The Effects of Nitrogen and Phosphorus Deficiency on the Main Physiology of Ilex chinensis and Transcriptomic Analysis

The Effects of Nitrogen and Phosphorus Deficiency on the Main Physiology of Ilex chinensis and Transcriptomic Analysis | 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 The Effects of Nitrogen and Phosphorus Deficiency on the Main Physiology of Ilex chinensis and Transcriptomic Analysis Jing Liu, Gong Cheng, Jiejie Jiao, Jiaxin Hu, Bingsong Zheng, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7390500/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 11 Feb, 2026 Read the published version in BMC Plant Biology → Version 1 posted 10 You are reading this latest preprint version Abstract The elements nitrogen (N) and phosphorus (P) are essential nutrients required for normal growth and photosynthesis in plants. However, nitrogen and phosphorus deficiencies can have a significant impact on plant physiology and metabolism. In this study, the two-year-old Ilex chinensis seedlings were used as the research object, the growth environment of low nitrogen (LN) and low phosphorus (LP) was simulated, and two treatment gradients of mild and severe stress were set up, and the changes of the growth and development of wintergreen and the main physiological indexes were studied under normal growth conditions as the control. Differentially expressed genes (DEGs) were identified by transcriptomic analysis at 10 weeks of severe stress. The results showed that nitrogen and phosphorus deficiency inhibited the growth of shoots and promoted root development of I. chinensis , and LN 2 and LP 2 treatments had the most serious effects. The physiological indexes showed that the contents of nitrate reductase (NR), glutamine synthetase (GS), superoxide dismutase (SOD) and peroxidase (POD) and malondialdehyde (MDA) in the LN 2 group were significantly increased by 112.36%, 290.19%, 67.56%, 151.79% and 248.04%, respectively, compared with the CK group after 10 weeks of treatment. The activities of acid phosphatase (ACP), SOD and POD, anthocyanins and MDA increased by 77.1%, 65.46%, 97.81%, 144.43% and 134.25%. Transcriptome analysis revealed that the key differentially expressed genes under the nitrogen and phosphorus deficit of wintergreen were mainly involved in the regulation of plant growth, root development, nitrogen and phosphorus uptake and other biological processes. These findings provide insights into the adaptation mechanisms of I. chinensis under nitrogen and phosphorus deficits and highlight potential target genes for improving nutrient use efficiency. This study contributes to a better understanding of the physiological and molecular responses of I. chinensis to nitrogen and phosphorus deficiency stress, and provides valuable information for optimizing its cultivation under nutrient-constrained conditions. Ilex chinensis Low nitrogen Low phosphorus Physiological properties Transcriptome Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 1 Introduction Nitrogen (N) and phosphorus (P) are key macroelements for plant growth and development, and are important components of biological macromolecules such as nucleic acids and proteins in plants. They are widely involved in physiological processes such as photosynthesis, respiration, and signal transduction in plants. The demand for nitrogen and phosphorus in plants runs through their entire life cycle [ 1 , 2 ] . Lack of nitrogen and phosphorus can lead to significant changes in plant morphology, growth, and development, ultimately resulting in reduced plant yield, decreased quality, poor ornamental value, and decreased economic value. Plants subjected to nitrogen and phosphorus deficiency stress will initiate a series of complex molecular response mechanisms to enhance the absorption and utilization of limited nutrients. In nitrogen deficient environments, plants enhance nitrogen uptake and transport by upregulating the expression of nitrate transporter genes ( NRT1 and NRT2 family genes), which are typically tissue-specific. The root system is often the primary site in response to low nitrogen stress [ 3 , 4 ] . In addition, up-regulation of GS1 [ 5 ] , BnaGLNs and BnaNIAs [ 6 ] gene expression promotes nitrogen assimilation and utilization. Under low phosphorus conditions, plants induce up-regulation of the purple acid phosphatase gene PpAPs to promote the synthesis and secretion of acid phosphatase (ACP) in roots, enhancing the activation and absorption capacity of insoluble phosphorus in soil [ 7 ] . Meanwhile, up-regulation of phosphorus transport related genes ( PHT1 , PHT1.10 , SPX6 , and SPX-MFS2 ) contributes to the effective transport and redistribution of phosphorus in plants [ 8 – 10 ] . These molecular response mechanisms reveal how plants optimize nutrient absorption and utilization through gene expression regulation under nutritional stress, providing important molecular targets for cultivating crop varieties with high nutrient utilization efficiency. Under nitrogen and phosphorus deficiency, plant root growth is restricted and photosynthesis is hindered. Therefore, plants optimize root growth and maintain photosynthetic efficiency by regulating the expression of a series of genes. Under low nitrogen and low phosphorus conditions, plants regulate root development by modulating plant hormone signals and interacting with related genes. For example, under low phosphorus treatment, GmGDPD2 regulates the levels of root hormones such as gibberellin, auxin, and ethylene by sensing the upstream signal GmMyb73 , inhibiting main root elongation, inducing lateral root and root hair formation, thereby affecting phosphorus absorption and utilization [ 11 ] ; The expression of GmEIL4 in soybean roots is significantly upregulated, which can promote the growth and development of hairy roots, increase root hair density, root length, and root surface area, improve phosphorus absorption and utilization ability, and GmEIL4 and GmEBF1 jointly participate in regulating the ethylene metabolism pathway of soybean, thereby affecting the response of roots to low phosphorus stress [ 12 ] . Under low nitrogen stress, the expression of auxin synthesis gene YUCCA and auxin inward transporter AUX1 related genes in peanuts is downregulated, leading to a decrease in auxin content and limiting the occurrence and elongation of lateral roots [ 13 ] ; The BnIPTs gene in rapeseed is downregulated in the roots, leading to a decrease in cytokinin concentration in the roots, which promotes root elongation and increases the number of root tips, enhancing their ability to absorb nutrients from the soil and increasing their resistance to low nitrogen stress [ 14 ] . In addition, due to adversity stress, plants cope with the increase in reactive oxygen species content in their bodies by upregulating the expression of antioxidant enzyme related genes ( SOD9 and CAT2 ), thereby increasing the activity of antioxidant enzymes in plants and maintaining the balance of intracellular reactive oxygen species [ 15 ] . However, the expression levels of chlorophyll catabolism related genes ( MdNYC1 , MdPAO , and MdSGR1 ) significantly increased [ 16 ] , while the expression levels of PSII related genes ( PsbO , PsbP , PsbQ , and PsbY ) were downregulated [ 17 , 18 ] , resulting in a decrease in chlorophyll content and photosynthetic efficiency. These molecular regulatory mechanisms reveal how plants balance growth needs and resource constraints through precise regulation of gene expression under nutritional stress, providing important molecular targets for cultivating stress tolerant crop varieties. Ilex chinensis is widely used in urban greening and disease treatment due to its excellent ornamental value and significant medicinal value [ 19 – 21 ] . However, the significant variations in climatic conditions and soil characteristics across different regions may lead to deficiencies or imbalances of N and P in the soil. For example, some areas lack essential N and P elements due to climate drought or soil barrenness, while others are affected by industrial pollution, frequent acid rain, and soil pH decline, which in turn reduce the availability of N and P. All of these factors have adverse effects on the growth and survival rate of I. chinensis . Therefore, investigating the optimal concentrations of N and P for the growth of I. chinensis , clarifying the roles of N and P in its growth and development, and understanding the impacts of N and P deficiency on plant growth are of great significance for the cultivation management and sustainable development of this species. The aim of this study is to evaluate the effects of different nitrogen and phosphorus concentrations on the growth status and physiological indicators of I. chinensis by simulating nitrogen and phosphorus deficit conditions and comparing them with normal growth conditions. The aim is to clarify the phenotypic changes of I. chinensis under nitrogen and phosphorus deficit conditions. The research results will provide theoretical support for screening suitable nitrogen and phosphorus concentrations for I. chinensis growth. In addition, this study will explore the gene response mechanism of I. chinensis under nitrogen and phosphorus deficiency through transcriptome analysis, and identify key response genes. The research results will provide scientific basis for creating transgenic I. chinensis varieties with high nitrogen and phosphorus utilization efficiency, and provide theoretical references for efficient plant cultivation, water and fertilizer management, and development and utilization. 2 Materials and Methods 2.1 Plant Materials and Growth Conditions The plant materials for this experiment were wild species of Ilex chinensis Sims, collected from Tianmu Mountain in Hangzhou, Zhejiang Province, eastern China. The collected seeds were stored in low-temperature sand, and seedlings of I. chinensis were obtained through seedling cultivation. Two-year-old seedlings with a uniform height of 1.0-1.10 m were selected for the study. Two weeks before the start of the experiment, the seedlings were transplanted into pots (height 30 cm, diameter 20 cm) filled with river sand, with one seedling per pot. During the experimental period, the seedlings were maintained in a greenhouse with a temperature of (25 ± 2)°C during the day and (20 ± 2)°C at night, a relative humidity of (70 ± 5)%, and natural light intensity. The plant species were identified by plant classification experts Yan Daoliang and Lou Luhuan. Voucher specimens have been deposited at the Herbarium of the Institute of Botany, Chinese Academy of Sciences (PE), with the accession number 4111. 2.1.1 Compliance with Guidelines All experimental research on plants, including the collection of plant materials, was conducted in compliance with institutional, national, and international guidelines. Field studies were conducted in accordance with local legislation. The necessary permissions and/or licenses were obtained for the collection of wild plant materials. 2.1.2 Compliance with the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) This study adheres to the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES). The plant species used in this study are not listed as endangered under CITES, and all collection and use of plant materials were conducted in accordance with the guidelines provided by CITES. 2.2 Experimental Design The experiment utilized a modified Hoagland nutrient solution with the following composition (mg/L): Ca(NO 3 ) 2 ·4H 2 O 616.64, KNO 3 376.34, KH 2 PO 4 101.49 and MgSO 4 60. Iron was supplied as Fe-EDTA solution (2.5 mL/L), and micronutrient solution (5 mL/L) was added, containing (mg/L): KI 0.83, H 3 BO 3 6.2, MnSO 4 ·4H 2 O 22.3, ZnSO 4 ·7H₂O 8.6, Na 2 MoO 4 ·2H 2 O 0.25, CuSO 4 ·5H 2 O 0.025 and CoCl 2 ·6H 2 O 0.025. Five treatment groups were established: (1) normal nutrient supply (CK, nitrogen (N) concentration 125 mg/L, phosphorus (P) concentration 0.75 mmol/L), (2) mild low-N (LN 1 , N concentration 25 mg/L, P concentration 0.75 mmol/L), (3) severe low-N (LN 2 , N concentration 8.33 mg/L, P concentration 0.75 mmol/L), (4) mild low-P (LP 1 , N concentration 125 mg/L, P concentration 0.15 mmol/L), and (5) severe low-P (LP 2 , N concentration 125 mg/L, P concentration 0.05 mmol/L). Other essential elements were maintained at standard levels to meet plant growth requirements. Each treatment was replicated three times, with 10 plants per replicate. The corresponding nutrient solution was applied every 7 days until leaching from the bottom of the pots, for a total of 10 weeks. Sampling was conducted at week 0 (T 0 ), week 5 (T 1 ), and week 10 (T 2 ) to measure plant growth indicators and physiological parameters. 2.3 Measurements of Growth and Physiological Parameters 2.3.1 Total Biomass and Root-Shoot Ratio Three plants were randomly selected from each treatment group for de-potting. The above-ground and below-ground parts were separated and thoroughly washed with deionized water to remove adhering soil and impurities. Subsequently, the samples were placed in a constant-temperature oven at 80°C until a constant weight was achieved. The dry weights of the above-ground and below-ground parts were measured using an electronic balance with a precision of 0.0001 g (ME104E, Mettler Toledo, Greifensee, Switzerland). The root-shoot ratio (RSR) was then calculated. RSR = Root Dry Weight / Shoot Dry Weight 2.3.2 Determination of Plant Root System Parameters Three plants were randomly selected from each treatment group for de-potting. The roots were carefully washed to remove any adhering soil and impurities. The total root length and root branch number were then measured using the LA-S plant image analysis system (LA-S, Hangzhou Wanshen Detection Technology Co., Ltd., Hangzhou, China). 2.3.3 Determination of leaf antioxidant enzymatic system index Enzyme extracts were prepared using phosphate buffer (pH 7.8), and enzyme activities were measured using standard colorimetric methods. The content of malondialdehyde (MDA) was determined using the thiobarbituric acid reaction method [ 22 ] . The activity of superoxide dismutase (SOD) was assessed by its ability to inhibit the reduction of nitroblue tetrazolium (NBT) [ 23 ] . The activity of peroxidase (POD) was measured by its capacity to oxidize guaiacol [ 24 ] . 2.3.4 Determination of enzyme activity and anthocyanin content related to nitrogen and phosphorus metabolism in leaves The activities of acid phosphatase (ACP), nitrate reductase (NR), and glutamine synthetase (GS) in leaves, as well as anthocyanin content, were measured using commercial kits from Suzhou Mengxi Biotechnology Co., Ltd. (Suzhou, China). 2.4 Transcriptomic Analysis Leaves from I. chinensis plants under N and P deficiency at time point T 2 were collected from three treatment groups: CK, LN 2 , and LP 2 . The leaves were immediately frozen in liquid nitrogen and stored at -80°C for subsequent analysis, with three biological replicates per sample. Total RNA was extracted from I. chinensis leaves by TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA), and its purity and quantity were checked via a bioanalyzer (Agilent, Santa Clara, CA, USA). Due to the low alignment rate of I. chinensis to existing genomes, the analysis of transcriptome was conducted without the reference genome. NovaseqTM 6000 (Illumina, San Diego, CA, USA) was used to sequence samples in PE150 mode, and Trinity was used to perform gene de novo assembly on sequencing data to obtain unigenes and transcripts (gene isoforms). Using DIAMOND for functional annotation of unigenes, and six databases (NCBI_NR, Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), Pfam, SwissProt, and eggNOG) were used to perform the annotation, with an E-value < 0.00001. Differential expression analysis was conducted using the criteria of fold change (FC) ≥ 2 or FC ≤ 0.5 (i.e., |log2FC| ≥ 1) and false discovery rate (FDR) < 0.05 to identify differentially expressed genes (DEGs). For multiple group comparisons, genes with FDR < 0.05 were selected to identify statistically significant differences across groups. To further classify the functions of the DEGs, GO functional annotation and KEGG pathway enrichment analysis were conducted. 2.5 Statistical Analysis All data were subjected to statistical analysis using SPSS 22.0 software. One-way analysis of variance (ANOVA) was employed to compare differences among different treatment groups, with the significance level set at P < 0.05. 3 Results 3.1 Effects of low-nitrogen (LN) and low phosphorus (LP) on growth traits of Ilex chinensis As shown in Fig. 1 , at time point T 2 , significant changes in leaf area and color were observed among the different treatment groups. Compared with the CK, under low-nitrogen (LN) stress, the color of I. chinensis leaves gradually lightened from dark green to light green with increasing stress intensity. The leaves also became thinner and exhibited reduced leaf area. Under LP 1 , the leaf color of I. chinensis only slightly lightened, with no significant difference in leaf area. However, under LP 2 , the leaves showed obvious chlorosis and yellowing, along with the appearance of dry, yellow spots. As shown in Fig. 2 , nitrogen and phosphorus deficiency significantly inhibited the growth and development of I. chinensis . Under CK conditions, the total biomass of I. chinensis increased by 11.05% and 14.97% at T 1 and T 2 , respectively, compared with T 0 . However, under LN and LP stress, the increase in total biomass was lower than that of CK and showed a continuous downward trend with prolonged stress duration. At T 2 , compared with T0, the total biomass of the LN 2 and LP 2 treatments only increased by 10.8% and 12.14%, respectively (Fig. 2 A). Moreover, N and P deficiency significantly increased the root-shoot ratio (RSR) of I. chinensis (Fig. 2 B). At T 2 , the RSR of the LN 2 and LP 2 treatments increased by 27.73% and 23.25%, respectively, compared with CK. Nevertheless, no significant differences were observed between the LN and LP treatment groups at T 1 and T 2 . Figures 2 C and 2 D indicate that N and P deficiency significantly affected the root growth of I. chinensis . At T 0 , there were no significant differences in primary root length and root branch number among the different treatment groups of I. chinensis . At T 1 and T 2 , no significant differences were observed in these indices within each LN and LP treatment group. However, compared with CK, the primary root length of the LN treatment groups LN 1 and LN 2 significantly increased by 14.77% and 7.44% at T 1 , and by 16.28% and 9.22% at T 2 , respectively. The root branch number significantly increased by 16.66% and 20.03% at T 1 , and by 10.33% and 12.97% at T 2 , respectively. For the LP treatment groups LP 1 and LP 2 , the primary root length significantly increased by 13.52% and 8.29% at T 1 , and by 15.53% and 8.9% at T 2 , respectively. The root branch number increased by 17.45% and 18.45% at T 1 , and by 11.62% and 12.88% at T 2 , respectively. Compared with T 0 , at T 1 , the primary root length of LN 1 and LN 2 increased by 21.58% and 23.81%, respectively, and the root branch number significantly increased by 22.16% and 15.71%, respectively. For LP 1 and LP 2 , the primary root length increased by 20.35% and 22.55%, respectively, and the root branch number significantly increased by 22.99% and 16.89%, respectively. 3.2 Effects of LN and LP on Antioxidant Enzyme Activities in I. chinensis As shown in Fig. 3 , at T 2 , the activities of superoxide dismutase (SOD) and peroxidase (POD), as well as the content of malondialdehyde (MDA), increased most significantly in the LN 2 and LP 2 groups. Specifically, in the LN 2 group, the activities of SOD and POD and the content of MDA were 1072.95 U·g − 1 , 153.8 U/g·min, and 23.98 µmol/g, respectively, which were significantly higher than those in the CK group by 67.56%, 151.79%, and 248.04%, respectively. In the LP 2 group, the activities of SOD and POD and the content of MDA were 1058.48 U/g, 120.8 U/g·min, and 16.14 µmol·g − 1 , respectively, which were significantly higher than those in the CK group by 65.46%, 97.81%, and 134.25%, respectively. At each treatment period, the activities of SOD and POD and the content of MDA in the LN 2 group were significantly higher than those in the LN 1 group. Under low-phosphorus stress, only the trends of SOD activity and MDA content were similar to those under low-nitrogen stress. At T 1 , there were no significant differences in POD activity between the LP 1 and LP 2 groups, but at T 2 , the POD activity in the LP 2 group was significantly lower than that in the LP 1 group. 3.3 Effects of LN Treatment on the Activities of Nitrate Reductase (NR) and Glutamine Synthetase (GS) in I. chinensis Leaves Under LN stress, the activities of NR and GS in the leaves of all treatment groups increased (Fig. 4 ), but the extent of the increase varied among groups. As the stress duration extended, the NR activity in the LN 1 and LN 2 groups was significantly higher than that in the CK group, reaching a peak at T 2 , with increases of 130.15% and 290.19% compared to CK, respectively. Additionally, the GS activity in LN 2 was significantly higher than that in LN 1 and much higher than CK throughout all treatment periods. Specifically, at T 1 and T 2 , the GS activity in LN 2 was 60.89% and 112.36% higher than CK, respectively, and 23.78% and 56.02% higher than LN 1 , respectively. 3.3 Effects of LP Treatment on Anthocyanin Content and Acid Phosphatase (ACP) Activity in I. chinensis Leaves Under LP treatment, both anthocyanin content and ACP activity exhibited a continuous upward trend, with the LP 2 group consistently showing higher levels than the LP 1 group across all treatment periods (Fig. 5 ). For instance, at T 1 , the anthocyanin content and ACP activity in the LP 2 group were 37.07% and 22.33% higher than those in the LP 1 group, respectively. By T 2 , the difference in anthocyanin content further increased, while the difference in ACP activity decreased, with the LP 2 group showing 52.08% higher anthocyanin content and 9.68% higher ACP activity compared to the LP 1 group. 3.4 Transcriptome Sequencing Analysis 3.4.1 Assessment and Assembly of Transcriptome Sequencing Data Illumina sequencing technology was employed to compare and analyze the transcriptome data of CK, LN 2 , and LP 2 samples. A total of 55.34 Gb of valid reads were obtained, comprising 376,227,290 reads. The effective data volume for each sample ranged from 5.05 Gb to 7.38 Gb. The proportion of bases with a quality value of ≥ 20 (Q20) was over 98.09%, and the average GC content was 45.22% (Table 1 ). Pearson correlation analysis of the samples (Fig. 6 ) revealed high similarity among biological replicates and minor differences in gene expression levels within each treatment group, while distinct differences were observed between different treatment groups. Therefore, the sequencing quality was satisfactory, and the data were suitable for subsequent comparative and analytical studies. Table 1 Sample sequencing data Sample Raw_Reads Raw_Bases Valid_Reads Valid_Bases Valid% Q20% GC% CK_1 43563974 6.53G 41599030 6.11G 95.49 98.31 45.17 CK_2 42925382 6.44G 40531846 5.94G 94.42 98.38 45.76 CK_3 42502884 6.38G 41094998 6.06G 96.69 98.34 44.97 LN2_1 41826864 6.27G 40459498 5.96G 96.73 98.29 44.79 LN2_2 41153310 6.17G 39746028 5.86G 96.58 98.32 44.55 LN2_3 52467644 7.87G 50180432 7.38G 95.64 98.32 44.85 LP2_1 48985036 7.35G 46849450 6.89G 95.64 98.37 45.64 LP2_2 43207630 6.48G 41426462 6.09G 95.88 98.31 45.59 LP2_3 35648318 5.35G 34339546 5.05G 96.33 98.09 45.74 3.4.2 Differential Gene Expression Analysis In this study, two comparisons were established: LN 2 vs. CK and LP 2 vs. CK. A total of 2480 differentially expressed genes (DEGs) were identified in the LN 2 vs. CK comparison, and 960 DEGs were identified in the LP 2 vs. CK comparison. Compared with the CK group, 845 DEGs were up-regulated and 1635 DEGs were down-regulated in the LN 2 treatment (LN 2 vs. CK). In the LP 2 treatment (LP 2 vs. CK), 401 DEGs were up-regulated and 559 DEGs were down-regulated. Under LN and LP, both comparison groups exhibited fewer up-regulated DEGs than down-regulated DEGs. Additionally, the total number of DEGs identified in the LN treatment group was higher than that in the LP treatment group (Fig. 7 A). This suggests that the response mechanisms of I. chinensis to LN conditions are more complex. Furthermore, a Venn diagram was constructed to compare the shared DEGs among the treatment groups (Fig. 7 B). A total of 445 common DEGs were identified in both LN and LP treatment groups. These shared DEGs likely play important roles in the resistance of I. chinensis to N and P deficiency stress. 3.4.3 GO and KEGG Enrichment Analysis of DEGs The GO enrichment analysis (Fig. 8 A) elucidated the functional profiles of differentially expressed genes (DEGs) in I. chinensis under LN and LP stress conditions. Under LN stress, DEGs in the LN 2 group were predominantly enriched in terms associated with chloroplast thylakoid membrane, ribosomal structure, thylakoids, photosynthesis, transition metal ion binding, and cellular transition metal ion homeostasis. In contrast, under LP stress, DEGs in the LP 2 group were primarily enriched in terms related to cell wall, heat response, ARF guanine nucleotide exchange factor, extrinsic component of the plasma membrane, plant-type hypersensitive response, and signal transduction. KEGG enrichment analysis (Fig. 8 B) further elucidates the metabolic regulatory pathways in I. chinensis under LN and LP stress. The results indicate that I. chinensis employs both common and unique metabolic regulatory pathways in response to LN and LP stress. In the LN treatment group, the KEGG metabolic pathways are primarily enriched in photosynthesis, ribosomes, cyanate metabolism, phenylpropanoid biosynthesis, flavonoid biosynthesis, photosynthesis - antenna proteins, nitrogen metabolism, and tyrosine metabolism. In the LP treatment group, the KEGG metabolic pathways are mainly enriched in flavonoid biosynthesis, arachidonic acid metabolism, plant-pathogen interaction, isoquinoline alkaloid biosynthesis, cutin and suberin biosynthesis, and tyrosine metabolism. The metabolic pathways commonly enriched by both LN and LP treatment groups include cyanate metabolism, phenylpropanoid biosynthesis, and flavonoid biosynthesis. These results indicate that I. chinensis adapts to LN and LP stress by modulating the expression of different genes. Under LN conditions, the DEGs in I. chinensis are primarily involved in processes related to photosynthesis and protein synthesis. In contrast, under LP conditions, the DEGs are more extensively involved in biosynthetic processes and cellular development-related processes. 3.4.4 Expression Patterns of DEGs in Key KEGG Pathways Based on the KEGG-related metabolic pathway enrichment results in response to nitrogen and phosphorus deficiency in I . chinensis , the significantly enriched key KEGG pathways in each treatment group were selected for further analysis. A heatmap analysis was performed to illustrate the expression patterns of the differentially expressed genes in the key KEGG pathways in the low-nitrogen treatment group (Fig. 9 ). The results showed that most of the differentially expressed genes involved in the key KEGG pathways exhibited down-regulated expression, with only a small proportion of genes showing up-regulated expression. Specifically, genes involved in photosynthesis included TRINITY_DN17859_c0_g1 ( PSBO ), TRINITY_DN4395_c0_g1 ( PSBO ), TRINITY_DN9156_c0_g1 ( XYLA ), TRINITY_DN15339_c0_g1 ( PETE ), TRINITY_DN2383_c0_g1 ( PSBW ), TRINITY_DN6719_c0_g1 ( PSBW ), TRINITY_DN2042_c2_g1 ( SEND33 ), TRINITY_DN14464_c0_g1 ( fdxH1 ), TRINITY_DN4423_c0_g1 ( PSAK ), TRINITY_DN383_c1_g1 ( ATPC ), and TRINITY_DN51_c0_g1 ( PSAO ), with the exception of TRINITY_DN9156_c0_g1 ( XYLA ), which was up-regulated, while the others were down-regulated. Genes involved in ribosome metabolism included TRINITY_DN5698_c0_g1 ( RCA ), TRINITY_DN320_c1_g1 ( SOG1 ), TRINITY_DN4378_c0_g1 ( APS1 ), TRINITY_DN37697_c0_g1 ( mrpl19 ), TRINITY_DN16074_c0_g1 ( RPP1A ), TRINITY_DN2782_c0_g1 ( UBI3 ), TRINITY_DN7984_c0_g1 ( ALTA5 ), TRINITY_DN412_c0_g1 ( UBQ8 ), TRINITY_DN2973_c0_g1 ( RPS28 ), TRINITY_DN9933_c0_g1 ( RPL18B ), TRINITY_DN6931_c0_g2 ( ycf3 ), TRINITY_DN4920_c0_g1 ( RAN1A ), and TRINITY_DN3843_c0_g1 ( RPL24 ), with the exception of TRINITY_DN320_c1_g1 ( SOG1 ), TRINITY_DN9933_c0_g1 ( RPL18B ), and TRINITY_DN3843_c0_g1 (RPL24), which were up-regulated, while the others were down-regulated. Genes involved in cyanamide metabolism included TRINITY_DN11758_c0_g1 ( CAS2 ), TRINITY_DN26728_c0_g1 ( SABP2 ), TRINITY_DN5704_c0_g1 ( MES10 ), TRINITY_DN29012_c0_g2 ( AtMg00310 ), TRINITY_DN32055_c0_g1 ( AtMg01250 ), TRINITY_DN8246_c0_g1 ( At1g65750 ), and TRINITY_DN4045_c0_g1 ( BGLU12 ), with the exception of TRINITY_DN11758_c0_g1 ( CAS2 ) and TRINITY_DN4045_c0_g1 ( BGLU12 ), which were down-regulated, while the others were up-regulated. Genes involved in phenylpropanoid biosynthesis included TRINITY_DN9992_c0_g1 ( F6'H1 ), TRINITY_DN8210_c0_g1 ( 4CL1 ), TRINITY_DN9046_c0_g1 ( PER42 ), TRINITY_DN3267_c0_g2 ( PAL ), TRINITY_DN24156_c1_g1 ( 10HGO ), TRINITY_DN4868_c0_g1 ( PNC1 ), and TRINITY_DN9532_c0_g1 ( PER43 ), with the exception of TRINITY_DN4868_c0_g1 ( PNC1 ), which was up-regulated, while the others were down-regulated. Genes involved in flavonoid biosynthesis included TRINITY_DN5453_c0_g1 ( ANS ), TRINITY_DN5906_c0_g1 ( DLO2 ), TRINITY_DN28994_c0_g1 ( DFR ), TRINITY_DN5516_c1_g1 ( NCS1 ), TRINITY_DN938_c0_g1 ( SRG1 ), TRINITY_DN10020_c0_g2 ( HST ), TRINITY_DN37560_c0_g1 ( CHAT ), TRINITY_DN13010_c0_g1 ( ACT ), TRINITY_DN1253_c0_g2 ( mdmC ), TRINITY_DN10247_c0_g1 ( LAR ), and TRINITY_DN8461_c0_g1 ( BP80 ), with the exception of TRINITY_DN5516_c1_g1 ( NCS1 ), which was up-regulated, while the others were down-regulated. The gene TRINITY_DN9112_c0_g1 (RLP30), involved in plant stress resistance, was significantly up-regulated. Genes involved in nitrogen metabolism included TRINITY_DN26004_c0_g1 ( ACA1 ), TRINITY_DN747_c0_g1 ( GS1-2 ), TRINITY_DN2414_c0_g1 ( GLT1 ), TRINITY_DN1838_c0_g1 ( CYN ), TRINITY_DN2438_c0_g1 ( NIA2 ), and TRINITY_DN3357_c0_g1 ( BCA5 ), with the exception of TRINITY_DN2438_c0_g1 ( NIA2 ), which was up-regulated, while the others were down-regulated. Genes involved in tyrosine metabolism included TRINITY_DN3259_c0_g1 ( ADH1 ), TRINITY_DN4933_c2_g1 ( ASP1 ), TRINITY_DN8458_c0_g1 ( At1g62810 ), and TRINITY_DN4153_c0_g3 ( co-2 ), with the exception of TRINITY_DN8458_c0_g1( At1g62810 ), which was up-regulated, while the others were down-regulated. all of which were down-regulated. A heatmap analysis was conducted to illustrate the expression patterns of the differentially expressed genes in the key KEGG pathways in the low-phosphorus treatment group (Fig. 9 ). The results indicated that the majority of the differentially expressed genes involved in the key KEGG pathways were down-regulated, while a portion of the genes exhibited up-regulated expression. Specifically, genes involved in flavonoid biosynthesis included TRINITY_DN38553_c0_g1 ( At5g05600 ), TRINITY_DN44_c1_g1 ( CODM ), TRINITY_DN28994_c0_g1 ( DFR ), TRINITY_DN938_c0_g1 ( SRG1 ), TRINITY_DN44_c3_g1 ( FHT ), and TRINITY_DN10247_c0_g1 ( LAR ). Except for TRINITY_DN38553_c0_g1 ( At5g05600 ), TRINITY_DN44_c1_g1 ( CODM ), and TRINITY_DN44_c3_g1 ( FHT ), all others were down-regulated. Genes involved in the biosynthesis of stilbenes, diarylheptanoids, and gingerols included TRINITY_DN13010_c0_g1 ( ACT ) and TRINITY_DN37560_c0_g1 ( CHAT ), both of which were down-regulated. Genes involved in arachidonic acid metabolism included TRINITY_DN23164_c0_g1 ( EPHX2 ), TRINITY_DN8168_c0_g1 ( OsI_027940 ), and TRINITY_DN5361_c0_g1 ( yfhM ), all of which were down-regulated. Genes involved in plant-pathogen interaction included TRINITY_DN10034_c0_g2 ( At1g59620 ), TRINITY_DN36138_c0_g1 ( EFR ), TRINITY_DN22339_c1_g1 ( R1A-10 ), TRINITY_DN12377_c0_g2 ( R1A-3 ), TRINITY_DN16250_c0_g1 ( At4g27190 ), TRINITY_DN29459_c0_g3 ( RPM1 ), TRINITY_DN37700_c0_g1 ( MKK6 ), TRINITY_DN5041_c0_g3 ( RPP8L4 ), TRINITY_DN3065_c0_g1 ( WRKY53 ), TRINITY_DN33342_c0_g1 ( CRF3 ), and TRINITY_DN8177_c0_g1 ( CG5412 ). Except for TRINITY_DN36138_c0_g1 ( EFR ), TRINITY_DN16250_c0_g1 ( At4g27190 ), TRINITY_DN29459_c0_g3 ( RPM1 ), TRINITY_DN37700_c0_g1 ( MKK6 ), TRINITY_DN5041_c0_g3 ( RPP8L4 ), and TRINITY_DN33342_c0_g1 ( CRF3 ), all others were down-regulated. Genes involved in the biosynthesis of cutin, suberin, and wax included TRINITY_DN6750_c1_g1 ( PXG4 ), TRINITY_DN6366_c0_g1 ( CER1 ), and TRINITY_DN9279_c0_g1 ( CYP704C1 ). Except for TRINITY_DN6750_c1_g1 ( PXG4 ), all others were down-regulated. Genes involved in endocytosis included TRINITY_DN30224_c0_g2 ( AtMg00810 ), TRINITY_DN8151_c0_g1 ( MED37D ), TRINITY_DN446_c1_g1 ( HSP70 ), TRINITY_DN13409_c0_g1 ( GN ), TRINITY_DN17085_c0_g1 ( GN ), TRINITY_DN18740_c1_g1 ( GN ), TRINITY_DN4537_c0_g1 ( HSC-2 ), TRINITY_DN9093_c0_g1 ( VPS2.3 ), and TRINITY_DN32516_c0_g1 ( ARF1 ). Except for TRINITY_DN30224_c0_g2 ( AtMg00810 ), TRINITY_DN13409_c0_g1 ( GN ), and TRINITY_DN9093_c0_g1 ( VPS2.3 ), all others were down-regulated. Genes involved in sulfur metabolism included TRINITY_DN573_c1_g1 ( APR3 ) and TRINITY_DN125_c0_g2 ( APS1 ), both of which were up-regulated. Genes involved in cyanamide metabolism included TRINITY_DN3601_c0_g1 ( BGLU12 ), TRINITY_DN5704_c0_g2 ( SABP2 ), and TRINITY_DN8246_c0_g1 ( At1g65750 ). Except for TRINITY_DN3601_c0_g1 ( BGLU12 ), all others were up-regulated. The gene involved in tyrosine metabolism was TRINITY_DN4153_c0_g3 ( co-2 ), which was down-regulated. Genes involved in RNA degradation included TRINITY_DN10720_c0_g1 ( PAB7 ), TRINITY_DN9881_c0_g1 ( PAB2 ), TRINITY_DN9881_c0_g2 ( PAB6 ), TRINITY_DN4343_c0_g1 ( PUP1 ), TRINITY_DN22471_c0_g1 ( NAC021 ), and TRINITY_DN7967_c0_g1 ( NAC029 ). Except for TRINITY_DN4343_c0_g1 ( PUP1 ), all others were down-regulated. Genes involved in phenylpropanoid biosynthesis included TRINITY_DN5846_c1_g1 ( TKPR1 ), TRINITY_DN6578_c1_g1 ( CAD1 ), and TRINITY_DN6456_c0_g1 ( gluA ). Except for TRINITY_DN5846_c1_g1 ( TKPR1 ), all others were down-regulated. 4 Discussion Nitrogen (N) and phosphorus (P) are essential macronutrients for plant growth and development and are common limiting factors in nutrient availability. The concentrations of nitrogen and phosphorus in the soil significantly influence various vital activities of plants, including the synthesis of proteins, phospholipids, nucleic acids, and overall growth and development. However, in practical agricultural and ecological settings, the availability of nitrogen and phosphorus in the soil is often affected by a variety of factors, such as human activities, soil type, natural conditions, and soil microbial communities. These factors can lead to low levels of available N and P in the soil, subjecting plants to P and N deficiency stresses, which are detrimental to plant growth and development. To effectively cope with phosphorus and nitrogen deficiency stresses, plants employ a range of complex mechanisms to maximize their resilience. These mechanisms include phenotypic changes in plant morphology and alterations in physiological and biochemical indicators, all of which help to mitigate the adverse effects of nitrogen and phosphorus deficiency on plant growth and development. Building on this understanding, the present study primarily investigates the morphological, physiological, and molecular responses of Ilex chinensis to nitrogen and phosphorus deficiency and provides theoretical insights into the tolerance mechanisms of I. chinensis under nitrogen and phosphorus deficiency conditions. 4.1 Morphological Adaptations of I. chinensis Facilitate Acclimation to LN and LP The phenotypic characteristics of the plant and the growth, morphology, and physiological traits of the root system are directly influenced by the nitrogen and phosphorus concentrations in the soil. In this study, compared with the CK group, the low-nitrogen (LN) treatment group exhibited significant phenotypic changes at T 2 : the leaves of I. chinensis became thinner, and the color changed from dark green to light green, with yellowing signs observed in the LN 2 group. In contrast, in the low-phosphorus (LP) treatment group, the leaves in both LP 1 and LP 2 groups lacked luster. The leaf color in the LP 1 group was not significantly different from that in the CK group, while the LP 2 group showed obvious chlorosis and yellowing. These results are consistent with previous studies in Solanum lycopersicum [ 25 ] , Arabidopsis thaliana [ 26 ] , and Malus pumila [ 27 ] , indicating that LP and LN stresses significantly affect the leaf development of I. chinensis . Other studies have shown that under LN and LP stresses, the total plant biomass decreases, while the root-shoot ratio significantly increases, along with an increase in the length of the primary root and the number of root branches [ 28 – 30 ] . This conclusion is consistent with the results of the present study, where LN and LP significantly inhibited the growth of I. chinensis , manifested as reduced biomass but an increased root-shoot ratio. This suggests that I. chinensis allocates more resources to the root system to enhance the plant's absorption and utilization efficiency of N and P from the soil, thereby maintaining plant growth under N and Pdeficiency conditions. 4.2 Resistance of I. chinensis to LN and LP Stresses is Enhanced through the Antioxidant Enzyme System The increase in malondialdehyde (MDA) content is indicative of lipid peroxidation of cell membranes and the accumulation of reactive oxygen species (ROS), reflecting the degree of membrane damage. Numerous studies have demonstrated that under low-nitrogen and LP stresses, the MDA content in plant leaves significantly increases [ 30 – 33 ] . The results of this study show that in I. chinensis leaves subjected to LN and LP treatments, the MDA content continuously rose with extended treatment duration, and this increase was more pronounced under severe stress conditions. This finding is consistent with previous research and indicates that the cell membranes of I. chinensis leaves are severely damaged under N and P deficiency. To cope with ROS accumulation and membrane oxidative damage, plants enhance the activities of antioxidant enzymes (such as SOD, POD, and CAT) to scavenge excess ROS within the plant, thereby strengthening their ability to withstand adverse conditions [ 17 , 33 , 34 ] . In this study, under low-nitrogen stress, the activities of SOD and POD in I. chinensis leaves were significantly elevated and positively correlated with the duration of stress, with greater increases observed in the LN 2 group compared to the LN 1 group. Under low-phosphorus stress, the changes in SOD activity were similar to those under low-nitrogen stress. However, while POD activity was higher than that of the CK group at T 1 , no significant differences were observed between the LP 1 and LP 2 groups. At T 2 , POD activity significantly increased in both LP 1 and LP 2 groups, with LP 1 showing significantly higher POD activity than LP 2 . This phenomenon may be attributed to the more severe phosphorus deficiency under severe stress conditions, which could inhibit the enhancement of enzyme activity. These conclusions indicate that I. chinensis can mitigate damage under low-nitrogen and low-phosphorus stresses by stimulating the synthesis and activity of antioxidant enzymes, activating the antioxidant system, and scavenging excess free radicals within the plant. 4.3 Accumulation of Nitrate reductase (NR) and glutamine synthetase (GS) Activities Enhances LN Stress Resistance in I. chinensis NR and GS are key enzymes in plant nitrogen metabolism and nitrogen absorption, and their activities are closely related to changes in nitrogen content. Their activities not only directly reflect the plant's ability to utilize and assimilate inorganic nitrogen but also indirectly affect the plant's ability to absorb and accumulate nitrate [ 35 , 36 ] . The trends in NR and GS activities under low-nitrogen stress have been confirmed in many plants, such as Populus simonii [ 37 ] , Triticum aestivum [ 38 ] , Camellia sinensis [ 3 ] , and Spinacia oleracea [ 39 ] . The results of this study are consistent with previous research. Under low-nitrogen stress, the activities of NR and GS in I. chinensis leaves increased continuously with prolonged stress duration, indicating enhanced nitrogen metabolism and assimilation capabilities within the plant, which in turn improved the utilization of limited nitrogen resources. Additionally, GS activity was higher in the LN 2 group than in the LN 1 group at both T 1 and T 2 , whereas NR activity showed no significant difference between the two treatment groups at T 1 but was significantly higher in the LN 2 group than in the LN 1 group at T 2 . This suggests that GS activity in I. chinensis may be more sensitive to nitrogen deficiency than NR activity. Under short-term severe stress, I. chinensis may first increase GS activity to improve the utilization of limited nitrogen sources, thereby stabilizing the nitrogen content within the plant to cope with stress. 4.4 Accumulation of Acid phosphatase (ACP) Activity and Anthocyanin Content Enhances LP Stress Resistance in I. chinensis ACP is activated in response to phosphorus starvation signals in plants, promoting the decomposition of organic, insoluble phosphorus in the environment and thereby enhancing the plant's phosphorus utilization efficiency. This mechanism plays a significant role in plant phosphorus metabolism and is one of the important strategies for plants to cope with low-phosphorus stress [ 40 – 43 ] . In this study, under low-phosphorus stress, the activity of ACP in I. chinensis leaves continuously increased with prolonged stress duration, with the most significant increase observed at T 2 . Moreover, the ACP activity in the LP 2 group was significantly higher than that in the LP1 group at all treatment periods, which is consistent with previous studies in Gossypium hirsutum [ 44 ] and Zea mays [ 45 ] . These findings indicate that I. chinensis enhances its absorption and utilization efficiency of phosphorus from the environment under low-phosphorus conditions, thereby improving its adaptability to phosphorus-deficient environments and alleviating the adverse effects caused by phosphorus deficiency. Anthocyanins are one of the important secondary metabolites in plants and are widely distributed in various plant organs, including roots, stems, leaves, flowers, and fruits. Under biotic and abiotic stresses, the synthesis and accumulation of anthocyanins are crucial for plant stress resistance, as they can enhance the plant's ability to scavenge ROS and mitigate damage to nucleic acids and chloroplasts caused by phosphorus deficiency [ 46 ] . In this study, the anthocyanin content in I. chinensis subjected to low-phosphorus stress continuously increased with prolonged stress duration. This finding is consistent with previous studies, which have shown that low-phosphorus stress promotes the synthesis and accumulation of anthocyanins in plants [ 17 , 47 – 50 ] . In this study, the trends in anthocyanin content and ACP activity were consistent. This coordinated change may be related to the mechanisms by which plants adapt to environmental changes under stress conditions through the regulation of metabolic pathways. 4.5 Molecular Response Mechanisms of I. chinensis to LN and LP Stresses Revealed by Transcriptomic Analysis Previous studies have revealed the critical roles of multiple genes in plant growth and stress responses. Specifically, genes such as TRINITY_DN9156_c0_g1 ( XYLA ), TRINITY_DN320_c1_g1 ( SOG1 ), TRINITY_DN3843_c0_g1 ( RPL24 ), TRINITY_DN5516_c1_g1 ( NCS1 ), and TRINITY_DN2438_c0_g1 ( NIA2 ) are involved in the regulation of plant growth and nitrogen metabolism [ 51 , 52 ] . Additionally, TRINITY_DN9112_c0_g1 ( RLP30 ) and TRINITY_DN29459_c0_g3 ( RPM1 ) participate in plant immune responses and modulate tolerance to abiotic stresses [ 53 – 55 ] . Furthermore, genes including TRINITY_DN38553_c0_g1 ( At5g05600 ), TRINITY_DN44_c3_g1 ( FHT ), TRINITY_DN37700_c0_g1 ( MKK6 ), TRINITY_DN573_c1_g1 ( APR3 ), TRINITY_DN125_c0_g2 ( APS1 ), and TRINITY_DN6750_c1_g1 ( PXG4 ) are implicated in the regulation of phosphorus uptake and utilization, enhancing plant adaptability to phosphorus-deficient environments [ 56 – 58 ] . Additionally, TRINITY_DN33342_c0_g1 ( CRF3 ) and TRINITY_DN4343_c0_g1 ( PUP1 ) influence root development, thereby improving phosphorus uptake and utilization efficiency [ 59 – 61 ] . These genes may play significant roles in plant adaptation to nitrogen and phosphorus deficiency stresses. In this study, based on the quantitative expression results, we found that the differentially expressed genes in I. chinensis under low-nitrogen and low-phosphorus treatments were predominantly down-regulated. However, under nitrogen deficiency stress, genes related to plant growth and nitrogen metabolism regulation, including TRINITY_DN9156_c0_g1 ( XYLA ), TRINITY_DN320_c1_g1 ( SOG1 ), TRINITY_DN3843_c0_g1 ( RPL24 ), and TRINITY_DN2438_c0_g1 ( NIA2 ), as well as the stress-responsive gene TRINITY_DN9112_c0_g1 ( RLP30 ), were significantly up-regulated. Under phosphorus deficiency stress, genes associated with phosphorus uptake and utilization, such as TRINITY_DN38553_c0_g1 ( At5g05600 ), TRINITY_DN44_c3_g1 ( FHT ), TRINITY_DN37700_c0_g1 ( MKK6 ), TRINITY_DN573_c1_g1 ( APR3 ), TRINITY_DN125_c0_g2 ( APS1 ), TRINITY_DN6750_c1_g1 ( PXG4 ), TRINITY_DN29459_c0_g3 ( RPM1 ), TRINITY_DN33342_c0_g1 ( CRF3 ), and TRINITY_DN4343_c0_g1 ( PUP1 ), were also up-regulated. These findings indicate that these genes may play crucial roles in the adaptation of I. chinensis to nitrogen and phosphorus deficiency stresses. Combining the results of GO functional enrichment analysis and KEGG metabolic pathway enrichment analysis, we further elucidated the molecular mechanisms of these genes in the adaptation of I. chinensis to nitrogen and phosphorus deficiency stresses. Under nitrogen-deficient conditions, the up-regulated genes were primarily enriched in pathways related to nitrogen metabolism, photosynthesis, and antioxidant responses. This is consistent with the strategies employed by plants such as Camellia sinensis [ 3 ] , Brassica napus [ 6 ] , and Malus pumila [ 16 ] , which enhance nitrogen metabolism and antioxidant defenses to maintain growth under nitrogen stress. Under phosphorus-deficient conditions, the up-regulated genes were mainly enriched in pathways related to phosphorus metabolism, plant-pathogen interactions, and root development. This aligns with the strategies of plants like Pinus massoniana [ 62 ] , Zea mays [ 15 ] , and Gossypium hirsutum [ 63 ] , which optimize phosphorus absorption and utilization as well as root structure to adapt to phosphorus stress. These results indicate that I. chinensis adapts to nitrogen and phosphorus deficiency stresses by regulating the expression of specific genes and metabolic pathways. 5 Conclusions In this study, the physiological and molecular response mechanisms of Ilex chinensis to low-nitrogen and low-phosphorus conditions were analyzed through physiological indices and transcriptomic analysis (Fig. 10 ). The increased activities of SOD and POD enhanced tolerance to nitrogen and phosphorus deficiency. The expression of genes responsive to nitrogen and phosphorus deficiency was regulated through pathways involved in plant growth, plant immune response, phosphorus uptake, and nitrogen metabolism, thereby improving tolerance to nitrogen and phosphorus deficiency. This study elucidates the differences in growth traits, antioxidant defense systems, and nitrogen and phosphorus metabolism of I. chinensis under nitrogen and phosphorus deficiency stress, providing important physiological and molecular data for understanding the mechanisms by which plants enhance their tolerance to nitrogen and phosphorus deficiency. Declarations Ethics approval and consent to participate: Not applicable. Consent for publication: Not applicable. Availability of data and materials: The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding authors. Competing interests: The authors declare that they have no conflict of interest. Funding: This work was supported by Zhejiang Province Major Science and Technology Project for Agriculture (Breeding of New Tree Species) and New Variety Breeding (grant/award Number:2021C02070-5-4). Authors' contributions: Conceptualization, D.Y.and J.J. ; data curation, J.L. , G.C. and J.H. ; funding acquisition, D.Y.and J.J..; methodology, G.C.; project administration, D.Y. , J.J. and B.Z.; supervision, D.Y.; writing—original draft, J.L., G.C. and J.J.; writing—review and editing, D.Y. All authors have read and agreed to the published version of the manuscript. Acknowledgements: This work was supported by Zhejiang Province Major Science and Technology Project for Agriculture (Breeding of New Tree Species) and New Variety Breeding (grant/award Number: 2021C02070-5-4). Authors' information: Jing Liu & Gong Cheng & Jiejie Jiao co-authored as the first authors. References Oldroyd, G.E.D., Leyser, O., 2020. A plant's diet, surviving in a variable nutrient environment. Science, 368(6486), eaba0196. https://doi.org/10.1126/science.aba0196. Lu, Z., Ren, T., Li, Y., Cakmak, I., Lu, J. , 2025. Nutrient limitations on photosynthesis: from individual to combinational stresses. Trends in plant science, 30(8), 872–885. https://doi.org/10.1016/j.tplants.2025.03.006. Lin, Z. H., Chen, C. S., Zhao, S. Q., Liu, Y., Zhong, Q. S., Ruan, Q. C., Chen, Z. H., You, X. M., Shan, R. Y., Li, X. L., Zhang, Y. Z., 2023. Molecular and physiological mechanisms of tea ( Camellia sinensis (L.) O. Kuntze) leaf and root in response to nitrogen deficiency. BMC genomics, 24(1), 27. https://doi.org/10.1186/s12864-023-09112-y. Zhuo, M., Sakuraba, Y., Yanagisawa, S., 2024. Dof1.7 and NIGT1 transcription factors mediate multilayered transcriptional regulation for different expression patterns of NITRATE TRANSPORTER2 genes under nitrogen deficiency stress. The New phytologist, 242(5), 2132–2147. https://doi.org/10.1111/nph.19695. Li, H.Q., Yu, M.Q., Zhu, X.B., Nai, F.R., Yang, R.R., Wang, L.L., Liu Y.P., Wei, Y.H., Ma, X.M., Yu, H.D., Wang, X.C., 2024. TaGSr contributes to low-nitrogen tolerance by optimizing nitrogen uptake. Environmental and Experimental Botany, 219: 105657. https://doi.org/10.1016/j.envexpbot.2024.105657. Xiao, Y., Yao J.Y., Liu D. Song, H.X., Zhang, Z.H., 2020 Expression profile analysis of low nitrogen stress in Brassica napus . Acta Agronomica Sinica , 46(10): 1526-1538. https://doi.org/10.3724/SP.J.1006.2020.94197. Huang, R., Luo, J.J., Wu, Y.H., Liu, P.D., Liu, G.D., 2021. Molecular Cloning of PpPAP Genes and Their Expression Responses to Phosphorus Deficiency in Elephant Grass ( Pennisetum purpureum ). Acta Agrestia Sinica, 29(3): 425-433. https://doi.org/10.11733/j.issn.1007-0435.2021.03.002. Luo, J., Liu, Z., Yan, J., Shi, W., Ying, Y., 2023. Genome-Wide Identification of SPX Family Genes and Functional Characterization of PeSPX6 and PeSPX-MFS2 in Response to Low Phosphorus in Phyllostachys edulis . Plants (Basel, Switzerland), 12(7), 1496. https://doi.org/10.3390/plants12071496. Meng, X.X., Li, W.F., Shen, R.F., Lan, P., 2021. Time-course response of phenotype and the expression of Pi-starvation responsive genes in high and low Pi-efficient wheat genotypes to Pi starvation. Plant Nutrition and Fertilizer Science, 27(11): 1883-1893. https://doi.org/10.11674/zwyf.2021176. Yang, S.Y., Lin, W.Y., Hsiao, Y.M., Chiou, T. J., 2024. Milestones in understanding transport, sensing, and signaling of the plant nutrient phosphorus. The Plant cell, 36(5), 1504–1523. https://doi.org/10.1093/plcell/koad326. Hu, D., Cui, R., Wang, K., Yang, Y., Wang, R., Zhu, H., He, M., Fan, Y., Wang, L., Wang, L., Chu, S., Zhang, J., Zhang, S., Yang, Y., Zhai, X., Lü, H., Zhang, D., Wang, J., Kong, F., Yu, D., … Zhang, D., 2024. The Myb73-GDPD2-GA2ox1 transcriptional regulatory module confers phosphate deficiency tolerance in soybean. The Plant cell, 36(6), 2176–2200. https://doi.org/10.1093/plcell/koae041. Yang, Y., Wang, R., Wang, L., Cui, R., Zhang, H., Che, Z., Hu, D., Chu, S., Jiao, Y., Yu, D., Zhang, D., 2023. GmEIL4 enhances soybean ( Glycine max ) phosphorus efficiency by improving root system development. Plant, cell & environment, 46(2), 592–606. https://doi.org/10.1111/pce.14497. Li, L., Cheng, X., Kong, X., Jia, P., Wang, X., Zhang, L., Zhang, X., Zhang, Y., Zhang, Z., Zhang, B., 2023. Comparative Transcriptomic Analysis Reveals the Negative Response Mechanism of Peanut Root Morphology and Nitrate Assimilation to Nitrogen Deficiency. Plants (Basel, Switzerland), 12(4), 732. https://doi.org/10.3390/plants12040732. Chen, J., Wan, H., Zhu, W., Dai, X., Yu, Y., Zeng, C., 2023. Identification and Expression Analysis of the Isopentenyl Transferase (IPT) Gene Family under Lack of Nitrogen Stress in Oilseed ( Brassica napus L.). Plants (Basel, Switzerland), 12(11), 2166. https://doi.org/10.3390/plants12112166. Tian, M., Zhang, C., Zhang, Z., Jiang, T., Hu, X., Qiu, H., Li, Z., 2023. Aspergillus niger Fermentation Broth Promotes Maize Germination and Alleviates Low Phosphorus Stress. Microorganisms, 11(7), 1737. https://doi.org/10.3390/microorganisms11071737. Wen, B., Gong, X., Chen, X., Tan, Q., Li, L., Wu, H., 2022. Transcriptome analysis reveals candidate genes involved in nitrogen deficiency stress in apples. Journal of plant physiology, 279, 153822. https://doi.org/10.1016/j.jplph.2022.153822. Qin, C., Shen, J., Qi, M.D., He, Z., Lian H.D., 2023. Effects of lanthanum chloride on physiological characteristics and related gene expression of mung bean under low-phosphorus stress. Jiangsu Agricultural Sciences, 51(8): 105-110. https://doi.org/10.15889/j.issn.1002-1302.2023.08.015. Li, J., Liu, X., Xu, L., Li, W., Yao, Q., Yin, X., Wang, Q., Tan, W., Xing, W., & Liu, D., 2023. Low nitrogen stress-induced transcriptome changes revealed the molecular response and tolerance characteristics in maintaining the C/N balance of sugar beet ( Beta vulgaris L.). Frontiers in plant science, 14, 1164151. https://doi.org/10.3389/fpls.2023.1164151. Li, R. F., Xia, W. Q., Liu, J. B., Cui, B. S., Hou, Q., Sun, H., Li, S., 2018. New triterpenoid saponins from the leaves of Ilex chinensis . Fitoterapia, 131, 134–140. https://doi.org/10.1016/j.fitote.2018.10.007. Shao, S. Y., Li, R. F., Sun, H., & Li, S., 2021. New triterpenoid saponins from the leaves of Ilex chinensis and their hepatoprotective activity. Chinese journal of natural medicines, 19(5), 376–384. https://doi.org/10.1016/S1875-5364(21)60036-5. Shao, S., Li, R., Wang, K., Xia, W., Cui, B., Li, S., 2023. Ilexchinene, a new seco-ursane triterpenoid from the leaves of Ilex chinensis with therapeutic effect on neuroinflammation by attenuating the MAPK/NF-κB signaling pathway. Phytomedicine : international journal of phytotherapy and phytopharmacology, 121, 155110. https://doi.org/10.1016/j.phymed.2023.155110. Reitznerová, A., Šuleková, M., Nagy, J., Marcinčák, S., Semjon, B., Čertík, M., & Klempová, T., 2017. Lipid Peroxidation Process in Meat and Meat Products: A Comparison Study of Malondialdehyde Determination between Modified 2-Thiobarbituric Acid Spectrophotometric Method and Reverse-Phase High-Performance Liquid Chromatography. Molecules (Basel, Switzerland), 22(11), 1988. https://doi.org/10.3390/molecules22111988. Giannopolitis, C.N., & Ries, S.K., 1977. Superoxide dismutases: I. Occurrence in higher plants. Plant physiology, 59(2), 309–314. https://doi.org/10.1104/pp.59.2.309. Chen, J.X., Wang, X.F., 2015). Experimental Guide to Plant Physiology. South China University of Technology Press. p72-73. Zheng, X., Yang, H., Zou, J., Jin, W., Qi, Z., Yang, P., Yu, J., & Zhou, J., 2024. SnRK1α1-mediated RBOH1 phosphorylation regulates reactive oxygen species to enhance tolerance to low nitrogen in tomato. The Plant cell, 37(1), koae321. https://doi.org/10.1093/plcell/koae321. Zhang, J.F., Wang, Y.Y., He, L., Yan, J.Y., Liu, Y.Y., Ruan, Z.Y., Liu, W.C., Yi, L., Ren, F., 2024. PHR1 involved in the regulation of low phosphate-induced leaf senescence by modulating phosphorus homeostasis in Arabidopsis. Plant, cell & environment, 47(3), 799–816. https://doi.org/10.1111/pce.14790. Wang, X., Chai, X., Gao, B., Deng, C., Günther, C. S., Wu, T., Zhang, X., Xu, X., Han, Z., & Wang, Y., 2023. Multi-omics analysis reveals the mechanism of bHLH130 responding to low-nitrogen stress of apple rootstock. Plant physiology, 191(2), 1305–1323. https://doi.org/10.1093/plphys/kiac519. Seidel, S.J., Ahmadi, S.H., Weihermüller, L., Couëdel, A., Lopez, G., Behrend, D., Kamali, B., Gaiser, T., Hernández-Ochoa, I.M., 2024. The overlooked effects of environmental impacts on root:shoot ratio in experiments and soil-crop models. The Science of the total environment, 955, 176738. https://doi.org/10.1016/j.scitotenv.2024.176738. Li, M., Zhou, J., Lang, X., Han, D., Hu, Y., Ding, Y., Wang, G., Guo, J., Shi, L., 2022. Integrating transcriptomic and metabolomic analysis in roots of wild soybean seedlings in response to low-phosphorus stress. Frontiers in plant science, 13, 1006806. https://doi.org/10.3389/fpls.2022.1006806. Xia, Z., Gong, Y., Yang, Y., Wu, M., Bai, J., Zhang, S., Lu, H., 2024. Effects of root-zone warming, nitrogen supply and their interactions on root-shoot growth, nitrogen uptake and photosynthetic physiological characteristics of maize. Plant physiology and biochemistry : PPB, 214, 108887. https://doi.org/10.1016/j.plaphy.2024.108887. Chen, L.H., Xu, M., Cheng, Z., Yang, L. T., 2024. Effects of Nitrogen Deficiency on the Photosynthesis, Chlorophyll a Fluorescence, Antioxidant System, and Sulfur Compounds in Oryza sativa . International journal of molecular sciences, 25(19), 10409. https://doi.org/10.3390/ijms251910409. Zhang, Z.W., Yang, X.Y., Zheng, X.J., Fu, Y.F., Lan, T., Tang, X.Y., Wang, C.Q., Chen, G.D., Zeng, J., Yuan, S., 2020. Vitamin E Is Superior to Vitamin C in Delaying Seedling Senescence and Improving Resistance in Arabidopsis Deficient in Macro-Elements. International journal of molecular sciences, 21(19), 7429. https://doi.org/10.3390/ijms21197429. Cai, Q., Zou, Y., Zhong, N., Jia, G., 2024. Effects of nutritional stress on soil fertility and antioxidant enzymes of rice in different growth periods. Frontiers in plant science, 15, 1471682. https://doi.org/10.3389/fpls.2024.1471682. Ali, M.F., Mahmood, I., Shamsi, I.H., Asad, S.A., Ali, H., Shah, G.A., Tung, S.A., Raja, N.I., Demirkıran, A.R., Hussain, M.I., 2025. Integrated application of nitrogen and mepiquat chloride improves yield of Brassica napus L. by modulating shattering, lodging and nitrogen use efficiency under rainfed conditions. Plant Growth Regul. https://doi.org/10.1007/s10725-025-01343-1. Cheng, L.L., Pan, Y., Lin, Y., Lin, S.X., Tong, Z.K., 2020. Effect of Low Nitrogen Stress on the Growth, Physiological, and Biochemical Characteristics of Different Betula luminifera Genotypes. Journal of Nuclear Agricultural Sciences, 34(11): 2435-2443. https://doi.org/10.11869/j.issn.100-8551.2020.11.2435. Luo, J., Hang, J.N., Wu, B.L., Wei, X.L., Zhao, Q.Z., Fang, Z.M., 2023. Co-overexpression of genes for nitrogen transport, assimilation, and utilization boosts rice grain yield and nitrogen use efficiency. The Crop Journal, 11(4): 785-799. https://doi.org/10.1016/j.cj.2023.01.005. Zhang, C., Meng, S., Li, M., Zhao, Z., 2018. Transcriptomic insight into nitrogen uptake and metabolism of Populus simonii in response to drought and low nitrogen stresses. Tree physiology, 38(11), 1672–1684. https://doi.org/10.1093/treephys/tpy085. Lv, X.M., Zhang, Y.X., Hu, L., Zhang, B., Xia, H.Y., Du, W.Y., Fan, S.J., Kong, L.G., 2021. Low-nitrogen stress stimulates lateral root initiation and nitrogen assimilation in wheat: roles of phytohormone signaling. Journal of Plant Growth Regulation, 40(2): 436-450. https://doi.org/10.1007/s00344-020-10112-5. Li, J., Yang, P., Sohail, H., Du, H.B., Li, J., 2023. The impact of short-term nitrogen starvation and replenishment on the nitrate metabolism of hydroponically grown spinach. Scientia Horticulturae, 309: 111632. https://doi.org/10.1016/j.scienta.2022.111632. Deng, S., Li, J., Du, Z., Wu, Z., Yang, J., Cai, H., Wu, G., Xu, F., Huang, Y., Wang, S., Wang, C., 2022. Rice ACID PHOSPHATASE 1 regulates Pi stress adaptation by maintaining intracellular Pi homeostasis. Plant, cell & environment, 45(1), 191–205. https://doi.org/10.1111/pce.14191. Xu, J.H., Qian, S.Y., Wang, S.Z., Shen, X.Y., Zhuang, C.C., 2022. Effects of low phosphorus stress on the growth and development in pe-cans ( Carya illinoinensis ). Journal of Fruit Science, 39(8): 1432-1442. https://doi.org/10.13925/j.cnki.gsxb.20210683. Liu, S., Xu, Z., Essemine, J., Liu, Y., Liu, C., Zhang, F., Iqbal, Z., Qu, M., 2024. GWAS unravels acid phosphatase ACP2 as a photosynthesis regulator under phosphate starvation conditions through modulating serine metabolism in rice. Plant communications, 5(7), 100885. https://doi.org/10.1016/j.xplc.2024.100885. Xie, B., Zhao, D.Y., Chen, Y.H., Cheng, C.G., An, X.H., 2022. Physiological response of young trees of different apple cultivars to nitrogen and phosphorus deficiency. Plant Nutrition and Fertilizer Science, 28(11): 2106-2117. https://doi.org/10.11674/zwyf.2022078. Asif, I., Dong, Q., Wang, X.R., Gui, H.P., Zhang, H.H., Zhang, X.L. , Song, M.Z., 2023. Phosphorus and carbohydrate metabolism contributes to low phosphorus tolerance in cotton. BMC plant biology, 23(1), 97. https://doi.org/10.1186/s12870-023-04100-6. Shen, Q., Xiao, J.T., Wang, L.Y., Feng, J.G., Chen, Y., Zhu, B., Li, H.G., Lambers, H., 2025. Maize shows intraspecific facilitation under phosphorus deficiency but competition under nitrogen deficiency when grown under increased plant densities in alkaline soil. Plant Soil. 2025. https://doi.org/10.1007/s11104-024-07188-3. Wang, H.X., Liu, T.Y., Zhuang, W.B., Wang, Z., Zhu, L., 2020. Research Advances in the Function of Anthocyanin in Plant Stress Response. Journal of Agricultural Biotechnology, 28(1): 174-183. https://doi.org/10.3969/j.issn.1674-7968.2020.01.018. Tao, H., Gao, F., Linying Li, He, Y., Zhang, X., Wang, M., Wei, J., Zhao, Y., Zhang, C., Wang, Q., Hong, G., 2024. WRKY33 negatively regulates anthocyanin biosynthesis and cooperates with PHR1 to mediate acclimation to phosphate starvation. Plant communications, 5(5), 100821. https://doi.org/10.1016/j.xplc.2024.100821. An, J. P., Li, H. L., Liu, Z. Y., Wang, D. R., You, C. X., Han, Y., 2023. The E3 ubiquitin ligase SINA1 and the protein kinase BIN2 cooperatively regulate PHR1 in apple anthocyanin biosynthesis. Journal of integrative plant biology, 65(9), 2175–2193. https://doi.org/10.1111/jipb.13538 Jia, X.Y., Liu, H., Shen, J., Li, F., Ding, N.,2015. Negative Regulation of Anthocyanin Biosynthesis in Tomato by MicroRNA828 Under Phosphate Deficiency. Scientia Agricultura Sinica, 2015, 48(15): 2911-2924. Yang, B., Peng, Y., Zhang, G., Liu, R., Hao, S., Ren, Y., Lu, S., Wang, X., Guo, L., 2025. Regulation of glucosylceramide synthase and sphingolipid remodeling in the plant response to phosphate deficiency. The Plant cell, 37(6), koaf138. https://doi.org/10.1093/plcell/koaf138. Ji, M.J., Cao, ZY, Wang YT, Lu, J.R., Wang, B.H., 2023. Genome-wide identification and analysis of NCS1 gene family in upland cotton. Scientia Agricultura Sinica, 2023, 35(2): 249-258. https://doi.org/10.3969/j.issn.1004-1524.2023.02.01. Liu, R.X., Li, H.L., Rui, L., Liu, G.D., Wang, T., Wang, X.F., Li, L.G., Zhang, Z., You, C.X., 2023. An apple NITRATE REDUCTASE 2 gene positively regulates nitrogen utilization and abiotic stress tolerance in Arabidopsis and apple callus. Plant physiology and biochemistry : PPB, 196, 23–32. https://doi.org/10.1016/j.plaphy.2023.01.026. Yang, Y., Steidele, C.E., Rössner, C., Löffelhardt, B., Kolb, D., Leisen, T., Zhang, W., Ludwig, C., Felix, G., Seidl, M. F., Becker, A., Nürnberger, T., Hahn, M., Gust, B., Gross, H., Hückelhoven, R., Gust, A. A., 2023. Convergent evolution of plant pattern recognition receptors sensing cysteine-rich patterns from three microbial kingdoms. Nature communications, 14(1), 3621. https://doi.org/10.1038/s41467-023-39208-8. Huang, Y., Gong, X., Shi, H., Wang, P., Yuan, Y., Kong, C., Zhou, J., Wu, D., Liang, Y., Wang, Y., Wang, J., 2025. OsHYPK/NatA-mediated N-terminal acetylation regulates the homeostasis of NLR immune protein to fine-tune rice immune responses and growth. Cell reports, 44(5), 115719. https://doi.org/10.1016/j.celrep.2025.115719. Dong, Q., Duan, D., Wang, F., Yang, K., Song, Y., Wang, Y., Wang, D., Ji, Z., Xu, C., Jia, P., Luan, H., Guo, S., Qi, G., Mao, K., Zhang, X., Tian, Y., Ma, Y., Ma, F., 2024. The MdVQ37-MdWRKY100 complex regulates salicylic acid content and MdRPM1 expression to modulate resistance to Glomerella leaf spot in apples. Plant biotechnology journal, 22(8), 2364–2376. https://doi.org/10.1111/pbi.14351. Yu, S.W., Zhang, X.Q., Chen, S.J., Ye, S.F., Zhang, Y., 2021. Analysis of the expression characteristics of OsMKKs gene under abiotic stress during development. Acta Agriculturae Shanghai, 37(3): 29-34. https://doi.org/10.15955/j.issn1000-3924.2021.03.06. Zhang, X.X., Ji, G.H., Mu. Y., Jiang, W.G., Liu, Q.Y., 2025. Identification and Abiotic Stress Response of Adenosine 5'-phosphosulfate Reductase (APR) Gene Family in Brassica napus . Acta Agriculturae Shanghai, 33(1): 15-29. https://doi.org/10.3969/j.issn.1674-7968.2025.01.002. Tran, A.D., Cho, K., Han, O., 2023. Rice peroxygenase catalyzes lipoxygenase-dependent regiospecific epoxidation of lipid peroxides in the response to abiotic stressors. Bioorganic chemistry, 131, 106285. https://doi.org/10.1016/j.bioorg.2022.106285. Rashotte, A. M., Mason, M. G., Hutchison, C. E., Ferreira, F. J., Schaller, G. E., Kieber, J. J., 2006. A subset of Arabidopsis AP2 transcription factors mediates cytokinin responses in concert with a two-component pathway. Proceedings of the National Academy of Sciences of the United States of America, 103(29), 11081–11085. https://doi.org/10.1073/pnas.0602038103. Prathap, V., Kumar, S., Tyagi, A., Mohapatra, T., 2024. Integrated transcriptome and proteome analysis of near-isogenic line provides insights on regulatory function of Pup1 QTL in rice under phosphorus-starvation stress. Environmental and Experimental Botany, 221: 105726. https://doi.org/10.1016/j.envexpbot.2024.105726. Gentile, D., Serino, G., Frugis, G., 2024. CRF transcription factors in the trade-off between abiotic stress response and plant developmental processes. Front Genet, 15: 1377204. Published 2024 Apr 16. https://doi.org/10.3389/fgene.2024.1377204. Wang, C.Y., Fan, F.H., 2023. Identification of SPL Gene Family in Pinus massoniana and Their Expression in Response to Low Phosphorus Stress. Journal of Agricultural Biotechnology, 31(3): 509-517. https://doi.org/10.3969/j.issn.1674-7968.2023.03.006. Meng, C.M., Geng, F.F., Qing, G.X., Li, X.L., Zhang, F.H., 2023. Cloning and Expression Analysis of Phosphorus Deficiency Stress Gene GhERF5 in Gossypium hirsutum L.. Acta Botanica Boreali-Occidentalia Sinica, 43(3): 382-388. https://doi.org/10.7606/j.issn.1000-4025.2023.03.0382. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 11 Feb, 2026 Read the published version in BMC Plant Biology → Version 1 posted Editorial decision: Revision requested 22 Oct, 2025 Reviews received at journal 18 Oct, 2025 Reviewers agreed at journal 17 Oct, 2025 Reviews received at journal 11 Oct, 2025 Reviewers agreed at journal 21 Sep, 2025 Reviewers invited by journal 19 Sep, 2025 Editor assigned by journal 14 Sep, 2025 Editor invited by journal 11 Sep, 2025 Submission checks completed at journal 11 Sep, 2025 First submitted to journal 11 Sep, 2025 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-7390500","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":522269610,"identity":"4d54dc15-ac70-4911-a063-038c245fcd85","order_by":0,"name":"Jing Liu","email":"","orcid":"","institution":"National Key Laboratory for Development and Utilization of Forest Food Resources, Zhejiang A\u0026F University","correspondingAuthor":false,"prefix":"","firstName":"Jing","middleName":"","lastName":"Liu","suffix":""},{"id":522269611,"identity":"d2efba59-467c-44ca-9d3e-9b8c684869fd","order_by":1,"name":"Gong Cheng","email":"","orcid":"","institution":"National Key Laboratory for Development and Utilization of Forest Food Resources, Zhejiang A\u0026F University","correspondingAuthor":false,"prefix":"","firstName":"Gong","middleName":"","lastName":"Cheng","suffix":""},{"id":522269612,"identity":"2fa96e02-fc3e-4f34-b22a-2617967cd23a","order_by":2,"name":"Jiejie Jiao","email":"","orcid":"","institution":"Zhejiang Hangzhou Urban Ecosystem Research Station, Zhejiang Academy of Forestry","correspondingAuthor":false,"prefix":"","firstName":"Jiejie","middleName":"","lastName":"Jiao","suffix":""},{"id":522269613,"identity":"5eab2394-58c6-400f-90cc-1d7bc5648590","order_by":3,"name":"Jiaxin Hu","email":"","orcid":"","institution":"National Key Laboratory for Development and Utilization of Forest Food Resources, Zhejiang A\u0026F University","correspondingAuthor":false,"prefix":"","firstName":"Jiaxin","middleName":"","lastName":"Hu","suffix":""},{"id":522269614,"identity":"6493e45d-8a0a-4f8f-95dc-cf08abf54c00","order_by":4,"name":"Bingsong Zheng","email":"","orcid":"","institution":"National Key Laboratory for Development and Utilization of Forest Food Resources, Zhejiang A\u0026F University","correspondingAuthor":false,"prefix":"","firstName":"Bingsong","middleName":"","lastName":"Zheng","suffix":""},{"id":522269615,"identity":"c11d354f-1122-4c14-a449-dd3ff6e99690","order_by":5,"name":"Daoliang Yan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4UlEQVRIiWNgGAWjYBACPmYwJcHADyQ/ADFjAyEtbDAtkg0MjDOI0wJjGBwgWgs787OHX/5YyBmfP/ywmYfBRnbDAeZnD/A7jM3cWLZNwtjswDFDoJY04w0H2MwNCPjFTFqyQSJx28EG88c8DIcTNxzgYZPAr4X9m7TEH4n6zc3sH4G2/CdGC4+Z5Ac2iQQDNh6Qww4QpaVMmrFNwnDGGZ7CxjkGycYzD7OZ4dXCz398m+SPP3Xy/P3HNza8qbCT7Tve/AyvFhBg5oEzQUHFTEg9EDD+IELRKBgFo2AUjGAAADvGP7kLJ9l1AAAAAElFTkSuQmCC","orcid":"","institution":"National Key Laboratory for Development and Utilization of Forest Food Resources, Zhejiang A\u0026F University","correspondingAuthor":true,"prefix":"","firstName":"Daoliang","middleName":"","lastName":"Yan","suffix":""}],"badges":[],"createdAt":"2025-08-17 05:23:06","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7390500/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7390500/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12870-026-08360-w","type":"published","date":"2026-02-11T15:56:59+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":92598757,"identity":"e6f818a8-e695-4739-a1ff-33b0f9582d3a","added_by":"auto","created_at":"2025-10-01 13:49:20","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":4442174,"visible":true,"origin":"","legend":"","description":"","filename":"TheEffectsofNitrogenandPhosphorusDeficiencyontheMainPhysiologyofIlexchinensisandTranscriptomicAnalysis.docx","url":"https://assets-eu.researchsquare.com/files/rs-7390500/v1/4d7c19a883f1ba0c8e633c4f.docx"},{"id":92598634,"identity":"bc9ac02b-c507-467a-acdb-262268b321b3","added_by":"auto","created_at":"2025-10-01 13:49:16","extension":"json","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":8153,"visible":true,"origin":"","legend":"","description":"","filename":"0a6926bf836a4b8db8e7fa1502aa74cf.json","url":"https://assets-eu.researchsquare.com/files/rs-7390500/v1/4464ab71c0cc1282f7c98e68.json"},{"id":92598636,"identity":"ea7676ed-b771-4919-ae18-04f6df09c169","added_by":"auto","created_at":"2025-10-01 13:49:17","extension":"xml","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":210512,"visible":true,"origin":"","legend":"","description":"","filename":"0a6926bf836a4b8db8e7fa1502aa74cf1enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-7390500/v1/f44d86ba423a238fb638faf5.xml"},{"id":92598645,"identity":"33a3b990-33c7-4a86-82d4-3f3a2ca088df","added_by":"auto","created_at":"2025-10-01 13:49:17","extension":"png","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":120822,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7390500/v1/6eba63da8ea705e555eac147.png"},{"id":92600003,"identity":"81b7aa99-4ed4-451a-9f94-e1721099c076","added_by":"auto","created_at":"2025-10-01 13:57:21","extension":"jpeg","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":654760,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7390500/v1/09698bc4fbe57eb8e851e659.jpeg"},{"id":92598639,"identity":"9b536ab3-1e26-4300-a6d7-66120f8ef4ba","added_by":"auto","created_at":"2025-10-01 13:49:17","extension":"png","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":197439,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7390500/v1/72a3f5f47a6435dbb15587f3.png"},{"id":92599999,"identity":"2f8839b1-e86c-4bc7-a162-f48a703e35fa","added_by":"auto","created_at":"2025-10-01 13:57:17","extension":"jpeg","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":12085,"visible":true,"origin":"","legend":"","description":"","filename":"groupimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7390500/v1/832d55d563604333d3cb62c0.jpeg"},{"id":92598637,"identity":"02b0f9d9-1f5f-4e03-8d48-8fe0fd2b74f3","added_by":"auto","created_at":"2025-10-01 13:49:17","extension":"jpeg","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":103788,"visible":true,"origin":"","legend":"","description":"","filename":"groupimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7390500/v1/1f3ad769ddeb48303e2c37d1.jpeg"},{"id":92598651,"identity":"6229cbe3-b3c8-4a0d-9d67-038f81b32b46","added_by":"auto","created_at":"2025-10-01 13:49:17","extension":"jpeg","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":67297,"visible":true,"origin":"","legend":"","description":"","filename":"groupimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7390500/v1/5803d04cf11b767c3af7864e.jpeg"},{"id":92598652,"identity":"8fbaa993-7870-4bc3-9a88-f8d6ab74358f","added_by":"auto","created_at":"2025-10-01 13:49:17","extension":"jpeg","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":26140,"visible":true,"origin":"","legend":"","description":"","filename":"groupimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7390500/v1/b18296e60760546dbd8f4f50.jpeg"},{"id":92598661,"identity":"6af14c41-9e2e-416f-8e7b-3829a8f6aa4b","added_by":"auto","created_at":"2025-10-01 13:49:18","extension":"jpeg","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":24420,"visible":true,"origin":"","legend":"","description":"","filename":"groupimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7390500/v1/ca585c2a6bddb9f1217d6497.jpeg"},{"id":92600001,"identity":"7616bb32-512b-4f03-a7f8-8d740392f79f","added_by":"auto","created_at":"2025-10-01 13:57:20","extension":"jpeg","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":24395,"visible":true,"origin":"","legend":"","description":"","filename":"groupimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7390500/v1/665ef2bd56e6fc8cd2e8149c.jpeg"},{"id":92598648,"identity":"9c37c21c-8e73-4e6f-9928-b6592b52117a","added_by":"auto","created_at":"2025-10-01 13:49:17","extension":"jpeg","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":61569,"visible":true,"origin":"","legend":"","description":"","filename":"groupimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7390500/v1/ee701d6c3fad5f29d7b39031.jpeg"},{"id":92598649,"identity":"46209ed1-e093-48d0-aacc-3e576b649192","added_by":"auto","created_at":"2025-10-01 13:49:17","extension":"png","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":33544,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7390500/v1/8a17b56d0627e1a309b279f5.png"},{"id":92598646,"identity":"db63fd48-39b1-4921-89bc-0007a2695db9","added_by":"auto","created_at":"2025-10-01 13:49:17","extension":"png","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":118009,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7390500/v1/cd76bcc5cd17ddd1a76870b0.png"},{"id":92600004,"identity":"7070c485-ea08-41d5-85b7-6eb3ba8f1f08","added_by":"auto","created_at":"2025-10-01 13:57:21","extension":"png","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":45813,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7390500/v1/c8fa3e1e7681a5165cd914c0.png"},{"id":92600002,"identity":"40b89557-967c-440e-be78-b11e68f68945","added_by":"auto","created_at":"2025-10-01 13:57:20","extension":"png","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":7661,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinegroupimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7390500/v1/0386a6c6ddf6697f59370720.png"},{"id":92598665,"identity":"06031f22-b8d4-4d02-b05a-c849d16d8ae2","added_by":"auto","created_at":"2025-10-01 13:49:18","extension":"png","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":31452,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinegroupimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7390500/v1/e8db6f9888e0bfebfab2d688.png"},{"id":92598664,"identity":"d0b4219a-1668-468d-bb8c-6661f278b750","added_by":"auto","created_at":"2025-10-01 13:49:18","extension":"png","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":20944,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinegroupimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7390500/v1/b81325aa2c2655ac6ff13e4b.png"},{"id":92598663,"identity":"68be68ef-5c0e-4678-b95b-a070d748ded2","added_by":"auto","created_at":"2025-10-01 13:49:18","extension":"png","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":8769,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinegroupimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7390500/v1/7a3442a57c27655683a911cd.png"},{"id":92598654,"identity":"b01eb4dd-d2ea-484e-ac14-0c439de0dcdd","added_by":"auto","created_at":"2025-10-01 13:49:17","extension":"png","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":7435,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinegroupimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7390500/v1/d73b4185d7a7e4f60d2edd24.png"},{"id":92598666,"identity":"752ff518-fb05-46be-8178-5d7c0bf81538","added_by":"auto","created_at":"2025-10-01 13:49:18","extension":"png","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":13062,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinegroupimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7390500/v1/ee294764303b114a8f1a5abc.png"},{"id":92598659,"identity":"aba6e1f2-8dc0-4fe8-a98e-1be35fef18f5","added_by":"auto","created_at":"2025-10-01 13:49:17","extension":"png","order_by":22,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":14924,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinegroupimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7390500/v1/f95ce81bef538860d4cb6e47.png"},{"id":92598667,"identity":"56103258-f1bc-4b91-a031-881a8091647d","added_by":"auto","created_at":"2025-10-01 13:49:18","extension":"xml","order_by":23,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":209197,"visible":true,"origin":"","legend":"","description":"","filename":"0a6926bf836a4b8db8e7fa1502aa74cf1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7390500/v1/29f9884b566d935deef2a4d8.xml"},{"id":92598668,"identity":"2ab4b6c9-ce72-4f80-adb3-192ae5da721b","added_by":"auto","created_at":"2025-10-01 13:49:18","extension":"html","order_by":24,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":225462,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7390500/v1/e3c029b62e3c1bf08baa1bb4.html"},{"id":92598640,"identity":"48c41c94-c090-45d4-99bc-8a16327e4e1c","added_by":"auto","created_at":"2025-10-01 13:49:17","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":68445,"visible":true,"origin":"","legend":"\u003cp\u003ePhenotypic changes of \u003cem\u003eI. chinensis\u003c/em\u003e leaves after T\u003csub\u003e2\u003c/sub\u003e treatment with LN and LP.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7390500/v1/deadb5b3cda7a01bef16c0fd.png"},{"id":92598635,"identity":"b7f67cce-16ff-4d56-860e-4e636da493a4","added_by":"auto","created_at":"2025-10-01 13:49:16","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":31036,"visible":true,"origin":"","legend":"\u003cp\u003eThe effects of LN (left) and LP (right) treatments on the main growth indicators of \u003cem\u003eI. chinensis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eNotes: T\u003csub\u003e0\u003c/sub\u003e=week 0, T\u003csub\u003e1\u003c/sub\u003e=week 5, T\u003csub\u003e2\u003c/sub\u003e=week 10; Different letters represent significant differences between the treatment groups (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7390500/v1/74eb4f7157345b8e6cfea3c8.png"},{"id":92600000,"identity":"d9d74aae-0027-48b9-89c7-ea92d7bdefea","added_by":"auto","created_at":"2025-10-01 13:57:18","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":35604,"visible":true,"origin":"","legend":"\u003cp\u003eDetermination of antioxidant enzyme system indexes of \u003cem\u003eI. chinensis\u003c/em\u003e leaves under LN (left) and LP (right) treatments\u003c/p\u003e\n\u003cp\u003eNotes: T\u003csub\u003e0\u003c/sub\u003e=week 0, T\u003csub\u003e1\u003c/sub\u003e=week 5, T\u003csub\u003e2\u003c/sub\u003e=week 10; Different letters represent significant differences between the treatment groups (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7390500/v1/64766f3780522305c371c71a.png"},{"id":92599998,"identity":"566fd5aa-3c14-4b5f-bbaf-e7491a3396f7","added_by":"auto","created_at":"2025-10-01 13:57:17","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":22214,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of low nitrogen treatment on the activities of NR (A) and GS (B) in \u003cem\u003eI. chinensis\u003c/em\u003e leaves\u003c/p\u003e\n\u003cp\u003eNotes: T\u003csub\u003e0\u003c/sub\u003e=week 0, T\u003csub\u003e1\u003c/sub\u003e=week 5, T\u003csub\u003e2\u003c/sub\u003e=week 10; Different letters represent significant differences between the treatment groups (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7390500/v1/58631aed415611ddcaa05836.png"},{"id":92598643,"identity":"926b8c6c-e459-4d36-8351-0889513d70f7","added_by":"auto","created_at":"2025-10-01 13:49:17","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":22898,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of low phosphorus treatment on anthocyanin content (A) and ACP (B) activity in \u003cem\u003eI. chinensis\u003c/em\u003e leaves\u003c/p\u003e\n\u003cp\u003eNotes: T\u003csub\u003e0\u003c/sub\u003e=week 0, T\u003csub\u003e1\u003c/sub\u003e=week 5, T\u003csub\u003e2\u003c/sub\u003e=week 10; Different letters represent significant differences between the treatment groups (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7390500/v1/0ae508394ec1d7c00d89c2c3.png"},{"id":92598644,"identity":"5396d1e3-0bba-4559-bd4a-031549fe04e7","added_by":"auto","created_at":"2025-10-01 13:49:17","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":122256,"visible":true,"origin":"","legend":"\u003cp\u003ePearson correlation coefficients among all samples\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7390500/v1/b25a996ced23ea4533bb29b2.png"},{"id":92598656,"identity":"4311f9ea-3f83-418e-950f-2bf6b595577c","added_by":"auto","created_at":"2025-10-01 13:49:17","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":87758,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of differentially expressed genes in the transcriptome of \u003cem\u003eI. chinensis\u003c/em\u003e under LN and LP treatment and control\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-7390500/v1/720fda128f6c71da3bb94f0a.png"},{"id":92598647,"identity":"6ecb3a51-580b-4b5e-a21b-08308990f767","added_by":"auto","created_at":"2025-10-01 13:49:17","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":273343,"visible":true,"origin":"","legend":"\u003cp\u003eBubble plot analysis of GO enrichment (A) and KEGG enrichment of differentially expressed genes under LN (left) and LP (right) treatment\u003c/p\u003e\n\u003cp\u003eNotes:\u003c/p\u003e\n\u003cp\u003eA: Results of the first 20 pathways for GO enrichment analysis of DEGs in LN\u003csub\u003e2\u003c/sub\u003e vs CK and LP\u003csub\u003e2\u003c/sub\u003e vs CK. The horizontal coordinate Rich Factor represents the proportion of the difference between the Unigenes located in the GO to the total Unigenes located in the GO (Rich.Factor = S/B). The larger the Rich factor, the higher the GO enrichment; the vertical coordinate is GO Term, that is, the GO function annotation; in the bubble chart, the size of the bubble represents the number of S, and the color of the bubble represents the P value of the enrichment analysis, that is, the significance of the enrichment. The smaller the P value, the more significant the enrichment is.\u003c/p\u003e\n\u003cp\u003eB: Results of the first 20 pathways for KEGG enrichment analysis of DEGs in LN2 vs CK and LP2 vs CK. The horizontal coordinate Rich Factor represents the proportion of the difference Unigene number located in the Pathway to the total Unigene number located in the Pathway. The larger the Rich Factor, the higher the degree of enrichment of the Pathway; the vertical coordinate is KEGG Pathway; in the bubble chart, the size of the bubble represents S, and the color of the bubble represents the P value of the enrichment analysis, that is, the significance of the enrichment. The smaller the P value, the more significant the enrichment is.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-7390500/v1/05954a5d384661b4c4867c41.png"},{"id":92598653,"identity":"a85f6afe-48fa-4742-ab4f-7dee63c16a1f","added_by":"auto","created_at":"2025-10-01 13:49:17","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":358149,"visible":true,"origin":"","legend":"\u003cp\u003eHeat map of differential gene expression in key KEGG pathways in the LN(left) and LP(right) treatment control group\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-7390500/v1/c11aa47db51decad9d6c120b.png"},{"id":92598655,"identity":"33342b01-7ea3-44db-a07d-ee08d8941c81","added_by":"auto","created_at":"2025-10-01 13:49:17","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":216516,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic diagram of the multiple regulatory mechanisms of \u003cem\u003eI. chinensis\u003c/em\u003e in response to nitrogen and phosphorus deficiency.\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-7390500/v1/29f8235370b248da1c175a34.png"},{"id":102785720,"identity":"a69d0466-a754-4cde-9161-d7edaa483f8f","added_by":"auto","created_at":"2026-02-16 16:09:12","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2712578,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7390500/v1/7c31e506-e1f2-4197-b9eb-1d41786995f5.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"The Effects of Nitrogen and Phosphorus Deficiency on the Main Physiology of Ilex chinensis and Transcriptomic Analysis","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eNitrogen (N) and phosphorus (P) are key macroelements for plant growth and development, and are important components of biological macromolecules such as nucleic acids and proteins in plants. They are widely involved in physiological processes such as photosynthesis, respiration, and signal transduction in plants. The demand for nitrogen and phosphorus in plants runs through their entire life cycle\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. Lack of nitrogen and phosphorus can lead to significant changes in plant morphology, growth, and development, ultimately resulting in reduced plant yield, decreased quality, poor ornamental value, and decreased economic value.\u003c/p\u003e\u003cp\u003ePlants subjected to nitrogen and phosphorus deficiency stress will initiate a series of complex molecular response mechanisms to enhance the absorption and utilization of limited nutrients. In nitrogen deficient environments, plants enhance nitrogen uptake and transport by upregulating the expression of nitrate transporter genes (\u003cem\u003eNRT1\u003c/em\u003e and \u003cem\u003eNRT2\u003c/em\u003e family genes), which are typically tissue-specific. The root system is often the primary site in response to low nitrogen stress \u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. In addition, up-regulation of \u003cem\u003eGS1\u003c/em\u003e \u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e, \u003cem\u003eBnaGLNs\u003c/em\u003e and \u003cem\u003eBnaNIAs\u003c/em\u003e\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003egene expression promotes nitrogen assimilation and utilization. Under low phosphorus conditions, plants induce up-regulation of the purple acid phosphatase gene \u003cem\u003ePpAPs\u003c/em\u003e to promote the synthesis and secretion of acid phosphatase (ACP) in roots, enhancing the activation and absorption capacity of insoluble phosphorus in soil \u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. Meanwhile, up-regulation of phosphorus transport related genes (\u003cem\u003ePHT1\u003c/em\u003e, \u003cem\u003ePHT1.10\u003c/em\u003e, \u003cem\u003eSPX6\u003c/em\u003e, and \u003cem\u003eSPX-MFS2\u003c/em\u003e) contributes to the effective transport and redistribution of phosphorus in plants \u003csup\u003e[\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. These molecular response mechanisms reveal how plants optimize nutrient absorption and utilization through gene expression regulation under nutritional stress, providing important molecular targets for cultivating crop varieties with high nutrient utilization efficiency.\u003c/p\u003e\u003cp\u003eUnder nitrogen and phosphorus deficiency, plant root growth is restricted and photosynthesis is hindered. Therefore, plants optimize root growth and maintain photosynthetic efficiency by regulating the expression of a series of genes. Under low nitrogen and low phosphorus conditions, plants regulate root development by modulating plant hormone signals and interacting with related genes. For example, under low phosphorus treatment, \u003cem\u003eGmGDPD2\u003c/em\u003e regulates the levels of root hormones such as gibberellin, auxin, and ethylene by sensing the upstream signal \u003cem\u003eGmMyb73\u003c/em\u003e, inhibiting main root elongation, inducing lateral root and root hair formation, thereby affecting phosphorus absorption and utilization \u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e; The expression of \u003cem\u003eGmEIL4\u003c/em\u003e in soybean roots is significantly upregulated, which can promote the growth and development of hairy roots, increase root hair density, root length, and root surface area, improve phosphorus absorption and utilization ability, and \u003cem\u003eGmEIL4\u003c/em\u003e and \u003cem\u003eGmEBF1\u003c/em\u003e jointly participate in regulating the ethylene metabolism pathway of soybean, thereby affecting the response of roots to low phosphorus stress \u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. Under low nitrogen stress, the expression of auxin synthesis gene \u003cem\u003eYUCCA\u003c/em\u003e and auxin inward transporter \u003cem\u003eAUX1\u003c/em\u003e related genes in peanuts is downregulated, leading to a decrease in auxin content and limiting the occurrence and elongation of lateral roots \u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e; The \u003cem\u003eBnIPTs\u003c/em\u003e gene in rapeseed is downregulated in the roots, leading to a decrease in cytokinin concentration in the roots, which promotes root elongation and increases the number of root tips, enhancing their ability to absorb nutrients from the soil and increasing their resistance to low nitrogen stress \u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. In addition, due to adversity stress, plants cope with the increase in reactive oxygen species content in their bodies by upregulating the expression of antioxidant enzyme related genes (\u003cem\u003eSOD9\u003c/em\u003e and \u003cem\u003eCAT2\u003c/em\u003e), thereby increasing the activity of antioxidant enzymes in plants and maintaining the balance of intracellular reactive oxygen species \u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. However, the expression levels of chlorophyll catabolism related genes (\u003cem\u003eMdNYC1\u003c/em\u003e, \u003cem\u003eMdPAO\u003c/em\u003e, and \u003cem\u003eMdSGR1\u003c/em\u003e) significantly increased \u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e, while the expression levels of PSII related genes (\u003cem\u003ePsbO\u003c/em\u003e, \u003cem\u003ePsbP\u003c/em\u003e, \u003cem\u003ePsbQ\u003c/em\u003e, and \u003cem\u003ePsbY\u003c/em\u003e) were downregulated \u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e, resulting in a decrease in chlorophyll content and photosynthetic efficiency. These molecular regulatory mechanisms reveal how plants balance growth needs and resource constraints through precise regulation of gene expression under nutritional stress, providing important molecular targets for cultivating stress tolerant crop varieties.\u003c/p\u003e\u003cp\u003e\u003cem\u003eIlex chinensis\u003c/em\u003e is widely used in urban greening and disease treatment due to its excellent ornamental value and significant medicinal value \u003csup\u003e[\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. However, the significant variations in climatic conditions and soil characteristics across different regions may lead to deficiencies or imbalances of N and P in the soil. For example, some areas lack essential N and P elements due to climate drought or soil barrenness, while others are affected by industrial pollution, frequent acid rain, and soil pH decline, which in turn reduce the availability of N and P. All of these factors have adverse effects on the growth and survival rate of \u003cem\u003eI. chinensis\u003c/em\u003e. Therefore, investigating the optimal concentrations of N and P for the growth of \u003cem\u003eI. chinensis\u003c/em\u003e, clarifying the roles of N and P in its growth and development, and understanding the impacts of N and P deficiency on plant growth are of great significance for the cultivation management and sustainable development of this species.\u003c/p\u003e\u003cp\u003eThe aim of this study is to evaluate the effects of different nitrogen and phosphorus concentrations on the growth status and physiological indicators of \u003cem\u003eI. chinensis\u003c/em\u003e by simulating nitrogen and phosphorus deficit conditions and comparing them with normal growth conditions. The aim is to clarify the phenotypic changes of \u003cem\u003eI. chinensis\u003c/em\u003e under nitrogen and phosphorus deficit conditions. The research results will provide theoretical support for screening suitable nitrogen and phosphorus concentrations for \u003cem\u003eI. chinensis\u003c/em\u003e growth. In addition, this study will explore the gene response mechanism of \u003cem\u003eI. chinensis\u003c/em\u003e under nitrogen and phosphorus deficiency through transcriptome analysis, and identify key response genes. The research results will provide scientific basis for creating transgenic \u003cem\u003eI. chinensis\u003c/em\u003e varieties with high nitrogen and phosphorus utilization efficiency, and provide theoretical references for efficient plant cultivation, water and fertilizer management, and development and utilization.\u003c/p\u003e"},{"header":"2 Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Plant Materials and Growth Conditions\u003c/h2\u003e\u003cp\u003eThe plant materials for this experiment were wild species of \u003cem\u003eIlex chinensis\u003c/em\u003e Sims, collected from Tianmu Mountain in Hangzhou, Zhejiang Province, eastern China. The collected seeds were stored in low-temperature sand, and seedlings of \u003cem\u003eI. chinensis\u003c/em\u003e were obtained through seedling cultivation. Two-year-old seedlings with a uniform height of 1.0-1.10 m were selected for the study. Two weeks before the start of the experiment, the seedlings were transplanted into pots (height 30 cm, diameter 20 cm) filled with river sand, with one seedling per pot. During the experimental period, the seedlings were maintained in a greenhouse with a temperature of (25\u0026thinsp;\u0026plusmn;\u0026thinsp;2)\u0026deg;C during the day and (20\u0026thinsp;\u0026plusmn;\u0026thinsp;2)\u0026deg;C at night, a relative humidity of (70\u0026thinsp;\u0026plusmn;\u0026thinsp;5)%, and natural light intensity.\u003c/p\u003e\u003cp\u003eThe plant species were identified by plant classification experts Yan Daoliang and Lou Luhuan. Voucher specimens have been deposited at the Herbarium of the Institute of Botany, Chinese Academy of Sciences (PE), with the accession number 4111.\u003c/p\u003e\u003cdiv id=\"Sec4\" class=\"Section3\"\u003e\u003ch2\u003e2.1.1 Compliance with Guidelines\u003c/h2\u003e\u003cp\u003e All experimental research on plants, including the collection of plant materials, was conducted in compliance with institutional, national, and international guidelines. Field studies were conducted in accordance with local legislation. The necessary permissions and/or licenses were obtained for the collection of wild plant materials.\u003c/p\u003e\u003cp\u003e\u003cb\u003e2.1.2 Compliance with the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES)\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThis study adheres to the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES). The plant species used in this study are not listed as endangered under CITES, and all collection and use of plant materials were conducted in accordance with the guidelines provided by CITES.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Experimental Design\u003c/h2\u003e\u003cp\u003eThe experiment utilized a modified Hoagland nutrient solution with the following composition (mg/L): Ca(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026middot;4H\u003csub\u003e2\u003c/sub\u003eO 616.64, KNO\u003csub\u003e3\u003c/sub\u003e 376.34, KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e 101.49 and MgSO\u003csub\u003e4\u003c/sub\u003e 60. Iron was supplied as Fe-EDTA solution (2.5 mL/L), and micronutrient solution (5 mL/L) was added, containing (mg/L): KI 0.83, H\u003csub\u003e3\u003c/sub\u003eBO\u003csub\u003e3\u003c/sub\u003e 6.2, MnSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;4H\u003csub\u003e2\u003c/sub\u003eO 22.3, ZnSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;7H₂O 8.6, Na\u003csub\u003e2\u003c/sub\u003eMoO\u003csub\u003e4\u003c/sub\u003e\u0026middot;2H\u003csub\u003e2\u003c/sub\u003eO 0.25, CuSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;5H\u003csub\u003e2\u003c/sub\u003eO 0.025 and CoCl\u003csub\u003e2\u003c/sub\u003e\u0026middot;6H\u003csub\u003e2\u003c/sub\u003eO 0.025. Five treatment groups were established: (1) normal nutrient supply (CK, nitrogen (N) concentration 125 mg/L, phosphorus (P) concentration 0.75 mmol/L), (2) mild low-N (LN\u003csub\u003e1\u003c/sub\u003e, N concentration 25 mg/L, P concentration 0.75 mmol/L), (3) severe low-N (LN\u003csub\u003e2\u003c/sub\u003e, N concentration 8.33 mg/L, P concentration 0.75 mmol/L), (4) mild low-P (LP\u003csub\u003e1\u003c/sub\u003e, N concentration 125 mg/L, P concentration 0.15 mmol/L), and (5) severe low-P (LP\u003csub\u003e2\u003c/sub\u003e, N concentration 125 mg/L, P concentration 0.05 mmol/L). Other essential elements were maintained at standard levels to meet plant growth requirements. Each treatment was replicated three times, with 10 plants per replicate. The corresponding nutrient solution was applied every 7 days until leaching from the bottom of the pots, for a total of 10 weeks. Sampling was conducted at week 0 (T\u003csub\u003e0\u003c/sub\u003e), week 5 (T\u003csub\u003e1\u003c/sub\u003e), and week 10 (T\u003csub\u003e2\u003c/sub\u003e) to measure plant growth indicators and physiological parameters.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Measurements of Growth and Physiological Parameters\u003c/h2\u003e\u003cdiv id=\"Sec7\" class=\"Section3\"\u003e\u003ch2\u003e2.3.1 Total Biomass and Root-Shoot Ratio\u003c/h2\u003e\u003cp\u003eThree plants were randomly selected from each treatment group for de-potting. The above-ground and below-ground parts were separated and thoroughly washed with deionized water to remove adhering soil and impurities. Subsequently, the samples were placed in a constant-temperature oven at 80\u0026deg;C until a constant weight was achieved. The dry weights of the above-ground and below-ground parts were measured using an electronic balance with a precision of 0.0001 g (ME104E, Mettler Toledo, Greifensee, Switzerland). The root-shoot ratio (RSR) was then calculated.\u003c/p\u003e\u003cp\u003eRSR\u0026thinsp;=\u0026thinsp;Root Dry Weight / Shoot Dry Weight\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section3\"\u003e\u003ch2\u003e2.3.2 Determination of Plant Root System Parameters\u003c/h2\u003e\u003cp\u003eThree plants were randomly selected from each treatment group for de-potting. The roots were carefully washed to remove any adhering soil and impurities. The total root length and root branch number were then measured using the LA-S plant image analysis system (LA-S, Hangzhou Wanshen Detection Technology Co., Ltd., Hangzhou, China).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section3\"\u003e\u003ch2\u003e2.3.3 Determination of leaf antioxidant enzymatic system index\u003c/h2\u003e\u003cp\u003eEnzyme extracts were prepared using phosphate buffer (pH 7.8), and enzyme activities were measured using standard colorimetric methods. The content of malondialdehyde (MDA) was determined using the thiobarbituric acid reaction method \u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. The activity of superoxide dismutase (SOD) was assessed by its ability to inhibit the reduction of nitroblue tetrazolium (NBT) \u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. The activity of peroxidase (POD) was measured by its capacity to oxidize guaiacol \u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section3\"\u003e\u003ch2\u003e2.3.4 Determination of enzyme activity and anthocyanin content related to nitrogen and phosphorus metabolism in leaves\u003c/h2\u003e\u003cp\u003eThe activities of acid phosphatase (ACP), nitrate reductase (NR), and glutamine synthetase (GS) in leaves, as well as anthocyanin content, were measured using commercial kits from Suzhou Mengxi Biotechnology Co., Ltd. (Suzhou, China).\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Transcriptomic Analysis\u003c/h2\u003e\u003cp\u003eLeaves from \u003cem\u003eI. chinensis\u003c/em\u003e plants under N and P deficiency at time point T\u003csub\u003e2\u003c/sub\u003e were collected from three treatment groups: CK, LN\u003csub\u003e2\u003c/sub\u003e, and LP\u003csub\u003e2\u003c/sub\u003e. The leaves were immediately frozen in liquid nitrogen and stored at -80\u0026deg;C for subsequent analysis, with three biological replicates per sample. Total RNA was extracted from \u003cem\u003eI. chinensis\u003c/em\u003e leaves by TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA), and its purity and quantity were checked via a bioanalyzer (Agilent, Santa Clara, CA, USA). Due to the low alignment rate of \u003cem\u003eI. chinensis\u003c/em\u003e to existing genomes, the analysis of transcriptome was conducted without the reference genome. NovaseqTM 6000 (Illumina, San Diego, CA, USA) was used to sequence samples in PE150 mode, and Trinity was used to perform gene de novo assembly on sequencing data to obtain unigenes and transcripts (gene isoforms). Using DIAMOND for functional annotation of unigenes, and six databases (NCBI_NR, Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), Pfam, SwissProt, and eggNOG) were used to perform the annotation, with an E-value\u0026thinsp;\u0026lt;\u0026thinsp;0.00001.\u003c/p\u003e\u003cp\u003eDifferential expression analysis was conducted using the criteria of fold change (FC)\u0026thinsp;\u0026ge;\u0026thinsp;2 or FC\u0026thinsp;\u0026le;\u0026thinsp;0.5 (i.e., |log2FC| \u0026ge; 1) and false discovery rate (FDR)\u0026thinsp;\u0026lt;\u0026thinsp;0.05 to identify differentially expressed genes (DEGs). For multiple group comparisons, genes with FDR\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were selected to identify statistically significant differences across groups. To further classify the functions of the DEGs, GO functional annotation and KEGG pathway enrichment analysis were conducted.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Statistical Analysis\u003c/h2\u003e\u003cp\u003eAll data were subjected to statistical analysis using SPSS 22.0 software. One-way analysis of variance (ANOVA) was employed to compare differences among different treatment groups, with the significance level set at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\u003c/div\u003e"},{"header":"3 Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003e3.1 Effects of low-nitrogen (LN) and low phosphorus (LP) on growth traits of \u003cem\u003eIlex chinensis\u003c/em\u003e\u003c/h2\u003e\n \u003cp\u003eAs shown in Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, at time point T\u003csub\u003e2\u003c/sub\u003e, significant changes in leaf area and color were observed among the different treatment groups. Compared with the CK, under low-nitrogen (LN) stress, the color of \u003cem\u003eI. chinensis\u003c/em\u003e leaves gradually lightened from dark green to light green with increasing stress intensity. The leaves also became thinner and exhibited reduced leaf area. Under LP\u003csub\u003e1\u003c/sub\u003e, the leaf color of \u003cem\u003eI. chinensis\u003c/em\u003e only slightly lightened, with no significant difference in leaf area. However, under LP\u003csub\u003e2\u003c/sub\u003e, the leaves showed obvious chlorosis and yellowing, along with the appearance of dry, yellow spots.\u003c/p\u003e\n \u003cp\u003eAs shown in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e, nitrogen and phosphorus deficiency significantly inhibited the growth and development of \u003cem\u003eI. chinensis\u003c/em\u003e. Under CK conditions, the total biomass of \u003cem\u003eI. chinensis\u003c/em\u003e increased by 11.05% and 14.97% at T\u003csub\u003e1\u003c/sub\u003e and T\u003csub\u003e2\u003c/sub\u003e, respectively, compared with T\u003csub\u003e0\u003c/sub\u003e. However, under LN and LP stress, the increase in total biomass was lower than that of CK and showed a continuous downward trend with prolonged stress duration. At T\u003csub\u003e2\u003c/sub\u003e, compared with T0, the total biomass of the LN\u003csub\u003e2\u003c/sub\u003e and LP\u003csub\u003e2\u003c/sub\u003e treatments only increased by 10.8% and 12.14%, respectively (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA). Moreover, N and P deficiency significantly increased the root-shoot ratio (RSR) of \u003cem\u003eI. chinensis\u003c/em\u003e (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB). At T\u003csub\u003e2\u003c/sub\u003e, the RSR of the LN\u003csub\u003e2\u003c/sub\u003e and LP\u003csub\u003e2\u003c/sub\u003e treatments increased by 27.73% and 23.25%, respectively, compared with CK. Nevertheless, no significant differences were observed between the LN and LP treatment groups at T\u003csub\u003e1\u003c/sub\u003e and T\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e\n \u003cp\u003eFigures \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC and \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eD indicate that N and P deficiency significantly affected the root growth of \u003cem\u003eI. chinensis\u003c/em\u003e. At T\u003csub\u003e0\u003c/sub\u003e, there were no significant differences in primary root length and root branch number among the different treatment groups of \u003cem\u003eI. chinensis\u003c/em\u003e. At T\u003csub\u003e1\u003c/sub\u003e and T\u003csub\u003e2\u003c/sub\u003e, no significant differences were observed in these indices within each LN and LP treatment group. However, compared with CK, the primary root length of the LN treatment groups LN\u003csub\u003e1\u003c/sub\u003e and LN\u003csub\u003e2\u003c/sub\u003e significantly increased by 14.77% and 7.44% at T\u003csub\u003e1\u003c/sub\u003e, and by 16.28% and 9.22% at T\u003csub\u003e2\u003c/sub\u003e, respectively. The root branch number significantly increased by 16.66% and 20.03% at T\u003csub\u003e1\u003c/sub\u003e, and by 10.33% and 12.97% at T\u003csub\u003e2\u003c/sub\u003e, respectively. For the LP treatment groups LP\u003csub\u003e1\u003c/sub\u003e and LP\u003csub\u003e2\u003c/sub\u003e, the primary root length significantly increased by 13.52% and 8.29% at T\u003csub\u003e1\u003c/sub\u003e, and by 15.53% and 8.9% at T\u003csub\u003e2\u003c/sub\u003e, respectively. The root branch number increased by 17.45% and 18.45% at T\u003csub\u003e1\u003c/sub\u003e, and by 11.62% and 12.88% at T\u003csub\u003e2\u003c/sub\u003e, respectively. Compared with T\u003csub\u003e0\u003c/sub\u003e, at T\u003csub\u003e1\u003c/sub\u003e, the primary root length of LN\u003csub\u003e1\u003c/sub\u003e and LN\u003csub\u003e2\u003c/sub\u003e increased by 21.58% and 23.81%, respectively, and the root branch number significantly increased by 22.16% and 15.71%, respectively. For LP\u003csub\u003e1\u003c/sub\u003e and LP\u003csub\u003e2\u003c/sub\u003e, the primary root length increased by 20.35% and 22.55%, respectively, and the root branch number significantly increased by 22.99% and 16.89%, respectively.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003e3.2 Effects of LN and LP on Antioxidant Enzyme Activities in \u003cem\u003eI. chinensis\u003c/em\u003e\u003c/h2\u003e\n \u003cp\u003eAs shown in Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e, at T\u003csub\u003e2\u003c/sub\u003e, the activities of superoxide dismutase (SOD) and peroxidase (POD), as well as the content of malondialdehyde (MDA), increased most significantly in the LN\u003csub\u003e2\u003c/sub\u003e and LP\u003csub\u003e2\u003c/sub\u003e groups. Specifically, in the LN\u003csub\u003e2\u003c/sub\u003e group, the activities of SOD and POD and the content of MDA were 1072.95 U\u0026middot;g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 153.8 U/g\u0026middot;min, and 23.98 \u0026micro;mol/g, respectively, which were significantly higher than those in the CK group by 67.56%, 151.79%, and 248.04%, respectively. In the LP\u003csub\u003e2\u003c/sub\u003e group, the activities of SOD and POD and the content of MDA were 1058.48 U/g, 120.8 U/g\u0026middot;min, and 16.14 \u0026micro;mol\u0026middot;g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively, which were significantly higher than those in the CK group by 65.46%, 97.81%, and 134.25%, respectively. At each treatment period, the activities of SOD and POD and the content of MDA in the LN\u003csub\u003e2\u003c/sub\u003e group were significantly higher than those in the LN\u003csub\u003e1\u003c/sub\u003e group. Under low-phosphorus stress, only the trends of SOD activity and MDA content were similar to those under low-nitrogen stress. At T\u003csub\u003e1\u003c/sub\u003e, there were no significant differences in POD activity between the LP\u003csub\u003e1\u003c/sub\u003e and LP\u003csub\u003e2\u003c/sub\u003e groups, but at T\u003csub\u003e2\u003c/sub\u003e, the POD activity in the LP\u003csub\u003e2\u003c/sub\u003e group was significantly lower than that in the LP\u003csub\u003e1\u003c/sub\u003e group.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e3.3 Effects of LN Treatment on the Activities of Nitrate Reductase (NR) and Glutamine Synthetase (GS) in\u003c/strong\u003e \u003cstrong\u003eI. chinensis\u003c/strong\u003e \u003cstrong\u003eLeaves\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eUnder LN stress, the activities of NR and GS in the leaves of all treatment groups increased (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e), but the extent of the increase varied among groups. As the stress duration extended, the NR activity in the LN\u003csub\u003e1\u003c/sub\u003e and LN\u003csub\u003e2\u003c/sub\u003e groups was significantly higher than that in the CK group, reaching a peak at T\u003csub\u003e2\u003c/sub\u003e, with increases of 130.15% and 290.19% compared to CK, respectively. Additionally, the GS activity in LN\u003csub\u003e2\u003c/sub\u003e was significantly higher than that in LN\u003csub\u003e1\u003c/sub\u003e and much higher than CK throughout all treatment periods. Specifically, at T\u003csub\u003e1\u003c/sub\u003e and T\u003csub\u003e2\u003c/sub\u003e, the GS activity in LN\u003csub\u003e2\u003c/sub\u003e was 60.89% and 112.36% higher than CK, respectively, and 23.78% and 56.02% higher than LN\u003csub\u003e1\u003c/sub\u003e, respectively.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e3.3 Effects of LP Treatment on Anthocyanin Content and Acid Phosphatase (ACP) Activity in\u003c/strong\u003e \u003cstrong\u003eI. chinensis\u003c/strong\u003e \u003cstrong\u003eLeaves\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eUnder LP treatment, both anthocyanin content and ACP activity exhibited a continuous upward trend, with the LP\u003csub\u003e2\u003c/sub\u003e group consistently showing higher levels than the LP\u003csub\u003e1\u003c/sub\u003e group across all treatment periods (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e). For instance, at T\u003csub\u003e1\u003c/sub\u003e, the anthocyanin content and ACP activity in the LP\u003csub\u003e2\u003c/sub\u003e group were 37.07% and 22.33% higher than those in the LP\u003csub\u003e1\u003c/sub\u003e group, respectively. By T\u003csub\u003e2\u003c/sub\u003e, the difference in anthocyanin content further increased, while the difference in ACP activity decreased, with the LP\u003csub\u003e2\u003c/sub\u003e group showing 52.08% higher anthocyanin content and 9.68% higher ACP activity compared to the LP\u003csub\u003e1\u003c/sub\u003e group.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003e3.4 Transcriptome Sequencing Analysis\u003c/h2\u003e\n \u003cdiv id=\"Sec17\" class=\"Section3\"\u003e\n \u003ch2\u003e3.4.1 Assessment and Assembly of Transcriptome Sequencing Data\u003c/h2\u003e\n \u003cp\u003eIllumina sequencing technology was employed to compare and analyze the transcriptome data of CK, LN\u003csub\u003e2\u003c/sub\u003e, and LP\u003csub\u003e2\u003c/sub\u003e samples. A total of 55.34 Gb of valid reads were obtained, comprising 376,227,290 reads. The effective data volume for each sample ranged from 5.05 Gb to 7.38 Gb. The proportion of bases with a quality value of \u0026ge;\u0026thinsp;20 (Q20) was over 98.09%, and the average GC content was 45.22% (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Pearson correlation analysis of the samples (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e) revealed high similarity among biological replicates and minor differences in gene expression levels within each treatment group, while distinct differences were observed between different treatment groups. Therefore, the sequencing quality was satisfactory, and the data were suitable for subsequent comparative and analytical studies.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eSample sequencing data\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSample\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eRaw_Reads\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eRaw_Bases\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eValid_Reads\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eValid_Bases\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eValid%\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eQ20%\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGC%\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCK_1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e43563974\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.53G\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e41599030\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.11G\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e95.49\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e98.31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e45.17\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCK_2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e42925382\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.44G\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e40531846\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.94G\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e94.42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e98.38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e45.76\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCK_3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e42502884\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.38G\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e41094998\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.06G\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e96.69\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e98.34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e44.97\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLN2_1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e41826864\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.27G\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e40459498\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.96G\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e96.73\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e98.29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e44.79\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLN2_2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e41153310\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.17G\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e39746028\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.86G\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e96.58\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e98.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e44.55\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLN2_3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e52467644\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.87G\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e50180432\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.38G\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e95.64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e98.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e44.85\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLP2_1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e48985036\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.35G\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e46849450\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.89G\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e95.64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e98.37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e45.64\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLP2_2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e43207630\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.48G\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e41426462\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.09G\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e95.88\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e98.31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e45.59\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLP2_3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e35648318\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.35G\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e34339546\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.05G\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e96.33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e98.09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e45.74\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec18\" class=\"Section3\"\u003e\n \u003ch2\u003e3.4.2 Differential Gene Expression Analysis\u003c/h2\u003e\n \u003cp\u003eIn this study, two comparisons were established: LN\u003csub\u003e2\u003c/sub\u003e vs. CK and LP\u003csub\u003e2\u003c/sub\u003e vs. CK. A total of 2480 differentially expressed genes (DEGs) were identified in the LN\u003csub\u003e2\u003c/sub\u003e vs. CK comparison, and 960 DEGs were identified in the LP\u003csub\u003e2\u003c/sub\u003e vs. CK comparison. Compared with the CK group, 845 DEGs were up-regulated and 1635 DEGs were down-regulated in the LN\u003csub\u003e2\u003c/sub\u003e treatment (LN\u003csub\u003e2\u003c/sub\u003e vs. CK). In the LP\u003csub\u003e2\u003c/sub\u003e treatment (LP\u003csub\u003e2\u003c/sub\u003e vs. CK), 401 DEGs were up-regulated and 559 DEGs were down-regulated. Under LN and LP, both comparison groups exhibited fewer up-regulated DEGs than down-regulated DEGs. Additionally, the total number of DEGs identified in the LN treatment group was higher than that in the LP treatment group (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eA). This suggests that the response mechanisms of \u003cem\u003eI. chinensis\u003c/em\u003e to LN conditions are more complex. Furthermore, a Venn diagram was constructed to compare the shared DEGs among the treatment groups (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eB). A total of 445 common DEGs were identified in both LN and LP treatment groups. These shared DEGs likely play important roles in the resistance of \u003cem\u003eI. chinensis\u003c/em\u003e to N and P deficiency stress.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec19\" class=\"Section3\"\u003e\n \u003ch2\u003e3.4.3 GO and KEGG Enrichment Analysis of DEGs\u003c/h2\u003e\n \u003cp\u003eThe GO enrichment analysis (Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eA) elucidated the functional profiles of differentially expressed genes (DEGs) in \u003cem\u003eI. chinensis\u003c/em\u003e under LN and LP stress conditions. Under LN stress, DEGs in the LN\u003csub\u003e2\u003c/sub\u003e group were predominantly enriched in terms associated with chloroplast thylakoid membrane, ribosomal structure, thylakoids, photosynthesis, transition metal ion binding, and cellular transition metal ion homeostasis. In contrast, under LP stress, DEGs in the LP\u003csub\u003e2\u003c/sub\u003e group were primarily enriched in terms related to cell wall, heat response, ARF guanine nucleotide exchange factor, extrinsic component of the plasma membrane, plant-type hypersensitive response, and signal transduction.\u003c/p\u003e\n \u003cp\u003eKEGG enrichment analysis (Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eB) further elucidates the metabolic regulatory pathways in \u003cem\u003eI. chinensis\u003c/em\u003e under LN and LP stress. The results indicate that \u003cem\u003eI. chinensis\u003c/em\u003e employs both common and unique metabolic regulatory pathways in response to LN and LP stress. In the LN treatment group, the KEGG metabolic pathways are primarily enriched in photosynthesis, ribosomes, cyanate metabolism, phenylpropanoid biosynthesis, flavonoid biosynthesis, photosynthesis - antenna proteins, nitrogen metabolism, and tyrosine metabolism. In the LP treatment group, the KEGG metabolic pathways are mainly enriched in flavonoid biosynthesis, arachidonic acid metabolism, plant-pathogen interaction, isoquinoline alkaloid biosynthesis, cutin and suberin biosynthesis, and tyrosine metabolism. The metabolic pathways commonly enriched by both LN and LP treatment groups include cyanate metabolism, phenylpropanoid biosynthesis, and flavonoid biosynthesis.\u003c/p\u003e\n \u003cp\u003eThese results indicate that \u003cem\u003eI. chinensis\u003c/em\u003e adapts to LN and LP stress by modulating the expression of different genes. Under LN conditions, the DEGs in \u003cem\u003eI. chinensis\u003c/em\u003e are primarily involved in processes related to photosynthesis and protein synthesis. In contrast, under LP conditions, the DEGs are more extensively involved in biosynthetic processes and cellular development-related processes.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec20\" class=\"Section3\"\u003e\n \u003ch2\u003e3.4.4 Expression Patterns of DEGs in Key KEGG Pathways\u003c/h2\u003e\n \u003cp\u003eBased on the KEGG-related metabolic pathway enrichment results in response to nitrogen and phosphorus deficiency in \u003cem\u003eI\u003c/em\u003e. \u003cem\u003echinensis\u003c/em\u003e, the significantly enriched key KEGG pathways in each treatment group were selected for further analysis. A heatmap analysis was performed to illustrate the expression patterns of the differentially expressed genes in the key KEGG pathways in the low-nitrogen treatment group (Fig. \u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003e). The results showed that most of the differentially expressed genes involved in the key KEGG pathways exhibited down-regulated expression, with only a small proportion of genes showing up-regulated expression.\u003c/p\u003e\n \u003cp\u003eSpecifically, genes involved in photosynthesis included TRINITY_DN17859_c0_g1 (\u003cem\u003ePSBO\u003c/em\u003e), TRINITY_DN4395_c0_g1 (\u003cem\u003ePSBO\u003c/em\u003e), TRINITY_DN9156_c0_g1 (\u003cem\u003eXYLA\u003c/em\u003e), TRINITY_DN15339_c0_g1 (\u003cem\u003ePETE\u003c/em\u003e), TRINITY_DN2383_c0_g1 (\u003cem\u003ePSBW\u003c/em\u003e), TRINITY_DN6719_c0_g1 (\u003cem\u003ePSBW\u003c/em\u003e), TRINITY_DN2042_c2_g1 (\u003cem\u003eSEND33\u003c/em\u003e), TRINITY_DN14464_c0_g1 (\u003cem\u003efdxH1\u003c/em\u003e), TRINITY_DN4423_c0_g1 (\u003cem\u003ePSAK\u003c/em\u003e), TRINITY_DN383_c1_g1 (\u003cem\u003eATPC\u003c/em\u003e), and TRINITY_DN51_c0_g1 (\u003cem\u003ePSAO\u003c/em\u003e), with the exception of TRINITY_DN9156_c0_g1 (\u003cem\u003eXYLA\u003c/em\u003e), which was up-regulated, while the others were down-regulated.\u003c/p\u003e\n \u003cp\u003eGenes involved in ribosome metabolism included TRINITY_DN5698_c0_g1 (\u003cem\u003eRCA\u003c/em\u003e), TRINITY_DN320_c1_g1 (\u003cem\u003eSOG1\u003c/em\u003e), TRINITY_DN4378_c0_g1 (\u003cem\u003eAPS1\u003c/em\u003e), TRINITY_DN37697_c0_g1 (\u003cem\u003emrpl19\u003c/em\u003e), TRINITY_DN16074_c0_g1 (\u003cem\u003eRPP1A\u003c/em\u003e), TRINITY_DN2782_c0_g1 (\u003cem\u003eUBI3\u003c/em\u003e), TRINITY_DN7984_c0_g1 (\u003cem\u003eALTA5\u003c/em\u003e), TRINITY_DN412_c0_g1 (\u003cem\u003eUBQ8\u003c/em\u003e), TRINITY_DN2973_c0_g1 (\u003cem\u003eRPS28\u003c/em\u003e), TRINITY_DN9933_c0_g1 (\u003cem\u003eRPL18B\u003c/em\u003e), TRINITY_DN6931_c0_g2 (\u003cem\u003eycf3\u003c/em\u003e), TRINITY_DN4920_c0_g1 (\u003cem\u003eRAN1A\u003c/em\u003e), and TRINITY_DN3843_c0_g1 (\u003cem\u003eRPL24\u003c/em\u003e), with the exception of TRINITY_DN320_c1_g1 (\u003cem\u003eSOG1\u003c/em\u003e), TRINITY_DN9933_c0_g1 (\u003cem\u003eRPL18B\u003c/em\u003e), and TRINITY_DN3843_c0_g1 (RPL24), which were up-regulated, while the others were down-regulated.\u003c/p\u003e\n \u003cp\u003eGenes involved in cyanamide metabolism included TRINITY_DN11758_c0_g1 (\u003cem\u003eCAS2\u003c/em\u003e), TRINITY_DN26728_c0_g1 (\u003cem\u003eSABP2\u003c/em\u003e), TRINITY_DN5704_c0_g1 (\u003cem\u003eMES10\u003c/em\u003e), TRINITY_DN29012_c0_g2 (\u003cem\u003eAtMg00310\u003c/em\u003e), TRINITY_DN32055_c0_g1 (\u003cem\u003eAtMg01250\u003c/em\u003e), TRINITY_DN8246_c0_g1 (\u003cem\u003eAt1g65750\u003c/em\u003e), and TRINITY_DN4045_c0_g1 (\u003cem\u003eBGLU12\u003c/em\u003e), with the exception of TRINITY_DN11758_c0_g1 (\u003cem\u003eCAS2\u003c/em\u003e) and TRINITY_DN4045_c0_g1 (\u003cem\u003eBGLU12\u003c/em\u003e), which were down-regulated, while the others were up-regulated.\u003c/p\u003e\n \u003cp\u003eGenes involved in phenylpropanoid biosynthesis included TRINITY_DN9992_c0_g1 (\u003cem\u003eF6\u0026apos;H1\u003c/em\u003e), TRINITY_DN8210_c0_g1 (\u003cem\u003e4CL1\u003c/em\u003e), TRINITY_DN9046_c0_g1 (\u003cem\u003ePER42\u003c/em\u003e), TRINITY_DN3267_c0_g2 (\u003cem\u003ePAL\u003c/em\u003e), TRINITY_DN24156_c1_g1 (\u003cem\u003e10HGO\u003c/em\u003e), TRINITY_DN4868_c0_g1 (\u003cem\u003ePNC1\u003c/em\u003e), and TRINITY_DN9532_c0_g1 (\u003cem\u003ePER43\u003c/em\u003e), with the exception of TRINITY_DN4868_c0_g1 (\u003cem\u003ePNC1\u003c/em\u003e), which was up-regulated, while the others were down-regulated.\u003c/p\u003e\n \u003cp\u003eGenes involved in flavonoid biosynthesis included TRINITY_DN5453_c0_g1 (\u003cem\u003eANS\u003c/em\u003e), TRINITY_DN5906_c0_g1 (\u003cem\u003eDLO2\u003c/em\u003e), TRINITY_DN28994_c0_g1 (\u003cem\u003eDFR\u003c/em\u003e), TRINITY_DN5516_c1_g1 (\u003cem\u003eNCS1\u003c/em\u003e), TRINITY_DN938_c0_g1 (\u003cem\u003eSRG1\u003c/em\u003e), TRINITY_DN10020_c0_g2 (\u003cem\u003eHST\u003c/em\u003e), TRINITY_DN37560_c0_g1 (\u003cem\u003eCHAT\u003c/em\u003e), TRINITY_DN13010_c0_g1 (\u003cem\u003eACT\u003c/em\u003e), TRINITY_DN1253_c0_g2 (\u003cem\u003emdmC\u003c/em\u003e), TRINITY_DN10247_c0_g1 (\u003cem\u003eLAR\u003c/em\u003e), and TRINITY_DN8461_c0_g1 (\u003cem\u003eBP80\u003c/em\u003e), with the exception of TRINITY_DN5516_c1_g1 (\u003cem\u003eNCS1\u003c/em\u003e), which was up-regulated, while the others were down-regulated.\u003c/p\u003e\n \u003cp\u003eThe gene TRINITY_DN9112_c0_g1 (RLP30), involved in plant stress resistance, was significantly up-regulated. Genes involved in nitrogen metabolism included TRINITY_DN26004_c0_g1 (\u003cem\u003eACA1\u003c/em\u003e), TRINITY_DN747_c0_g1 (\u003cem\u003eGS1-2\u003c/em\u003e), TRINITY_DN2414_c0_g1 (\u003cem\u003eGLT1\u003c/em\u003e), TRINITY_DN1838_c0_g1 (\u003cem\u003eCYN\u003c/em\u003e), TRINITY_DN2438_c0_g1 (\u003cem\u003eNIA2\u003c/em\u003e), and TRINITY_DN3357_c0_g1 (\u003cem\u003eBCA5\u003c/em\u003e), with the exception of TRINITY_DN2438_c0_g1 (\u003cem\u003eNIA2\u003c/em\u003e), which was up-regulated, while the others were down-regulated. Genes involved in tyrosine metabolism included TRINITY_DN3259_c0_g1 (\u003cem\u003eADH1\u003c/em\u003e), TRINITY_DN4933_c2_g1 (\u003cem\u003eASP1\u003c/em\u003e), TRINITY_DN8458_c0_g1 (\u003cem\u003eAt1g62810\u003c/em\u003e), and TRINITY_DN4153_c0_g3 (\u003cem\u003eco-2\u003c/em\u003e), with the exception of TRINITY_DN8458_c0_g1(\u003cem\u003eAt1g62810\u003c/em\u003e), which was up-regulated, while the others were down-regulated. all of which were down-regulated.\u003c/p\u003e\n \u003cp\u003eA heatmap analysis was conducted to illustrate the expression patterns of the differentially expressed genes in the key KEGG pathways in the low-phosphorus treatment group (Fig. \u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003e). The results indicated that the majority of the differentially expressed genes involved in the key KEGG pathways were down-regulated, while a portion of the genes exhibited up-regulated expression.\u003c/p\u003e\n \u003cp\u003eSpecifically, genes involved in flavonoid biosynthesis included TRINITY_DN38553_c0_g1 (\u003cem\u003eAt5g05600\u003c/em\u003e), TRINITY_DN44_c1_g1 (\u003cem\u003eCODM\u003c/em\u003e), TRINITY_DN28994_c0_g1 (\u003cem\u003eDFR\u003c/em\u003e), TRINITY_DN938_c0_g1 (\u003cem\u003eSRG1\u003c/em\u003e), TRINITY_DN44_c3_g1 (\u003cem\u003eFHT\u003c/em\u003e), and TRINITY_DN10247_c0_g1 (\u003cem\u003eLAR\u003c/em\u003e). Except for TRINITY_DN38553_c0_g1 (\u003cem\u003eAt5g05600\u003c/em\u003e), TRINITY_DN44_c1_g1 (\u003cem\u003eCODM\u003c/em\u003e), and TRINITY_DN44_c3_g1 (\u003cem\u003eFHT\u003c/em\u003e), all others were down-regulated.\u003c/p\u003e\n \u003cp\u003eGenes involved in the biosynthesis of stilbenes, diarylheptanoids, and gingerols included TRINITY_DN13010_c0_g1 (\u003cem\u003eACT\u003c/em\u003e) and TRINITY_DN37560_c0_g1 (\u003cem\u003eCHAT\u003c/em\u003e), both of which were down-regulated. Genes involved in arachidonic acid metabolism included TRINITY_DN23164_c0_g1 (\u003cem\u003eEPHX2\u003c/em\u003e), TRINITY_DN8168_c0_g1 (\u003cem\u003eOsI_027940\u003c/em\u003e), and TRINITY_DN5361_c0_g1 (\u003cem\u003eyfhM\u003c/em\u003e), all of which were down-regulated.\u003c/p\u003e\n \u003cp\u003eGenes involved in plant-pathogen interaction included TRINITY_DN10034_c0_g2 (\u003cem\u003eAt1g59620\u003c/em\u003e), TRINITY_DN36138_c0_g1 (\u003cem\u003eEFR\u003c/em\u003e), TRINITY_DN22339_c1_g1 (\u003cem\u003eR1A-10\u003c/em\u003e), TRINITY_DN12377_c0_g2 (\u003cem\u003eR1A-3\u003c/em\u003e), TRINITY_DN16250_c0_g1 (\u003cem\u003eAt4g27190\u003c/em\u003e), TRINITY_DN29459_c0_g3 (\u003cem\u003eRPM1\u003c/em\u003e), TRINITY_DN37700_c0_g1 (\u003cem\u003eMKK6\u003c/em\u003e), TRINITY_DN5041_c0_g3 (\u003cem\u003eRPP8L4\u003c/em\u003e), TRINITY_DN3065_c0_g1 (\u003cem\u003eWRKY53\u003c/em\u003e), TRINITY_DN33342_c0_g1 (\u003cem\u003eCRF3\u003c/em\u003e), and TRINITY_DN8177_c0_g1 (\u003cem\u003eCG5412\u003c/em\u003e). Except for TRINITY_DN36138_c0_g1 (\u003cem\u003eEFR\u003c/em\u003e), TRINITY_DN16250_c0_g1 (\u003cem\u003eAt4g27190\u003c/em\u003e), TRINITY_DN29459_c0_g3 (\u003cem\u003eRPM1\u003c/em\u003e), TRINITY_DN37700_c0_g1 (\u003cem\u003eMKK6\u003c/em\u003e), TRINITY_DN5041_c0_g3 (\u003cem\u003eRPP8L4\u003c/em\u003e), and TRINITY_DN33342_c0_g1 (\u003cem\u003eCRF3\u003c/em\u003e), all others were down-regulated.\u003c/p\u003e\n \u003cp\u003eGenes involved in the biosynthesis of cutin, suberin, and wax included TRINITY_DN6750_c1_g1 (\u003cem\u003ePXG4\u003c/em\u003e), TRINITY_DN6366_c0_g1 (\u003cem\u003eCER1\u003c/em\u003e), and TRINITY_DN9279_c0_g1 (\u003cem\u003eCYP704C1\u003c/em\u003e). Except for TRINITY_DN6750_c1_g1 (\u003cem\u003ePXG4\u003c/em\u003e), all others were down-regulated. Genes involved in endocytosis included TRINITY_DN30224_c0_g2 (\u003cem\u003eAtMg00810\u003c/em\u003e), TRINITY_DN8151_c0_g1 (\u003cem\u003eMED37D\u003c/em\u003e), TRINITY_DN446_c1_g1 (\u003cem\u003eHSP70\u003c/em\u003e), TRINITY_DN13409_c0_g1 (\u003cem\u003eGN\u003c/em\u003e), TRINITY_DN17085_c0_g1 (\u003cem\u003eGN\u003c/em\u003e), TRINITY_DN18740_c1_g1 (\u003cem\u003eGN\u003c/em\u003e), TRINITY_DN4537_c0_g1 (\u003cem\u003eHSC-2\u003c/em\u003e), TRINITY_DN9093_c0_g1 (\u003cem\u003eVPS2.3\u003c/em\u003e), and TRINITY_DN32516_c0_g1 (\u003cem\u003eARF1\u003c/em\u003e). Except for TRINITY_DN30224_c0_g2 (\u003cem\u003eAtMg00810\u003c/em\u003e), TRINITY_DN13409_c0_g1 (\u003cem\u003eGN\u003c/em\u003e), and TRINITY_DN9093_c0_g1 (\u003cem\u003eVPS2.3\u003c/em\u003e), all others were down-regulated.\u003c/p\u003e\n \u003cp\u003eGenes involved in sulfur metabolism included TRINITY_DN573_c1_g1 (\u003cem\u003eAPR3\u003c/em\u003e) and TRINITY_DN125_c0_g2 (\u003cem\u003eAPS1\u003c/em\u003e), both of which were up-regulated. Genes involved in cyanamide metabolism included TRINITY_DN3601_c0_g1 (\u003cem\u003eBGLU12\u003c/em\u003e), TRINITY_DN5704_c0_g2 (\u003cem\u003eSABP2\u003c/em\u003e), and TRINITY_DN8246_c0_g1 (\u003cem\u003eAt1g65750\u003c/em\u003e). Except for TRINITY_DN3601_c0_g1 (\u003cem\u003eBGLU12\u003c/em\u003e), all others were up-regulated. The gene involved in tyrosine metabolism was TRINITY_DN4153_c0_g3 (\u003cem\u003eco-2\u003c/em\u003e), which was down-regulated.\u003c/p\u003e\n \u003cp\u003eGenes involved in RNA degradation included TRINITY_DN10720_c0_g1 (\u003cem\u003ePAB7\u003c/em\u003e), TRINITY_DN9881_c0_g1 (\u003cem\u003ePAB2\u003c/em\u003e), TRINITY_DN9881_c0_g2 (\u003cem\u003ePAB6\u003c/em\u003e), TRINITY_DN4343_c0_g1 (\u003cem\u003ePUP1\u003c/em\u003e), TRINITY_DN22471_c0_g1 (\u003cem\u003eNAC021\u003c/em\u003e), and TRINITY_DN7967_c0_g1 (\u003cem\u003eNAC029\u003c/em\u003e). Except for TRINITY_DN4343_c0_g1 (\u003cem\u003ePUP1\u003c/em\u003e), all others were down-regulated. Genes involved in phenylpropanoid biosynthesis included TRINITY_DN5846_c1_g1 (\u003cem\u003eTKPR1\u003c/em\u003e), TRINITY_DN6578_c1_g1 (\u003cem\u003eCAD1\u003c/em\u003e), and TRINITY_DN6456_c0_g1 (\u003cem\u003egluA\u003c/em\u003e). Except for TRINITY_DN5846_c1_g1 (\u003cem\u003eTKPR1\u003c/em\u003e), all others were down-regulated.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eNitrogen (N) and phosphorus (P) are essential macronutrients for plant growth and development and are common limiting factors in nutrient availability. The concentrations of nitrogen and phosphorus in the soil significantly influence various vital activities of plants, including the synthesis of proteins, phospholipids, nucleic acids, and overall growth and development. However, in practical agricultural and ecological settings, the availability of nitrogen and phosphorus in the soil is often affected by a variety of factors, such as human activities, soil type, natural conditions, and soil microbial communities. These factors can lead to low levels of available N and P in the soil, subjecting plants to P and N deficiency stresses, which are detrimental to plant growth and development.\u003c/p\u003e\u003cp\u003eTo effectively cope with phosphorus and nitrogen deficiency stresses, plants employ a range of complex mechanisms to maximize their resilience. These mechanisms include phenotypic changes in plant morphology and alterations in physiological and biochemical indicators, all of which help to mitigate the adverse effects of nitrogen and phosphorus deficiency on plant growth and development. Building on this understanding, the present study primarily investigates the morphological, physiological, and molecular responses of \u003cem\u003eIlex chinensis\u003c/em\u003e to nitrogen and phosphorus deficiency and provides theoretical insights into the tolerance mechanisms of \u003cem\u003eI. chinensis\u003c/em\u003e under nitrogen and phosphorus deficiency conditions.\u003c/p\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003e4.1 Morphological Adaptations of \u003cem\u003eI. chinensis\u003c/em\u003e Facilitate Acclimation to LN and LP\u003c/h2\u003e\u003cp\u003eThe phenotypic characteristics of the plant and the growth, morphology, and physiological traits of the root system are directly influenced by the nitrogen and phosphorus concentrations in the soil. In this study, compared with the CK group, the low-nitrogen (LN) treatment group exhibited significant phenotypic changes at T\u003csub\u003e2\u003c/sub\u003e: the leaves of \u003cem\u003eI. chinensis\u003c/em\u003e became thinner, and the color changed from dark green to light green, with yellowing signs observed in the LN\u003csub\u003e2\u003c/sub\u003e group. In contrast, in the low-phosphorus (LP) treatment group, the leaves in both LP\u003csub\u003e1\u003c/sub\u003e and LP\u003csub\u003e2\u003c/sub\u003e groups lacked luster. The leaf color in the LP\u003csub\u003e1\u003c/sub\u003e group was not significantly different from that in the CK group, while the LP\u003csub\u003e2\u003c/sub\u003e group showed obvious chlorosis and yellowing. These results are consistent with previous studies in \u003cem\u003eSolanum lycopersicum\u003c/em\u003e \u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e, \u003cem\u003eArabidopsis thaliana\u003c/em\u003e \u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e, and \u003cem\u003eMalus pumila\u003c/em\u003e \u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e, indicating that LP and LN stresses significantly affect the leaf development of \u003cem\u003eI. chinensis\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eOther studies have shown that under LN and LP stresses, the total plant biomass decreases, while the root-shoot ratio significantly increases, along with an increase in the length of the primary root and the number of root branches \u003csup\u003e[\u003cspan additionalcitationids=\"CR29\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e. This conclusion is consistent with the results of the present study, where LN and LP significantly inhibited the growth of \u003cem\u003eI. chinensis\u003c/em\u003e, manifested as reduced biomass but an increased root-shoot ratio. This suggests that \u003cem\u003eI. chinensis\u003c/em\u003e allocates more resources to the root system to enhance the plant's absorption and utilization efficiency of N and P from the soil, thereby maintaining plant growth under N and Pdeficiency conditions.\u003c/p\u003e\u003cp\u003e\u003cb\u003e4.2 Resistance of\u003c/b\u003e \u003cb\u003eI. chinensis\u003c/b\u003e \u003cb\u003eto LN and LP Stresses is Enhanced through the Antioxidant Enzyme System\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe increase in malondialdehyde (MDA) content is indicative of lipid peroxidation of cell membranes and the accumulation of reactive oxygen species (ROS), reflecting the degree of membrane damage. Numerous studies have demonstrated that under low-nitrogen and LP stresses, the MDA content in plant leaves significantly increases \u003csup\u003e[\u003cspan additionalcitationids=\"CR31 CR32\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e. The results of this study show that in \u003cem\u003eI. chinensis\u003c/em\u003e leaves subjected to LN and LP treatments, the MDA content continuously rose with extended treatment duration, and this increase was more pronounced under severe stress conditions. This finding is consistent with previous research and indicates that the cell membranes of \u003cem\u003eI. chinensis\u003c/em\u003e leaves are severely damaged under N and P deficiency.\u003c/p\u003e\u003cp\u003eTo cope with ROS accumulation and membrane oxidative damage, plants enhance the activities of antioxidant enzymes (such as SOD, POD, and CAT) to scavenge excess ROS within the plant, thereby strengthening their ability to withstand adverse conditions \u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e. In this study, under low-nitrogen stress, the activities of SOD and POD in \u003cem\u003eI. chinensis\u003c/em\u003e leaves were significantly elevated and positively correlated with the duration of stress, with greater increases observed in the LN\u003csub\u003e2\u003c/sub\u003e group compared to the LN\u003csub\u003e1\u003c/sub\u003e group. Under low-phosphorus stress, the changes in SOD activity were similar to those under low-nitrogen stress. However, while POD activity was higher than that of the CK group at T\u003csub\u003e1\u003c/sub\u003e, no significant differences were observed between the LP\u003csub\u003e1\u003c/sub\u003e and LP\u003csub\u003e2\u003c/sub\u003e groups. At T\u003csub\u003e2\u003c/sub\u003e, POD activity significantly increased in both LP\u003csub\u003e1\u003c/sub\u003e and LP\u003csub\u003e2\u003c/sub\u003e groups, with LP\u003csub\u003e1\u003c/sub\u003e showing significantly higher POD activity than LP\u003csub\u003e2\u003c/sub\u003e. This phenomenon may be attributed to the more severe phosphorus deficiency under severe stress conditions, which could inhibit the enhancement of enzyme activity.\u003c/p\u003e\u003cp\u003eThese conclusions indicate that \u003cem\u003eI. chinensis\u003c/em\u003e can mitigate damage under low-nitrogen and low-phosphorus stresses by stimulating the synthesis and activity of antioxidant enzymes, activating the antioxidant system, and scavenging excess free radicals within the plant.\u003c/p\u003e\u003cp\u003e\u003cb\u003e4.3 Accumulation of Nitrate reductase (NR) and glutamine synthetase (GS) Activities Enhances LN Stress Resistance in\u003c/b\u003e \u003cb\u003eI. chinensis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eNR and GS are key enzymes in plant nitrogen metabolism and nitrogen absorption, and their activities are closely related to changes in nitrogen content. Their activities not only directly reflect the plant's ability to utilize and assimilate inorganic nitrogen but also indirectly affect the plant's ability to absorb and accumulate nitrate \u003csup\u003e[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e. The trends in NR and GS activities under low-nitrogen stress have been confirmed in many plants, such as \u003cem\u003ePopulus simonii\u003c/em\u003e \u003csup\u003e[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e, \u003cem\u003eTriticum aestivum\u003c/em\u003e \u003csup\u003e[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e, \u003cem\u003eCamellia sinensis\u003c/em\u003e\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e, and \u003cem\u003eSpinacia oleracea\u003c/em\u003e \u003csup\u003e[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe results of this study are consistent with previous research. Under low-nitrogen stress, the activities of NR and GS in \u003cem\u003eI. chinensis\u003c/em\u003e leaves increased continuously with prolonged stress duration, indicating enhanced nitrogen metabolism and assimilation capabilities within the plant, which in turn improved the utilization of limited nitrogen resources. Additionally, GS activity was higher in the LN\u003csub\u003e2\u003c/sub\u003e group than in the LN\u003csub\u003e1\u003c/sub\u003e group at both T\u003csub\u003e1\u003c/sub\u003e and T\u003csub\u003e2\u003c/sub\u003e, whereas NR activity showed no significant difference between the two treatment groups at T\u003csub\u003e1\u003c/sub\u003e but was significantly higher in the LN\u003csub\u003e2\u003c/sub\u003e group than in the LN\u003csub\u003e1\u003c/sub\u003e group at T\u003csub\u003e2\u003c/sub\u003e. This suggests that GS activity in \u003cem\u003eI. chinensis\u003c/em\u003e may be more sensitive to nitrogen deficiency than NR activity. Under short-term severe stress, \u003cem\u003eI. chinensis\u003c/em\u003e may first increase GS activity to improve the utilization of limited nitrogen sources, thereby stabilizing the nitrogen content within the plant to cope with stress.\u003c/p\u003e\u003cp\u003e\u003cb\u003e4.4 Accumulation of Acid phosphatase (ACP) Activity and Anthocyanin Content Enhances LP Stress Resistance in\u003c/b\u003e \u003cb\u003eI. chinensis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eACP is activated in response to phosphorus starvation signals in plants, promoting the decomposition of organic, insoluble phosphorus in the environment and thereby enhancing the plant's phosphorus utilization efficiency. This mechanism plays a significant role in plant phosphorus metabolism and is one of the important strategies for plants to cope with low-phosphorus stress \u003csup\u003e[\u003cspan additionalcitationids=\"CR41 CR42\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]\u003c/sup\u003e. In this study, under low-phosphorus stress, the activity of ACP in \u003cem\u003eI. chinensis\u003c/em\u003e leaves continuously increased with prolonged stress duration, with the most significant increase observed at T\u003csub\u003e2\u003c/sub\u003e. Moreover, the ACP activity in the LP\u003csub\u003e2\u003c/sub\u003e group was significantly higher than that in the LP1 group at all treatment periods, which is consistent with previous studies in \u003cem\u003eGossypium hirsutum\u003c/em\u003e \u003csup\u003e[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]\u003c/sup\u003e and \u003cem\u003eZea mays\u003c/em\u003e \u003csup\u003e[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]\u003c/sup\u003e. These findings indicate that \u003cem\u003eI. chinensis\u003c/em\u003e enhances its absorption and utilization efficiency of phosphorus from the environment under low-phosphorus conditions, thereby improving its adaptability to phosphorus-deficient environments and alleviating the adverse effects caused by phosphorus deficiency.\u003c/p\u003e\u003cp\u003eAnthocyanins are one of the important secondary metabolites in plants and are widely distributed in various plant organs, including roots, stems, leaves, flowers, and fruits. Under biotic and abiotic stresses, the synthesis and accumulation of anthocyanins are crucial for plant stress resistance, as they can enhance the plant's ability to scavenge ROS and mitigate damage to nucleic acids and chloroplasts caused by phosphorus deficiency \u003csup\u003e[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]\u003c/sup\u003e. In this study, the anthocyanin content in \u003cem\u003eI. chinensis\u003c/em\u003e subjected to low-phosphorus stress continuously increased with prolonged stress duration. This finding is consistent with previous studies, which have shown that low-phosphorus stress promotes the synthesis and accumulation of anthocyanins in plants \u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan additionalcitationids=\"CR48 CR49\" citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIn this study, the trends in anthocyanin content and ACP activity were consistent. This coordinated change may be related to the mechanisms by which plants adapt to environmental changes under stress conditions through the regulation of metabolic pathways.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e\u003ch2\u003e4.5 Molecular Response Mechanisms of \u003cem\u003eI. chinensis\u003c/em\u003e to LN and LP Stresses Revealed by Transcriptomic Analysis\u003c/h2\u003e\u003cp\u003ePrevious studies have revealed the critical roles of multiple genes in plant growth and stress responses. Specifically, genes such as TRINITY_DN9156_c0_g1 (\u003cem\u003eXYLA\u003c/em\u003e), TRINITY_DN320_c1_g1 (\u003cem\u003eSOG1\u003c/em\u003e), TRINITY_DN3843_c0_g1 (\u003cem\u003eRPL24\u003c/em\u003e), TRINITY_DN5516_c1_g1 (\u003cem\u003eNCS1\u003c/em\u003e), and TRINITY_DN2438_c0_g1 (\u003cem\u003eNIA2\u003c/em\u003e) are involved in the regulation of plant growth and nitrogen metabolism \u003csup\u003e[\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]\u003c/sup\u003e. Additionally, TRINITY_DN9112_c0_g1 (\u003cem\u003eRLP30\u003c/em\u003e) and TRINITY_DN29459_c0_g3 (\u003cem\u003eRPM1\u003c/em\u003e) participate in plant immune responses and modulate tolerance to abiotic stresses \u003csup\u003e[\u003cspan additionalcitationids=\"CR54\" citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]\u003c/sup\u003e. Furthermore, genes including TRINITY_DN38553_c0_g1 (\u003cem\u003eAt5g05600\u003c/em\u003e), TRINITY_DN44_c3_g1 (\u003cem\u003eFHT\u003c/em\u003e), TRINITY_DN37700_c0_g1 (\u003cem\u003eMKK6\u003c/em\u003e), TRINITY_DN573_c1_g1 (\u003cem\u003eAPR3\u003c/em\u003e), TRINITY_DN125_c0_g2 (\u003cem\u003eAPS1\u003c/em\u003e), and TRINITY_DN6750_c1_g1 (\u003cem\u003ePXG4\u003c/em\u003e) are implicated in the regulation of phosphorus uptake and utilization, enhancing plant adaptability to phosphorus-deficient environments \u003csup\u003e[\u003cspan additionalcitationids=\"CR57\" citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]\u003c/sup\u003e. Additionally, TRINITY_DN33342_c0_g1 (\u003cem\u003eCRF3\u003c/em\u003e) and TRINITY_DN4343_c0_g1 (\u003cem\u003ePUP1\u003c/em\u003e) influence root development, thereby improving phosphorus uptake and utilization efficiency \u003csup\u003e[\u003cspan additionalcitationids=\"CR60\" citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]\u003c/sup\u003e. These genes may play significant roles in plant adaptation to nitrogen and phosphorus deficiency stresses.\u003c/p\u003e\u003cp\u003eIn this study, based on the quantitative expression results, we found that the differentially expressed genes in \u003cem\u003eI. chinensis\u003c/em\u003e under low-nitrogen and low-phosphorus treatments were predominantly down-regulated. However, under nitrogen deficiency stress, genes related to plant growth and nitrogen metabolism regulation, including TRINITY_DN9156_c0_g1 (\u003cem\u003eXYLA\u003c/em\u003e), TRINITY_DN320_c1_g1 (\u003cem\u003eSOG1\u003c/em\u003e), TRINITY_DN3843_c0_g1 (\u003cem\u003eRPL24\u003c/em\u003e), and TRINITY_DN2438_c0_g1 (\u003cem\u003eNIA2\u003c/em\u003e), as well as the stress-responsive gene TRINITY_DN9112_c0_g1 (\u003cem\u003eRLP30\u003c/em\u003e), were significantly up-regulated. Under phosphorus deficiency stress, genes associated with phosphorus uptake and utilization, such as TRINITY_DN38553_c0_g1 (\u003cem\u003eAt5g05600\u003c/em\u003e), TRINITY_DN44_c3_g1 (\u003cem\u003eFHT\u003c/em\u003e), TRINITY_DN37700_c0_g1 (\u003cem\u003eMKK6\u003c/em\u003e), TRINITY_DN573_c1_g1 (\u003cem\u003eAPR3\u003c/em\u003e), TRINITY_DN125_c0_g2 (\u003cem\u003eAPS1\u003c/em\u003e), TRINITY_DN6750_c1_g1 (\u003cem\u003ePXG4\u003c/em\u003e), TRINITY_DN29459_c0_g3 (\u003cem\u003eRPM1\u003c/em\u003e), TRINITY_DN33342_c0_g1 (\u003cem\u003eCRF3\u003c/em\u003e), and TRINITY_DN4343_c0_g1 (\u003cem\u003ePUP1\u003c/em\u003e), were also up-regulated. These findings indicate that these genes may play crucial roles in the adaptation of \u003cem\u003eI. chinensis\u003c/em\u003e to nitrogen and phosphorus deficiency stresses.\u003c/p\u003e\u003cp\u003eCombining the results of GO functional enrichment analysis and KEGG metabolic pathway enrichment analysis, we further elucidated the molecular mechanisms of these genes in the adaptation of \u003cem\u003eI. chinensis\u003c/em\u003e to nitrogen and phosphorus deficiency stresses. Under nitrogen-deficient conditions, the up-regulated genes were primarily enriched in pathways related to nitrogen metabolism, photosynthesis, and antioxidant responses. This is consistent with the strategies employed by plants such as \u003cem\u003eCamellia sinensis\u003c/em\u003e \u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e, \u003cem\u003eBrassica napus\u003c/em\u003e \u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e, and \u003cem\u003eMalus pumila\u003c/em\u003e \u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e, which enhance nitrogen metabolism and antioxidant defenses to maintain growth under nitrogen stress. Under phosphorus-deficient conditions, the up-regulated genes were mainly enriched in pathways related to phosphorus metabolism, plant-pathogen interactions, and root development. This aligns with the strategies of plants like \u003cem\u003ePinus massoniana\u003c/em\u003e \u003csup\u003e[\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]\u003c/sup\u003e, \u003cem\u003eZea mays\u003c/em\u003e \u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e, and \u003cem\u003eGossypium hirsutum\u003c/em\u003e \u003csup\u003e[\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]\u003c/sup\u003e, which optimize phosphorus absorption and utilization as well as root structure to adapt to phosphorus stress. These results indicate that \u003cem\u003eI. chinensis\u003c/em\u003e adapts to nitrogen and phosphorus deficiency stresses by regulating the expression of specific genes and metabolic pathways.\u003c/p\u003e\u003c/div\u003e"},{"header":"5 Conclusions","content":"\u003cp\u003eIn this study, the physiological and molecular response mechanisms of \u003cem\u003eIlex chinensis\u003c/em\u003e to low-nitrogen and low-phosphorus conditions were analyzed through physiological indices and transcriptomic analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e). The increased activities of SOD and POD enhanced tolerance to nitrogen and phosphorus deficiency. The expression of genes responsive to nitrogen and phosphorus deficiency was regulated through pathways involved in plant growth, plant immune response, phosphorus uptake, and nitrogen metabolism, thereby improving tolerance to nitrogen and phosphorus deficiency. This study elucidates the differences in growth traits, antioxidant defense systems, and nitrogen and phosphorus metabolism of \u003cem\u003eI. chinensis\u003c/em\u003e under nitrogen and phosphorus deficiency stress, providing important physiological and molecular data for understanding the mechanisms by which plants enhance their tolerance to nitrogen and phosphorus deficiency.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate:\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication:\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials:\u003c/strong\u003e The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u0026nbsp;\u003c/strong\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e This work was supported by Zhejiang Province Major Science and Technology Project for Agriculture (Breeding of New Tree Species) and New Variety Breeding (grant/award Number:2021C02070-5-4).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions:\u003c/strong\u003e Conceptualization, D.Y.and J.J. ; data curation, J.L. , G.C. and J.H. ; funding acquisition, D.Y.and J.J..; methodology, G.C.; project administration, D.Y. , J.J. and B.Z.; supervision, D.Y.; writing\u0026mdash;original draft, J.L., G.C. and J.J.; writing\u0026mdash;review and editing, D.Y. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements:\u0026nbsp;\u003c/strong\u003eThis work was supported by Zhejiang Province Major Science and Technology Project for Agriculture (Breeding of New Tree Species) and New Variety Breeding (grant/award Number: 2021C02070-5-4).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; information:\u0026nbsp;\u003c/strong\u003eJing Liu \u0026amp; Gong Cheng \u0026amp; Jiejie Jiao co-authored as the first authors.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eOldroyd, G.E.D., Leyser, O., 2020. A plant\u0026apos;s diet, surviving in a variable nutrient environment. Science, 368(6486), eaba0196. https://doi.org/10.1126/science.aba0196.\u003c/li\u003e\n\u003cli\u003eLu, Z., Ren, T., Li, Y., Cakmak, I., Lu, J. , 2025. Nutrient limitations on photosynthesis: from individual to combinational stresses. Trends in plant science, 30(8), 872\u0026ndash;885. https://doi.org/10.1016/j.tplants.2025.03.006.\u003c/li\u003e\n\u003cli\u003eLin, Z. H., Chen, C. S., Zhao, S. Q., Liu, Y., Zhong, Q. S., Ruan, Q. C., Chen, Z. H., You, X. M., Shan, R. Y., Li, X. L., Zhang, Y. Z., 2023. Molecular and physiological mechanisms of tea (\u003cem\u003eCamellia sinensis\u003c/em\u003e (L.) O. Kuntze) leaf and root in response to nitrogen deficiency. BMC genomics, 24(1), 27. https://doi.org/10.1186/s12864-023-09112-y.\u003c/li\u003e\n\u003cli\u003eZhuo, M., Sakuraba, Y., Yanagisawa, S., 2024. Dof1.7 and NIGT1 transcription factors mediate multilayered transcriptional regulation for different expression patterns of NITRATE TRANSPORTER2 genes under nitrogen deficiency stress. The New phytologist, 242(5), 2132\u0026ndash;2147. https://doi.org/10.1111/nph.19695.\u003c/li\u003e\n\u003cli\u003eLi, H.Q., Yu, M.Q., Zhu, X.B., Nai, F.R., Yang, R.R., Wang, L.L., Liu Y.P., Wei, Y.H., Ma, X.M., Yu, H.D., Wang, X.C., 2024. TaGSr contributes to low-nitrogen tolerance by optimizing nitrogen uptake. Environmental and Experimental Botany, 219: 105657. https://doi.org/10.1016/j.envexpbot.2024.105657.\u003c/li\u003e\n\u003cli\u003eXiao, Y., Yao J.Y., Liu D. Song, H.X., Zhang, Z.H., 2020 Expression profile analysis of low nitrogen stress in \u003cem\u003eBrassica napus\u003c/em\u003e. Acta Agronomica Sinica , 46(10): 1526-1538. https://doi.org/10.3724/SP.J.1006.2020.94197.\u003c/li\u003e\n\u003cli\u003eHuang, R., Luo, J.J., Wu, Y.H., Liu, P.D., Liu, G.D., 2021. Molecular Cloning of \u003cem\u003ePpPAP\u003c/em\u003e Genes and Their Expression Responses to Phosphorus Deficiency in Elephant Grass (\u003cem\u003ePennisetum purpureum\u003c/em\u003e). Acta Agrestia Sinica, 29(3): 425-433. https://doi.org/10.11733/j.issn.1007-0435.2021.03.002.\u003c/li\u003e\n\u003cli\u003eLuo, J., Liu, Z., Yan, J., Shi, W., Ying, Y., 2023. Genome-Wide Identification of \u003cem\u003eSPX\u003c/em\u003e Family Genes and Functional Characterization of \u003cem\u003ePeSPX6\u003c/em\u003e and \u003cem\u003ePeSPX-MFS2\u003c/em\u003e in Response to Low Phosphorus in \u003cem\u003ePhyllostachys edulis\u003c/em\u003e. Plants (Basel, Switzerland), 12(7), 1496. https://doi.org/10.3390/plants12071496.\u003c/li\u003e\n\u003cli\u003eMeng, X.X., Li, W.F., Shen, R.F., Lan, P., 2021. Time-course response of phenotype and the expression of Pi-starvation responsive genes in high and low Pi-efficient wheat genotypes to Pi starvation. Plant Nutrition and Fertilizer Science, 27(11): 1883-1893. https://doi.org/10.11674/zwyf.2021176.\u003c/li\u003e\n\u003cli\u003eYang, S.Y., Lin, W.Y., Hsiao, Y.M., Chiou, T. J., 2024. Milestones in understanding transport, sensing, and signaling of the plant nutrient phosphorus. The Plant cell, 36(5), 1504\u0026ndash;1523. https://doi.org/10.1093/plcell/koad326.\u003c/li\u003e\n\u003cli\u003eHu, D., Cui, R., Wang, K., Yang, Y., Wang, R., Zhu, H., He, M., Fan, Y., Wang, L., Wang, L., Chu, S., Zhang, J., Zhang, S., Yang, Y., Zhai, X., L\u0026uuml;, H., Zhang, D., Wang, J., Kong, F., Yu, D., \u0026hellip; Zhang, D., 2024. The Myb73-GDPD2-GA2ox1 transcriptional regulatory module confers phosphate deficiency tolerance in soybean. The Plant cell, 36(6), 2176\u0026ndash;2200. https://doi.org/10.1093/plcell/koae041.\u003c/li\u003e\n\u003cli\u003eYang, Y., Wang, R., Wang, L., Cui, R., Zhang, H., Che, Z., Hu, D., Chu, S., Jiao, Y., Yu, D., Zhang, D., 2023. GmEIL4 enhances soybean (\u003cem\u003eGlycine max\u003c/em\u003e) phosphorus efficiency by improving root system development. Plant, cell \u0026amp; environment, 46(2), 592\u0026ndash;606. https://doi.org/10.1111/pce.14497.\u003c/li\u003e\n\u003cli\u003eLi, L., Cheng, X., Kong, X., Jia, P., Wang, X., Zhang, L., Zhang, X., Zhang, Y., Zhang, Z., Zhang, B., 2023. Comparative Transcriptomic Analysis Reveals the Negative Response Mechanism of Peanut Root Morphology and Nitrate Assimilation to Nitrogen Deficiency. Plants (Basel, Switzerland), 12(4), 732. https://doi.org/10.3390/plants12040732.\u003c/li\u003e\n\u003cli\u003eChen, J., Wan, H., Zhu, W., Dai, X., Yu, Y., Zeng, C., 2023. Identification and Expression Analysis of the Isopentenyl Transferase (IPT) Gene Family under Lack of Nitrogen Stress in Oilseed (\u003cem\u003eBrassica napus\u003c/em\u003e L.). Plants (Basel, Switzerland), 12(11), 2166. https://doi.org/10.3390/plants12112166.\u003c/li\u003e\n\u003cli\u003eTian, M., Zhang, C., Zhang, Z., Jiang, T., Hu, X., Qiu, H., Li, Z., 2023. Aspergillus niger Fermentation Broth Promotes Maize Germination and Alleviates Low Phosphorus Stress. Microorganisms, 11(7), 1737. https://doi.org/10.3390/microorganisms11071737.\u003c/li\u003e\n\u003cli\u003eWen, B., Gong, X., Chen, X., Tan, Q., Li, L., Wu, H., 2022. Transcriptome analysis reveals candidate genes involved in nitrogen deficiency stress in apples. Journal of plant physiology, 279, 153822. https://doi.org/10.1016/j.jplph.2022.153822.\u003c/li\u003e\n\u003cli\u003eQin, C., Shen, J., Qi, M.D., He, Z., Lian H.D., 2023. Effects of lanthanum chloride on physiological characteristics and related gene expression of mung bean under low-phosphorus stress. Jiangsu Agricultural Sciences, 51(8): 105-110. https://doi.org/10.15889/j.issn.1002-1302.2023.08.015.\u003c/li\u003e\n\u003cli\u003eLi, J., Liu, X., Xu, L., Li, W., Yao, Q., Yin, X., Wang, Q., Tan, W., Xing, W., \u0026amp; Liu, D., 2023. Low nitrogen stress-induced transcriptome changes revealed the molecular response and tolerance characteristics in maintaining the C/N balance of sugar beet (\u003cem\u003eBeta vulgaris\u003c/em\u003e L.). Frontiers in plant science, 14, 1164151. https://doi.org/10.3389/fpls.2023.1164151.\u003c/li\u003e\n\u003cli\u003eLi, R. F., Xia, W. Q., Liu, J. B., Cui, B. S., Hou, Q., Sun, H., Li, S., 2018. New triterpenoid saponins from the leaves of \u003cem\u003eIlex chinensis\u003c/em\u003e. Fitoterapia, 131, 134\u0026ndash;140. https://doi.org/10.1016/j.fitote.2018.10.007.\u003c/li\u003e\n\u003cli\u003eShao, S. Y., Li, R. F., Sun, H., \u0026amp; Li, S., 2021. New triterpenoid saponins from the leaves of \u003cem\u003eIlex chinensis\u003c/em\u003e and their hepatoprotective activity. Chinese journal of natural medicines, 19(5), 376\u0026ndash;384. https://doi.org/10.1016/S1875-5364(21)60036-5.\u003c/li\u003e\n\u003cli\u003eShao, S., Li, R., Wang, K., Xia, W., Cui, B., Li, S., 2023. Ilexchinene, a new seco-ursane triterpenoid from the leaves of \u003cem\u003eIlex chinensis\u003c/em\u003e with therapeutic effect on neuroinflammation by attenuating the MAPK/NF-\u0026kappa;B signaling pathway. Phytomedicine : international journal of phytotherapy and phytopharmacology, 121, 155110. https://doi.org/10.1016/j.phymed.2023.155110.\u003c/li\u003e\n\u003cli\u003eReitznerov\u0026aacute;, A., \u0026Scaron;ulekov\u0026aacute;, M., Nagy, J., Marcinč\u0026aacute;k, S., Semjon, B., Čert\u0026iacute;k, M., \u0026amp; Klempov\u0026aacute;, T., 2017. Lipid Peroxidation Process in Meat and Meat Products: A Comparison Study of Malondialdehyde Determination between Modified 2-Thiobarbituric Acid Spectrophotometric Method and Reverse-Phase High-Performance Liquid Chromatography. Molecules (Basel, Switzerland), 22(11), 1988. https://doi.org/10.3390/molecules22111988.\u003c/li\u003e\n\u003cli\u003eGiannopolitis, C.N., \u0026amp; Ries, S.K., 1977. Superoxide dismutases: I. Occurrence in higher plants. Plant physiology, 59(2), 309\u0026ndash;314. https://doi.org/10.1104/pp.59.2.309.\u003c/li\u003e\n\u003cli\u003eChen, J.X., Wang, X.F., 2015). Experimental Guide to Plant Physiology. South China University of Technology Press. p72-73.\u003c/li\u003e\n\u003cli\u003eZheng, X., Yang, H., Zou, J., Jin, W., Qi, Z., Yang, P., Yu, J., \u0026amp; Zhou, J., 2024. SnRK1\u0026alpha;1-mediated RBOH1 phosphorylation regulates reactive oxygen species to enhance tolerance to low nitrogen in tomato. The Plant cell, 37(1), koae321. https://doi.org/10.1093/plcell/koae321.\u003c/li\u003e\n\u003cli\u003eZhang, J.F., Wang, Y.Y., He, L., Yan, J.Y., Liu, Y.Y., Ruan, Z.Y., Liu, W.C., Yi, L., Ren, F., 2024. PHR1 involved in the regulation of low phosphate-induced leaf senescence by modulating phosphorus homeostasis in Arabidopsis. Plant, cell \u0026amp; environment, 47(3), 799\u0026ndash;816. https://doi.org/10.1111/pce.14790.\u003c/li\u003e\n\u003cli\u003eWang, X., Chai, X., Gao, B., Deng, C., G\u0026uuml;nther, C. S., Wu, T., Zhang, X., Xu, X., Han, Z., \u0026amp; Wang, Y., 2023. Multi-omics analysis reveals the mechanism of bHLH130 responding to low-nitrogen stress of apple rootstock. Plant physiology, 191(2), 1305\u0026ndash;1323. https://doi.org/10.1093/plphys/kiac519.\u003c/li\u003e\n\u003cli\u003eSeidel, S.J., Ahmadi, S.H., Weiherm\u0026uuml;ller, L., Cou\u0026euml;del, A., Lopez, G., Behrend, D., Kamali, B., Gaiser, T., Hern\u0026aacute;ndez-Ochoa, I.M., 2024. The overlooked effects of environmental impacts on root:shoot ratio in experiments and soil-crop models. The Science of the total environment, 955, 176738. https://doi.org/10.1016/j.scitotenv.2024.176738.\u003c/li\u003e\n\u003cli\u003eLi, M., Zhou, J., Lang, X., Han, D., Hu, Y., Ding, Y., Wang, G., Guo, J., Shi, L., 2022. Integrating transcriptomic and metabolomic analysis in roots of wild soybean seedlings in response to low-phosphorus stress. Frontiers in plant science, 13, 1006806. https://doi.org/10.3389/fpls.2022.1006806.\u003c/li\u003e\n\u003cli\u003eXia, Z., Gong, Y., Yang, Y., Wu, M., Bai, J., Zhang, S., Lu, H., 2024. Effects of root-zone warming, nitrogen supply and their interactions on root-shoot growth, nitrogen uptake and photosynthetic physiological characteristics of maize. Plant physiology and biochemistry : PPB, 214, 108887. https://doi.org/10.1016/j.plaphy.2024.108887.\u003c/li\u003e\n\u003cli\u003eChen, L.H., Xu, M., Cheng, Z., Yang, L. T., 2024. Effects of Nitrogen Deficiency on the Photosynthesis, Chlorophyll a Fluorescence, Antioxidant System, and Sulfur Compounds in \u003cem\u003eOryza sativa\u003c/em\u003e. International journal of molecular sciences, 25(19), 10409. https://doi.org/10.3390/ijms251910409.\u003c/li\u003e\n\u003cli\u003eZhang, Z.W., Yang, X.Y., Zheng, X.J., Fu, Y.F., Lan, T., Tang, X.Y., Wang, C.Q., Chen, G.D., Zeng, J., Yuan, S., 2020. Vitamin E Is Superior to Vitamin C in Delaying Seedling Senescence and Improving Resistance in Arabidopsis Deficient in Macro-Elements. International journal of molecular sciences, 21(19), 7429. https://doi.org/10.3390/ijms21197429.\u003c/li\u003e\n\u003cli\u003eCai, Q., Zou, Y., Zhong, N., Jia, G., 2024. Effects of nutritional stress on soil fertility and antioxidant enzymes of rice in different growth periods. Frontiers in plant science, 15, 1471682. https://doi.org/10.3389/fpls.2024.1471682.\u003c/li\u003e\n\u003cli\u003eAli, M.F., Mahmood, I., Shamsi, I.H., Asad, S.A., Ali, H., Shah, G.A., Tung, S.A., Raja, N.I., Demirkıran, A.R., Hussain, M.I., 2025. Integrated application of nitrogen and mepiquat chloride improves yield of \u003cem\u003eBrassica napus\u003c/em\u003e L. by modulating shattering, lodging and nitrogen use efficiency under rainfed conditions. Plant Growth Regul. https://doi.org/10.1007/s10725-025-01343-1.\u003c/li\u003e\n\u003cli\u003eCheng, L.L., Pan, Y., Lin, Y., Lin, S.X., Tong, Z.K., 2020. Effect of Low Nitrogen Stress on the Growth, Physiological, and Biochemical Characteristics of Different Betula luminifera Genotypes. Journal of Nuclear Agricultural Sciences, 34(11): 2435-2443. https://doi.org/10.11869/j.issn.100-8551.2020.11.2435.\u003c/li\u003e\n\u003cli\u003eLuo, J., Hang, J.N., Wu, B.L., Wei, X.L., Zhao, Q.Z., Fang, Z.M., 2023. Co-overexpression of genes for nitrogen transport, assimilation, and utilization boosts rice grain yield and nitrogen use efficiency. The Crop Journal, 11(4): 785-799. https://doi.org/10.1016/j.cj.2023.01.005.\u003c/li\u003e\n\u003cli\u003eZhang, C., Meng, S., Li, M., Zhao, Z., 2018. Transcriptomic insight into nitrogen uptake and metabolism of Populus simonii in response to drought and low nitrogen stresses. Tree physiology, 38(11), 1672\u0026ndash;1684. https://doi.org/10.1093/treephys/tpy085.\u003c/li\u003e\n\u003cli\u003eLv, X.M., Zhang, Y.X., Hu, L., Zhang, B., Xia, H.Y., Du, W.Y., Fan, S.J., Kong, L.G., 2021. Low-nitrogen stress stimulates lateral root initiation and nitrogen assimilation in wheat: roles of phytohormone signaling. Journal of Plant Growth Regulation, 40(2): 436-450. https://doi.org/10.1007/s00344-020-10112-5.\u003c/li\u003e\n\u003cli\u003eLi, J., Yang, P., Sohail, H., Du, H.B., Li, J., 2023. The impact of short-term nitrogen starvation and replenishment on the nitrate metabolism of hydroponically grown spinach. Scientia Horticulturae, 309: 111632. https://doi.org/10.1016/j.scienta.2022.111632.\u003c/li\u003e\n\u003cli\u003eDeng, S., Li, J., Du, Z., Wu, Z., Yang, J., Cai, H., Wu, G., Xu, F., Huang, Y., Wang, S., Wang, C., 2022. Rice ACID PHOSPHATASE 1 regulates Pi stress adaptation by maintaining intracellular Pi homeostasis. Plant, cell \u0026amp; environment, 45(1), 191\u0026ndash;205. https://doi.org/10.1111/pce.14191.\u003c/li\u003e\n\u003cli\u003eXu, J.H., Qian, S.Y., Wang, S.Z., Shen, X.Y., Zhuang, C.C., 2022. Effects of low phosphorus stress on the growth and development in pe-cans (\u003cem\u003eCarya illinoinensis\u003c/em\u003e). Journal of Fruit Science, 39(8): 1432-1442. https://doi.org/10.13925/j.cnki.gsxb.20210683.\u003c/li\u003e\n\u003cli\u003eLiu, S., Xu, Z., Essemine, J., Liu, Y., Liu, C., Zhang, F., Iqbal, Z., Qu, M., 2024. GWAS unravels acid phosphatase ACP2 as a photosynthesis regulator under phosphate starvation conditions through modulating serine metabolism in rice. Plant communications, 5(7), 100885. https://doi.org/10.1016/j.xplc.2024.100885.\u003c/li\u003e\n\u003cli\u003eXie, B., Zhao, D.Y., Chen, Y.H., Cheng, C.G., An, X.H., 2022. Physiological response of young trees of different apple cultivars to nitrogen and phosphorus deficiency. Plant Nutrition and Fertilizer Science, 28(11): 2106-2117. https://doi.org/10.11674/zwyf.2022078.\u003c/li\u003e\n\u003cli\u003eAsif, I., Dong, Q., Wang, X.R., Gui, H.P., Zhang, H.H., Zhang, X.L. , Song, M.Z., 2023. Phosphorus and carbohydrate metabolism contributes to low phosphorus tolerance in cotton. BMC plant biology, 23(1), 97. https://doi.org/10.1186/s12870-023-04100-6.\u003c/li\u003e\n\u003cli\u003eShen, Q., Xiao, J.T., Wang, L.Y., Feng, J.G., Chen, Y., Zhu, B., Li, H.G., Lambers, H., 2025. Maize shows intraspecific facilitation under phosphorus deficiency but competition under nitrogen deficiency when grown under increased plant densities in alkaline soil. Plant Soil. 2025. https://doi.org/10.1007/s11104-024-07188-3.\u003c/li\u003e\n\u003cli\u003eWang, H.X., Liu, T.Y., Zhuang, W.B., Wang, Z., Zhu, L., 2020. Research Advances in the Function of Anthocyanin in Plant Stress Response. Journal of Agricultural Biotechnology, 28(1): 174-183. https://doi.org/10.3969/j.issn.1674-7968.2020.01.018.\u003c/li\u003e\n\u003cli\u003eTao, H., Gao, F., Linying Li, He, Y., Zhang, X., Wang, M., Wei, J., Zhao, Y., Zhang, C., Wang, Q., Hong, G., 2024. WRKY33 negatively regulates anthocyanin biosynthesis and cooperates with PHR1 to mediate acclimation to phosphate starvation. Plant communications, 5(5), 100821. https://doi.org/10.1016/j.xplc.2024.100821.\u003c/li\u003e\n\u003cli\u003eAn, J. P., Li, H. L., Liu, Z. Y., Wang, D. R., You, C. X., Han, Y., 2023. The E3 ubiquitin ligase SINA1 and the protein kinase BIN2 cooperatively regulate PHR1 in apple anthocyanin biosynthesis. Journal of integrative plant biology, 65(9), 2175\u0026ndash;2193. https://doi.org/10.1111/jipb.13538\u003c/li\u003e\n\u003cli\u003eJia, X.Y., Liu, H., Shen, J., Li, F., Ding, N.,2015. Negative Regulation of Anthocyanin Biosynthesis in Tomato by MicroRNA828 Under Phosphate Deficiency. Scientia Agricultura Sinica, 2015, 48(15): 2911-2924. \u003c/li\u003e\n\u003cli\u003eYang, B., Peng, Y., Zhang, G., Liu, R., Hao, S., Ren, Y., Lu, S., Wang, X., Guo, L., 2025. Regulation of glucosylceramide synthase and sphingolipid remodeling in the plant response to phosphate deficiency. The Plant cell, 37(6), koaf138. https://doi.org/10.1093/plcell/koaf138.\u003c/li\u003e\n\u003cli\u003eJi, M.J., Cao, ZY, Wang YT, Lu, J.R., Wang, B.H., 2023. Genome-wide identification and analysis of \u003cem\u003eNCS1\u003c/em\u003e gene family in upland cotton. Scientia Agricultura Sinica, 2023, 35(2): 249-258. https://doi.org/10.3969/j.issn.1004-1524.2023.02.01.\u003c/li\u003e\n\u003cli\u003eLiu, R.X., Li, H.L., Rui, L., Liu, G.D., Wang, T., Wang, X.F., Li, L.G., Zhang, Z., You, C.X., 2023. An apple NITRATE REDUCTASE 2 gene positively regulates nitrogen utilization and abiotic stress tolerance in \u003cem\u003eArabidopsis\u003c/em\u003e and apple callus. Plant physiology and biochemistry : PPB, 196, 23\u0026ndash;32. https://doi.org/10.1016/j.plaphy.2023.01.026.\u003c/li\u003e\n\u003cli\u003eYang, Y., Steidele, C.E., R\u0026ouml;ssner, C., L\u0026ouml;ffelhardt, B., Kolb, D., Leisen, T., Zhang, W., Ludwig, C., Felix, G., Seidl, M. F., Becker, A., N\u0026uuml;rnberger, T., Hahn, M., Gust, B., Gross, H., H\u0026uuml;ckelhoven, R., Gust, A. A., 2023. Convergent evolution of plant pattern recognition receptors sensing cysteine-rich patterns from three microbial kingdoms. Nature communications, 14(1), 3621. https://doi.org/10.1038/s41467-023-39208-8.\u003c/li\u003e\n\u003cli\u003eHuang, Y., Gong, X., Shi, H., Wang, P., Yuan, Y., Kong, C., Zhou, J., Wu, D., Liang, Y., Wang, Y., Wang, J., 2025. OsHYPK/NatA-mediated N-terminal acetylation regulates the homeostasis of NLR immune protein to fine-tune rice immune responses and growth. Cell reports, 44(5), 115719. https://doi.org/10.1016/j.celrep.2025.115719.\u003c/li\u003e\n\u003cli\u003eDong, Q., Duan, D., Wang, F., Yang, K., Song, Y., Wang, Y., Wang, D., Ji, Z., Xu, C., Jia, P., Luan, H., Guo, S., Qi, G., Mao, K., Zhang, X., Tian, Y., Ma, Y., Ma, F., 2024. The MdVQ37-MdWRKY100 complex regulates salicylic acid content and MdRPM1 expression to modulate resistance to Glomerella leaf spot in apples. Plant biotechnology journal, 22(8), 2364\u0026ndash;2376. https://doi.org/10.1111/pbi.14351.\u003c/li\u003e\n\u003cli\u003eYu, S.W., Zhang, X.Q., Chen, S.J., Ye, S.F., Zhang, Y., 2021. Analysis of the expression characteristics of \u003cem\u003eOsMKKs\u003c/em\u003e gene under abiotic stress during development. Acta Agriculturae Shanghai, 37(3): 29-34. https://doi.org/10.15955/j.issn1000-3924.2021.03.06.\u003c/li\u003e\n\u003cli\u003eZhang, X.X., Ji, G.H., Mu. Y., Jiang, W.G., Liu, Q.Y., 2025. Identification and Abiotic Stress Response of Adenosine 5\u0026apos;-phosphosulfate Reductase (APR) Gene Family in \u003cem\u003eBrassica napus\u003c/em\u003e. Acta Agriculturae Shanghai, 33(1): 15-29. https://doi.org/10.3969/j.issn.1674-7968.2025.01.002.\u003c/li\u003e\n\u003cli\u003eTran, A.D., Cho, K., Han, O., 2023. Rice peroxygenase catalyzes lipoxygenase-dependent regiospecific epoxidation of lipid peroxides in the response to abiotic stressors. Bioorganic chemistry, 131, 106285. https://doi.org/10.1016/j.bioorg.2022.106285.\u003c/li\u003e\n\u003cli\u003eRashotte, A. M., Mason, M. G., Hutchison, C. E., Ferreira, F. J., Schaller, G. E., Kieber, J. J., 2006. A subset of \u003cem\u003eArabidopsis\u003c/em\u003e AP2 transcription factors mediates cytokinin responses in concert with a two-component pathway. Proceedings of the National Academy of Sciences of the United States of America, 103(29), 11081\u0026ndash;11085. https://doi.org/10.1073/pnas.0602038103.\u003c/li\u003e\n\u003cli\u003ePrathap, V., Kumar, S., Tyagi, A., Mohapatra, T., 2024. Integrated transcriptome and proteome analysis of near-isogenic line provides insights on regulatory function of \u003cem\u003ePup1 \u003c/em\u003eQTL in rice under phosphorus-starvation stress. Environmental and Experimental Botany, 221: 105726. https://doi.org/10.1016/j.envexpbot.2024.105726.\u003c/li\u003e\n\u003cli\u003eGentile, D., Serino, G., Frugis, G., 2024. CRF transcription factors in the trade-off between abiotic stress response and plant developmental processes. Front Genet, 15: 1377204. Published 2024 Apr 16. https://doi.org/10.3389/fgene.2024.1377204.\u003c/li\u003e\n\u003cli\u003eWang, C.Y., Fan, F.H., 2023. Identification of SPL Gene Family in Pinus massoniana and Their Expression in Response to Low Phosphorus Stress. Journal of Agricultural Biotechnology, 31(3): 509-517. https://doi.org/10.3969/j.issn.1674-7968.2023.03.006.\u003c/li\u003e\n\u003cli\u003eMeng, C.M., Geng, F.F., Qing, G.X., Li, X.L., Zhang, F.H., 2023. Cloning and Expression Analysis of Phosphorus Deficiency Stress Gene GhERF5 in\u003cem\u003e Gossypium hirsutum \u003c/em\u003eL.. Acta Botanica Boreali-Occidentalia Sinica, 43(3): 382-388. https://doi.org/10.7606/j.issn.1000-4025.2023.03.0382.\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":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Ilex chinensis, Low nitrogen, Low phosphorus, Physiological properties, Transcriptome","lastPublishedDoi":"10.21203/rs.3.rs-7390500/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7390500/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe elements nitrogen (N) and phosphorus (P) are essential nutrients required for normal growth and photosynthesis in plants. However, nitrogen and phosphorus deficiencies can have a significant impact on plant physiology and metabolism. In this study, the two-year-old \u003cem\u003eIlex chinensis\u003c/em\u003e seedlings were used as the research object, the growth environment of low nitrogen (LN) and low phosphorus (LP) was simulated, and two treatment gradients of mild and severe stress were set up, and the changes of the growth and development of wintergreen and the main physiological indexes were studied under normal growth conditions as the control. Differentially expressed genes (DEGs) were identified by transcriptomic analysis at 10 weeks of severe stress. The results showed that nitrogen and phosphorus deficiency inhibited the growth of shoots and promoted root development of \u003cem\u003eI. chinensis\u003c/em\u003e, and LN\u003csub\u003e2\u003c/sub\u003e and LP\u003csub\u003e2\u003c/sub\u003e treatments had the most serious effects. The physiological indexes showed that the contents of nitrate reductase (NR), glutamine synthetase (GS), superoxide dismutase (SOD) and peroxidase (POD) and malondialdehyde (MDA) in the LN\u003csub\u003e2\u003c/sub\u003e group were significantly increased by 112.36%, 290.19%, 67.56%, 151.79% and 248.04%, respectively, compared with the CK group after 10 weeks of treatment. The activities of acid phosphatase (ACP), SOD and POD, anthocyanins and MDA increased by 77.1%, 65.46%, 97.81%, 144.43% and 134.25%. Transcriptome analysis revealed that the key differentially expressed genes under the nitrogen and phosphorus deficit of wintergreen were mainly involved in the regulation of plant growth, root development, nitrogen and phosphorus uptake and other biological processes. These findings provide insights into the adaptation mechanisms of \u003cem\u003eI. chinensis\u003c/em\u003e under nitrogen and phosphorus deficits and highlight potential target genes for improving nutrient use efficiency. This study contributes to a better understanding of the physiological and molecular responses of \u003cem\u003eI. chinensis\u003c/em\u003e to nitrogen and phosphorus deficiency stress, and provides valuable information for optimizing its cultivation under nutrient-constrained conditions.\u003c/p\u003e","manuscriptTitle":"The Effects of Nitrogen and Phosphorus Deficiency on the Main Physiology of Ilex chinensis and Transcriptomic Analysis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-01 13:49:12","doi":"10.21203/rs.3.rs-7390500/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-22T13:53:54+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-19T02:56:50+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"194135920471388042310093672585812567779","date":"2025-10-18T02:31:53+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-11T11:14:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"161663814801745173440728494717685564601","date":"2025-09-22T02:34:18+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-19T12:56:25+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-14T07:13:28+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-09-12T03:44:45+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-11T05:17:22+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Plant Biology","date":"2025-09-11T05:13:22+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"40064aee-5e04-4c7c-880a-f38aac6f0c9c","owner":[],"postedDate":"October 1st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-02-16T16:05:48+00:00","versionOfRecord":{"articleIdentity":"rs-7390500","link":"https://doi.org/10.1186/s12870-026-08360-w","journal":{"identity":"bmc-plant-biology","isVorOnly":false,"title":"BMC Plant Biology"},"publishedOn":"2026-02-11 15:56:59","publishedOnDateReadable":"February 11th, 2026"},"versionCreatedAt":"2025-10-01 13:49:12","video":"","vorDoi":"10.1186/s12870-026-08360-w","vorDoiUrl":"https://doi.org/10.1186/s12870-026-08360-w","workflowStages":[]},"version":"v1","identity":"rs-7390500","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7390500","identity":"rs-7390500","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}