ZnO NPs Enhanced Photosynthetic Capacity, Promoted New Shoot Development, and Improved the Community Composition of Phyllosphere Epiphytic and Endophytic Microorganisms in Tea Plants

ZnO NPs Enhanced Photosynthetic Capacity, Promoted New Shoot Development, and Improved the Community Composition of Phyllosphere Epiphytic and Endophytic Microorganisms in Tea Plants | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article ZnO NPs Enhanced Photosynthetic Capacity, Promoted New Shoot Development, and Improved the Community Composition of Phyllosphere Epiphytic and Endophytic Microorganisms in Tea Plants Hao Chen, Yujie Song, Yu Wang, Huan Wang, Zhaotang Ding, Kai Fan This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4019055/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 01 Jul, 2024 Read the published version in Journal of Nanobiotechnology → Version 1 posted 7 You are reading this latest preprint version Abstract Background Nanotechnology holds revolutionary potential in the field of agriculture, with zinc oxide nanoparticles (ZnO NPs) demonstrating advantages in promoting crop growth. Photosynthesis is a key process in the growth and quality formation of tea plants, and phyllosphere microorganisms also have a significant impact on plant growth and health. However, the effects of ZnO NPs on the photosynthesis of tea plants, the sprouting of new shoots, and the community of phyllosphere microorganisms are not yet clear. Results This study investigated the photosynthetic physiological parameters of tea plants under the influence of ZnO NPs, the content of key photosynthetic enzymes such as RubisCO, chlorophyll content, chlorophyll fluorescence parameters, transcriptomes (leaves and new shoots), extensively targeted metabolomes (leaves and new shoots), mineral element content (leaves and new shoots), and the communities of epiphytic and endophytic microorganisms in the phyllosphere. The results indicated that ZnO NPs could enhance the photosynthesis of tea plants, upregulate the expression of some genes related to photosynthesis, increase the accumulation of photosynthetic products, promote the development of new shoots, and alter the content of various mineral elements in the leaves and new shoots of tea plants. Additionally, ZnO NPs improved the community composition of epiphytic and endophytic microorganisms in the phyllosphere of tea plants, inhibited potential pathogenic microorganisms, and allowed various beneficial microorganisms with potential growth-promoting properties to become dominant species. Conclusion This study demonstrates that ZnO NPs have a positive impact on the photosynthesis of tea plants, the sprouting of new shoots, and the community of phyllosphere microorganisms, which can improve the growth condition of tea plants. These findings provide new scientific evidence for the application of ZnO NPs in sustainable agricultural development and contribute to advancing research in nanobiotechnology aimed at enhancing crop yield and quality. Camellia sinensis (L.) O. Kuntze ZnO NPs Photosynthesis Sprouting of new shoots Epiphytic microorganisms Endophytic microorganisms Phyllosphere Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Background Nanotechnology is becoming an innovative tool in the field of agriculture, with broad application prospects. Compared to traditional materials, nanomaterials' advantages in agriculture are mainly reflected in their unique size effects and surface effects. These effects endow nanomaterials with better solubility, stability, and bioavailability, which play significant roles in promoting crop growth, pest and disease control, and environmental pollution remediation [ 1 – 3 ]. Photosynthesis is the foundation of growth, development, and yield formation in tea plants. It is not only the key process to produce biomass and energy storage in tea plants but also directly affects the quality and yield of tea leaves [ 4 ]. During the growth cycle of tea plants, the efficiency of photosynthesis determines the ability to convert light energy into chemical energy, thereby affecting the growth rate of tea plants and the final yield of tea leaves. The importance of photosynthesis for tea plants is also reflected in its impact on the synthesis of secondary metabolites. For example, the strength of photosynthetic capacity directly affects the synthesis of bioactive substances such as catechins in tea leaves, which are key factors determining the quality of tea [ 5 ]. At the same time, the sprouting of tea buds plays an important role in the life cycle of tea plants. From a biological perspective, the budding process is the cornerstone of plant growth, overall development, and reproduction [ 6 ]. From an economic perspective, tea buds are the main source of the economic value of tea plants and the raw material for producing premium finished tea. The earlier the tea buds sprout, the greater the economic value they can generate to some extent. Therefore, regulating the better sprouting of tea buds is our goal [ 7 ]. Thus, improving the efficiency of photosynthesis and promoting the sprouting of new shoots are crucial for tea production. The positive effects of ZnO NPs on plants have been confirmed in multiple studies. For instance, ZnO NPs have been shown to increase the growth rate and biomass of crops [ 8 ], enhance the efficiency of photosynthesis in plants [ 9 ], and improve the antioxidant system of plants [ 10 ]. These positive effects not only improve the overall health of plants but may also increase crop yield by enhancing the absorption and utilization rate of nutrients. At the same time, ZnO NPs have been found to increase the zinc content and quality of wheat grains, enhance the aroma and nutritional components of rice, and strengthen rice resistance to blast disease [ 11 – 13 ]. Zinc plays multiple biological roles within plants, being a component of many enzymes and involved in nitrogen metabolism, protein synthesis, plant hormone regulation, and antioxidative defense [ 14 ]. Therefore, the supply of zinc is crucial for plant growth and development. As a novel source of zinc, ZnO NPs not only provide the zinc required by plants but also enhance the utilization efficiency of zinc through nanoscale effects [ 15 ]. However, the impact of ZnO NPs on tea plants, especially in relation to photosynthesis, remains unknown. Microorganisms residing in the phyllosphere play a crucial role in plant productivity and health [ 16 ] The microbial composition living on the surface of the phyllosphere is known as epiphytic microorganisms, while those residing within the phyllosphere tissues (either in the intercellular spaces, i.e., the apoplast, or inside plant cells) are referred to as endophytic microorganisms (or phyllosphere endophytes). The same microorganism may be both an epiphyte and an endophyte, occupying both ecological niches [ 17 ]. Phyllosphere microorganisms can directly or indirectly affect plant growth through nitrogen fixation, phosphate solubilization, synthesis of growth hormones, and production of disease-resistant substances [ 18 ]. Additionally, phyllosphere microorganisms can affect plant photosynthesis and influence the plant's efficiency in utilizing light energy through interactions with the plant [ 19 ]. Regulating the microbial community of the tea plant phyllosphere is very important, but compared to soil microorganisms, there is less research on tea plant phyllosphere microorganisms. Phyllosphere microorganisms (especially epiphytes) are highly susceptible to external environmental influences. Environmental factors such as climate conditions, atmospheric pollution, soil quality, and human agricultural activities can have direct or indirect effects on phyllosphere microorganisms. Chemicals used in agricultural activities can affect the diversity and activity of microbial communities by altering the chemical environment of the plant leaf surface [ 20 ]. The application of nanomaterials is considered an effective means to regulate the composition of phyllosphere microbial communities. Studies have found that nanomaterials can affect plant growth by altering the community structure and function of phyllosphere microorganisms [ 21 ]. However, the impact of ZnO NPs on the microbial community of the tea plant phyllosphere remains unknown. This article explores the effects of ZnO NPs on tea plant photosynthesis, sprouting of new shoots, mineral element content, and phyllosphere microbial communities (including both epiphytic and endophytic microorganisms). We hypothesize that ZnO NPs will have a positive impact on the growth of tea plants. At the same time, due to the strong bactericidal properties of ZnO NPs, we speculate that they will change the composition of the tea plant phyllosphere microbial community, potentially eliminating some potential plant pathogens residing in the phyllosphere. This study helps to understand the behavior and effects of ZnO NPs within plants, and through this research, we hope to provide a more in-depth and comprehensive scientific basis for the application of nanomaterials in sustainable agricultural development. Materials and Methods Plant Materials In this study, one-year-old Camellia sinensis cv. Shuchazao tea plants were selected as the research subjects. The tea plants were planted in a nutrient-rich nursery substrate with an organic matter content of about 60%, a total porosity of about 75%, a bulk density of about 0.35%, and a pH of about 5.5. The nursery substrate was provided by Shouguang Yixiandu Agricultural Science and Technology Co., Ltd. (Heze, China). The tea plants were grown under a photoperiod of 14 hours of light and 10 hours of darkness, with a daytime temperature of 28°C, a nighttime temperature of 22°C, light intensity of 10000lx, and air humidity of 75%. Experimental Treatments Referring to the application of ZnO NPs on plants, three concentrations were selected for foliar spraying on tea plants: 0 mg L − 1 (CK), 50 mg L − 1 (T1), and 100 mg L − 1 (T2) [ 12 , 22 , 23 ]. A total of 432 tea plants were planted, with 144 plants for each treatment, divided into 8 groups, with 18 plants per group. The zinc oxide nanoparticles used in this experiment had a particle size of 30 nm and a purity of more than 99.9%, provided by Shanghai Macklin Biochemical Co., Ltd. (Shanghai, China). Seven days after planting the tea plants, foliar spraying treatments were conducted every three days, for a total of three times. In this study, the first mature leaf below the new shoot was selected for research. Measurement of Photosynthetic Physiological Parameters, Photosynthetic Enzyme Content, Nitrogen Content, and Sprouting Rate of New Shoots The photosynthetic physiological parameters of tea plants (net photosynthetic rate (Photo), stomatal conductance (Cond), intercellular CO2 concentration (Ci), and transpiration rate (Trmmol)) were measured using a portable photosynthesis system (LI-6400XT, LI-COR, Inc. Lincoln, NE, USA). The measurements were taken at 9:00 AM, with an air flow rate set to 500 µmol m − 2 s − 1 , a CO2 concentration of 400 µmol m − 2 s − 1 , and light intensity consistent with the growth conditions of the tea plants. Twelve plants were randomly selected from each treatment for measurement. The contents of ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO), fructose-1,6-bisphosphatase (FBP), and phosphoenolpyruvate carboxylase (PEPC) were measured by Genepioneer Biotechnologies (Nanjing, China) using research reagent kits (ELISA method), provided by Jiangsu Jingmei Biotechnology Co., Ltd. (Yancheng, China) (product numbers A-P0018B, A-P0357B, and A-P0104B), with ten replicates for each treatment. Leaf nitrogen content was measured using a plant nutrition analyzer (TYS-4H, Top Cloud-Agri, Zhejiang, China), with sixteen replicates for each treatment. The sprouting rate of new shoots was determined by counting the number of sprouted shoots in each of the eight groups of tea plants (each group consisting of 18 plants) per treatment, with a shoot considered sprouted once it was fully expanded. Measurement of Chlorophyll Content and Chlorophyll Fluorescence Parameters Chlorophyll content was measured using a research reagent kit provided by Suzhou Grace Biotechnology Co., Ltd. (Suzhou, China) (product number G0601W), with ten replicates for each treatment. The soil plant analysis development (SPAD) value was measured using a plant nutrition analyzer (TYS-4H, Top Cloud-Agri, Zhejiang, China), with sixteen replicates for each treatment. Chlorophyll fluorescence parameters were measured using a chlorophyll fluorescence imaging system (IMAGING-PAM, WALZ, Effeltrich, Germany). Leaves were dark-adapted for 30 minutes, and the instrument was calibrated for light intensity and Abs. The actinic light was set to 5, with other parameters set to the default values of the program. Four plants were randomly selected from each treatment for measurement. Measurement of Mineral Elements in Leaves and New Shoots The content of mineral elements in leaves and new shoots was determined by atomic absorption spectrophotometry, with three biological replicates per treatment. Weigh 1.0 g (accurate to 0.0001 g) of fresh tea plant samples into a polytetrafluoroethylene digestion vessel, add 5 mL of nitric acid, and soak overnight. Cover with the inner lid, tighten the stainless-steel jacket, and place in a constant temperature drying oven at 80°C for 1.5 hours, 120°C for 1.5 hours, then raise to 160°C for 4 hours, and allow to cool naturally to room temperature in the oven. Transfer the digestion liquid into a 25 mL volumetric flask, rinse the digestion vessel and inner lid three times with a small amount of 1% nitric acid solution, combine the rinses in the volumetric flask, and make up to the mark with 1% nitric acid, mix well and set aside. A reagent blank is also prepared. The sample solution is measured for element concentration c1 (mg L − 1 ) by an inductively coupled plasma emission spectrometer (iCAP7400, Thermo Fisher Scientific, Waltham, USA). The content of elements in the sample (mg kg − 1 ) = c1V1 m − 1 , where c1 is the concentration of the element measured by the instrument, V1 is the volume of digestion (mL), and m is the mass of the sample (g). Transcriptome Measurement of Leaves and New Shoots The transcriptome sequencing process includes total RNA extraction, mRNA enrichment, double-stranded cDNA synthesis, end repair, A-tailing and adapter ligation, fragment selection and PCR amplification, library quality assessment, and Illumina sequencing. Three biological replicates were measured for each treatment. To ensure high-quality RNA and the absence of DNA contamination, the extracted RNA was rigorously analyzed for integrity and precisely tested for purity and concentration. The research process began with RNA samples prepared using the NEBNext Ultra RNA Library Prep Kit for Illumina. Purified fragments underwent PCR, and their quality was evaluated using the Agilent Bioanalyzer 2100 system. Index-coded samples were clustered on a cBot Cluster Generation System and sequenced to generate 150 bp paired-end reads. The data was filtered using fastp to produce 'clean reads'. Clean reads were compared to the reference genome using HISAT. Gene prediction was performed with StringTie, which provided faster and more accurate transcript splicing. FeatureCounts assisted in gene alignment, and the FPKM of each gene was calculated based on their specific length. Extensive Targeted Metabolome Measurement of Leaves and New Shoots Biological samples were processed using vacuum freeze-drying technology in a lyophilizer (Scientz-100F), followed by grinding at 30 Hz for 1.5 minutes using a grinder (MM 400, Retsch). 50 mg of the resulting sample powder was weighed with an electronic balance (MS105DΜ) and mixed with 1200 µL of pre-cooled 70% methanolic aqueous solution containing an internal standard. The mixture was subjected to intermittent vortexing and centrifugation. After centrifugation, the supernatant was separated, filtered, and prepared for UPLC-MS/MS analysis. Under UPLC conditions, an UPLC-ESI-MS/MS system (UPLC, ExionLC™ AD, https://sciex.com.cn/ ) and a tandem mass spectrometry system ( https://sciex.com.cn/ ) were used to analyze the sample extracts. A precise mobile phase gradient was employed using solvent A (pure water with 0.1% formic acid) and solvent B (acetonitrile with 0.1% formic acid). Precise flow rate, temperature, and injection volumes were maintained throughout the process. The operating parameters of the ESI-Q TRAP-MS/MS included preset values for source temperature, ion spray voltage, gas pressures, and collision-activated dissociation levels. The QQQ scans were performed as MRM experiments, with DP and CE adjusted for individual MRM transitions. Specific MRM transitions were closely monitored throughout the experiment, aimed at identifying the metabolites eluted during the period. Measurement of Epiphytic and Endophytic Microorganisms in the Phyllosphere This study utilized 16S rDNA amplicon sequencing to measure epiphytic and endophytic bacteria and Internal Transcribed Spacer (ITS) sequencing for epiphytic and endophytic fungi [ 24 ]. Extraction of Epiphytic Microorganisms For each sample, 15 g of tea leaves (approximately 50 leaves) were placed in a sterile tube, to which 150 mL of potassium phosphate buffer (0.1 mol L − 1 , pH = 8) was added. The samples were ultrasonicated for 1 minute, vortexed for 10 seconds, and the ultrasonication and vortexing were repeated twice. The washed leaves were removed, potassium phosphate buffer was added again, and the ultrasonication and vortexing steps were repeated. The washing liquid was filtered through a 0.22 µm filter membrane, which was then flash-frozen in liquid nitrogen and stored at -80°C. Extraction of Endophytic Microorganisms The leaf surfaces were successively soaked in 75% ethanol for 1 minute, 3.25% sodium hypochlorite for 3 minutes, and 75% ethanol for 30 seconds, followed by rinsing three times with sterile distilled water. The leaves were then freeze-dried in liquid nitrogen [ 25 ]. Extraction of Genomic DNA and PCR Amplification Genomic DNA was extracted from the samples using the CTAB method. The DNA's purity and concentration were checked using agarose gel electrophoresis. An appropriate amount of sample DNA was taken in a centrifuge tube and diluted with sterile water to 1 ng µL − 1 . The diluted genomic DNA was used as a template, and PCR was performed using barcode-specific primers, Phusion® High-Fidelity PCR Master Mix with GC Buffer from New England Biolabs, and a high-efficiency high-fidelity enzyme to ensure amplification efficiency and accuracy. Mixing and Purification of PCR Products The PCR products were checked by electrophoresis on a 2% agarose gel; qualified PCR products were purified with magnetic beads, quantified enzymatically, and mixed in equal amounts based on the concentration of the PCR products. After thorough mixing, the PCR products were checked again on a 2% agarose gel, and the target bands were recovered using a gel recovery kit provided by Qiagen. Library Construction and Sequencing Libraries were constructed using the TruSeq® DNA PCR-Free Sample Preparation Kit, quantified by Qubit and Q-PCR, and sequenced on a NovaSeq6000 after passing quality control. Sequencing Data Processing Sample data were demultiplexed from the raw data based on barcode sequences and PCR primer sequences, and the barcode and primer sequences were trimmed. High-quality reads were obtained by filtering the raw reads using fastp (v0.22.0, https://github.com/OpenGene/fastp ). Quantitative Real-Time PCR Validation Reverse transcription amplification was performed using the SynScript® III RT SuperMix for qPCR reverse transcription kit produced by Beijing Biotech Co., Ltd. (Beijing, China). The resulting cDNA products were diluted fourfold and used as templates for qPCR amplification with Tsingke ArtiCanCEO SYBR qPCR Mix. CsGAPDH was used as the reference gene, and gene expression was calculated using the 2 −ΔΔCT method. The qRT-PCR samples underwent three biological replicates and three technical replicates. All primers used in this study are listed in Table.S1. Results Effects of ZnO NPs on Photosynthetic Physiological Parameters, Photosynthetic Enzyme Content, Nitrogen Content, and Sprouting Rate of New Shoots in Tea Plants To investigate the effects of ZnO NPs on photosynthesis and new shoot development in tea plants, we measured the Photo, Cond, Ci, Trmmol, RubisCO content, FBP content, PEPC content, nitrogen content, and sprouting rate of new shoots. As shown in Fig. 1 , except for Ci, ZnO NPs significantly increased the Photo, Cond, and Trmmol of tea plants, with the most significant effects observed at a concentration of 100 mg L − 1 (Fig. 1 A). Simultaneously, ZnO NPs significantly increased the content of photosynthetic enzymes in the leaves, indirectly indicating stronger activities of RubisCO, FBP, and PEPC (Fig. 1 B). Additionally, the nitrogen content in tea leaves also increased under the influence of ZnO NPs (Fig. 1 C). Interestingly, ZnO NPs also improved the sprouting rate of new shoots, although the two different concentrations did not show significant differences in their effects on the sprouting rate (Fig. 1 D). In summary, ZnO NPs significantly enhanced the photosynthetic capacity of tea leaves and the sprouting rate of new shoots. Effects of ZnO NPs on Chlorophyll Content and Chlorophyll Fluorescence Parameters in Tea Plants To explore the impact of ZnO NPs on the chlorophyll content and chlorophyll fluorescence parameters in tea plants, we measured the chlorophyll content, SPAD values, and chlorophyll fluorescence parameters in tea leaves (Fig. 2 ). The combined measurements of total chlorophyll, chlorophyll a (chl a), chlorophyll b (chl b), and SPAD values indicate that ZnO NPs can increase the chlorophyll content in tea leaves (Fig. 2 A). In terms of chlorophyll fluorescence parameters, aside from the relative electron transport rate (ETR), ZnO NPs significantly enhanced the maximum photochemical efficiency of photosystem II (Fv/Fm), the photochemical quenching coefficient (qP), and the actual photochemical efficiency of photosystem II (Y(II)) (Fig. 2 B). In summary, both the chlorophyll content and chlorophyll fluorescence parameters demonstrate that ZnO NPs can significantly improve the photosynthetic capacity of tea leaves. Effects of ZnO NPs on the Content of Mineral Elements in Tea Leaves and New Shoots To explore the impact of applying one mineral element (ZnO NPs) on other minerals in tea leaves and new shoots, we measured the content of eight mineral elements related to the growth and quality of tea plants (Fig. 3 ). Under the influence of nano zinc oxide, the zinc content in tea leaves increased, but a significant difference was only observed at the concentration of 100 mg L − 1 (T2). Interestingly, the zinc content in new shoots did not significantly increase, which could be speculated that ZnO NPs were sprayed before the sprouting of new shoots, and nano zinc oxide did not directly contact the new shoots, nor was there sufficient time for the zinc element to be transported from the leaves to the new shoots. Selenium showed significant differences under the influence of nano zinc oxide; at the concentration of 50 mg L − 1 (T1), the selenium content in the leaves significantly decreased, while it significantly increased in the new shoots, suggesting that at this concentration, ZnO NPs promoted the transfer of selenium from the leaves to the new shoots. This difference was not observed at T2. Meanwhile, as the concentration of nano zinc oxide increased, the molybdenum content in the leaves significantly increased, but the molybdenum content in the new shoots significantly decreased at T2. The copper content in the leaves significantly increased under T2, but no significant difference was observed in the copper content in the new shoots. At the same time, no significant changes were found in the iron content in the leaves, but the iron content in the new shoots significantly increased at T1. The magnesium content in the leaves significantly increased at T1, with no significant differences observed in the new shoots. Similar to magnesium, the calcium content in both leaves and new shoots showed a consistent expression trend, with a significant increase in calcium content in the leaves only at T1. Interestingly, calcium and magnesium, as macronutrients required by tea plants, exhibited a consistent expression trend. Unlike the other mineral elements, the aluminum content in the leaves significantly decreased at T2, with no significant differences observed in the new shoots. In summary, ZnO NPs had a significant impact on the content of mineral elements in tea leaves, with a greater effect on the leaves than on the new shoots. Integrated Analysis of Differential Gene Expression and Differential Metabolite Expression Reveals the Intrinsic Mechanism Behind ZnO NPs Enhancing Photosynthetic Capacity and Promoting New Shoot Development in Tea Plants Photosynthesis-Related Pathways and Endogenous Auxin Pathway Metabolites and Gene Expression Differential gene expression in tea shoots and leaves under the influence of ZnO NPs, KEGG enrichment analysis of differential genes, GO enrichment analysis, and qRT-PCR analysis; differential metabolites in leaves and new shoots and KEGG enrichment analysis of differential metabolites are presented in Additional file 1. To comprehensively elucidate the response of tea plants to ZnO NPs from both gene expression and metabolite perspectives, we conducted an integrated analysis of the transcriptome and metabolome. Under the treatment of two concentrations of ZnO NPs, tea leaves were significantly enriched in pathways related to photosynthesis, specifically Photosynthesis - antenna proteins (Fig. 4 A). In this pathway, all the differentially expressed genes compared to the control were significantly upregulated, mainly regulating the light-harvesting complex II chlorophyll a/b binding proteins (Lhca2, Lhca4, Lhca5, Lhcb1, Lhcb2, Lhcb3, and Lhcb6). Interestingly, in the Starch and sucrose metabolism pathway closely related to photosynthesis, all differential metabolites such as Sucrose, D-Glucose-6P, D-Fructose-6P, D-Glucose-6P, etc., showed a significant increase in content. We also presented the significant gene expression levels related to this pathway (Fig. 4 B). Furthermore, under both concentrations of ZnO NPs treatment, the endogenous auxin content was significantly increased, and the gene and metabolite expression levels in the related metabolic pathway are shown in Fig. 4 C. Weighted Gene Co-expression Network Analysis (WGCNA) of Differential Genes and Key Differential Metabolites (Identification of coexpressed gene networks and key candidates) To further understand the regulation of sucrose metabolism changes in tea leaves caused by ZnO NPs, WGCNA was conducted to study the co-expression network of differentially expressed genes (DEGs). Based on similar expression patterns, a total of 14 co-expressed modules were identified (Fig. 5 ). Genes preferentially expressed in sugar metabolism-related significant differential metabolites were mainly concentrated in the MEblue module, with 11 metabolites having a correlation R 2 greater than 0.8 with this module. The key product of sugar metabolism, Sucrose, also had the highest correlation with this module (R 2 = 0.86, p = 0.0027). Within the MEblue module, 54 genes related to starch and sucrose metabolism were identified, including 11 regulating glucose-1-phosphate adenylyltransferase ( CSS0000089, CSS0026130, CSS0026865, CSS0032913, CSS0035261, CSS0035419, CSS0050226, novel.1469, novel.4393, novel.4625, novel.7765 ); 2 regulating glucose-6-phosphate isomerase ( CSS0002435, CSS0045493 ); 2 regulating trehalose 6-phosphate synthase/phosphatase ( CSS0002735, CSS0035721 ); 5 regulating beta-glucosidase ( CSS0003115, CSS0006278, CSS0016696, CSS0017477, CSS0036292 ); 6 regulating beta-amylase ( CSS0003801, CSS0018030, CSS0027311, CSS0032302, CSS0043627, CSS0047759 ); 3 regulating granule-bound starch synthase ( CSS0004941, CSS0037992, CSS0045869 ); 5 regulating starch synthase ( CSS0005914, CSS0021129, CSS0023626, CSS00248447, novel.5440 ); 5 regulating glucan endo-1,3-beta-glucosidase 1/2/3 ( CSS0008156, CSS0012190, CSS0043010, CSS0048079, CSS0050142 ); 1 regulating alpha,alpha-trehalase ( CSS0008297 ); 1 regulating alpha-amylase ( CSS0008836 ); 3 regulating trehalose 6-phosphate phosphatase ( CSS0009951, CSS0045703, novel.736 ); 3 regulating glycogen phosphorylase ( CSS0015277, CSS0022613, CSS0038552 ); 2 regulating 1,4-alpha-glucan branching enzyme ( CSS0016120, novel.877 ); 1 regulating sucrose-phosphate synthase ( CSS0024623 ); 1 regulating sucrose-6-phosphatase ( CSS0028296 ); 2 regulating beta-fructofuranosidase ( CSS0033878, novel.2722 ); 1 regulating ADP-sugar diphosphatase ( CSS0042147 ). Two-way Orthogonal Partial Least Squares (O2PLS) Analysis To uncover the internal connections between gene expression and metabolite changes in tea plants in response to ZnO NPs, and to determine the degree of association between them, as well as to identify the main genes and metabolites causing this association, we conducted an O2PLS analysis (Fig. 6 ). O2PLS is an unsupervised modeling technique that can objectively describe whether there is a correlation trend between two sets of data, avoiding false-positive associations from the outset. Compared to traditional supervised models such as PCA (Principal Component Analysis), PLS (Partial Least Squares), and CCA (Canonical Correlation Analysis), O2PLS considers factors such as the size, scale, distribution, and experimental errors of datasets in different scenarios. The modeling process takes into account the joint, specific, and residual parts between different datasets, making it suitable for data mining in complex scenarios. Under the influence of ZnO NPs, the top ten genes affecting changes in metabolites in tea leaves and their expression levels are shown in Fig. 6 A, while the top ten metabolites affecting transcript expression in tea leaves and their expression levels are shown in Fig. 6 B. Similarly, the top ten genes affecting changes in metabolites in new shoots and their expression levels are shown in Fig. 6 C, and the top ten metabolites affecting transcript expression in new shoots and their expression levels are shown in Fig. 6 D. Interestingly, among the top ten metabolites with the greatest impact on transcript expression in tea leaves, seven are related to starch and sucrose metabolism. Analysis of the Phyllosphere Epiphytic Microbial Structure of Tea Plants Under the Influence of ZnO NPs ASV Clustering Analysis of Epiphytic Bacteria and Fungi To investigate the community composition of epiphytic microbes in the phyllosphere of tea plants under the influence of ZnO NPs, we performed denoising analysis on effective data to generate Amplicon Sequence Variants (ASVs). Based on the ASV analysis results, each ASV sequence was annotated to obtain corresponding species information. Compared to traditional Operational Taxonomic Units (OTUs) clustering analysis, ASV analysis does not lose taxonomic resolution and can accurately distinguish sequences above the similarity threshold. As shown in Fig. 7 A, under the influence of different concentrations of ZnO NPs, tea plants shared 692 ASVs of epiphytic bacteria, with 3901 unique ASVs in CKEP, 4966 in T1EP, and 3901 in T2EP. As shown in Fig. 7 B, under the influence of different concentrations of ZnO NPs, tea plants shared 55 ASVs of epiphytic fungi, with 270 unique ASVs in CKED, 346 in T1ED, and 115 in T2ED. The clustering results indicate that different concentrations of ZnO NPs significantly changed the community structure of epiphytic bacteria and fungi. For the selection of differential epiphytic microbes under the influence of ZnO NPs, random forest analysis, and functional prediction, see Additional file 2. Genus-level Clustering Analysis of Epiphytic Bacteria and Fungi To analyze the differences in the community composition of epiphytic microbes in the phyllosphere of tea plants under different concentrations of ZnO NPs, we performed clustering analysis at the genus level based on quantitative information, facilitating the discovery of microbial population aggregation under different treatments. As shown in Fig. 8 A, the bacterial communities in CKEP were mainly clustered in genera such as Marinococcus, Devosia, Pseudomonas, Hyphomicrobium, Bryobacter, Acinetobacter, Bradyrhizobium, Lactococcus, Sphingomonas, Cutibacterium, Paenibacillus , and Bacillus . The bacterial communities in T1EP were mainly clustered in Rhodococcus , unidentified Beijerinckiaceae , Salana, Truepera, Mycobacterium, Chryseobacterium, Aureimonas, Staphylococcus , unidentified Cyanobacteria , Stenotrophomonas , unidentified Halomonadaceae, Aquipuribacter , unidentified Rhizobiaceae, Patulibacter, Arthrobacter, Nocardioides, Paracoccus, Arsenophonus, Microbacterium , and Brevundimonas . The bacterial communities in T2EP were enriched in Arsenophonus and Microbacterium. As shown in Fig. 8 B, the fungal communities in CKEP were mainly clustered in Cylindrocladiella , unclassified Hypocreales, Cladosporium, Sterigmatomyces, Aspergillus, Penicillium, Xylaria, Phyllosticta, Acremonium, Arthothelium , and Colletotrichum . The fungal communities in T1EP were mainly clustered in Candida, Fusarium, Mortierella, Cryptococcus ( f Filobasidiaceae ), Guehomyces , unclassified Phaeosphaeriaceae, Schizophyllum, Trametes, Cryptococcus ( Tremellales family Incertae sedis ), Lophiostoma, Marasmius , unclassified Fungi, Rhodotorula, Gibberella , and Pyrenochaeta . The fungal communities in T2EP were mainly enriched in unclassified Pleosporales, Cystofilobasidium, Monographella, Mrakia, Mrakiella, and Malassezia . The clustering results show significant differences in the dominant microbial species, indicating that different concentrations of ZnO NPs have selective effects on epiphytic bacteria and fungi. Analysis of the Phyllosphere Endophytic Microbial Structure of Tea Plants Under the Influence of ZnO NPs ASV Clustering Analysis of Endophytic Bacteria and Fungi To study the community composition of endophytic microbes in the phyllosphere of tea plants under the influence of ZnO NPs, similar to epiphytic microbes, we performed ASV clustering analysis. As shown in Fig. 9 , under the influence of different concentrations of ZnO NPs, tea plants shared 16 ASVs of endophytic bacteria, with 58 unique ASVs in CKED, 47 in T1ED, and 161 in T2ED. The figure also shows that under the influence of different concentrations of ZnO NPs, tea plants shared 112 ASVs of endophytic fungi, with 595 unique ASVs in CKED, 186 in T1ED, and 317 in T2ED. The clustering results indicate that different concentrations of ZnO NPs significantly changed the community structure of endophytic bacteria and fungi. For the selection of differential endophytic microbes under the influence of ZnO NPs, random forest analysis, and functional prediction, see Additional file 3. Genus-level Clustering Analysis of Endophytic Bacteria and Fungi and Their Association with Starch and Sucrose Metabolism Products To analyze the differences in the community composition of endophytic microbes in the phyllosphere of tea plants under different concentrations of ZnO NPs, we performed clustering analysis at the genus level. As shown in Fig. 10 A, the bacterial communities in CKED were mainly clustered in Marinococcus, Leifsonia, Duganella, Bacteroides , and Delftia . The bacterial communities in T1ED were mainly clustered in Bacillus, Exiguobacterium, Delftia, Enterobacter, Mycobacterium, and unidentified Burkholderiaceae . The bacterial communities in T2ED were mainly clustered in Massilia, Rhodococcus, Paenibacillus, Salana, Paracoccus, Brevundimonas, Aureimonas, Sphingomonas, Ralstonia, Pseudomonas, Pantoea, Corynebacterium, Truepera, Nocardioides, Chryseobacterium, Microbacterium , unidentified Rhizobiaceae, Stenotrophomonas , and Acinetobacter . As shown in Fig. 10 B, the fungal communities in CKED were mainly clustered in Aureobasidium, Lalaria , unclassified Taphrinaceae, Uwebraunia, Ascochyta, Ophiocordyceps, Microcyclospora, Acremonium, Cryptococcus (Filobasidiaceae), Sporobolomyces, Lophiostoma, Monographella , and Cryptococcus ( Tremellales family Incertae sedis ). The fungal communities in T1ED were mainly clustered in S terigmatomyces, Humicola, Candida, Tilletiopsis, Colletotrichum , and Arthothelium . The fungal communities in T2ED were mainly enriched in Davidiella, Fusarium, Pyrenochaeta, Penicillium , unclassified Hypocreales, Boletus, Malassezia, and Aspergillus . The clustering results show significant differences in the dominant microbial species, indicating that different concentrations of ZnO NPs have selective effects on endophytic bacteria and fungi. To investigate the correlation between endophytic microbes in the phyllosphere of tea plants and differential starch and sucrose metabolism products under the influence of ZnO NPs, we conducted a Spearman correlation hierarchical clustering analysis (Fig. 11 ). Several endophytic microbes showed significant correlations with sugar metabolism (with fungi demonstrating stronger correlations). Specific correlation coefficients and significance values between each microbe and sugars are presented in Table.S2. Notably, Taphrina, Cylindrocladiella, Aspergillus, Boletus, Malassezia, Cladosporium, Xenocylindrocladium, Cordyceps , and Pyrenochaeta showed significant positive correlations with sucrose. Discussion Photosynthesis plays a crucial role in plant energy storage, biomass accumulation, growth and development, and resistance to abiotic stress, and enhancing photosynthesis is fundamental to crop quality and high yield [ 26 , 27 ]. This study, integrating photosynthetic physiological parameters, chlorophyll fluorescence parameters, the content of key photosynthetic enzymes such as RubisCO, the expression of genes related to photosynthesis, and photosynthetic energy reserves, indicates that ZnO NPs have a positive effect on the photosynthesis of tea plants. ZnO NPs improved the photosynthetic physiological parameters of tea plants. Photosynthetic physiological parameters are important indicators that directly reflect the photosynthetic capacity of plants [ 28 ], with Photo being an important indicator of plant growth capacity; Cond reflects the chloroplast's ability to utilize external CO2, and Trmmol reflects the important process of plant water circulation, which also to some extent reflects the intensity of plant growth metabolism [ 29 – 31 ]. Studies have shown that ZnO NPs can improve plant photosynthetic physiological parameters [ 32 ], and our research also shows that, except for Ci, 100mg L − 1 ZnO NPs significantly increased the Photo, Cond, and Trmmol of tea plant leaves. Unlike Cond and Trmmol, Photo showed significant differences at a concentration of 50mg L − 1 . ZnO NPs can increase the content of key photosynthetic enzymes such as RubisCO in tea plant leaves. Photosynthetic enzymes such as RubisCO, FBP, and PEPC are important hubs limiting the rate of photosynthesis. RubisCO is a major bottleneck in the photosynthesis of C3 plants, catalyzing the assimilation of CO2 in the dark reactions of photosynthesis [ 33 ]. The rate of CO2 assimilation by RubisCO in leaves depends on the amount of this enzyme [ 34 ]. In this study, the content of RubisCO in tea plant leaves was significantly increased under the influence of two concentrations of ZnO NPs. FBP plays an important role in sugar metabolism and photosynthesis. In plants, there are two isoenzymes of FBP that are essential for photosynthesis: one is located in the cytosol, involved in the synthesis of sucrose from triose phosphate; the other is in the chloroplast, involved in the regeneration of ribulose bisphosphate in the photosynthetic carbon reduction cycle [ 35 , 36 ]. ZnO NPs significantly increased the FBP content in tea plant leaves, and interestingly, the sucrose content in leaves, which is closely related to FBP, was significantly increased, and genes regulating the starch-sucrose synthesis metabolic pathway were significantly upregulated. Although PEPC is a key enzyme in the photosynthetic pathway of C4 plants, it also participates in important processes in C3 plants, such as malate synthesis, pH regulation, and replenishing tricarboxylic acid (TCA) cycle intermediates [ 37 , 38 ]. The use of ZnO NPs also increased the content of PEPC in tea plant leaves. ZnO NPs can promote the synthesis of chlorophyll in tea plant leaves and improve chlorophyll fluorescence parameters. In the light-dependent stage of photosynthesis, chlorophyll absorbs light energy by exciting electrons, allowing plants to convert light energy into chemical energy. The excited electrons are transferred to the primary electron acceptor of photosystem II reaction center, then move through the electron transport chain, ultimately leading to the reduction of the primary electron acceptor I. This process results in the synthesis of adenosine triphosphate (ATP) and reduced nicotinamide adenine dinucleotide phosphate (NADPH), which provide energy and reducing power for the synthesis of organic matter in the light-independent stage of photosynthesis [ 39 ]. The use of ZnO NPs increased the chlorophyll content in tea plant leaves, which is consistent with the results of studies on wheat, maize, and cucumber [ 22 , 40 , 41 ]. At the same time, ZnO NPs significantly increased the values of Fv/Fm, qP, and Y(II) in tea plant leaves, reflecting the positive effect of ZnO NPs on the photosystem II of tea plants. After spraying ZnO NPs, we found differential expression of many genes related to photosynthesis. KEGG enrichment analysis showed that differentially expressed genes were significantly enriched in Photosynthesis - antenna proteins, with genes regulating light-harvesting complex II chlorophyll a/b binding protein (LHCB) such as Lhca2, Lhca4, Lhca5, Lhcb1, Lhcb2, Lhcb3, and Lhcb6 all upregulated under the influence of ZnO NPs. LHCB, as the main light-harvesting complex on the thylakoid membranes of chloroplasts, accounts for about half of the total chlorophyll and is an important component of non-pigment proteins on the thylakoid membranes of green plants [ 42 ]. Its main function is to absorb and transfer light energy, mainly in the form of photons absorbed by chlorophyll pigments, to the photosystems, where a light-induced electron transport chain reaction occurs, driving photosynthesis [ 43 ]. Additionally, LHCII is involved in state transitions, a mechanism used by plants to balance the distribution of light energy between photosystem II (PSII) and photosystem I (PSI). Specifically, the activity of LHCII has been shown to affect the rate of state transitions and the macrostructure of PSII [ 44 ]. Differentially expressed genes were also significantly enriched in GO terms such as photosynthesis, light harvesting, chlorophyll binding, photosynthesis, light reaction, photosystem I, and photosynthesis, light harvesting in photosystem I, all of which are closely related to photosynthesis. ZnO NPs can promote the accumulation of photosynthetic products in tea plants. Sucrose is the main product of photosynthesis in many plants. Biochemically, as the main transport sugar, it transports carbon and energy from source leaves to several non-photosynthetic tissues (sink tissues), including roots, flowers, seeds, and fruits. Its role in photosynthesis and subsequent processes is significant and multifaceted. Moreover, studies on sugar sensing and signaling pathways indicate that sucrose can regulate the expression of genes related to photosynthesis and growth in many plant tissues [ 45 ]. Under the influence of ZnO NPs, differentially abundant metabolites in tea plant leaves were significantly enriched in Starch and sucrose metabolism, with sucrose content significantly increased under the treatment of two concentrations of ZnO NPs, and more importantly, the content of all differentially abundant metabolites in this pathway increased more than twofold. WGCNA analysis also showed that sucrose had the highest correlation with the identified key module, which also found 54 genes regulating Starch and sucrose metabolism. O2PLS identified that 7 out of the top 10 most influential metabolites were related to starch and sucrose metabolism. Various research results indicate that the use of ZnO NPs significantly increased the energy (carbon) reserves of photosynthesis. Photosynthesis and sucrose also drive the development of buds. The products of photosynthesis in green plants (sugars) act as nutritional signals to regulate the development of plant bud stem cells through the target-of-rapamycin (TOR) signaling pathway, thus promoting the sprouting of new shoots [ 46 ]. Sucrose is an early hormonal signal regulator that controls bud growth, and it determines the sustained growth of buds in a concentration-dependent manner. Additionally, sucrose can upregulate early auxin synthesis genes, increasing auxin content, thereby promoting bud development [ 47 ]. Our study also indicates that ZnO NPs can induce an increase in auxin content in tea plant leaves. Auxins play a key role in stimulating various aspects of plant growth, including bud development and germination [ 48 ]. Fundamentally, auxins trigger the breaking of dormancy by initiating cell elongation and division, essential processes in breaking bud dormancy and beginning germination. The role of auxin in breaking dormancy is central to ensuring the continuation of plant developmental cycles [ 49 ]. In this study, the content of auxin significantly increased under the influence of two concentrations, suggesting that ZnO NPs may promote the development of tea buds by regulating the increase in endogenous auxin content. The use of ZnO NPs also affected the content of other mineral elements, including zinc, in the leaves and new shoots of tea plants. The use of ZnO NPs increased the content of zinc, molybdenum, and copper elements in the leaves. Zinc mainly acts as a metal activator of enzymes in plants and plays an important role in the metabolism of carbohydrates and the synthesis of auxins. Molybdenum is also an essential trace element for plants, having a positive impact on plant nitrogen fixation, chlorophyll synthesis, photosynthesis, and sugar metabolism [ 50 ]. The presence of copper is closely related to plant photosynthesis; copper can improve the absorption and utilization efficiency of light energy in plants, thereby promoting photosynthesis and increasing the synthesis of chlorophyll [ 51 ]. Calcium and magnesium, as macronutrients required by tea plants, play an indispensable role in plant growth and development [ 52 ], and they showed a consistent expression trend under the influence of ZnO NPs. Tea plants are aluminum-accumulating plants, and excessive aluminum can cause aluminum toxicity, leading to stunted growth, yellowing of leaf tips, leaves, stems, and significantly reducing crop yield and quality [ 53 , 54 ]. The use of ZnO NPs reduced the content of aluminum in the leaves, indicating the potential of ZnO NPs in alleviating plant aluminum toxicity. The content of selenium and iron in the new shoots increased under the influence of 50mg L − 1 ZnO NPs, and selenium and iron have positive effects on tea quality and human health [ 55 , 56 ], suggesting the potential to also enhance the quality of finished tea products. The use of ZnO NPs altered the dominant community composition of epiphytic microorganisms on tea plant phyllosphere, and the dominant microbial community structure varied at different concentrations. The dominant epiphytic bacterial communities on tea plant phyllosphere without ZnO NPs treatment included Bradyrhizobium, Pseudomonas, Bacillus, Paenibacillus, Sphingomonas, Acinetobacter , and Lactococcus . Bradyrhizobium is well-known for its ability to fix atmospheric nitrogen in symbiosis with leguminous plants, enhancing their growth [ 57 ]. Pseudomonas and Bacillus are known to promote plant growth by enhancing nutrient uptake, producing phytohormones, and assisting in disease resistance [ 58 ]. Paenibacillus has also been noted to promote plant growth, improve plant nutrition, and help plants resist pathogens [ 59 ]. Sphingomonas is well characterized for its plant growth promotion ability and degradation of complex organic compounds [ 60 ]. Acinetobacter and Lactococcus have been implicated in both positive and negative interactions with plants, with some strains of Acinetobacter known for plant growth-promoting abilities, while others have been associated with plant diseases [ 61 ].The dominant epiphytic fungal communities on tea plant phyllosphere without ZnO NPs treatment included Cylindrocladiella, Cladosporium, Sterigmatomyces, Aspergillus, Penicillium, Xylaria, Phyllosticta, Acremonium, Arthothelium , and Colletotrichum . Cylindrocladiella, Acremonium, Cladosporium , and Colletotrichum are known to include plant pathogenic species that can cause diseases, impacting the health and productivity of a host plant [ 62 – 64 ]. Aspergillus and Penicillium include species that can be harmful to plants as pathogens [ 65 , 66 ]. Phyllosticta is also a plant pathogenic fungus [ 67 ]. Conversely, Xylaria possibly plays roles in nutrient cycling and potentially even growth promotion [ 68 ]. The dominant epiphytic bacterial communities on tea plant phyllosphere with 50mg L − 1 ZnO NPs treatment included Rhodococcus, Salana, Truepera, Mycobacterium, Chryseobacterium, Aureimonas, Staphylococcus, Stenotrophomonas, Aquipuribacter, Patulibacter, Arthrobacter, Nocardioides, Paracoccus, Arsenophonus, Microbacterium , and Brevundimonas. Rhodococcus and Arthrobacter can decompose plant residues, playing an important role in maintaining plant health [ 69 ]. Stenotrophomonas and Microbacterium are known beneficial organisms for plants, improving plant living conditions through hormone production and nitrogen fixation [ 70 , 71 ]. Chryseobacterium and Brevundimonas have species known to form biofilms on plant leaf surfaces, which may be related to antagonistic responses to pathogens [ 72 , 73 ]. The role of Mycobacterium on plant leaf surfaces is mainly related to its ability to degrade environmental pollutants [ 74 ].The dominant epiphytic fungal communities on tea plant phyllosphere with 50mg L − 1 ZnO NPs treatment included Candida, Fusarium, Mortierella, Guehomyces, Schizophyllum, Trametes, Lophiostoma, Marasmius, Rhodotorula, Gibberella , and Pyrenochaeta . Candida and Rhodotorula may be beneficial in dealing with environmental stress or promoting plant growth [ 75 , 76 ]. Mortierella is generally found in soil and may have a lesser impact on phyllosphere microorganisms, but reports suggest Mortierella may be beneficial for the growth of certain trees [ 77 ].The dominant epiphytic bacterial communities on tea plant phyllosphere with 100mg L − 1 ZnO NPs treatment included Arsenophonus and Microbacterium . The role of Microbacterium in plant phyllosphere microbial communities varies, with some species known to be beneficial to plants, for example, by producing various bioactive compounds beneficial to plant growth and repairing damaged plants [ 78 ]. They help to improve the plant's environment, increasing plant adaptability to environmental stress.The dominant epiphytic fungal communities on tea plant phyllosphere with 100mg L − 1 ZnO NPs treatment included Cystofilobasidium, Monographella, Mrakia, Mrakiella , and Malassezia . Cystofilobasidium 's role in plants is mainly reflected in its biocontrol potential, such as being used as a biological control agent to combat postharvest diseases of apples and citrus fruits [ 79 ]. The use of ZnO NPs also changed the dominant community composition of endophytic microorganisms in the tea plant phyllosphere, and the dominant microbial community structure varied at different concentrations. The dominant endophytic bacterial communities on tea plant phyllosphere without ZnO NPs treatment included Marinococcus, Leifsonia, Duganella, Bacteroides , and Delftia . Marinococcus can interact with plants in high-salt environments to help reduce the adverse effects of salt stress [ 80 ]. Leifsonia is a microbe related to plant growth and photosynthesis, found to affect photosynthetic parameters and the activity of defense enzymes in sugarcane [ 81 ]. Duganella can also act as a potential bioinoculant by solubilizing phosphates and promoting plant growth [ 82 ].The dominant endophytic fungal communities on tea plant phyllosphere without ZnO NPs treatment included Aureobasidium, Lalaria, Uwebraunia, Ascochyta, Ophiocordyceps, Microcyclospora, Acremonium, Cryptococcus, Sporobolomyces, Lophiostoma , and Monographella . Aureobasidium is a beneficial fungus with potential biocontrol effects against various pathogens [ 83 ]. Ascochyta is usually associated with diseases in plants, causing tissue necrosis and affecting plant growth and development, potentially leading to significant yield loss. Ascochyta fungi spread through spores between plants and can survive in the phyllosphere environment [ 84 ]. Microcyclospora may act as a pathogen in its interactions with plants; it is one of the fungi causing sooty blotch on apples and can produce triazolone compounds with significant bioactivity [ 85 ]. Acremonium species have been reported to promote plant growth and enhance plant disease resistance [ 86 ]. Sporobolomyces has been found to form symbiotic relationships on plant surfaces and may provide protection against various environmental stresses [ 87 ]. Lophiostoma exhibits antimicrobial activity and potential biocontrol activity, suggesting they may play an important role in maintaining plant health [ 88 ]. The dominant endophytic bacterial communities on tea plant phyllosphere with 50mg L − 1 ZnO NPs treatment included Bacillus, Exiguobacterium, Delftia, Enterobacter , and Mycobacterium . Bacillus is a beneficial phyllosphere microbe that can promote plant growth and photosynthesis through various mechanisms. These bacteria function by producing growth hormones, improving the absorption and utilization efficiency of nutrients, and enhancing plant stress resistance. Studies have found that Bacillus can promote the growth and yield of cucumbers by accelerating the absorption of plant nutrients and improving plant photosynthesis [ 89 ], and under salt stress conditions, Bacillus improves the growth of radish plants by increasing growth and photosynthetic pigment content [ 90 ]. Plants inoculated with Bacillus can improve gas exchange, increase CO2 assimilation (photosynthesis), and improve all growth parameters of pepper plants [ 91 ]. At the same time, plants inoculated with Exiguobacterium bacteria showed better growth and photosynthesis [ 92 ]. Delftia also has the potential to promote plant growth [ 93 ]. Enterobacter can significantly improve the growth attributes and photosynthetic mechanisms of wheat while reducing the use of chemical fertilizers without affecting the normal growth of plants [ 94 ]. Interestingly, Mycobacterium and Bacillus can promote seed germination when co-cultivated with plants, which may have a role in breaking dormancy, potentially similar to the mechanisms promoting tea bud germination [ 95 ].The dominant endophytic fungal communities on tea plant phyllosphere with 50mg L − 1 ZnO NPs treatment included S terigmatomyces, Humicola, Candida, Tilletiopsis, Colletotrichum , and Arthothelium . Apart from the potential application of Tilletiopsis in plant disease management, the other dominant fungi have not been found to play a role in plant growth [ 96 ].The dominant endophytic bacterial communities on tea plant phyllosphere with 100mg L − 1 ZnO NPs treatment included Massilia, Rhodococcus, Paenibacillus, Salana, Paracoccus, Brevundimonas, Aureimonas, Sphingomonas, Ralstonia, Pseudomonas, Pantoea, Corynebacterium, Truepera, Nocardioides, Chryseobacterium, Microbacterium, Stenotrophomonas , and Acinetobacter . Rhodococcus has shown plant growth-promoting abilities [ 97 ]. Bacteria of the Paenibacillus genus are widely studied and are of interest due to their potential in promoting plant growth and development. These bacteria can enhance plant growth and help plants resist diseases through various mechanisms, including nitrogen fixation, production of plant hormones (such as indole-3-acetic acid), and solubilization of phosphates [ 98 ]. Brevundimonas has been found to promote plant growth through various mechanisms [ 99 ]. Sphingomonas plays a positive role in plant growth and development and photosynthesis. These microbes have been found to increase plant stress resistance, accelerate plant growth, and also play a role in environmental remediation [ 100 – 102 ]. Pseudomonas has been proven to significantly promote plant growth and improve photosynthesis. These microbes can produce a variety of plant growth-promoting substances and induce responses in the host plant's defense network, primary metabolism, photosynthesis, etc. [ 103 ]. For example, Pseudomonas has been found to increase plant photosynthetic efficiency and carbon fixation capacity, as well as promote phytoremediation of heavy metals [ 104 ]. Additionally, Pseudomonas can also improve plant growth characteristics and photosynthetic pigments under salt stress conditions [ 105 ]. These studies indicate that microbes of the Pseudomonas genus are important plant growth-promoting bacteria that can improve plant growth and photosynthesis through various mechanisms. Pantoea , as an endophytic bacterium, can enhance the photosynthesis of the host plant, promote the growth of the host plant, and significantly increase the transport of photosynthetic assimilates from source to sink. At the same time, Pantoea can also enhance plant drought resistance [ 106 , 107 ]. Corynebacterium has also shown its ability to improve the photosynthetic capacity of crops [ 108 , 109 ]. Chryseobacterium can solubilize phosphates, and its combined use with nitrogen and phosphorus fertilizers can promote plant growth and increase crop yield [ 110 ]; studies have also shown that Chryseobacterium can increase the content of photosynthetic pigments in plant leaves and produce indole-3-acetic acid [ 111 , 112 ]. Microbacterium can regulate the physiological hormones and molecular characteristics of tomatoes under drought stress, significantly improving plant photosynthetic activity and biomass, indicating that Microbacterium has a positive impact on plant growth and development under adverse conditions [ 113 ]. Acinetobacter has a positive impact on plant photosynthetic characteristics (photosynthetic rate, stomatal conductance, internal CO2 concentration, and total chlorophyll content), indicating that this endophytic bacterium may promote plant growth by affecting photosynthesis [ 114 , 115 ].The dominant endophytic fungal communities on tea plant phyllosphere with 100mg L − 1 ZnO NPs treatment included Davidiella, Fusarium, Pyrenochaeta, Penicillium, Boletus, Malassezia , and Aspergillus . Penicillium is a widely present fungus that can promote plant growth and enhance plant resistance to stress by producing plant hormones and other bioactive substances [ 116 ]. Aspergillus is considered a plant growth-promoting fungus, capable of promoting plant growth and enhancing plant resistance to stress by producing plant hormones and other bioactive substances [ 117 ]. In summary, the use of ZnO NPs improved the community composition of epiphytic and endophytic microorganisms on tea plants, with many growth-promoting and photosynthesis-enhancing microbes becoming dominant populations and eliminating potentially pathogenic plant pathogens, which may be related to the antimicrobial properties of ZnO NPs. Conclusions This study investigated the effects of ZnO NPs on tea plants, focusing on photosynthesis (photosynthetic physiological parameters, photosynthetic enzymes, chlorophyll fluorescence parameters), new shoot germination, transcriptome (leaves and new shoots), metabolome (leaves and new shoots), mineral element content (leaves and new shoots), and phyllosphere microbial communities (epiphytic and endophytic microorganisms). The study showed that (1). ZnO NPs enhanced the photosynthesis of tea plants by upregulating the expression of some genes related to photosynthesis and increasing the accumulation of photosynthetic products. (2). ZnO NPs increased the auxin content in tea plants, promoting the development of new shoots. (3). ZnO NPs altered the mineral element composition of tea plants, increasing the content of zinc, molybdenum, and copper elements in the leaves, and changing the content of selenium and iron elements in the new shoots, with a minor impact on the mineral content of the new shoots. (4). ZnO NPs improved the community composition of both epiphytic and endophytic microorganisms in the tea plant phyllosphere, suppressing some potentially pathogenic microorganisms and promoting many beneficial microbes that potentially enhance plant photosynthesis and growth to become dominant populations. This research provides new insights into how ZnO NPs can improve the growth of tea plants. Abbreviations ZnO NPs, zinc oxide nanoparticles; CK, foliar spraying with pure water; CKL, tea leaves under CK treatment; CKS, tea shoots under CK treatment; CKEP, phyllosphere epiphytic microorganisms in tea leaf under CK treatment; CKED, phyllosphere endophytic microorganisms in tea leaf under CK treatment; T1, foliar spraying with 50mg L -1 ZnO NPs; T1L, tea leaves under T1 treatment; T1S, tea shoots under T1 treatment; T1EP, phyllosphere epiphytic microorganisms in tea leaf under T1 treatment; T1ED, phyllosphere endophytic microorganisms in tea leaf under T1 treatment; T2, foliar spraying with 100mg L -1 ZnO NPs; T2L, tea leaves under T2 treatment; T2S, tea shoots under T2 treatment; T2EP, phyllosphere epiphytic microorganisms in tea leaf under T2 treatment; T2ED, phyllosphere endophytic microorganisms in tea leaf under T2 treatment; Photo, net photosynthetic rate; Cond, stomatal conductance; Ci, intercellular CO2 concentration; Trmmol, transpiration rate; RubisCO, ribulose-1,5-bisphosphate carboxylase/oxygenase; FBP, fructose-1,6-bisphosphatase; PEPC, phosphoenolpyruvate carboxylase; SPAD, soil plant analysis development; chl a, chlorophyll a; chl b, chlorophyll b; ETR, relative electron transport rate; Fv/Fm, maximum photochemical efficiency of photosystem II; qP, photochemical quenching coefficient; Y(II), actual photochemical efficiency of photosystem II; WGCNA, weighted gene co-expression network analysis; DEGs, differentially expressed genes; O2PLS, two-way orthogonal partial least squares; PCA, principal component analysis; PLS, partial least squares; CCA, canonical correlation analysis; ASVs, amplicon sequence variants; OTUs, operational taxonomic units; TCA, tricarboxylic acid; ATP, adenosine triphosphate; NADPH, nicotinamide adenine dinucleotide phosphate; LHCB, light-harvesting complex II chlorophyll a/b binding protein; TOR, target-of-rapamycin. Declarations Ethics approval and consent to participate We confirmed that all methods involving the plant and its material complied with relevant institutional, national, and international guidelines and legislation. Consent for publication Not applicable Availability of data and materials The raw sequencing data were deposited in NCBI Sequence Read Archive (SRA) under accession number SUB14254443 for transcriptome, SUB14256009 for bacteria and SUB14256035 for fungi. Competing interests The authors declare that they have no competing interests. Funding The research was funded by the Natural Science Foundation of China (32002087), the Technology System of Modern Agricultural Industry in Shandong Province (SDAIT-19-01), the Livelihood Project of Qingdao City (21-1-4-ny-2-nsh), the Special Talent Program of SAAS (CXGC2023A11), the Agricultural Improved Variety Project of Shandong Province(2020LZGC010). 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Plant Physiology and Biochemistry. 2011;49:852–61. Additional Declarations No competing interests reported. Supplementary Files Additionalfile1.pdf Additionalfile2.pdf Additionalfile3.pdf Table.S1.xlsx Table.S2.xlsx Table.S3.xls Table.S4.xlsx Table.S5.xlsx Cite Share Download PDF Status: Published Journal Publication published 01 Jul, 2024 Read the published version in Journal of Nanobiotechnology → Version 1 posted Editorial decision: Revision requested 09 Apr, 2024 Reviews received at journal 27 Mar, 2024 Reviewers agreed at journal 17 Mar, 2024 Reviewers invited by journal 16 Mar, 2024 Submission checks completed at journal 07 Mar, 2024 Editor assigned by journal 07 Mar, 2024 First submitted to journal 05 Mar, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4019055","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":277099870,"identity":"57ee7ea4-c985-4139-be69-590a112d330a","order_by":0,"name":"Hao Chen","email":"","orcid":"","institution":"Qingdao Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Hao","middleName":"","lastName":"Chen","suffix":""},{"id":277099871,"identity":"710b1619-acfd-4ed8-a986-2546569dd5ae","order_by":1,"name":"Yujie Song","email":"","orcid":"","institution":"Qingdao Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Yujie","middleName":"","lastName":"Song","suffix":""},{"id":277099872,"identity":"663c5dfa-e7e4-4fd9-ab14-c5b304a6a921","order_by":2,"name":"Yu Wang","email":"","orcid":"","institution":"Qingdao Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Yu","middleName":"","lastName":"Wang","suffix":""},{"id":277099873,"identity":"44ea5c1c-31e6-47f7-8b79-7880a90c6c2f","order_by":3,"name":"Huan Wang","email":"","orcid":"","institution":"Qingdao Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Huan","middleName":"","lastName":"Wang","suffix":""},{"id":277099874,"identity":"2ed192fd-3c43-4331-9313-b9529323749f","order_by":4,"name":"Zhaotang Ding","email":"","orcid":"","institution":"Shandong Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Zhaotang","middleName":"","lastName":"Ding","suffix":""},{"id":277099875,"identity":"346ef45d-f51d-4e71-8270-a6f5fe1042a9","order_by":5,"name":"Kai Fan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyklEQVRIiWNgGAWjYBAC9gYGZgYGAxCrsfHhB2K08BwAawHq4TncbCxBvBaQNRLpbQI8RGlhP3vY4EfBHzn+mQ/bGCQY7OR0Gwhp4clLTuwxMDCWuJ3Y9qCAIdnY7AABLfYMOcYHeAwMEhtuJ7YbSDAcSNxGSAsP/xvjg38MDOrn3zzYJsFDlBaJHONkoC0JBjcYidbyxthYxsDYcOOZRGAgGxDhFx7+HGPJN3/k5OWOH3/48EOFnRxBLWjAgDTlo2AUjIJRMApwAADfWT4cN8QDawAAAABJRU5ErkJggg==","orcid":"","institution":"Qingdao Agricultural University","correspondingAuthor":true,"prefix":"","firstName":"Kai","middleName":"","lastName":"Fan","suffix":""}],"badges":[],"createdAt":"2024-03-06 02:48:59","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4019055/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4019055/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12951-024-02667-2","type":"published","date":"2024-07-02T00:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":52516282,"identity":"b5b6d01f-f4f5-4081-a18f-e94de2f5046a","added_by":"auto","created_at":"2024-03-12 12:59:52","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1652753,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of ZnO NPs on photosynthetic physiological parameters (A), photosynthetic enzyme content (B), nitrogen content, sprouting rate, and sprouting status of new shoots (C) in tea plants. Data were analyzed using one-way ANOVA, where \"ns\" indicates p ≥ 0.05 (not significant); \"*\" indicates p \u0026lt; 0.05 (significant); \"**\" indicates p \u0026lt; 0.01 (more significant); \"***\" indicates p \u0026lt; 0.001 (highly significant); \"****\" indicates p \u0026lt; 0.0001 (extremely significant).\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-4019055/v1/04c6eaa867b8447a09f1a4f8.png"},{"id":52517255,"identity":"2b234736-a967-4aaa-9a4f-ca44f7f93f65","added_by":"auto","created_at":"2024-03-12 13:23:52","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":646079,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of ZnO NPs on chlorophyll content (A) and chlorophyll fluorescence parameters (B) in tea plants. Data were analyzed using one-way ANOVA, where \"ns\" indicates p ≥ 0.05 (not significant); \"*\" indicates p \u0026lt; 0.05 (significant); \"**\" indicates p \u0026lt; 0.01 (more significant); \"***\" indicates p \u0026lt; 0.001 (highly significant); \"****\" indicates p \u0026lt; 0.0001 (extremely significant).\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-4019055/v1/ba40f31ac40a2faf5720a54c.png"},{"id":52516281,"identity":"1bd38391-973d-4e2b-ae4a-89169fb29ef6","added_by":"auto","created_at":"2024-03-12 12:59:52","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1211461,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of ZnO NPs on the content of mineral elements in tea leaves (A) and new shoots (B). Data were analyzed using one-way ANOVA, where \"ns\" indicates p ≥ 0.05 (not significant); \"*\" indicates p \u0026lt; 0.05 (significant); \"**\" indicates p \u0026lt; 0.01 (more significant); \"***\" indicates p \u0026lt; 0.001 (highly significant); \"****\" indicates p \u0026lt; 0.0001 (extremely significant). \"FW\" stands for fresh weight. The content of elements in tea leaves (C) and new shoots (D) under the influence of ZnO NPs is normalized at the sample level, reflecting the trends and similarities in substance variation across different samples.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-4019055/v1/2bc1722ae24f43c292de3b1d.png"},{"id":52516954,"identity":"9f55562f-25b1-4dc0-8acd-e0b55669125f","added_by":"auto","created_at":"2024-03-12 13:15:52","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2019098,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression levels of genes related to Photosynthesis - antenna proteins under the influence of ZnO NPs (A). Expression levels of genes and metabolites related to starch and sucrose metabolism under the influence of ZnO NPs (B). Expression levels of genes and metabolites related to the auxin synthesis pathway under the influence of ZnO NPs (C).\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-4019055/v1/fe51449bc8ffa4f2a7b12a07.png"},{"id":52516499,"identity":"08e166ed-41b2-4363-a464-62325c0199ca","added_by":"auto","created_at":"2024-03-12 13:07:52","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2042351,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHierarchical clustering of gene networks in tea leaves under the influence of ZnO NPs and key candidate genes with co-expressed genes in 14 modules. Each leaflet on the tree corresponds to an individual gene (A), with the horizontal axis representing metabolite names and the vertical axis with each color representing a module. The deeper the color (deep red or deep blue), the stronger the correlation between the metabolite and the module, and the lighter the color, the weaker the correlation between the sample and the module. Red indicates a positive correlation, while blue indicates a negative correlation (B).\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-4019055/v1/b115807691448b2402b99933.png"},{"id":52516287,"identity":"a30d34fe-c426-40a1-9c71-4ef6bd4459c6","added_by":"auto","created_at":"2024-03-12 12:59:52","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":3410442,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIntegrated analysis of gene and metabolite expression in tea leaves and new shoots based on O2PLS under the influence of ZnO NPs. The top ten genes with the greatest impact on metabolite changes in tea leaves and their expression levels (A); the top ten metabolites with the greatest impact on gene expression in tea leaves and their expression levels (B); the top ten genes with the greatest impact on metabolite changes in new shoots and their expression levels (C); the top ten metabolites with the greatest impact on gene expression in new shoots and their expression levels (D); white boxes represent metabolites not detected in the treatment group (ZnO NPs); black boxes represent substances not detected in the control group; gray boxes represent substances not detected in both the treatment and control groups.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Fig.6.png","url":"https://assets-eu.researchsquare.com/files/rs-4019055/v1/aeacea45c545051189b9a9c1.png"},{"id":52516502,"identity":"4fcbbd35-af18-47b6-b26c-c98600e110ac","added_by":"auto","created_at":"2024-03-12 13:07:52","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":423601,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVenn diagrams showing the ASV clustering of epiphytic bacteria (A) and fungi (B) in the phyllosphere of tea plants under the influence of ZnO NPs.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Fig.7.png","url":"https://assets-eu.researchsquare.com/files/rs-4019055/v1/3af60a77dc769c9f7e83ee02.png"},{"id":52516294,"identity":"6cc5d7d5-5f1c-4c64-becc-2e0b376c628a","added_by":"auto","created_at":"2024-03-12 12:59:52","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1009727,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHeatmaps of ASV-based clustering of epiphytic bacterial (A) and fungal (B) communities in the phyllosphere of tea plants under the influence of ZnO NPs. Vertically, the heatmaps display sample information, and horizontally, they show species classification information. The clustering trees within the figure represent species clustering; the values in the heatmap correspond to Z-Score normalized relative quantitative data.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Fig.8.png","url":"https://assets-eu.researchsquare.com/files/rs-4019055/v1/1ddbc9e7fb899b48d9f500c2.png"},{"id":52516505,"identity":"6e172c77-4359-453e-b3a6-203922c15e86","added_by":"auto","created_at":"2024-03-12 13:07:53","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":352924,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVenn diagrams showing the ASV clustering of endophytic bacteria (A) and fungi (B) in the phyllosphere of tea plants under the influence of ZnO NPs.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Fig.9.png","url":"https://assets-eu.researchsquare.com/files/rs-4019055/v1/19a9ff94cca44cd572b0e919.png"},{"id":52516286,"identity":"b59fbb6e-3459-4cc1-b959-c420d8a8f8ae","added_by":"auto","created_at":"2024-03-12 12:59:52","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":984885,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHeatmaps of ASV-based clustering of endophytic bacterial (A) and fungal (B) communities in the phyllosphere of tea plants under the influence of ZnO NPs. Vertically, the heatmaps display sample information, and horizontally, they show species classification information. The clustering trees within the figure represent species clustering; the values in the heatmap correspond to Z-Score normalized relative quantitative data.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Fig.10.png","url":"https://assets-eu.researchsquare.com/files/rs-4019055/v1/7f7cef2cb2c0aa4253bac069.png"},{"id":52516291,"identity":"599c9d53-6555-4470-82b7-c70065749665","added_by":"auto","created_at":"2024-03-12 12:59:52","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":1279692,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSpearman correlation between endophytic bacteria (A) and fungi (B) in the phyllosphere of tea plants under the influence of ZnO NPs and differential starch and sucrose metabolism products. The selected threshold for p-value is 0.05, and for cor.value is 0.80.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Fig.11.png","url":"https://assets-eu.researchsquare.com/files/rs-4019055/v1/33ff933ee84e1bff23cbafbf.png"},{"id":96636837,"identity":"ceaf4999-e248-49cb-99c1-838b1f6772c3","added_by":"auto","created_at":"2025-11-24 13:39:14","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":18979061,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4019055/v1/c197bc65-f2dd-4b07-a9eb-c6181ec9236b.pdf"},{"id":52516292,"identity":"4969f459-374c-429c-8241-8ee93fe8a9e6","added_by":"auto","created_at":"2024-03-12 12:59:52","extension":"pdf","order_by":13,"title":"","display":"","copyAsset":false,"role":"supplement","size":1841591,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4019055/v1/81b8ef8749467887a55486c0.pdf"},{"id":52516504,"identity":"b41930f8-10c6-4ec4-a02b-784469673004","added_by":"auto","created_at":"2024-03-12 13:07:52","extension":"pdf","order_by":14,"title":"","display":"","copyAsset":false,"role":"supplement","size":816859,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4019055/v1/5a0680b92c36b6ccf50745cc.pdf"},{"id":52516300,"identity":"5a065878-8d80-4673-bd81-f627b6420aae","added_by":"auto","created_at":"2024-03-12 12:59:53","extension":"pdf","order_by":15,"title":"","display":"","copyAsset":false,"role":"supplement","size":809225,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile3.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4019055/v1/9fb440744402b6965cfb94b8.pdf"},{"id":52516503,"identity":"5c5f9c54-d409-4cbf-bf8d-7397c16e0cef","added_by":"auto","created_at":"2024-03-12 13:07:52","extension":"xlsx","order_by":16,"title":"","display":"","copyAsset":false,"role":"supplement","size":18761,"visible":true,"origin":"","legend":"","description":"","filename":"Table.S1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4019055/v1/d616de9c1fda96ff78782d9b.xlsx"},{"id":52516956,"identity":"3cdc7586-0d85-4b6f-82a2-12f96dd786df","added_by":"auto","created_at":"2024-03-12 13:15:53","extension":"xlsx","order_by":17,"title":"","display":"","copyAsset":false,"role":"supplement","size":74580,"visible":true,"origin":"","legend":"","description":"","filename":"Table.S2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4019055/v1/9e0fdc31610096ffc1b2d1d8.xlsx"},{"id":52516290,"identity":"58110a88-0579-49fc-a2e0-5d33394e69e8","added_by":"auto","created_at":"2024-03-12 12:59:52","extension":"xls","order_by":18,"title":"","display":"","copyAsset":false,"role":"supplement","size":1010,"visible":true,"origin":"","legend":"","description":"","filename":"Table.S3.xls","url":"https://assets-eu.researchsquare.com/files/rs-4019055/v1/641fc9729299966768f0385d.xls"},{"id":52516295,"identity":"970f73df-1e93-4414-ad0f-0dd6f34d571b","added_by":"auto","created_at":"2024-03-12 12:59:52","extension":"xlsx","order_by":19,"title":"","display":"","copyAsset":false,"role":"supplement","size":131690,"visible":true,"origin":"","legend":"","description":"","filename":"Table.S4.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4019055/v1/a0104776aaa3b493db432d4f.xlsx"},{"id":52516297,"identity":"ba62151c-a702-447a-90b7-2e0667abd556","added_by":"auto","created_at":"2024-03-12 12:59:53","extension":"xlsx","order_by":20,"title":"","display":"","copyAsset":false,"role":"supplement","size":69802,"visible":true,"origin":"","legend":"","description":"","filename":"Table.S5.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4019055/v1/bab32fbe15966db8f20caac2.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"ZnO NPs Enhanced Photosynthetic Capacity, Promoted New Shoot Development, and Improved the Community Composition of Phyllosphere Epiphytic and Endophytic Microorganisms in Tea Plants","fulltext":[{"header":"Background","content":"\u003cp\u003eNanotechnology is becoming an innovative tool in the field of agriculture, with broad application prospects. Compared to traditional materials, nanomaterials' advantages in agriculture are mainly reflected in their unique size effects and surface effects. These effects endow nanomaterials with better solubility, stability, and bioavailability, which play significant roles in promoting crop growth, pest and disease control, and environmental pollution remediation [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePhotosynthesis is the foundation of growth, development, and yield formation in tea plants. It is not only the key process to produce biomass and energy storage in tea plants but also directly affects the quality and yield of tea leaves [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. During the growth cycle of tea plants, the efficiency of photosynthesis determines the ability to convert light energy into chemical energy, thereby affecting the growth rate of tea plants and the final yield of tea leaves. The importance of photosynthesis for tea plants is also reflected in its impact on the synthesis of secondary metabolites. For example, the strength of photosynthetic capacity directly affects the synthesis of bioactive substances such as catechins in tea leaves, which are key factors determining the quality of tea [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. At the same time, the sprouting of tea buds plays an important role in the life cycle of tea plants. From a biological perspective, the budding process is the cornerstone of plant growth, overall development, and reproduction [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. From an economic perspective, tea buds are the main source of the economic value of tea plants and the raw material for producing premium finished tea. The earlier the tea buds sprout, the greater the economic value they can generate to some extent. Therefore, regulating the better sprouting of tea buds is our goal [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Thus, improving the efficiency of photosynthesis and promoting the sprouting of new shoots are crucial for tea production.\u003c/p\u003e \u003cp\u003eThe positive effects of ZnO NPs on plants have been confirmed in multiple studies. For instance, ZnO NPs have been shown to increase the growth rate and biomass of crops [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], enhance the efficiency of photosynthesis in plants [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], and improve the antioxidant system of plants [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. These positive effects not only improve the overall health of plants but may also increase crop yield by enhancing the absorption and utilization rate of nutrients. At the same time, ZnO NPs have been found to increase the zinc content and quality of wheat grains, enhance the aroma and nutritional components of rice, and strengthen rice resistance to blast disease [\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Zinc plays multiple biological roles within plants, being a component of many enzymes and involved in nitrogen metabolism, protein synthesis, plant hormone regulation, and antioxidative defense [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Therefore, the supply of zinc is crucial for plant growth and development. As a novel source of zinc, ZnO NPs not only provide the zinc required by plants but also enhance the utilization efficiency of zinc through nanoscale effects [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. However, the impact of ZnO NPs on tea plants, especially in relation to photosynthesis, remains unknown.\u003c/p\u003e \u003cp\u003eMicroorganisms residing in the phyllosphere play a crucial role in plant productivity and health [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] The microbial composition living on the surface of the phyllosphere is known as epiphytic microorganisms, while those residing within the phyllosphere tissues (either in the intercellular spaces, i.e., the apoplast, or inside plant cells) are referred to as endophytic microorganisms (or phyllosphere endophytes). The same microorganism may be both an epiphyte and an endophyte, occupying both ecological niches [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Phyllosphere microorganisms can directly or indirectly affect plant growth through nitrogen fixation, phosphate solubilization, synthesis of growth hormones, and production of disease-resistant substances [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Additionally, phyllosphere microorganisms can affect plant photosynthesis and influence the plant's efficiency in utilizing light energy through interactions with the plant [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Regulating the microbial community of the tea plant phyllosphere is very important, but compared to soil microorganisms, there is less research on tea plant phyllosphere microorganisms.\u003c/p\u003e \u003cp\u003ePhyllosphere microorganisms (especially epiphytes) are highly susceptible to external environmental influences. Environmental factors such as climate conditions, atmospheric pollution, soil quality, and human agricultural activities can have direct or indirect effects on phyllosphere microorganisms. Chemicals used in agricultural activities can affect the diversity and activity of microbial communities by altering the chemical environment of the plant leaf surface [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The application of nanomaterials is considered an effective means to regulate the composition of phyllosphere microbial communities. Studies have found that nanomaterials can affect plant growth by altering the community structure and function of phyllosphere microorganisms [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. However, the impact of ZnO NPs on the microbial community of the tea plant phyllosphere remains unknown.\u003c/p\u003e \u003cp\u003eThis article explores the effects of ZnO NPs on tea plant photosynthesis, sprouting of new shoots, mineral element content, and phyllosphere microbial communities (including both epiphytic and endophytic microorganisms). We hypothesize that ZnO NPs will have a positive impact on the growth of tea plants. At the same time, due to the strong bactericidal properties of ZnO NPs, we speculate that they will change the composition of the tea plant phyllosphere microbial community, potentially eliminating some potential plant pathogens residing in the phyllosphere. This study helps to understand the behavior and effects of ZnO NPs within plants, and through this research, we hope to provide a more in-depth and comprehensive scientific basis for the application of nanomaterials in sustainable agricultural development.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlant Materials\u003c/h2\u003e \u003cp\u003eIn this study, one-year-old \u003cem\u003eCamellia sinensis\u003c/em\u003e cv. Shuchazao tea plants were selected as the research subjects. The tea plants were planted in a nutrient-rich nursery substrate with an organic matter content of about 60%, a total porosity of about 75%, a bulk density of about 0.35%, and a pH of about 5.5. The nursery substrate was provided by Shouguang Yixiandu Agricultural Science and Technology Co., Ltd. (Heze, China). The tea plants were grown under a photoperiod of 14 hours of light and 10 hours of darkness, with a daytime temperature of 28\u0026deg;C, a nighttime temperature of 22\u0026deg;C, light intensity of 10000lx, and air humidity of 75%.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eExperimental Treatments\u003c/h2\u003e \u003cp\u003eReferring to the application of ZnO NPs on plants, three concentrations were selected for foliar spraying on tea plants: 0 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (CK), 50 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (T1), and 100 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (T2) [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. A total of 432 tea plants were planted, with 144 plants for each treatment, divided into 8 groups, with 18 plants per group. The zinc oxide nanoparticles used in this experiment had a particle size of 30 nm and a purity of more than 99.9%, provided by Shanghai Macklin Biochemical Co., Ltd. (Shanghai, China). Seven days after planting the tea plants, foliar spraying treatments were conducted every three days, for a total of three times. In this study, the first mature leaf below the new shoot was selected for research.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMeasurement of Photosynthetic Physiological Parameters, Photosynthetic Enzyme Content, Nitrogen Content, and Sprouting Rate of New Shoots\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe photosynthetic physiological parameters of tea plants (net photosynthetic rate (Photo), stomatal conductance (Cond), intercellular CO2 concentration (Ci), and transpiration rate (Trmmol)) were measured using a portable photosynthesis system (LI-6400XT, LI-COR, Inc. Lincoln, NE, USA). The measurements were taken at 9:00 AM, with an air flow rate set to 500 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, a CO2 concentration of 400 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, and light intensity consistent with the growth conditions of the tea plants. Twelve plants were randomly selected from each treatment for measurement.\u003c/p\u003e \u003cp\u003eThe contents of ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO), fructose-1,6-bisphosphatase (FBP), and phosphoenolpyruvate carboxylase (PEPC) were measured by Genepioneer Biotechnologies (Nanjing, China) using research reagent kits (ELISA method), provided by Jiangsu Jingmei Biotechnology Co., Ltd. (Yancheng, China) (product numbers A-P0018B, A-P0357B, and A-P0104B), with ten replicates for each treatment.\u003c/p\u003e \u003cp\u003eLeaf nitrogen content was measured using a plant nutrition analyzer (TYS-4H, Top Cloud-Agri, Zhejiang, China), with sixteen replicates for each treatment.\u003c/p\u003e \u003cp\u003eThe sprouting rate of new shoots was determined by counting the number of sprouted shoots in each of the eight groups of tea plants (each group consisting of 18 plants) per treatment, with a shoot considered sprouted once it was fully expanded.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eMeasurement of Chlorophyll Content and Chlorophyll Fluorescence Parameters\u003c/h2\u003e \u003cp\u003eChlorophyll content was measured using a research reagent kit provided by Suzhou Grace Biotechnology Co., Ltd. (Suzhou, China) (product number G0601W), with ten replicates for each treatment. The soil plant analysis development (SPAD) value was measured using a plant nutrition analyzer (TYS-4H, Top Cloud-Agri, Zhejiang, China), with sixteen replicates for each treatment.\u003c/p\u003e \u003cp\u003eChlorophyll fluorescence parameters were measured using a chlorophyll fluorescence imaging system (IMAGING-PAM, WALZ, Effeltrich, Germany). Leaves were dark-adapted for 30 minutes, and the instrument was calibrated for light intensity and Abs. The actinic light was set to 5, with other parameters set to the default values of the program. Four plants were randomly selected from each treatment for measurement.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eMeasurement of Mineral Elements in Leaves and New Shoots\u003c/h2\u003e \u003cp\u003eThe content of mineral elements in leaves and new shoots was determined by atomic absorption spectrophotometry, with three biological replicates per treatment. Weigh 1.0 g (accurate to 0.0001 g) of fresh tea plant samples into a polytetrafluoroethylene digestion vessel, add 5 mL of nitric acid, and soak overnight. Cover with the inner lid, tighten the stainless-steel jacket, and place in a constant temperature drying oven at 80\u0026deg;C for 1.5 hours, 120\u0026deg;C for 1.5 hours, then raise to 160\u0026deg;C for 4 hours, and allow to cool naturally to room temperature in the oven. Transfer the digestion liquid into a 25 mL volumetric flask, rinse the digestion vessel and inner lid three times with a small amount of 1% nitric acid solution, combine the rinses in the volumetric flask, and make up to the mark with 1% nitric acid, mix well and set aside. A reagent blank is also prepared. The sample solution is measured for element concentration c1 (mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) by an inductively coupled plasma emission spectrometer (iCAP7400, Thermo Fisher Scientific, Waltham, USA). The content of elements in the sample (mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u0026thinsp;=\u0026thinsp;c1V1 m\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, where c1 is the concentration of the element measured by the instrument, V1 is the volume of digestion (mL), and m is the mass of the sample (g).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eTranscriptome Measurement of Leaves and New Shoots\u003c/h2\u003e \u003cp\u003eThe transcriptome sequencing process includes total RNA extraction, mRNA enrichment, double-stranded cDNA synthesis, end repair, A-tailing and adapter ligation, fragment selection and PCR amplification, library quality assessment, and Illumina sequencing. Three biological replicates were measured for each treatment.\u003c/p\u003e \u003cp\u003eTo ensure high-quality RNA and the absence of DNA contamination, the extracted RNA was rigorously analyzed for integrity and precisely tested for purity and concentration. The research process began with RNA samples prepared using the NEBNext Ultra RNA Library Prep Kit for Illumina. Purified fragments underwent PCR, and their quality was evaluated using the Agilent Bioanalyzer 2100 system. Index-coded samples were clustered on a cBot Cluster Generation System and sequenced to generate 150 bp paired-end reads. The data was filtered using fastp to produce 'clean reads'. Clean reads were compared to the reference genome using HISAT. Gene prediction was performed with StringTie, which provided faster and more accurate transcript splicing. FeatureCounts assisted in gene alignment, and the FPKM of each gene was calculated based on their specific length.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eExtensive Targeted Metabolome Measurement of Leaves and New Shoots\u003c/h2\u003e \u003cp\u003eBiological samples were processed using vacuum freeze-drying technology in a lyophilizer (Scientz-100F), followed by grinding at 30 Hz for 1.5 minutes using a grinder (MM 400, Retsch). 50 mg of the resulting sample powder was weighed with an electronic balance (MS105DΜ) and mixed with 1200 \u0026micro;L of pre-cooled 70% methanolic aqueous solution containing an internal standard. The mixture was subjected to intermittent vortexing and centrifugation. After centrifugation, the supernatant was separated, filtered, and prepared for UPLC-MS/MS analysis.\u003c/p\u003e \u003cp\u003eUnder UPLC conditions, an UPLC-ESI-MS/MS system (UPLC, ExionLC\u0026trade; AD, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://sciex.com.cn/\u003c/span\u003e\u003cspan address=\"https://sciex.com.cn/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and a tandem mass spectrometry system (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://sciex.com.cn/\u003c/span\u003e\u003cspan address=\"https://sciex.com.cn/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) were used to analyze the sample extracts. A precise mobile phase gradient was employed using solvent A (pure water with 0.1% formic acid) and solvent B (acetonitrile with 0.1% formic acid). Precise flow rate, temperature, and injection volumes were maintained throughout the process.\u003c/p\u003e \u003cp\u003eThe operating parameters of the ESI-Q TRAP-MS/MS included preset values for source temperature, ion spray voltage, gas pressures, and collision-activated dissociation levels. The QQQ scans were performed as MRM experiments, with DP and CE adjusted for individual MRM transitions. Specific MRM transitions were closely monitored throughout the experiment, aimed at identifying the metabolites eluted during the period.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eMeasurement of Epiphytic and Endophytic Microorganisms in the Phyllosphere\u003c/h2\u003e \u003cp\u003eThis study utilized 16S rDNA amplicon sequencing to measure epiphytic and endophytic bacteria and Internal Transcribed Spacer (ITS) sequencing for epiphytic and endophytic fungi [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eExtraction of Epiphytic Microorganisms\u003c/h2\u003e \u003cp\u003eFor each sample, 15 g of tea leaves (approximately 50 leaves) were placed in a sterile tube, to which 150 mL of potassium phosphate buffer (0.1 mol L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, pH\u0026thinsp;=\u0026thinsp;8) was added. The samples were ultrasonicated for 1 minute, vortexed for 10 seconds, and the ultrasonication and vortexing were repeated twice. The washed leaves were removed, potassium phosphate buffer was added again, and the ultrasonication and vortexing steps were repeated. The washing liquid was filtered through a 0.22 \u0026micro;m filter membrane, which was then flash-frozen in liquid nitrogen and stored at -80\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eExtraction of Endophytic Microorganisms\u003c/h2\u003e \u003cp\u003eThe leaf surfaces were successively soaked in 75% ethanol for 1 minute, 3.25% sodium hypochlorite for 3 minutes, and 75% ethanol for 30 seconds, followed by rinsing three times with sterile distilled water. The leaves were then freeze-dried in liquid nitrogen [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eExtraction of Genomic DNA and PCR Amplification\u003c/h2\u003e \u003cp\u003eGenomic DNA was extracted from the samples using the CTAB method. The DNA's purity and concentration were checked using agarose gel electrophoresis. An appropriate amount of sample DNA was taken in a centrifuge tube and diluted with sterile water to 1 ng \u0026micro;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The diluted genomic DNA was used as a template, and PCR was performed using barcode-specific primers, Phusion\u0026reg; High-Fidelity PCR Master Mix with GC Buffer from New England Biolabs, and a high-efficiency high-fidelity enzyme to ensure amplification efficiency and accuracy.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eMixing and Purification of PCR Products\u003c/h2\u003e \u003cp\u003eThe PCR products were checked by electrophoresis on a 2% agarose gel; qualified PCR products were purified with magnetic beads, quantified enzymatically, and mixed in equal amounts based on the concentration of the PCR products. After thorough mixing, the PCR products were checked again on a 2% agarose gel, and the target bands were recovered using a gel recovery kit provided by Qiagen.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eLibrary Construction and Sequencing\u003c/h2\u003e \u003cp\u003eLibraries were constructed using the TruSeq\u0026reg; DNA PCR-Free Sample Preparation Kit, quantified by Qubit and Q-PCR, and sequenced on a NovaSeq6000 after passing quality control.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eSequencing Data Processing\u003c/h2\u003e \u003cp\u003eSample data were demultiplexed from the raw data based on barcode sequences and PCR primer sequences, and the barcode and primer sequences were trimmed. High-quality reads were obtained by filtering the raw reads using fastp (v0.22.0, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/OpenGene/fastp\u003c/span\u003e\u003cspan address=\"https://github.com/OpenGene/fastp\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative Real-Time PCR Validation\u003c/h2\u003e \u003cp\u003eReverse transcription amplification was performed using the SynScript\u0026reg; III RT SuperMix for qPCR reverse transcription kit produced by Beijing Biotech Co., Ltd. (Beijing, China). The resulting cDNA products were diluted fourfold and used as templates for qPCR amplification with Tsingke ArtiCanCEO SYBR qPCR Mix. CsGAPDH was used as the reference gene, and gene expression was calculated using the 2\u003csup\u003e\u0026minus;ΔΔCT\u003c/sup\u003e method. The qRT-PCR samples underwent three biological replicates and three technical replicates. All primers used in this study are listed in Table.S1.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eEffects of ZnO NPs on Photosynthetic Physiological Parameters, Photosynthetic Enzyme Content, Nitrogen Content, and Sprouting Rate of New Shoots in Tea Plants\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the effects of ZnO NPs on photosynthesis and new shoot development in tea plants, we measured the Photo, Cond, Ci, Trmmol, RubisCO content, FBP content, PEPC content, nitrogen content, and sprouting rate of new shoots. As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, except for Ci, ZnO NPs significantly increased the Photo, Cond, and Trmmol of tea plants, with the most significant effects observed at a concentration of 100 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA). Simultaneously, ZnO NPs significantly increased the content of photosynthetic enzymes in the leaves, indirectly indicating stronger activities of RubisCO, FBP, and PEPC (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB). Additionally, the nitrogen content in tea leaves also increased under the influence of ZnO NPs (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC). Interestingly, ZnO NPs also improved the sprouting rate of new shoots, although the two different concentrations did not show significant differences in their effects on the sprouting rate (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eD). In summary, ZnO NPs significantly enhanced the photosynthetic capacity of tea leaves and the sprouting rate of new shoots.\u003c/p\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n\u003ch2\u003eEffects of ZnO NPs on Chlorophyll Content and Chlorophyll Fluorescence Parameters in Tea Plants\u003c/h2\u003e\n\u003cp\u003eTo explore the impact of ZnO NPs on the chlorophyll content and chlorophyll fluorescence parameters in tea plants, we measured the chlorophyll content, SPAD values, and chlorophyll fluorescence parameters in tea leaves (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). The combined measurements of total chlorophyll, chlorophyll a (chl a), chlorophyll b (chl b), and SPAD values indicate that ZnO NPs can increase the chlorophyll content in tea leaves (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA). In terms of chlorophyll fluorescence parameters, aside from the relative electron transport rate (ETR), ZnO NPs significantly enhanced the maximum photochemical efficiency of photosystem II (Fv/Fm), the photochemical quenching coefficient (qP), and the actual photochemical efficiency of photosystem II (Y(II)) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB). In summary, both the chlorophyll content and chlorophyll fluorescence parameters demonstrate that ZnO NPs can significantly improve the photosynthetic capacity of tea leaves.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffects of ZnO NPs on the Content of Mineral Elements in Tea Leaves and New Shoots\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo explore the impact of applying one mineral element (ZnO NPs) on other minerals in tea leaves and new shoots, we measured the content of eight mineral elements related to the growth and quality of tea plants (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). Under the influence of nano zinc oxide, the zinc content in tea leaves increased, but a significant difference was only observed at the concentration of 100 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (T2). Interestingly, the zinc content in new shoots did not significantly increase, which could be speculated that ZnO NPs were sprayed before the sprouting of new shoots, and nano zinc oxide did not directly contact the new shoots, nor was there sufficient time for the zinc element to be transported from the leaves to the new shoots. Selenium showed significant differences under the influence of nano zinc oxide; at the concentration of 50 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (T1), the selenium content in the leaves significantly decreased, while it significantly increased in the new shoots, suggesting that at this concentration, ZnO NPs promoted the transfer of selenium from the leaves to the new shoots. This difference was not observed at T2. Meanwhile, as the concentration of nano zinc oxide increased, the molybdenum content in the leaves significantly increased, but the molybdenum content in the new shoots significantly decreased at T2. The copper content in the leaves significantly increased under T2, but no significant difference was observed in the copper content in the new shoots. At the same time, no significant changes were found in the iron content in the leaves, but the iron content in the new shoots significantly increased at T1. The magnesium content in the leaves significantly increased at T1, with no significant differences observed in the new shoots. Similar to magnesium, the calcium content in both leaves and new shoots showed a consistent expression trend, with a significant increase in calcium content in the leaves only at T1. Interestingly, calcium and magnesium, as macronutrients required by tea plants, exhibited a consistent expression trend. Unlike the other mineral elements, the aluminum content in the leaves significantly decreased at T2, with no significant differences observed in the new shoots. In summary, ZnO NPs had a significant impact on the content of mineral elements in tea leaves, with a greater effect on the leaves than on the new shoots.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIntegrated Analysis of Differential Gene Expression and Differential Metabolite Expression Reveals the Intrinsic Mechanism Behind ZnO NPs Enhancing Photosynthetic Capacity and Promoting New Shoot Development in Tea Plants\u003c/strong\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\n\u003ch2\u003ePhotosynthesis-Related Pathways and Endogenous Auxin Pathway Metabolites and Gene Expression\u003c/h2\u003e\n\u003cp\u003eDifferential gene expression in tea shoots and leaves under the influence of ZnO NPs, KEGG enrichment analysis of differential genes, GO enrichment analysis, and qRT-PCR analysis; differential metabolites in leaves and new shoots and KEGG enrichment analysis of differential metabolites are presented in Additional file 1. To comprehensively elucidate the response of tea plants to ZnO NPs from both gene expression and metabolite perspectives, we conducted an integrated analysis of the transcriptome and metabolome.\u003c/p\u003e\n\u003cp\u003eUnder the treatment of two concentrations of ZnO NPs, tea leaves were significantly enriched in pathways related to photosynthesis, specifically Photosynthesis - antenna proteins (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA). In this pathway, all the differentially expressed genes compared to the control were significantly upregulated, mainly regulating the light-harvesting complex II chlorophyll a/b binding proteins (Lhca2, Lhca4, Lhca5, Lhcb1, Lhcb2, Lhcb3, and Lhcb6). Interestingly, in the Starch and sucrose metabolism pathway closely related to photosynthesis, all differential metabolites such as Sucrose, D-Glucose-6P, D-Fructose-6P, D-Glucose-6P, etc., showed a significant increase in content. We also presented the significant gene expression levels related to this pathway (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB). Furthermore, under both concentrations of ZnO NPs treatment, the endogenous auxin content was significantly increased, and the gene and metabolite expression levels in the related metabolic pathway are shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWeighted Gene Co-expression Network Analysis (WGCNA) of Differential Genes and Key Differential Metabolites (Identification of coexpressed gene networks and key candidates)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further understand the regulation of sucrose metabolism changes in tea leaves caused by ZnO NPs, WGCNA was conducted to study the co-expression network of differentially expressed genes (DEGs). Based on similar expression patterns, a total of 14 co-expressed modules were identified (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e). Genes preferentially expressed in sugar metabolism-related significant differential metabolites were mainly concentrated in the MEblue module, with 11 metabolites having a correlation R\u003csup\u003e2\u003c/sup\u003e greater than 0.8 with this module. The key product of sugar metabolism, Sucrose, also had the highest correlation with this module (R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.86, p\u0026thinsp;=\u0026thinsp;0.0027). Within the MEblue module, 54 genes related to starch and sucrose metabolism were identified, including 11 regulating glucose-1-phosphate adenylyltransferase (\u003cem\u003eCSS0000089, CSS0026130, CSS0026865, CSS0032913, CSS0035261, CSS0035419, CSS0050226, novel.1469, novel.4393, novel.4625, novel.7765\u003c/em\u003e); 2 regulating glucose-6-phosphate isomerase (\u003cem\u003eCSS0002435, CSS0045493\u003c/em\u003e); 2 regulating trehalose 6-phosphate synthase/phosphatase (\u003cem\u003eCSS0002735, CSS0035721\u003c/em\u003e); 5 regulating beta-glucosidase (\u003cem\u003eCSS0003115, CSS0006278, CSS0016696, CSS0017477, CSS0036292\u003c/em\u003e); 6 regulating beta-amylase (\u003cem\u003eCSS0003801, CSS0018030, CSS0027311, CSS0032302, CSS0043627, CSS0047759\u003c/em\u003e); 3 regulating granule-bound starch synthase (\u003cem\u003eCSS0004941, CSS0037992, CSS0045869\u003c/em\u003e); 5 regulating starch synthase (\u003cem\u003eCSS0005914, CSS0021129, CSS0023626, CSS00248447, novel.5440\u003c/em\u003e); 5 regulating glucan endo-1,3-beta-glucosidase 1/2/3 (\u003cem\u003eCSS0008156, CSS0012190, CSS0043010, CSS0048079, CSS0050142\u003c/em\u003e); 1 regulating alpha,alpha-trehalase (\u003cem\u003eCSS0008297\u003c/em\u003e); 1 regulating alpha-amylase (\u003cem\u003eCSS0008836\u003c/em\u003e); 3 regulating trehalose 6-phosphate phosphatase (\u003cem\u003eCSS0009951, CSS0045703, novel.736\u003c/em\u003e); 3 regulating glycogen phosphorylase (\u003cem\u003eCSS0015277, CSS0022613, CSS0038552\u003c/em\u003e); 2 regulating 1,4-alpha-glucan branching enzyme (\u003cem\u003eCSS0016120, novel.877\u003c/em\u003e); 1 regulating sucrose-phosphate synthase (\u003cem\u003eCSS0024623\u003c/em\u003e); 1 regulating sucrose-6-phosphatase (\u003cem\u003eCSS0028296\u003c/em\u003e); 2 regulating beta-fructofuranosidase (\u003cem\u003eCSS0033878, novel.2722\u003c/em\u003e); 1 regulating ADP-sugar diphosphatase (\u003cem\u003eCSS0042147\u003c/em\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\n\u003ch2\u003eTwo-way Orthogonal Partial Least Squares (O2PLS) Analysis\u003c/h2\u003e\n\u003cp\u003eTo uncover the internal connections between gene expression and metabolite changes in tea plants in response to ZnO NPs, and to determine the degree of association between them, as well as to identify the main genes and metabolites causing this association, we conducted an O2PLS analysis (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e). O2PLS is an unsupervised modeling technique that can objectively describe whether there is a correlation trend between two sets of data, avoiding false-positive associations from the outset. Compared to traditional supervised models such as PCA (Principal Component Analysis), PLS (Partial Least Squares), and CCA (Canonical Correlation Analysis), O2PLS considers factors such as the size, scale, distribution, and experimental errors of datasets in different scenarios. The modeling process takes into account the joint, specific, and residual parts between different datasets, making it suitable for data mining in complex scenarios.\u003c/p\u003e\n\u003cp\u003eUnder the influence of ZnO NPs, the top ten genes affecting changes in metabolites in tea leaves and their expression levels are shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eA, while the top ten metabolites affecting transcript expression in tea leaves and their expression levels are shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eB. Similarly, the top ten genes affecting changes in metabolites in new shoots and their expression levels are shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eC, and the top ten metabolites affecting transcript expression in new shoots and their expression levels are shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eD. Interestingly, among the top ten metabolites with the greatest impact on transcript expression in tea leaves, seven are related to starch and sucrose metabolism.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalysis of the Phyllosphere Epiphytic Microbial Structure of Tea Plants Under the Influence of ZnO NPs\u003c/strong\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\n\u003ch2\u003eASV Clustering Analysis of Epiphytic Bacteria and Fungi\u003c/h2\u003e\n\u003cp\u003eTo investigate the community composition of epiphytic microbes in the phyllosphere of tea plants under the influence of ZnO NPs, we performed denoising analysis on effective data to generate Amplicon Sequence Variants (ASVs). Based on the ASV analysis results, each ASV sequence was annotated to obtain corresponding species information. Compared to traditional Operational Taxonomic Units (OTUs) clustering analysis, ASV analysis does not lose taxonomic resolution and can accurately distinguish sequences above the similarity threshold. As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eA, under the influence of different concentrations of ZnO NPs, tea plants shared 692 ASVs of epiphytic bacteria, with 3901 unique ASVs in CKEP, 4966 in T1EP, and 3901 in T2EP. As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eB, under the influence of different concentrations of ZnO NPs, tea plants shared 55 ASVs of epiphytic fungi, with 270 unique ASVs in CKED, 346 in T1ED, and 115 in T2ED. The clustering results indicate that different concentrations of ZnO NPs significantly changed the community structure of epiphytic bacteria and fungi. For the selection of differential epiphytic microbes under the influence of ZnO NPs, random forest analysis, and functional prediction, see Additional file 2.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\n\u003ch2\u003eGenus-level Clustering Analysis of Epiphytic Bacteria and Fungi\u003c/h2\u003e\n\u003cp\u003eTo analyze the differences in the community composition of epiphytic microbes in the phyllosphere of tea plants under different concentrations of ZnO NPs, we performed clustering analysis at the genus level based on quantitative information, facilitating the discovery of microbial population aggregation under different treatments. As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eA, the bacterial communities in CKEP were mainly clustered in genera such as \u003cem\u003eMarinococcus, Devosia, Pseudomonas, Hyphomicrobium, Bryobacter, Acinetobacter, Bradyrhizobium, Lactococcus, Sphingomonas, Cutibacterium, Paenibacillus\u003c/em\u003e, and \u003cem\u003eBacillus\u003c/em\u003e. The bacterial communities in T1EP were mainly clustered in \u003cem\u003eRhodococcus\u003c/em\u003e, unidentified \u003cem\u003eBeijerinckiaceae\u003c/em\u003e, \u003cem\u003eSalana, Truepera, Mycobacterium, Chryseobacterium, Aureimonas, Staphylococcus\u003c/em\u003e, unidentified \u003cem\u003eCyanobacteria\u003c/em\u003e, \u003cem\u003eStenotrophomonas\u003c/em\u003e, unidentified \u003cem\u003eHalomonadaceae, Aquipuribacter\u003c/em\u003e, unidentified \u003cem\u003eRhizobiaceae, Patulibacter, Arthrobacter, Nocardioides, Paracoccus, Arsenophonus, Microbacterium\u003c/em\u003e, and \u003cem\u003eBrevundimonas\u003c/em\u003e. The bacterial communities in T2EP were enriched in Arsenophonus and Microbacterium. As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eB, the fungal communities in CKEP were mainly clustered in \u003cem\u003eCylindrocladiella\u003c/em\u003e, unclassified \u003cem\u003eHypocreales, Cladosporium, Sterigmatomyces, Aspergillus, Penicillium, Xylaria, Phyllosticta, Acremonium, Arthothelium\u003c/em\u003e, and \u003cem\u003eColletotrichum\u003c/em\u003e. The fungal communities in T1EP were mainly clustered in \u003cem\u003eCandida, Fusarium, Mortierella, Cryptococcus\u003c/em\u003e (\u003cem\u003ef Filobasidiaceae\u003c/em\u003e), \u003cem\u003eGuehomyces\u003c/em\u003e, unclassified \u003cem\u003ePhaeosphaeriaceae, Schizophyllum, Trametes, Cryptococcus\u003c/em\u003e (\u003cem\u003eTremellales family Incertae sedis\u003c/em\u003e), \u003cem\u003eLophiostoma, Marasmius\u003c/em\u003e, unclassified \u003cem\u003eFungi, Rhodotorula, Gibberella\u003c/em\u003e, and \u003cem\u003ePyrenochaeta\u003c/em\u003e. The fungal communities in T2EP were mainly enriched in unclassified \u003cem\u003ePleosporales, Cystofilobasidium, Monographella, Mrakia, Mrakiella, and Malassezia\u003c/em\u003e. The clustering results show significant differences in the dominant microbial species, indicating that different concentrations of ZnO NPs have selective effects on epiphytic bacteria and fungi.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalysis of the Phyllosphere Endophytic Microbial Structure of Tea Plants Under the Influence of ZnO NPs\u003c/strong\u003e\u003c/p\u003e\n\u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\n\u003ch2\u003eASV Clustering Analysis of Endophytic Bacteria and Fungi\u003c/h2\u003e\n\u003cp\u003eTo study the community composition of endophytic microbes in the phyllosphere of tea plants under the influence of ZnO NPs, similar to epiphytic microbes, we performed ASV clustering analysis. As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003e, under the influence of different concentrations of ZnO NPs, tea plants shared 16 ASVs of endophytic bacteria, with 58 unique ASVs in CKED, 47 in T1ED, and 161 in T2ED. The figure also shows that under the influence of different concentrations of ZnO NPs, tea plants shared 112 ASVs of endophytic fungi, with 595 unique ASVs in CKED, 186 in T1ED, and 317 in T2ED. The clustering results indicate that different concentrations of ZnO NPs significantly changed the community structure of endophytic bacteria and fungi. For the selection of differential endophytic microbes under the influence of ZnO NPs, random forest analysis, and functional prediction, see Additional file 3.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGenus-level Clustering Analysis of Endophytic Bacteria and Fungi and Their Association with Starch and Sucrose Metabolism Products\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo analyze the differences in the community composition of endophytic microbes in the phyllosphere of tea plants under different concentrations of ZnO NPs, we performed clustering analysis at the genus level. As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e10\u003c/span\u003eA, the bacterial communities in CKED were mainly clustered in \u003cem\u003eMarinococcus, Leifsonia, Duganella, Bacteroides\u003c/em\u003e, and \u003cem\u003eDelftia\u003c/em\u003e. The bacterial communities in T1ED were mainly clustered in \u003cem\u003eBacillus, Exiguobacterium, Delftia, Enterobacter, Mycobacterium, and\u003c/em\u003e unidentified \u003cem\u003eBurkholderiaceae\u003c/em\u003e. The bacterial communities in T2ED were mainly clustered in \u003cem\u003eMassilia, Rhodococcus, Paenibacillus, Salana, Paracoccus, Brevundimonas, Aureimonas, Sphingomonas, Ralstonia, Pseudomonas, Pantoea, Corynebacterium, Truepera, Nocardioides, Chryseobacterium, Microbacterium\u003c/em\u003e, unidentified \u003cem\u003eRhizobiaceae, Stenotrophomonas\u003c/em\u003e, and \u003cem\u003eAcinetobacter\u003c/em\u003e. As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e10\u003c/span\u003eB, the fungal communities in CKED were mainly clustered in \u003cem\u003eAureobasidium, Lalaria\u003c/em\u003e, unclassified \u003cem\u003eTaphrinaceae, Uwebraunia, Ascochyta, Ophiocordyceps, Microcyclospora, Acremonium, Cryptococcus (Filobasidiaceae), Sporobolomyces, Lophiostoma, Monographella\u003c/em\u003e, and \u003cem\u003eCryptococcus\u003c/em\u003e (\u003cem\u003eTremellales family Incertae sedis\u003c/em\u003e). The fungal communities in T1ED were mainly clustered in S\u003cem\u003eterigmatomyces, Humicola, Candida, Tilletiopsis, Colletotrichum\u003c/em\u003e, and \u003cem\u003eArthothelium\u003c/em\u003e. The fungal communities in T2ED were mainly enriched in \u003cem\u003eDavidiella, Fusarium, Pyrenochaeta, Penicillium\u003c/em\u003e, unclassified \u003cem\u003eHypocreales, Boletus, Malassezia, and Aspergillus\u003c/em\u003e. The clustering results show significant differences in the dominant microbial species, indicating that different concentrations of ZnO NPs have selective effects on endophytic bacteria and fungi.\u003c/p\u003e\n\u003cp\u003eTo investigate the correlation between endophytic microbes in the phyllosphere of tea plants and differential starch and sucrose metabolism products under the influence of ZnO NPs, we conducted a Spearman correlation hierarchical clustering analysis (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e11\u003c/span\u003e). Several endophytic microbes showed significant correlations with sugar metabolism (with fungi demonstrating stronger correlations). Specific correlation coefficients and significance values between each microbe and sugars are presented in Table.S2. \u003cem\u003eNotably, Taphrina, Cylindrocladiella, Aspergillus, Boletus, Malassezia, Cladosporium, Xenocylindrocladium, Cordyceps\u003c/em\u003e, and \u003cem\u003ePyrenochaeta\u003c/em\u003e showed significant positive correlations with sucrose.\u003c/p\u003e\n\u003c/div\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003ePhotosynthesis plays a crucial role in plant energy storage, biomass accumulation, growth and development, and resistance to abiotic stress, and enhancing photosynthesis is fundamental to crop quality and high yield [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. This study, integrating photosynthetic physiological parameters, chlorophyll fluorescence parameters, the content of key photosynthetic enzymes such as RubisCO, the expression of genes related to photosynthesis, and photosynthetic energy reserves, indicates that ZnO NPs have a positive effect on the photosynthesis of tea plants.\u003c/p\u003e \u003cp\u003eZnO NPs improved the photosynthetic physiological parameters of tea plants. Photosynthetic physiological parameters are important indicators that directly reflect the photosynthetic capacity of plants [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], with Photo being an important indicator of plant growth capacity; Cond reflects the chloroplast's ability to utilize external CO2, and Trmmol reflects the important process of plant water circulation, which also to some extent reflects the intensity of plant growth metabolism [\u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Studies have shown that ZnO NPs can improve plant photosynthetic physiological parameters [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], and our research also shows that, except for Ci, 100mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e ZnO NPs significantly increased the Photo, Cond, and Trmmol of tea plant leaves. Unlike Cond and Trmmol, Photo showed significant differences at a concentration of 50mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eZnO NPs can increase the content of key photosynthetic enzymes such as RubisCO in tea plant leaves. Photosynthetic enzymes such as RubisCO, FBP, and PEPC are important hubs limiting the rate of photosynthesis. RubisCO is a major bottleneck in the photosynthesis of C3 plants, catalyzing the assimilation of CO2 in the dark reactions of photosynthesis [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The rate of CO2 assimilation by RubisCO in leaves depends on the amount of this enzyme [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. In this study, the content of RubisCO in tea plant leaves was significantly increased under the influence of two concentrations of ZnO NPs. FBP plays an important role in sugar metabolism and photosynthesis. In plants, there are two isoenzymes of FBP that are essential for photosynthesis: one is located in the cytosol, involved in the synthesis of sucrose from triose phosphate; the other is in the chloroplast, involved in the regeneration of ribulose bisphosphate in the photosynthetic carbon reduction cycle [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. ZnO NPs significantly increased the FBP content in tea plant leaves, and interestingly, the sucrose content in leaves, which is closely related to FBP, was significantly increased, and genes regulating the starch-sucrose synthesis metabolic pathway were significantly upregulated. Although PEPC is a key enzyme in the photosynthetic pathway of C4 plants, it also participates in important processes in C3 plants, such as malate synthesis, pH regulation, and replenishing tricarboxylic acid (TCA) cycle intermediates [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. The use of ZnO NPs also increased the content of PEPC in tea plant leaves.\u003c/p\u003e \u003cp\u003eZnO NPs can promote the synthesis of chlorophyll in tea plant leaves and improve chlorophyll fluorescence parameters. In the light-dependent stage of photosynthesis, chlorophyll absorbs light energy by exciting electrons, allowing plants to convert light energy into chemical energy. The excited electrons are transferred to the primary electron acceptor of photosystem II reaction center, then move through the electron transport chain, ultimately leading to the reduction of the primary electron acceptor I. This process results in the synthesis of adenosine triphosphate (ATP) and reduced nicotinamide adenine dinucleotide phosphate (NADPH), which provide energy and reducing power for the synthesis of organic matter in the light-independent stage of photosynthesis [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. The use of ZnO NPs increased the chlorophyll content in tea plant leaves, which is consistent with the results of studies on wheat, maize, and cucumber [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. At the same time, ZnO NPs significantly increased the values of Fv/Fm, qP, and Y(II) in tea plant leaves, reflecting the positive effect of ZnO NPs on the photosystem II of tea plants.\u003c/p\u003e \u003cp\u003eAfter spraying ZnO NPs, we found differential expression of many genes related to photosynthesis. KEGG enrichment analysis showed that differentially expressed genes were significantly enriched in Photosynthesis - antenna proteins, with genes regulating light-harvesting complex II chlorophyll a/b binding protein (LHCB) such as Lhca2, Lhca4, Lhca5, Lhcb1, Lhcb2, Lhcb3, and Lhcb6 all upregulated under the influence of ZnO NPs. LHCB, as the main light-harvesting complex on the thylakoid membranes of chloroplasts, accounts for about half of the total chlorophyll and is an important component of non-pigment proteins on the thylakoid membranes of green plants [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Its main function is to absorb and transfer light energy, mainly in the form of photons absorbed by chlorophyll pigments, to the photosystems, where a light-induced electron transport chain reaction occurs, driving photosynthesis [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Additionally, LHCII is involved in state transitions, a mechanism used by plants to balance the distribution of light energy between photosystem II (PSII) and photosystem I (PSI). Specifically, the activity of LHCII has been shown to affect the rate of state transitions and the macrostructure of PSII [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Differentially expressed genes were also significantly enriched in GO terms such as photosynthesis, light harvesting, chlorophyll binding, photosynthesis, light reaction, photosystem I, and photosynthesis, light harvesting in photosystem I, all of which are closely related to photosynthesis.\u003c/p\u003e \u003cp\u003eZnO NPs can promote the accumulation of photosynthetic products in tea plants. Sucrose is the main product of photosynthesis in many plants. Biochemically, as the main transport sugar, it transports carbon and energy from source leaves to several non-photosynthetic tissues (sink tissues), including roots, flowers, seeds, and fruits. Its role in photosynthesis and subsequent processes is significant and multifaceted. Moreover, studies on sugar sensing and signaling pathways indicate that sucrose can regulate the expression of genes related to photosynthesis and growth in many plant tissues [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Under the influence of ZnO NPs, differentially abundant metabolites in tea plant leaves were significantly enriched in Starch and sucrose metabolism, with sucrose content significantly increased under the treatment of two concentrations of ZnO NPs, and more importantly, the content of all differentially abundant metabolites in this pathway increased more than twofold. WGCNA analysis also showed that sucrose had the highest correlation with the identified key module, which also found 54 genes regulating Starch and sucrose metabolism. O2PLS identified that 7 out of the top 10 most influential metabolites were related to starch and sucrose metabolism. Various research results indicate that the use of ZnO NPs significantly increased the energy (carbon) reserves of photosynthesis.\u003c/p\u003e \u003cp\u003ePhotosynthesis and sucrose also drive the development of buds. The products of photosynthesis in green plants (sugars) act as nutritional signals to regulate the development of plant bud stem cells through the target-of-rapamycin (TOR) signaling pathway, thus promoting the sprouting of new shoots [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Sucrose is an early hormonal signal regulator that controls bud growth, and it determines the sustained growth of buds in a concentration-dependent manner. Additionally, sucrose can upregulate early auxin synthesis genes, increasing auxin content, thereby promoting bud development [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Our study also indicates that ZnO NPs can induce an increase in auxin content in tea plant leaves. Auxins play a key role in stimulating various aspects of plant growth, including bud development and germination [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Fundamentally, auxins trigger the breaking of dormancy by initiating cell elongation and division, essential processes in breaking bud dormancy and beginning germination. The role of auxin in breaking dormancy is central to ensuring the continuation of plant developmental cycles [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. In this study, the content of auxin significantly increased under the influence of two concentrations, suggesting that ZnO NPs may promote the development of tea buds by regulating the increase in endogenous auxin content.\u003c/p\u003e \u003cp\u003eThe use of ZnO NPs also affected the content of other mineral elements, including zinc, in the leaves and new shoots of tea plants. The use of ZnO NPs increased the content of zinc, molybdenum, and copper elements in the leaves. Zinc mainly acts as a metal activator of enzymes in plants and plays an important role in the metabolism of carbohydrates and the synthesis of auxins. Molybdenum is also an essential trace element for plants, having a positive impact on plant nitrogen fixation, chlorophyll synthesis, photosynthesis, and sugar metabolism [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. The presence of copper is closely related to plant photosynthesis; copper can improve the absorption and utilization efficiency of light energy in plants, thereby promoting photosynthesis and increasing the synthesis of chlorophyll [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Calcium and magnesium, as macronutrients required by tea plants, play an indispensable role in plant growth and development [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e], and they showed a consistent expression trend under the influence of ZnO NPs. Tea plants are aluminum-accumulating plants, and excessive aluminum can cause aluminum toxicity, leading to stunted growth, yellowing of leaf tips, leaves, stems, and significantly reducing crop yield and quality [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. The use of ZnO NPs reduced the content of aluminum in the leaves, indicating the potential of ZnO NPs in alleviating plant aluminum toxicity. The content of selenium and iron in the new shoots increased under the influence of 50mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e ZnO NPs, and selenium and iron have positive effects on tea quality and human health [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e], suggesting the potential to also enhance the quality of finished tea products.\u003c/p\u003e \u003cp\u003eThe use of ZnO NPs altered the dominant community composition of epiphytic microorganisms on tea plant phyllosphere, and the dominant microbial community structure varied at different concentrations. The dominant epiphytic bacterial communities on tea plant phyllosphere without ZnO NPs treatment included \u003cem\u003eBradyrhizobium, Pseudomonas, Bacillus, Paenibacillus, Sphingomonas, Acinetobacter\u003c/em\u003e, and \u003cem\u003eLactococcus\u003c/em\u003e. \u003cem\u003eBradyrhizobium\u003c/em\u003e is well-known for its ability to fix atmospheric nitrogen in symbiosis with leguminous plants, enhancing their growth [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. \u003cem\u003ePseudomonas\u003c/em\u003e and \u003cem\u003eBacillus\u003c/em\u003e are known to promote plant growth by enhancing nutrient uptake, producing phytohormones, and assisting in disease resistance [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. \u003cem\u003ePaenibacillus\u003c/em\u003e has also been noted to promote plant growth, improve plant nutrition, and help plants resist pathogens [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. \u003cem\u003eSphingomonas\u003c/em\u003e is well characterized for its plant growth promotion ability and degradation of complex organic compounds [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. \u003cem\u003eAcinetobacter\u003c/em\u003e and \u003cem\u003eLactococcus\u003c/em\u003e have been implicated in both positive and negative interactions with plants, with some strains of Acinetobacter known for plant growth-promoting abilities, while others have been associated with plant diseases [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e].The dominant epiphytic fungal communities on tea plant phyllosphere without ZnO NPs treatment included \u003cem\u003eCylindrocladiella, Cladosporium, Sterigmatomyces, Aspergillus, Penicillium, Xylaria, Phyllosticta, Acremonium, Arthothelium\u003c/em\u003e, and \u003cem\u003eColletotrichum\u003c/em\u003e. \u003cem\u003eCylindrocladiella, Acremonium, Cladosporium\u003c/em\u003e, and \u003cem\u003eColletotrichum\u003c/em\u003e are known to include plant pathogenic species that can cause diseases, impacting the health and productivity of a host plant [\u003cspan additionalcitationids=\"CR63\" citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. \u003cem\u003eAspergillus\u003c/em\u003e and \u003cem\u003ePenicillium\u003c/em\u003e include species that can be harmful to plants as pathogens [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e, \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. \u003cem\u003ePhyllosticta\u003c/em\u003e is also a plant pathogenic fungus [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]. Conversely, \u003cem\u003eXylaria\u003c/em\u003e possibly plays roles in nutrient cycling and potentially even growth promotion [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe dominant epiphytic bacterial communities on tea plant phyllosphere with 50mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e ZnO NPs treatment included \u003cem\u003eRhodococcus, Salana, Truepera, Mycobacterium, Chryseobacterium, Aureimonas, Staphylococcus, Stenotrophomonas, Aquipuribacter, Patulibacter, Arthrobacter, Nocardioides, Paracoccus, Arsenophonus, Microbacterium\u003c/em\u003e, and \u003cem\u003eBrevundimonas. Rhodococcus\u003c/em\u003e and \u003cem\u003eArthrobacter\u003c/em\u003e can decompose plant residues, playing an important role in maintaining plant health [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]. \u003cem\u003eStenotrophomonas\u003c/em\u003e and \u003cem\u003eMicrobacterium\u003c/em\u003e are known beneficial organisms for plants, improving plant living conditions through hormone production and nitrogen fixation [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e, \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e]. \u003cem\u003eChryseobacterium\u003c/em\u003e and \u003cem\u003eBrevundimonas\u003c/em\u003e have species known to form biofilms on plant leaf surfaces, which may be related to antagonistic responses to pathogens [\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e, \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e]. The role of \u003cem\u003eMycobacterium\u003c/em\u003e on plant leaf surfaces is mainly related to its ability to degrade environmental pollutants [\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e].The dominant epiphytic fungal communities on tea plant phyllosphere with 50mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e ZnO NPs treatment included \u003cem\u003eCandida, Fusarium, Mortierella, Guehomyces, Schizophyllum, Trametes, Lophiostoma, Marasmius, Rhodotorula, Gibberella\u003c/em\u003e, and \u003cem\u003ePyrenochaeta\u003c/em\u003e. \u003cem\u003eCandida\u003c/em\u003e and \u003cem\u003eRhodotorula\u003c/em\u003e may be beneficial in dealing with environmental stress or promoting plant growth [\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e, \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e]. \u003cem\u003eMortierella\u003c/em\u003e is generally found in soil and may have a lesser impact on phyllosphere microorganisms, but reports suggest \u003cem\u003eMortierella\u003c/em\u003e may be beneficial for the growth of certain trees [\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e].The dominant epiphytic bacterial communities on tea plant phyllosphere with 100mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e ZnO NPs treatment included \u003cem\u003eArsenophonus\u003c/em\u003e and \u003cem\u003eMicrobacterium\u003c/em\u003e. The role of \u003cem\u003eMicrobacterium\u003c/em\u003e in plant phyllosphere microbial communities varies, with some species known to be beneficial to plants, for example, by producing various bioactive compounds beneficial to plant growth and repairing damaged plants [\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e]. They help to improve the plant's environment, increasing plant adaptability to environmental stress.The dominant epiphytic fungal communities on tea plant phyllosphere with 100mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e ZnO NPs treatment included \u003cem\u003eCystofilobasidium, Monographella, Mrakia, Mrakiella\u003c/em\u003e, and \u003cem\u003eMalassezia\u003c/em\u003e. \u003cem\u003eCystofilobasidium\u003c/em\u003e's role in plants is mainly reflected in its biocontrol potential, such as being used as a biological control agent to combat postharvest diseases of apples and citrus fruits [\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe use of ZnO NPs also changed the dominant community composition of endophytic microorganisms in the tea plant phyllosphere, and the dominant microbial community structure varied at different concentrations. The dominant endophytic bacterial communities on tea plant phyllosphere without ZnO NPs treatment included \u003cem\u003eMarinococcus, Leifsonia, Duganella, Bacteroides\u003c/em\u003e, and \u003cem\u003eDelftia\u003c/em\u003e. \u003cem\u003eMarinococcus\u003c/em\u003e can interact with plants in high-salt environments to help reduce the adverse effects of salt stress [\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e]. \u003cem\u003eLeifsonia\u003c/em\u003e is a microbe related to plant growth and photosynthesis, found to affect photosynthetic parameters and the activity of defense enzymes in sugarcane [\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e]. \u003cem\u003eDuganella\u003c/em\u003e can also act as a potential bioinoculant by solubilizing phosphates and promoting plant growth [\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e].The dominant endophytic fungal communities on tea plant phyllosphere without ZnO NPs treatment included \u003cem\u003eAureobasidium, Lalaria, Uwebraunia, Ascochyta, Ophiocordyceps, Microcyclospora, Acremonium, Cryptococcus, Sporobolomyces, Lophiostoma\u003c/em\u003e, and \u003cem\u003eMonographella\u003c/em\u003e. \u003cem\u003eAureobasidium\u003c/em\u003e is a beneficial fungus with potential biocontrol effects against various pathogens [\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e]. \u003cem\u003eAscochyta\u003c/em\u003e is usually associated with diseases in plants, causing tissue necrosis and affecting plant growth and development, potentially leading to significant yield loss. \u003cem\u003eAscochyta\u003c/em\u003e fungi spread through spores between plants and can survive in the phyllosphere environment [\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e]. \u003cem\u003eMicrocyclospora\u003c/em\u003e may act as a pathogen in its interactions with plants; it is one of the fungi causing sooty blotch on apples and can produce triazolone compounds with significant bioactivity [\u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e]. \u003cem\u003eAcremonium\u003c/em\u003e species have been reported to promote plant growth and enhance plant disease resistance [\u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e]. \u003cem\u003eSporobolomyces\u003c/em\u003e has been found to form symbiotic relationships on plant surfaces and may provide protection against various environmental stresses [\u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e87\u003c/span\u003e]. \u003cem\u003eLophiostoma\u003c/em\u003e exhibits antimicrobial activity and potential biocontrol activity, suggesting they may play an important role in maintaining plant health [\u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e88\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe dominant endophytic bacterial communities on tea plant phyllosphere with 50mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e ZnO NPs treatment included \u003cem\u003eBacillus, Exiguobacterium, Delftia, Enterobacter\u003c/em\u003e, and \u003cem\u003eMycobacterium\u003c/em\u003e. \u003cem\u003eBacillus\u003c/em\u003e is a beneficial phyllosphere microbe that can promote plant growth and photosynthesis through various mechanisms. These bacteria function by producing growth hormones, improving the absorption and utilization efficiency of nutrients, and enhancing plant stress resistance. Studies have found that \u003cem\u003eBacillus\u003c/em\u003e can promote the growth and yield of cucumbers by accelerating the absorption of plant nutrients and improving plant photosynthesis [\u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e89\u003c/span\u003e], and under salt stress conditions, \u003cem\u003eBacillus\u003c/em\u003e improves the growth of radish plants by increasing growth and photosynthetic pigment content [\u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e90\u003c/span\u003e]. Plants inoculated with \u003cem\u003eBacillus\u003c/em\u003e can improve gas exchange, increase CO2 assimilation (photosynthesis), and improve all growth parameters of pepper plants [\u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e91\u003c/span\u003e]. At the same time, plants inoculated with \u003cem\u003eExiguobacterium\u003c/em\u003e bacteria showed better growth and photosynthesis [\u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e92\u003c/span\u003e]. \u003cem\u003eDelftia\u003c/em\u003e also has the potential to promote plant growth [\u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e93\u003c/span\u003e]. \u003cem\u003eEnterobacter\u003c/em\u003e can significantly improve the growth attributes and photosynthetic mechanisms of wheat while reducing the use of chemical fertilizers without affecting the normal growth of plants [\u003cspan citationid=\"CR94\" class=\"CitationRef\"\u003e94\u003c/span\u003e]. Interestingly, \u003cem\u003eMycobacterium\u003c/em\u003e and \u003cem\u003eBacillus\u003c/em\u003e can promote seed germination when co-cultivated with plants, which may have a role in breaking dormancy, potentially similar to the mechanisms promoting tea bud germination [\u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e95\u003c/span\u003e].The dominant endophytic fungal communities on tea plant phyllosphere with 50mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e ZnO NPs treatment included S\u003cem\u003eterigmatomyces, Humicola, Candida, Tilletiopsis, Colletotrichum\u003c/em\u003e, and \u003cem\u003eArthothelium\u003c/em\u003e. Apart from the potential application of \u003cem\u003eTilletiopsis\u003c/em\u003e in plant disease management, the other dominant fungi have not been found to play a role in plant growth [\u003cspan citationid=\"CR96\" class=\"CitationRef\"\u003e96\u003c/span\u003e].The dominant endophytic bacterial communities on tea plant phyllosphere with 100mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e ZnO NPs treatment included \u003cem\u003eMassilia, Rhodococcus, Paenibacillus, Salana, Paracoccus, Brevundimonas, Aureimonas, Sphingomonas, Ralstonia, Pseudomonas, Pantoea, Corynebacterium, Truepera, Nocardioides, Chryseobacterium, Microbacterium, Stenotrophomonas\u003c/em\u003e, and \u003cem\u003eAcinetobacter\u003c/em\u003e. \u003cem\u003eRhodococcus\u003c/em\u003e has shown plant growth-promoting abilities [\u003cspan citationid=\"CR97\" class=\"CitationRef\"\u003e97\u003c/span\u003e]. \u003cem\u003eBacteria\u003c/em\u003e of the \u003cem\u003ePaenibacillus\u003c/em\u003e genus are widely studied and are of interest due to their potential in promoting plant growth and development. These bacteria can enhance plant growth and help plants resist diseases through various mechanisms, including nitrogen fixation, production of plant hormones (such as indole-3-acetic acid), and solubilization of phosphates [\u003cspan citationid=\"CR98\" class=\"CitationRef\"\u003e98\u003c/span\u003e]. \u003cem\u003eBrevundimonas\u003c/em\u003e has been found to promote plant growth through various mechanisms [\u003cspan citationid=\"CR99\" class=\"CitationRef\"\u003e99\u003c/span\u003e]. \u003cem\u003eSphingomonas\u003c/em\u003e plays a positive role in plant growth and development and photosynthesis. These microbes have been found to increase plant stress resistance, accelerate plant growth, and also play a role in environmental remediation [\u003cspan additionalcitationids=\"CR101\" citationid=\"CR100\" class=\"CitationRef\"\u003e100\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR102\" class=\"CitationRef\"\u003e102\u003c/span\u003e]. \u003cem\u003ePseudomonas\u003c/em\u003e has been proven to significantly promote plant growth and improve photosynthesis. These microbes can produce a variety of plant growth-promoting substances and induce responses in the host plant's defense network, primary metabolism, photosynthesis, etc. [\u003cspan citationid=\"CR103\" class=\"CitationRef\"\u003e103\u003c/span\u003e]. For example, \u003cem\u003ePseudomonas\u003c/em\u003e has been found to increase plant photosynthetic efficiency and carbon fixation capacity, as well as promote phytoremediation of heavy metals [\u003cspan citationid=\"CR104\" class=\"CitationRef\"\u003e104\u003c/span\u003e]. Additionally, \u003cem\u003ePseudomonas\u003c/em\u003e can also improve plant growth characteristics and photosynthetic pigments under salt stress conditions [\u003cspan citationid=\"CR105\" class=\"CitationRef\"\u003e105\u003c/span\u003e]. These studies indicate that microbes of the Pseudomonas genus are important plant growth-promoting bacteria that can improve plant growth and photosynthesis through various mechanisms. \u003cem\u003ePantoea\u003c/em\u003e, as an endophytic bacterium, can enhance the photosynthesis of the host plant, promote the growth of the host plant, and significantly increase the transport of photosynthetic assimilates from source to sink. At the same time, \u003cem\u003ePantoea\u003c/em\u003e can also enhance plant drought resistance [\u003cspan citationid=\"CR106\" class=\"CitationRef\"\u003e106\u003c/span\u003e, \u003cspan citationid=\"CR107\" class=\"CitationRef\"\u003e107\u003c/span\u003e]. \u003cem\u003eCorynebacterium\u003c/em\u003e has also shown its ability to improve the photosynthetic capacity of crops [\u003cspan citationid=\"CR108\" class=\"CitationRef\"\u003e108\u003c/span\u003e, \u003cspan citationid=\"CR109\" class=\"CitationRef\"\u003e109\u003c/span\u003e]. \u003cem\u003eChryseobacterium\u003c/em\u003e can solubilize phosphates, and its combined use with nitrogen and phosphorus fertilizers can promote plant growth and increase crop yield [\u003cspan citationid=\"CR110\" class=\"CitationRef\"\u003e110\u003c/span\u003e]; studies have also shown that \u003cem\u003eChryseobacterium\u003c/em\u003e can increase the content of photosynthetic pigments in plant leaves and produce indole-3-acetic acid [\u003cspan citationid=\"CR111\" class=\"CitationRef\"\u003e111\u003c/span\u003e, \u003cspan citationid=\"CR112\" class=\"CitationRef\"\u003e112\u003c/span\u003e]. \u003cem\u003eMicrobacterium\u003c/em\u003e can regulate the physiological hormones and molecular characteristics of tomatoes under drought stress, significantly improving plant photosynthetic activity and biomass, indicating that \u003cem\u003eMicrobacterium\u003c/em\u003e has a positive impact on plant growth and development under adverse conditions [\u003cspan citationid=\"CR113\" class=\"CitationRef\"\u003e113\u003c/span\u003e]. \u003cem\u003eAcinetobacter\u003c/em\u003e has a positive impact on plant photosynthetic characteristics (photosynthetic rate, stomatal conductance, internal CO2 concentration, and total chlorophyll content), indicating that this endophytic bacterium may promote plant growth by affecting photosynthesis [\u003cspan citationid=\"CR114\" class=\"CitationRef\"\u003e114\u003c/span\u003e, \u003cspan citationid=\"CR115\" class=\"CitationRef\"\u003e115\u003c/span\u003e].The dominant endophytic fungal communities on tea plant phyllosphere with 100mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e ZnO NPs treatment included \u003cem\u003eDavidiella, Fusarium, Pyrenochaeta, Penicillium, Boletus, Malassezia\u003c/em\u003e, and \u003cem\u003eAspergillus\u003c/em\u003e. \u003cem\u003ePenicillium\u003c/em\u003e is a widely present fungus that can promote plant growth and enhance plant resistance to stress by producing plant hormones and other bioactive substances [\u003cspan citationid=\"CR116\" class=\"CitationRef\"\u003e116\u003c/span\u003e]. \u003cem\u003eAspergillus\u003c/em\u003e is considered a plant growth-promoting fungus, capable of promoting plant growth and enhancing plant resistance to stress by producing plant hormones and other bioactive substances [\u003cspan citationid=\"CR117\" class=\"CitationRef\"\u003e117\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn summary, the use of ZnO NPs improved the community composition of epiphytic and endophytic microorganisms on tea plants, with many growth-promoting and photosynthesis-enhancing microbes becoming dominant populations and eliminating potentially pathogenic plant pathogens, which may be related to the antimicrobial properties of ZnO NPs.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study investigated the effects of ZnO NPs on tea plants, focusing on photosynthesis (photosynthetic physiological parameters, photosynthetic enzymes, chlorophyll fluorescence parameters), new shoot germination, transcriptome (leaves and new shoots), metabolome (leaves and new shoots), mineral element content (leaves and new shoots), and phyllosphere microbial communities (epiphytic and endophytic microorganisms). The study showed that (1). ZnO NPs enhanced the photosynthesis of tea plants by upregulating the expression of some genes related to photosynthesis and increasing the accumulation of photosynthetic products. (2). ZnO NPs increased the auxin content in tea plants, promoting the development of new shoots. (3). ZnO NPs altered the mineral element composition of tea plants, increasing the content of zinc, molybdenum, and copper elements in the leaves, and changing the content of selenium and iron elements in the new shoots, with a minor impact on the mineral content of the new shoots. (4). ZnO NPs improved the community composition of both epiphytic and endophytic microorganisms in the tea plant phyllosphere, suppressing some potentially pathogenic microorganisms and promoting many beneficial microbes that potentially enhance plant photosynthesis and growth to become dominant populations. This research provides new insights into how ZnO NPs can improve the growth of tea plants.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eZnO NPs, zinc oxide nanoparticles; CK, foliar spraying with pure water; CKL, tea leaves under CK treatment; CKS, tea shoots under CK treatment; CKEP, phyllosphere epiphytic microorganisms in tea leaf under CK treatment; CKED, phyllosphere endophytic microorganisms in tea leaf under CK treatment; T1, foliar spraying with 50mg L\u003csup\u003e-1\u003c/sup\u003eZnO NPs; T1L, tea leaves under T1 treatment; T1S, tea shoots under T1 treatment; T1EP, phyllosphere epiphytic microorganisms in tea leaf under T1 treatment; T1ED, phyllosphere endophytic microorganisms in tea leaf under T1 treatment; T2, foliar spraying with 100mg L\u003csup\u003e-1\u003c/sup\u003eZnO NPs; T2L, tea leaves under T2 treatment; T2S, tea shoots under T2 treatment; T2EP, phyllosphere epiphytic microorganisms in tea leaf under T2 treatment; T2ED, phyllosphere endophytic microorganisms in tea leaf under T2 treatment; Photo, net photosynthetic rate; Cond, stomatal conductance; Ci, intercellular CO2 concentration; Trmmol, transpiration rate; RubisCO, ribulose-1,5-bisphosphate carboxylase/oxygenase; FBP, fructose-1,6-bisphosphatase; PEPC, phosphoenolpyruvate carboxylase; SPAD, soil plant analysis development; chl a, chlorophyll a; chl b, chlorophyll b; ETR, relative electron transport rate; Fv/Fm, maximum photochemical efficiency of photosystem II; qP, photochemical quenching coefficient; Y(II), actual photochemical efficiency of photosystem II; WGCNA, weighted gene co-expression network analysis; DEGs, differentially expressed genes; O2PLS, two-way orthogonal partial least squares; PCA, principal component analysis; PLS, partial least squares; CCA, canonical correlation analysis; ASVs, amplicon sequence variants; OTUs, operational taxonomic units; TCA, tricarboxylic acid; ATP, adenosine triphosphate; NADPH, nicotinamide adenine dinucleotide phosphate; LHCB, light-harvesting complex II chlorophyll a/b binding protein; TOR, target-of-rapamycin.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eEthics approval and consent to participate\u003c/h2\u003e\n\u003cp\u003eWe confirmed that all methods involving the plant and its material complied with relevant institutional, national, and international guidelines and legislation.\u003c/p\u003e\n\u003ch2\u003eConsent for publication\u003c/h2\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003ch2\u003eAvailability of data and materials\u003c/h2\u003e\n\u003cp\u003eThe raw sequencing data were deposited in NCBI Sequence Read Archive (SRA) under accession number SUB14254443 for transcriptome,\u0026nbsp;SUB14256009 for bacteria and SUB14256035 for fungi.\u003c/p\u003e\n\u003ch2\u003eCompeting interests\u003c/h2\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThe research was funded by the Natural Science Foundation of China (32002087), the Technology System of Modern Agricultural Industry in Shandong Province (SDAIT-19-01), the Livelihood Project of Qingdao City (21-1-4-ny-2-nsh), the Special Talent Program of SAAS (CXGC2023A11), the Agricultural Improved Variety Project of Shandong Province(2020LZGC010).\u003c/p\u003e\n\u003ch2\u003eAuthors\u0026apos; contributions\u003c/h2\u003e\n\u003cp\u003eHC conducted an experiment, analyzed the data, and wrote a manuscript. YS and HW collected samples. ZD, YW and KF put forward hypotheses and designed experiments. All authors contributed to the article and approved the submission.\u003c/p\u003e\n\u003ch2\u003eAcknowledgements\u003c/h2\u003e\n\u003cp\u003eWe are grateful to Wuhan Metware Biotechnology Co., Ltd for assisting in sequencing.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eZhao L, Bai T, Wei H, Gardea-Torresdey JL, Keller A, White JC. Nanobiotechnology-based strategies for enhanced crop stress resilience. Nat Food. 2022;3:829\u0026ndash;36.\u003c/li\u003e\n\u003cli\u003eKhot LR, Sankaran S, Maja JM, Ehsani R, Schuster EW. Applications of nanomaterials in agricultural production and crop protection: A review. Crop Protection. 2012;35:64\u0026ndash;70.\u003c/li\u003e\n\u003cli\u003eMukherjee A, Majumdar S, Servin AD, Pagano L, Dhankher OP, White JC. Carbon Nanomaterials in Agriculture: A Critical Review. 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Plant Physiology and Biochemistry. 2011;49:852\u0026ndash;61.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-nanobiotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jnan","sideBox":"Learn more about [Journal of Nanobiotechnology](http://jnanobiotechnology.biomedcentral.com)","snPcode":"12951","submissionUrl":"https://submission.nature.com/new-submission/12951/3","title":"Journal of Nanobiotechnology","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Camellia sinensis (L.) O. Kuntze, ZnO NPs, Photosynthesis, Sprouting of new shoots, Epiphytic microorganisms, Endophytic microorganisms, Phyllosphere","lastPublishedDoi":"10.21203/rs.3.rs-4019055/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4019055/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eNanotechnology holds revolutionary potential in the field of agriculture, with zinc oxide nanoparticles (ZnO NPs) demonstrating advantages in promoting crop growth. Photosynthesis is a key process in the growth and quality formation of tea plants, and phyllosphere microorganisms also have a significant impact on plant growth and health. However, the effects of ZnO NPs on the photosynthesis of tea plants, the sprouting of new shoots, and the community of phyllosphere microorganisms are not yet clear.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThis study investigated the photosynthetic physiological parameters of tea plants under the influence of ZnO NPs, the content of key photosynthetic enzymes such as RubisCO, chlorophyll content, chlorophyll fluorescence parameters, transcriptomes (leaves and new shoots), extensively targeted metabolomes (leaves and new shoots), mineral element content (leaves and new shoots), and the communities of epiphytic and endophytic microorganisms in the phyllosphere. The results indicated that ZnO NPs could enhance the photosynthesis of tea plants, upregulate the expression of some genes related to photosynthesis, increase the accumulation of photosynthetic products, promote the development of new shoots, and alter the content of various mineral elements in the leaves and new shoots of tea plants. Additionally, ZnO NPs improved the community composition of epiphytic and endophytic microorganisms in the phyllosphere of tea plants, inhibited potential pathogenic microorganisms, and allowed various beneficial microorganisms with potential growth-promoting properties to become dominant species.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThis study demonstrates that ZnO NPs have a positive impact on the photosynthesis of tea plants, the sprouting of new shoots, and the community of phyllosphere microorganisms, which can improve the growth condition of tea plants. These findings provide new scientific evidence for the application of ZnO NPs in sustainable agricultural development and contribute to advancing research in nanobiotechnology aimed at enhancing crop yield and quality.\u003c/p\u003e","manuscriptTitle":"ZnO NPs Enhanced Photosynthetic Capacity, Promoted New Shoot Development, and Improved the Community Composition of Phyllosphere Epiphytic and Endophytic Microorganisms in Tea Plants","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-12 12:59:47","doi":"10.21203/rs.3.rs-4019055/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-04-09T17:25:46+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-03-27T11:38:06+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"e46ec284-f841-46a7-8df3-1e75b3c853cc","date":"2024-03-18T01:37:34+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-03-16T13:53:35+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-03-07T18:07:02+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-03-07T18:07:02+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Nanobiotechnology","date":"2024-03-06T02:39:23+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-nanobiotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jnan","sideBox":"Learn more about [Journal of Nanobiotechnology](http://jnanobiotechnology.biomedcentral.com)","snPcode":"12951","submissionUrl":"https://submission.nature.com/new-submission/12951/3","title":"Journal of Nanobiotechnology","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"86424535-9497-4bc8-bfd3-d7ebb19c43a0","owner":[],"postedDate":"March 12th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-11-24T13:38:45+00:00","versionOfRecord":{"articleIdentity":"rs-4019055","link":"https://doi.org/10.1186/s12951-024-02667-2","journal":{"identity":"journal-of-nanobiotechnology","isVorOnly":false,"title":"Journal of Nanobiotechnology"},"publishedOn":"2024-07-02 00:00:00","publishedOnDateReadable":"July 2nd, 2024"},"versionCreatedAt":"2024-03-12 12:59:47","video":"","vorDoi":"10.1186/s12951-024-02667-2","vorDoiUrl":"https://doi.org/10.1186/s12951-024-02667-2","workflowStages":[]},"version":"v1","identity":"rs-4019055","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4019055","identity":"rs-4019055","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}