Discovery of Nine Dipeptidyl Peptidase-4 Inhibitors from Coptis chinensis Using Virtual Screening, Bioactivity Evaluation, and Binding Studies.

The objective of this study was to identify multiple alkaloids in Coptis chinensis that demonstrate inhibitory activity against DPP-4 and systematically evaluate their activity and binding characteristics. A combined strategy that included molecular docking, a DPP-4 inhibition assay, surface plasmon resonance (SPR), and a molecular dynamics simulation technique was employed. The results showed that nine alkaloids in Coptis chinensis directly inhibited DPP-4, with IC50 values of 3.44-53.73 µM. SPR-based binding studies revealed that these alkaloids display rapid binding and dissociation characteristics when interacting with DPP-4, with KD values ranging from 8.11 to 29.97 µM. A molecular dynamics analysis revealed that equilibrium was rapidly reached by nine DPP-4-ligand systems with minimal fluctuations, while binding free energy calculations showed that the ∆Gbind values for the nine test compounds ranged from -31.84 to -16.06 kcal/mol. The most important forces for the binding of these alkaloids with DPP-4 are electrostatic interactions and van der Waals forces. Various important amino acid residues, such as Arg125, His126, Phe357, Arg358, and Tyr547, were involved in the inhibition of DPP-4 by the compounds, revealing a mechanistic basis for the further optimization of these alkaloids as DPP-4 inhibitors. This study confirmed nine alkaloids as direct inhibitors of DPP-4 and characterized their binding features, thereby providing a basis for further research and development on novel DPP-4 inhibitors.

Comparative transcriptome analysis of different tissues of Solanum khasianum reveals candidate genes involved in steroidal glycoalkaloid biosynthesis.

Fruits and leaves of Solanum khasianum C. B. Clarke have long been used as a common Chinese herbal medicine. Steroidal glycoalkaloids (SGAs), the main active ingredient in S. khasianum, exhibit various pharmacological effects. However, genes involved in the SGA biosynthetic pathway in S. khasianum have not yet been identified. Genes encoding potential key SGA biosynthesis enzymes were identified through comprehensive RNA sequencing analysis (RNA-seq) of S. khasianum leaves, stems, and fruits. A total of 123,704 unigenes were obtained, of which 109,775 (88.74%) were annotated in seven public databases. Among these, 54 unigenes potentially involved in SGA biosynthesis were identified. Additionally, 23,636 differentially expressed genes were identified by comparing gene expression levels among the fruits, stems, and leaves of S. khasianum. The structural characteristics and phylogenetic relationship of cycloartenol synthase involved in SGA biosynthesis were further analyzed. Solasodine constituent was detected by high-performance liquid chromatography. This is the first study to report the comparative transcriptome analysis of different tissues of S. khasianum that identifies valuable genes potentially involved in SGA biosynthesis in this species.

Transcriptome analyses reveal the expression profile of genes related to lignan biosynthesis in Anthriscus sylvestris L. Hoffm. Gen.

Anthriscus sylvestris L. Hoffm. Gen (A. sylvestris) is a perennial herb widely used for antitussive and diuretic purposes in traditional Korean and Chinese medicine. Lignans are critical secondary metabolites with widely pharmacological activities in A. sylvestris. Using transcriptome data of A. sylvestris, we identified genes related to lignan biosynthesis. In all, 123,852 unigenes were obtained from the flowers, leaves, roots, and stems of A. sylvestris with the Illumina HiSeq 4000 platform. The average length of unigenes was 1,123 bp and 91,217 (73.65%) of them were annotated in public databases. Differentially expressed genes and root-specific genes were analyzed between roots and the other three tissue types by comparing gene expression profiles. Specifically, the key enzyme genes involved in lignan biosynthesis were identified and analyzed. The expression levels of some of these genes were highest in the roots, consistent with the accumulation of deoxypodophyllotoxin. These expression levels were experimentally verified via quantitative real-time polymerase chain reaction (qRT-PCR). This research provides valuable information on the transcriptome data of A. sylvestris and the identification of candidate genes associated with the biosynthesis of lignans, laying the foundation for further research on genomics in A. sylvestris and related species. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-022-01156-w.

[Identification of key enzyme genes involved in biosynthesis pathways of lignan and lignin in Eucommia ulmoides based on transcriptome assembly].

Lignan is the main medicinal component of Eucommia ulmoides, and lignin is involved in the defense of plants against diseases and insect pests.They are synthesized from coniferyl alcohol with the help of dirigent(DIR) and peroxidase(POD), respectively.In this study, transcriptome assembly of stems and leaves of E.ulmoides was performed, yielding 112 578 unigenes.Among them, 70 459 were annotated in seven databases.A total of 59 unigenes encodes 11 key enzymes in the biosynthesis pathways of lignin and lignin, of which 11 encode POD and 8 encode DIR.A total of 13 unigenes encoding transcription factors are involved in phenylpropanoid metabolism. Compared with leaves of E.ulmoides, 7 575 unigenes were more highly expressed in stems, of which 462 were involved in phenylpropanoid biosynthesis.Our results extend the public transcriptome dataset of E.ulmoides, which provide valuable information for the analysis of biosynthesis pathways of lignan and lignin in E.ulmoides and lay a foundation for further study on the functions and regulation mechanism of key enzymes in lignan and lignin biosynthesis pathways.

Eutrophication likely prompts metal bioaccumulation in edible clams.

Coastal eutrophication is an indisputable reality and becoming a worldwide concern. However, whether and how eutrophication affects metal bioaccumulation in marine bivalves have not yet been elucidated. Here, we present the potential influence of coastal eutrophication on metal bioaccumulation in the Manila clam Ruditapes philippinarum. The degree of coastal eutrophication was examined monthly over a 1-year period at three sampling sites. The bioconcentration factor (BCF), biosediment accumulation factor (BSAF) and metal pollution index (MPI) were applied to evaluate the efficiency of metal bioaccumulation in R. philippinarum. BCF and BSAF indicated that eutrophication did not significantly affect the bioaccumulation of Cr, Cu, Zn, Cd, Pb, Hg, and As in R. philippinarum. However, up to 56% of MPI variation can be related to the level of eutrophication. Therefore, further research should address the synergistic effects of eutrophication and metal pollution on coastal ecosystems.

Protective effect of coumarin-pi against t-BHP-induced hepatotoxicity by upregulating antioxidant enzymes via enhanced Nrf2 signaling.

Coumarin-pi, a new coumarin derivative isolated from the mushroom Paxillus involutus, has antioxidative activity, but the underlying mechanism against intracellular oxidative stress is still unclear. This study investigated its cytoprotective effects and the antioxidative mechanism in tert-butyl hydroperoxide (t-BHP)-induced HepG2 cells. The results demonstrated that coumarin-pi suppressed t-BHP-stimulated cytotoxicity, cell apoptosis, and generation of reactive oxygen species (ROS). Additionally, coumarin-pi promoted nuclear factor erythroid 2-related factor 2 (Nrf2) expression and upregulated the protein expression of antioxidantenzymes, including heme oxygenase-1 (HO-1), NAD(P)H: quinone oxidase (NQO1), glutamyl cysteine ligase catalytic subunit (GCLC), and glutamate-cysteine ligase regulatory subunit (GCLM). After coumarin-pi treatment, transcriptome sequencing and bioinformatic analysis revealed that 256 genes were differentially expressed; interestingly, only 20 genes were downregulated, and the rest of the genes were upregulated. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional annotation were used to identify changes in metabolic pathways. Collectively, the results presented in this study indicate that coumarin-pi has a protective effect against t-BHP-induced cellular damage and oxidative stress.

[Identification of key enzyme genes involved in biosynthesis of steroidal saponins and analysis of biosynthesis pathway in Polygonatum cyrtonema].

Steroidal saponins, which are the characteristic and main active constituents of Polygonatum, exhibit a broad range of pharmacological functions, such as regulating blood sugar, preventing cardiovascular and cerebrovascular diseases and anti-tumor. In this study, we performed RNA sequencing(RNA-Seq) analysis for the flowers, leaves, roots, and rhizomes of Polygonatum cyrtonema using the BGISEQ-500 platform to understand the biosynthesis pathway of steroidal saponins and study their key enzyme genes. The assembly of transcripts for four tissues generated 129 989 unigenes, of which 88 958 were mapped to several public databases for functional annotation, 22 813 unigenes were assigned to 53 subcategories and 64 877 unigenes were annotated to 136 pathways in KEGG database. Furthermore, 502 unigenes involved in the biosynthesis pathway of steroidal saponins were identified, of which 97 unigenes encoding 12 key enzymes. Cycloartenol synthase, the first key enzyme in the pathway of phytosterol biosynthesis, showed conserved catalytic domain and substrate binding domain based on sequence analysis and homology modeling. Differentially expressed genes(DEGs) were identified in rhizomes as compared to other tissues(flowers, leaves or roots).The 2 437 unigenes annotated by KEGG showed rhizome-specific expression, of which 35 unigenes involved in the biosynthesis of steroidal saponins. Our results greatly extend the public transcriptome dataset of Polygonatum and provide valuable information for the identification of candidate genes involved in the biosynthesis of steroidal saponins and other important secondary metabolites.

A mutant of hydrophobin HGFI tuning the self-assembly behaviour and biosurfactant activity.

Hydrophobins are a series of low molecular weight proteins produced by filamentous fungi that play an important role in fungal growth. They have a globular structure and possess a unique hydrophobic patch on their surface that makes them amphiphilic, making them among the most surface-active proteins. Herein, the surface charge properties of HGFI, a class I hydrophobin from Grifola frondosa, were altered by replacing the negatively charged Glu24 with a positively charged Lys to generate the ME24 mutant. Pichia pastoris GS115 was used for recombinant expression of the ME24 mutant, which was purified by a two-step procedure. The function of the mutated residue in HGFI self-assembly was investigated. Reverse-phase high-performance liquid chromatography analysis revealed that the polarity of ME24 was enhanced compared with HGFI. Circular dichroism, thioflavin T assay, water contact angle and atomic force microscopy indicated that Glu24 participates in rodlet formation. Water solubility detection and dynamic light scattering showed that Glu24 affects the assembled state of HGFI in aqueous solution. The behaviour of the mutant in an emulsion, in the dispersion of insoluble materials and in large-scaled protein production suggests the functions of hydrophobins can be tuned for new applications.

High-yield fermentation and a novel heat-precipitation purification method for hydrophobin HGFI from Grifola frondosa in Pichia pastoris.

Hydrophobins are proteins produced by filamentous fungi with high natural-surfactant activities and that can self-assemble in interfaces of air-water or solid-water to form amphiphilic membranes. Here, we reported a high-yield fermentation method for hydrophobin HGFI from Grifola frondosa in Pichia pastoris, attaining production of 300 mg/L by keeping the dissolved oxygen level at 15%-25% by turning the methanol-feeding speed. We also developed a novel HGFI-purification method enabling large-scare purification of HGFI, with >90% recovery. Additionally, we observed that hydrophobin HGFI in fermentation broth precipitated at pH < 7.0 and temperatures >90 °C. We also identified the structure and properties of proteins purified by this method through atomic force microscopy, circular dichroism, X-ray photoelectron spectroscopy, and water-contact angle measurement, which is similar to protein purification by ultrafiltration without heating treatment that enables our method to maintain native HGFI structure and properties. Furthermore, the purification method presented here can be applied to large-scale purification of other type I hydrophobins.

Seawater acidification affects the physiological energetics and spawning capacity of the Manila clam Ruditapes philippinarum during gonadal maturation.

Ocean acidification is predicted to have widespread implications for marine bivalve mollusks. While our understanding of its impact on their physiological and behavioral responses is increasing, little is known about their reproductive responses under future scenarios of anthropogenic climate change. In this study, we examined the physiological energetics of the Manila clam Ruditapes philippinarum exposed to CO2-induced seawater acidification during gonadal maturation. Three recirculating systems filled with 600 L of seawater were manipulated to three pH levels (8.0, 7.7, and 7.4) corresponding to control and projected pH levels for 2100 and 2300. In each system, temperature was gradually increased ca. 0.3°C per day from 10 to 20°C for 30days and maintained at 20°C for the following 40days. Irrespective of seawater pH levels, clearance rate (CR), respiration rate (RR), ammonia excretion rate (ER), and scope for growth (SFG) increased after a 30-day stepwise warming protocol. When seawater pH was reduced, CR, ratio of oxygen to nitrogen, and SFG significantly decreased concurrently, whereas ammonia ER increased. RR was virtually unaffected under acidified conditions. Neither temperature nor acidification showed a significant effect on food absorption efficiency. Our findings indicate that energy is allocated away from reproduction under reduced seawater pH, potentially resulting in an impaired or suppressed reproductive function. This interpretation is based on the fact that spawning was induced in only 56% of the clams grown at pH 7.4. Seawater acidification can therefore potentially impair the physiological energetics and spawning capacity of R. philippinarum.

Unlocking the night: Exploring the health impacts of night-time walking environments on health outcomes of older adults.

Walking remains the primary form of physical activity for many older adults in China, and the quality of the walking environment at night may determine the frequency and duration of evening outings. This study reveals how night-time environmental features influence the health outcomes of older adults. Using the medical check-up records of 87,578 older adults from a public health service in the Beilin district of Xi'an city, China, the role of the night-time walking environment in managing chronic conditions was examined. A favorable night-time walking environment reduced the prevalence of chronic conditions and comorbidities among older adults. However, the health effects stemming from the night-time walking environment exhibited heterogeneity, with significant impacts only on metabolic conditions, such as hypertension and diabetes, while the effects on other conditions were not significant. Our findings supplement the theory of healthy aging by highlighting the potential value of the environment in managing chronic conditions, which may serve as a cost-effective health intervention for aging societies.

Transcriptomic insights into cessation of clam embryonic development following transgenerational exposure to ocean acidity extreme.

Ocean acidity extremes (OAX) events are becoming more frequent and intense in coastal areas in the context of climate change, generating widespread consequences on marine calcifying organisms and ecosystems they support. While transgenerational exposure to end-of-century scenario of ocean acidification (i.e., at pH 7.7) can confer calcifiers resilience, whether and to what extent such resilience holds true under OAX conditions is still poorly understood. Here, we found that transgenerational exposure of Ruditapes philippinarum to OAX resulted in cessation of embryonic development at the trochophore stage, implying devastating consequences of OAX on marine bivalves. We identified a large number of differentially expressed genes in embryos following transgenerationally exposed to OAX, which were mainly significantly enriched in KEGG pathways related to energy metabolism, immunity and apoptosis. These pathways were significantly activated, and genes involved in these processes were up-regulated, indicating strong cellular stress responses to OAX. These findings demonstrate that transgenerational exposure to OAX can result in embryonic developmental cessation by severe cellular damages, implying that transgenerational acclimation maybe not a panacea for marine bivalves to cope with OAX, and hence urgent efforts are required to understand consequences of intensifying OAX events in coastal ecosystems.

Elucidating maternal provisioning for bivalve larvae under ocean acidity extreme events.

Ocean acidity extreme (OAX) events, triggered by climate change and anthropogenic activities, are projected to become more intense and frequent in coastal ecosystems, devastating marine bivalves and ecosystems they support. Maternal effects adaptively modulate offspring performance in response to climatic stressors, but whether and to what extent they can confer offspring resistance to OAX remain largely unknown. Here, we investigated impacts of OAX on the parental and larval lipidomes of Manila clams (Ruditapes philippinarum) to add further insights into the energetic nature of maternal effects. A total of 177 significantly down-regulated lipid components (categorized into glycerolipids mainly) were shown in OAX-stressed adults compared with those reared under ambient conditions, and following parental conditioning, larvae also exhibited a further decreasing down-regulation of the glycerolipid components. Triacylglycerols were identified as the predominant composition of glycerolipids and the primary sources of energy for gonadal maturation and larvae development. Yet, larvae spawn from adults exposed to OAX had significantly lower contents of triacylglycerols than those without a prior history of parental conditioning, with the carbon chain length and unsaturation degree of the triacylglycerol components being significantly affected. The latter was also in line with significant increases in the production of triacylglycerol byproducts (diacylglycerols). Overall, our findings suggest that when OAX prevailed during reproductive seasons of Manila clams, maternal effects could be maladaptive by depressing the energetic deposition of larvae, and may not be a potential adaptive modulator of marine bivalves to cope with unprecedented environmental change.

Environmental and social framework to protect marine bivalves under extreme weather events.

Rising ocean temperatures, a consequence of anthropogenic climate change, are increasing the frequency, intensity, and magnitude of extreme marine heatwaves (MHWs). These persistent anomalous warming events can have severe ecological and socioeconomic impacts, threatening ecologically and economically vital organisms such as bivalves and the ecosystems they support. Developing robust environmental and social frameworks to enhance the resilience and adaptability of bivalve aquaculture is critical to ensuring the sustainability of this crucial food source. This review synthesizes the current understanding of the physiological and ecological impacts of MHWs on commercially important bivalve species farmed globally. We propose an integrated risk assessment framework that encompasses environmental monitoring, farm-level preparedness planning, and community-level social support systems to safeguard bivalve aquaculture. Specifically, we examine heatwave prediction models, local mitigation strategies, and social programs that could mitigate the impacts on bivalve farms and vulnerable coastal communities economically dependent on this fishery. At the farm level, adaptation strategies such as selective breeding for heat-tolerant strains, optimized site selection, and adjustments to culture practices can improve survival outcomes during MHWs. Robust disease surveillance and management programs are essential for early detection and rapid response. Furthermore, we highlight the importance of stakeholder engagement, knowledge exchange, and collaborative governance in developing context-specific, inclusive, and equitable safeguard systems. Proactive measures, such as advanced forecasting tools like the California Current Marine Heat Wave Tracker developed by NOAA's Southwest Fisheries Science Center, enable preemptive action before losses occur. Coordinated preparation and response, underpinned by continuous monitoring and adaptive management, promise to protect these climate-vulnerable food systems and coastal communities. However, sustained research, innovation, and cross-sector collaboration are imperative to navigate the challenges posed by our rapidly changing oceans.

Exploring the regulatory role of Linc00893 in asthenozoospermia: Insights into sperm motility and SSC viability.

The role of long intergenic noncoding RNA 00893 (Linc00893) in asthenozoospermia (AS) and its impact on sperm motility remains unclear The present study explored the effect of Linc00893 on AS, specifically its effect on sperm motility and its relationship with spermatogonial stem cell (SSC) vitality and myosin heavy chain 9 (MYH9) protein expression. Linc00893 expression was analyzed in semen samples using reverse transcription­quantitative PCR, revealing a significant downregulation in samples from individuals with AS compared with those from healthy subjects. This downregulation was found to be negatively correlated with parameters of sperm motility. To further understand the role of Linc00893, small interfering RNA was used to knockdown its expression in SSCs. This knockdown led to a marked decrease in cell vitality and an increase in apoptosis. Notably, Linc00893 knockdown was shown to inhibit MYH9 expression by competitively binding with microRNA­107, a finding verified by dual­luciferase reporter and RNA immunoprecipitation assays. Furthermore, using the GSE160749 dataset from the Gene Expression Omnibus database, it was revealed that MYH9 protein expression was downregulated in AS samples. Subsequently, lentiviral vectors were constructed to induce overexpression of MYH9, which in turn reduced SSC apoptosis and counteracted the apoptosis triggered by Linc00893 knockdown. In conclusion, the present study identified the role of Linc00893 in AS, particularly its regulatory impact on sperm motility, SSC vitality and MYH9 expression. These findings may provide information on the potential regulatory mechanisms in AS development, and identify Linc00893 and MYH9 as possible targets for diagnosing and treating AS­related disorders.

Mapping conservation priorities for wild yak (Bos mutus) habitats on the Tibetan Plateau, China.

The wild yak (Bos mutus) is a cold-tolerant herbivore native to the Tibetan Plateau and has been categorized as vulnerable by the International Union for Conservation of Nature and Natural Resources. Low population densities within currently fragmented habitats and unclear landscape conservation priorities warrant attention. Herein, we employed the maximum entropy (MaxEnt) model using over 900 wild yak occurrence records to model wild yak habitat suitability. Our analysis revealed unprotected wild yak landscapes covering 30.79 % of the habitat area, indicating a conservation gap between protected areas (PAs) and wild yak habitats. To protect metapopulation dynamics and mitigate high risks of poaching, habitat degradation and fragmentation, resource competition, and degenerated genetic characterization of wild yaks in fragmented and degraded habitat, we identified eight habitat patches as landscape conservation units (LCUs) and 14 linkages among the LCUs, enhancing the connectivity between LCUs to decrease negative effects of genetic threats. A centrality analysis demonstrated that Changtang, Arjinshan, and Hoh Xil national nature reserves and their linkages are all critical for the maintenance of habitat connectivity. Here, we suggest that habitat- and LCU-specific conservation strategies should be highlighted during the establishment of PAs and transboundary cooperation. Ultimately, our results can assist conservationists and land managers in comprehending wild yak distribution, movement, and habitat requirements, as well as for the development of effective protection strategies. Furthermore, the combined modeling method (MaxEnt-Zonation-InVEST) could be utilized as a component for identifying conservation priorities and linkages between core patches for species and assessing the efficiency of PAs, core habitats, and corridors in achieving conservation goals. Our study can provide a framework in identifying priority conservation and connectivity between habitat patches to facilitate effectively conservation and genetic resilience for endangered species in fragmented habitats.

Behavioral responses of intertidal clams to compound extreme weather and climate events.

Rapidly increasing concentration of carbon dioxide (CO2) in the atmosphere not only results in global warming, but also drives increasing seawater acidification. Infaunal bivalves play critical roles in benthic-pelagic coupling, but little is known about their behavioral responses to compound climate events. Here, we tested how heatwaves and acidification affected the behavior of Manila clams (Ruditapes philippinarum). Under acidified conditions, the clams remained capable of burrowing into sediments. Yet, when heatwaves attacked, significant decreases in burrowing ability occurred. Following two consecutive events of heatwaves, the clams exhibited rapid behavioral acclimation. The present study showed that the behavior of R. philippinarum is more sensitive to heatwaves than acidification. Given that the behavior can act as an early and sensitive indicator of the fitness of intertidal bivalves, whether, and to what extent, behavioral acclimation can persist under scenarios of intensifying heatwaves in the context of ocean acidification deserve further investigations.

Elucidating responses of the intertidal clam Ruditapes philippinarum to compound extreme oceanic events.

Ocean acidification and heatwaves caused by rising CO2 affect bivalves and other coastal organisms. Intertidal bivalves are vital to benthic ecosystems, but their physiological and metabolic responses to compound catastrophic climate events are unknown. Here, we examined Manila clam (Ruditapes philippinarum) responses to low pH and heatwaves. Biochemical and gene expression demonstrated that pH and heatwaves greatly affect physiological energy enzymes and genes expression. In the presence of heatwaves, Manila clams expressed more enzymes and genes involved in physiological energetics regardless of acidity, even more so than in the presence of both. In this study, calcifying organisms' biochemical and molecular reactions are more susceptible to temperature rises than acidity. Acclimation under harsh weather conditions was consistent with thermal stress increase at lower biological organization levels. These substantial temporal biochemical and molecular patterns illuminate clam tipping points. This study helps us understand how compound extreme weather and climate events affect coastal bivalves for future conservation efforts.

Bivalves under extreme weather events: A comparative study of five economically important species in the South China sea during marine heatwaves.

Marine heatwaves (MHWs) are increasing in frequency and intensity, threatening marine organisms and ecosystems they support. Yet, little is known about impacts of intensifying MHWs on ecologically and economically important bivalves cultured in the South China Sea. Here, we compared survival and physiological responses of five bivalve species, Pinctada fucata, Crassostrea angulata, Perna viridis, Argopecten irradians and Paphia undulata, to two consecutive MHWs events (3 days of thermal exposure to + 4 °C or + 8 °C, following 3 days of recovery under ambient conditions). While P. fucata, P. viridis, and P. undulata are native to the South China Sea region, C. angulata and A. irradians are not. Individuals of P. fucata, C. angulata and P. viridis had higher stress tolerance to MHWs than A. irradians and P. undulata, the latter already experiencing 100% mortality under +8 °C conditions during the first event. With increasing intensity of MHWs, standard metabolic rates of all five species increased significantly, in line with significant depressions of function-related energy-metabolizing enzymes (CMA, NKA, and T-ATP). Likewise, activities of antioxidant enzymes (SOD, CAT, and MDA) and shell mineralization-related enzymes (AKP and ACP) responded significantly to MHWs, despite species-specific performances observed. These findings demonstrate that some bivalve species can likely fail to accommodate intensifying MHWs events in the South China Sea, but some may persist. If this is the case, then one would expect substantial loss of fitness in bivalve aquaculture in the South China Sea under intensifying MHWs conditions.

Seasonal variations affect the ecosystem functioning and microbial assembly processes in plantation forest soils.

While afforestation mitigates climate concerns, the impact of afforestation on ecological assembly processes and multiple soil functions (multifunctionality) in afforested areas remains unclear. The Xiong'an New Area plantation forests (Pinus and Sophora forests) in North China were selected to examine the effects of plantation types across four distinct seasons on soil microbiomes. Three functional categories (nutrient stocks, organic matter decomposition, and microbial functional genes) of multifunctionality and the average (net) multifunctionality were quantified. All these categories are directly related to soil functions. The results showed that net soil multifunctionality as a broad function did not change seasonally, unlike other narrow functional categories. Bacterial communities were deterministically (variable selection and homogenous selection) structured, whereas the stochastic process of dispersal limitation was mainly responsible for the assembly and turnover of fungal and protist communities. In Pinus forests, winter initiates a sudden shift from deterministic to stochastic processes in bacterial community assembly, accompanied by decreased Shannon diversity and heightened nutrient cycling (nutrient stocks and organic matter decomposition). This indicates the potential vulnerability of deterministic assembly to seasonal fluctuations, particularly in environments rich in nutrients. The results predicted that protist community composition was uniquely structured with C-related functional activities relative to bacterial and fungal ß-diversity variations, which were mostly explained by seasonal variations. Our study highlighted the importance of the protist phagocytosis process on soil microbial interactions through the predicted impact of protist α-diversity on microbial cooccurrence network parameters. This association might be driven by the high abundance of protist consumers as the main predators of bacterial and fungal lineages in our sampling plots. Our findings reveal that the complexity of microbial co-occurrence interactions was considerably higher in spring, perhaps attributing thermal variability and increased resource availability within spring that foster microbial diversity and network complexity. This study contributes to local ecosystem prospects to model the behavior of soil biota seasonally and their implied effects on soil functioning and microbial assembly processes, which will benefit global-scale afforestation programs by promoting novel, precise, and rational plantation forests for future environmental sustainability and self-sufficiency.