCED) and microbial recruitment (e.g., LYK3, NFP), while rhizosphere microbes upregulated genes encoding stress-responsive enzymes (e.g., superoxide dismutase, peroxidase) and nutrient transporters (e.g., NRT2, PHT1); (3) Coevolutionary selection pressure drove the enrichment of microbial functional traits related to metabolite degradation and stress adaptation, and plant metabolic traits related to microbial recruitment. Moreover, the coevolutionary degree was positively correlated with AFS adaptability (r = 0.87, p < 0.01). Our findings demonstrate that the coevolution of rhizosphere microbiome and plant stress-resistant metabolism enhances the adaptability of subtropical AFSs to combined abiotic stresses, and plant diversity is a key driver of this coevolution. This provides a new theoretical basis for constructing stress-resistant AFSs by regulating plant-microbe coevolutionary relationships.

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Nitrogen (N) deposition has become a key global change factor affecting soil carbon (C) sink function in subtropical agroforestry systems (AFSs). Soil microbes are the core drivers of soil C cycling, and their community interactions (microbial networks) play critical roles in maintaining microbial functional stability and regulating C transformation processes. However, how N deposition affects soil microbial network complexity, keystone taxa, and their linkages with soil C sink function in subtropical AFSs remains unclear. Here, we conducted a 3-year N deposition manipulation experiment (0, 50, 100, 150 kg N ha⁻¹ yr⁻¹) in three typical subtropical AFSs (tea-oil camellia + clover, chestnut + wheat, and bamboo + peanut). High-throughput sequencing, co-occurrence network analysis, and soil C pool determination were used to explore the responses of bacterial and fungal networks to N deposition and their regulatory effects on soil C sink function. Results showed that: (1) N deposition significantly altered soil microbial network structure: low N deposition (50 kg N ha⁻¹ yr⁻¹) increased network complexity (nodes, edges, average degree) by 12.3%-18.7%, while high N deposition (100, 150 kg N ha⁻¹ yr⁻¹) decreased network complexity by 15.6%-24.5%, with the strongest inhibition in bamboo + peanut AFS; (2) Keystone taxa shifted under N deposition: low N favored copiotrophic taxa (e.g., Proteobacteria, Ascomycota), while high N promoted oligotrophic taxa (e.g., Acidobacteria, Basidiomycota); (3) Soil total organic C (SOC), microbial biomass C (MBC), and recalcitrant C contents first increased and then decreased with increasing N deposition, peaking at 50 kg N ha⁻¹ yr⁻¹ (increased by 8.9%-12.6% compared to control); (4) Microbial network complexity was significantly positively correlated with SOC and recalcitrant C contents (r = 0.76-0.83, p < 0.01), and keystone taxa abundance explained 42.3%-56.8% of the variation in soil C pool components; (5) Tea-oil camellia + clover AFS with high plant diversity had higher microbial network stability and C sequestration capacity under N deposition, which alleviated the negative effects of high N deposition. Our findings indicate that low N deposition enhances soil C sink function by increasing microbial network complexity and optimizing keystone taxa composition, while high N deposition weakens C sink function by simplifying microbial networks. Plant diversity mediates the response of microbial networks to N deposition, thereby regulating soil C sink function. This study provides a new perspective of microbial interactions for understanding the effects of N deposition on soil C cycling in subtropical AFSs and optimizing management practices to enhance C sequestration under global change.

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Microbial functional plasticity, the ability of microbial communities to adjust metabolic activities in response to environmental changes, is critical for maintaining ecosystem stability of subtropical agroforestry systems (AFSs) under increasing environmental disturbances. However, the role of rhizosphere metabolites in regulating microbial functional plasticity remains unclear. Here, we conducted a field experiment combined with controlled pot trials to investigate the effects of drought disturbance on rhizosphere metabolite profiles and microbial functional plasticity in three subtropical AFSs (alley cropping, silvopasture, forest garden). Multi-omics approaches (metabolomics, metagenomics, metatranscriptomics) were used to characterize rhizosphere metabolite dynamics, microbial taxonomic composition, and functional gene expression. Results showed that forest garden exhibited the highest rhizosphere metabolite diversity (286 metabolites) and strongest microbial functional plasticity (functional redundancy index = 0.82) under drought, compared to silvopasture (243 metabolites, redundancy index = 0.71) and alley cropping (198 metabolites, redundancy index = 0.58). Key rhizosphere metabolites (e.g., flavonoids, polyamines, amino acids) significantly correlated with the expression of microbial functional genes involved in drought resistance (e.g., osmolyte synthesis, antioxidant enzymes) and nutrient cycling (e.g., nitrogen fixation, phosphorus solubilization). Specifically, flavonoids in forest garden rhizosphere induced the upregulation of microbial genes encoding trehalose synthase (RPKM = 102.3) and catalase (RPKM = 89.7), enhancing microbial drought tolerance. Metabolic network analysis revealed that rhizosphere metabolites formed functional modules that mediated microbial functional shifts, with the flavonoid-amino acid module being the core regulator of microbial plasticity in forest garden. Our findings demonstrate that rhizosphere metabolites drive microbial functional plasticity in subtropical AFSs under drought disturbance, highlighting the importance of plant diversity in shaping rhizosphere metabolite profiles to enhance AFS resilience. This provides a theoretical basis for optimizing AFS design by selecting plant species with drought-adaptive rhizosphere metabolite traits.

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Litter decomposition is a core ecological process regulating soil carbon (C) cycling in subtropical agroforestry systems (AFSs), and its response to climate change (e.g., warming and altered precipitation) directly affects soil C pool stability and ecosystem C sequestration capacity. Soil microbes (bacteria and fungi) are the primary drivers of litter decomposition, but the specific mechanisms by which microbial communities, functional genes, and enzyme activities regulate litter decomposition and soil C pool stability under climate change remain unclear. Here, we conducted a 4-year field manipulation experiment (warming + reduced precipitation) in three typical subtropical AFSs (poplar-soybean intercropping, citrus-grass intercropping, and Chinese fir-peanut intercropping) to explore the microbial drivers of litter decomposition and soil C pool dynamics. We combined litterbag decomposition experiments, high-throughput sequencing, quantitative real-time PCR (qPCR), and extracellular enzyme activity (EEA) assays to characterize litter decomposition rates, soil C pool components (labile C: DOC, MBC; stable C: SOC, recalcitrant C), microbial community structure, functional gene abundance, and EEA. Results showed that: (1) Warming + reduced precipitation significantly decreased litter decomposition rates by 18.2%-25.7% across all AFSs, with the strongest inhibition in poplar-soybean intercropping (low litter quality) and the weakest in Chinese fir-peanut intercropping (high litter diversity); (2) The reduction in decomposition was closely associated with changes in microbial community composition: fungal/bacterial ratio decreased by 32.1%-45.3%, and the relative abundance of lignin-degrading fungi (e.g., Trichoderma, Phanerochaete) and cellulose-degrading bacteria (e.g., Cellulomonas, Bacillus) significantly declined; (3) Functional gene abundance (ligninase gene: lcc; cellulase gene: cel48) and EEA (lignin peroxidase, cellulase, β-glucosidase) were significantly reduced under climate change, and these indices were positively correlated with litter decomposition rates (r = 0.78-0.86, p < 0.01); (4) Soil labile C pool (DOC, MBC) decreased by 15.6%-22.3%, while stable C pool (SOC, recalcitrant C) increased by 8.9%-13.2% under climate change, and the stability of soil C pool (recalcitrant C/SOC ratio) was negatively correlated with microbial functional potential (sum of lcc and cel48 gene abundance, r = -0.83, p < 0.01); (5) Chinese fir-peanut intercropping with high litter diversity had higher microbial diversity, functional gene abundance, and EEA, which alleviated the negative impact of climate change on litter decomposition and soil C pool stability. Our findings demonstrate that climate change inhibits litter decomposition by altering microbial community structure and reducing microbial functional potential, thereby increasing soil C pool stability in subtropical AFSs, and litter diversity is a key regulating factor. This study provides a new microbial perspective for predicting soil C cycling responses to climate change and optimizing AFS management to enhance C sequestration capacity.

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Subtropical agroforestry systems (AFSs) harbor complex plant-microbe interactions that drive metabolic flux networks and support ecosystem stability. However, the metabolic regulatory mechanisms underlying these interactions remain poorly elucidated. Here, we integrated metabolomics, transcriptomics, metagenomics, and metatranscriptomics to characterize plant-microbe metabolic interactions in three typical subtropical AFSs (alley cropping, silvopasture, forest garden). Root exudate metabolomes, plant transcriptomes, rhizosphere microbial communities, and microbial metabolic pathways were systematically analyzed. Results showed that forest garden exhibited the most diverse root exudate profile (128 unique metabolites) and highest microbial alpha diversity (Shannon index = 7.82). Key metabolites (e.g., flavonoids, organic acids) in root exudates shaped rhizosphere microbial communities by selecting for specific taxa (e.g., Pseudomonas, Arbuscular Mycorrhizal Fungi). Transcriptomic analysis revealed that plant genes involved in flavonoid biosynthesis (e.g., CHS, FLS) were significantly upregulated in forest garden, which correlated with increased microbial genes encoding flavonoid degradation enzymes. Metabolic network reconstruction identified 18 core metabolic modules mediating plant-microbe interactions, including nitrogen cycling, carbon sequestration, and secondary metabolite synthesis. These modules were more interconnected in forest garden, indicating a more stable metabolic network. Our findings provide a comprehensive understanding of metabolic regulatory mechanisms in AFS plant-microbe interactions, offering novel insights for optimizing AFS design via metabolic engineering.

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