Abstract Vetiveria zizanioides, renowned for its robust stability and exceptional capacity to sequester heavy metals, has garnered widespread application in tailings ecological restoration efforts. Arbuscular mycorrhizal fungi (AMF), which are capable of forming symbiotic relationships with more than 80% of terrestrial plant roots, play a pivotal role in enhancing plant nutrient uptake and bolstering resilience. In this study, we conducted a comprehensive investigation into the physiological and biochemical responses of Vetiveria zizanioides subjected to varying levels of copper stress (with copper concentrations ranging from 0 mg/kg to 400 mg/kg), with or without AMF inoculation. Additionally, we performed nontargeted metabonomic analyses to gain deeper insights into the metabolic changes that occur in vetiver grass under AMF inoculation and copper stress. Our findings revealed that Vetiveria zizanioides inoculated with AMF consistently demonstrated superior growth performance across all copper stress levels compared with noninoculated counterparts. Using nontargeted metabonomic analyses, inoculation with AMF affects the metabolism of phenylalanine and related pathways in vetiver as well as contributing to the promotion of the formation of phytochelatins (PCs) from glutamate, thereby alleviating copper stress. The results highlight the potential of AMF-inoculated Vetiveria zizanioides as a promising bioremediation tool capable of effectively mitigating the adverse effects of heavy metal pollution. Keywords: Vetiveria zizanioides, Arbuscular mycorrhizal Fungi, Copper stress, Physiological and biochemical responses, Metabonomics Subject terms: Biotechnology, Plant sciences, Biogeochemistry, Environmental sciences Introduction Tailings are defined as the waste or nonvaluable byproduct generated during mining activities and the processing of minerals and other materials; they contain small amounts of residual valuable minerals, chemicals, water, and heavy metals^[34]1. According to a survey, the output of industrial solid waste in China exceeded 3 billion t/year in 2020, with an average annual growth rate of approximately 7%^[35]2. Tailings production accounts for approximately 80% of total industrial waste, and the total reserves exceed 60 billion tons^[36]3. Tailings contain a large number of metal elements, which can cause substantial harm to ecosystems and agricultural systems^[37]4,[38]5. For example, excessive copper can inhibit plant growth, reduce photosynthesis by affecting photosystem II^[39]6, reduce root activity, and inhibit the absorption of other mineral elements, reducing yield^[40]7. Therefore, it is necessary to formulate a solution to solve this serious problem. Phytoremediation is a sustainable, cost-effective and environmentally friendly method^[41]8,[42]9 in which plants are used to remove metals and organic pollutants in soil^[43]10. Vetiveria zizanioides is a perennial herbaceous plant of the genus Vetiveria in the family Gramineae that has been widely used in the field of ecological restoration of tailings because of its remarkable characteristics, such as resistance to many kinds of heavy metals, strong root system, high biomass, and rapid growth rate^[44]11,[45]12. However, plants growing in tailings are at risk of toxic stress and irreversible damage from heavy metals, which leads to low biomass, reducing remediation efficiency^[46]13. Therefore, effective techniques must be introduced to improve the efficiency of phytoremediation. Arbuscular mycorrhizal fungi (AMF) are plant-related microorganisms that establish a reciprocal relationship with 70–90% of land plant roots in various soils^[47]14,[48]15. In addition to having great potential for alleviating heavy metals in plants, AMF are also affordable and eco-friendly bioremediation tools for soils polluted by heavy metals^[49]16. AMF can greatly improve the ability of plants to absorb nutrients through mycelial networks, thus directly and indirectly affecting plant physiological characteristics and improving plant growth^[50]14,[51]17. AMF can not only improve the ability of plants to absorb mineral nutrients and trace metals but also affect the accumulation and transportation of metals in plants to reduce plant toxicity under heavy metal stress^[52]18,[53]19. In recent years, many reports have shown that AMF improve heavy metal tolerance in plants^[54]20–[55]24. However, the mechanism by which AMF inoculation improves the tolerance of Vetiveria zizanioides plants is still not clear. Therefore, this study aimed to explore the changes in the physiological, biochemical and metabolite contents of Vetiveria zizanioides with or without AMF inoculation under Cu stress. These results could help to address the following questions: (1) Does the tolerance range of Vetiveria zizanioides to Cu stress expand after inoculation with AMF? (2) What is the mechanism by which AMF enhance the Cu tolerance of Vetiveria zizanioides? At the same time, this study will help deepen the understanding of the mechanism of plant stress resistance, promote the implementation of precision agriculture, enhance ecosystem services, and expand the application of biotechnology. Materials and methods Experimental materials Vetiveria zizanioides was purchased from Shennong Baicao Company in Guanling County, Guizhou Province. The company has a good reputation and rich experience in the field of plant breeding and cultivation. The stems of Vetiveria zizanioides were inoculated on solid culture media via plant tissue culture and cultured for 30 d at 24–28 °C, 55% humidity and 3000–15,000 lx illumination. After Vetiveria zizanioides took root and emerged, it was transferred to a seedbed for domestication. The soil used in this study was collected from the grassland science experimental site of Guizhou University. The soil physical and chemical properties are shown in Table [56]1. Table 1. Physical and chemical properties of soil. Ingredient Content Total nitrogen 655 mg/kg Total phosphorus 762.67 mg/kg Total potassium 2.61 mg/kg Available nitrogen 33.23 mg/kg Available potassium 293.33 mg/kg Available phosphorus 4.74 mg/kg Total Cu 18.28 mg/kg pH 6.68 Organic matter 2.61% [57]Open in a new tab Fine sand (diameter < 1 mm) was purchased from Henan Xinyang Guotong Electronic Commerce Co., Ltd. The fine sand was rinsed with tap water three times. The air-dried soil was screened with a 2 mm sieve and mixed with sand at a mass ratio of 2:1. The mixture was sterilized in a high-temperature autoclave at 121 °C and 0.1 MPa for 2 h and then was removed and air-dried for later use. The test strain was Funneliformis mosseae, which was purchased from the Institute of Root Biology of Yangtze University. The company’s varieties were selected based on its reputation in the industry, technological advantages, market position, and adaptability to the environment and economic value. The inoculant was a rhizosphere sand mixture containing spores, hyphae, infected root segments and a propagation matrix obtained by the propagation of white clover as the host plant, and each gram of inoculum contained 25 spores. Experimental design A pot experiment was conducted in June 2022 at Guizhou University in Guiyang city, Guizhou Province, China. The experimental design included two distinct treatment groups: an AMF-inoculated group (M) and a noninoculated control group (NM), each subjected to four different concentrations of Cu^2+ (0 mg/kg, 100 mg/kg, 200 mg/kg, and 400 mg/kg). The specific amounts of CuSO[4]·5H[2]O required for each treatment are detailed in Table [58]2. To ensure reproducibility, each treatment was replicated three times. Table 2. Preparation of standard stock solution of Cu. Concentration grade (mg/kg) Soil requirement (kg) Cu demand (mg) CuSO[4]·5H[2]O demand (mg) 0 2.5 0 0 100 2.5 250 976.56 200 2.5 500 1953.13 400 2.5 1000 3906.25 [59]Open in a new tab Prior to planting, the pots were thoroughly sterilized with sodium hypochlorite and filled with 2.5 kg of soil up to 2/3rd of their capacity. After leaving the flower pot undisturbed for 10 days, for the inoculation treatment (M), 100 g of the microbial agent is evenly distributed on the soil surface, while the non-inoculated control (NM) is provided with an equal amount of inactivated fungicide in an identical manner. Vetiveria zizanioides seedlings were subsequently carefully transplanted into the pots, which were covered with the remaining soil, and the cultivation process was initiated. After three months of cultivation, the upper portions of the Vetiveria zizanioides plants were uniformly trimmed to a standardized height of 40 cm to prepare them for the application of copper. Copper stress was performed using a copper standard stock solution (Copper stress was performed using a copper standard stock solution (the amount of CuSO[4]·5H[2]O required for each treatment is shown in Table [60]2). throughout the copper stress period, the plants were maintained with daily irrigation via ultrapure water and biweekly supplementation with a tailored Hoagland’s nutrient solution (The configuration of the nutrient solution is shown in Table [61]3). The samples were systematically harvested after 30 days of exposure to monitor the physiological responses of the plants to stress. Table 3. Preparation of Hoagland nutrient solution. Ingredient Content (mg·L^− 1) KNO^3 607 Ca (NO[3])[2]·4H[2]O 945 MgSO[4]·7H[2]O 493 NH[4]H[2]PO[4] 115 Molysite solution 2.5 Trace element solution 5 [62]Open in a new tab Determination of indexes and methods Determination of morphological indexes The height of the plants under different treatments was measured before collection. After collection, fresh plants were baked in an oven at 105 °C for 30 min and then dried at 75 °C to a constant weight, after which the dried plants were weighed^[63]11. The roots and shoots of each plant under different treatments were separated and placed in water trays to promote root spread and keep the roots moist. The root systems were scanned via the WinRHIZO program on an Epson scanner (Epson Perfection V800 Photo) to determine the total root length, number of root tips, root surface area, root volume and root diameter^[64]25. These values were analyzed via three biological replicates. Determination of physiological and biochemical indexes The methodology for the quantification of soluble sugars was based on that outlined by Irigoyen et al.^[65]26, ensuring accuracy and reproducibility. For soluble proteins, the Bradford^[66]27 protocol was followed. Furthermore, fresh leaf samples were subjected to rigorous analysis to determine leaf proline content, malondialdehyde (MDA) content, and the activity of key antioxidant enzymes (CAT, POD, and SOD). These analyses were based on methods established by Bates et al.^[67]28 for proline, by Angelini et al.^[68]29 and Gong and Shahrooz Bahram^[69]30 for MDA, and by Agarwal and Pandey^[70]31 along with Yordanova et al.^[71]32 for the assessment of antioxidant enzymes. Determination of the Cu content Each sample was placed in an oven at 60–80 °C, dried to a constant weight, crushed and passed through a 0.2 mm nylon sieve. A 0.5 g sample was accurately weighed and placed in a Kjeldahl flask, 10 ml of HNO: HC1O[4] (4:1) mixture was added, the mixture was stirred well, covered with watch glass, left overnight, and digested on a 1000 W hot plate. When white smoke was no longer emitted and the solution became clear and colorless, and the sample was evaporated to near dryness. Then, heating was stopped, and the mixture was cooled to room temperature and transferred to a 25 mL volumetric flask. Finally, the Cu content was determined via ICP‒MS^[72]33. To evaluate the ability of plants to transport and enrich heavy metals from belowground to aboveground parts, the translocation factor (TF) was calculated as follows: graphic file with name M1.gif where C[1] and C[0] are the copper contents in the stem and roots of the plant, respectively^[73]34. Metabonomic analysis Sample preparation Vetiveria zizanioides in the AMF-inoculated (M-Cu) and noninoculated (Cu) treatment groups under 200 mg/kg copper stress were selected for metabonomics analysis on the basis of the phenotypes and physiological indices of the plants in the early stage of copper stress. The leaves of Vetiveria zizanioides were selected for metabolomics analysis. 50 mg of each sample was taken and dried lyophilized were ground in a 2 mL Eppendorf tube containing a 5 mm tungsten bead for 1 min at 65 Hz in a Grinding Mill. Metabolites were extracted using 1 mL precooled mixtures of methanol, acetonitrile and water (v/v/v, 2:2:1) and then placed for 1 h ultrasonic shaking in ice baths. Subsequently, the mixture was placed at −20 °C for 1 h and centrifuged at 14,000 g for 20 min at 4 °C. The supernatants were recovered and concentrated to dryness in vacuum.100 µL sample was thoroughly mixed with 400 µL of cold methanol acetonitrile (v/v, 1:1) via vortexing. And then the mixture were processed with sonication for 1 h in ice baths. The mixture was then incubated at −20 °C for 1 h, and centrifuged at 4 °C for 20 min with a speed of 14,000 g. The supernatants were then harvested and dried under vacuum LC-MS analysis. The metabolites were extracted from cell residue with 1 mL precooled methanol/acetonitrile/water (v/v, 2:2:1) under sonication for 1 h in ice baths. The mixture was incubated at -20 °C for 1 h followed by centrifugation at 14,000 g, 4 °C for 20 min, and then transferred to the sampling vial for LC-MS analysis. UHPLC-MS/MS analysis Metabolomics profiling was analyzed using a UPLC-ESI-Q-Orbitrap-MS system (UHPLC, Shimadzu Nexera X2 LC-30AD, Shimadzu, Japan) coupled with Q-Exactive Plus (Thermo Scientific, San Jose, USA).For hydrophilic interaction liquid chromatography (HILIC) separation, samples were analyzed using a 2.1 mm*100 mm ACQUIY UPLC BEH Amide 1.7 μm column (Waters, Ireland). The flow rate was 0.5 mL/min and the mobile phase contained: A: 25 mM ammonium acetate and 25 mM ammonium hydroxide in water and B: 100% acetonitrile (ACN). The gradient was 95% B for 1 min and was linearly reduced to 65% in 7 min, and then reduced to 35% in 2 min and maintained for 1 min, and then increased to 95% in 0.5 min, with 2 min re-equilibration period employed. Both electrospray ionization (ESI) positive-mode and negative mode were applied for MS data acquisition. The HESI source conditions were set as follows: Spray Voltage: + 3800v/− 3200v; Capillary Temperature: 320 ℃; Sheath Gas: 30 psi; Aux Gas: 5 psi; Probe Heater Temp: 350 ℃; S-Lens RF Level: 50. In MS only acquisition, the instrument was set to acquire over the m/z range 80-1200 Da. The full MS scans were acquired at a resolution of 70,000 at m/z 200, and 17,500 at m/z 200 for MS/MS scan. The maximum injection time was set to for 100 ms for MS and 50 ms for MS/MS. The isolation window for MS2 was set to 2 m/z and the normalized collision energy (stepped) was set as 27, 29 and 32 for fragmentation. Quality control (QC) samples were prepared by pooling aliquots of all samples that were representative of the samples under analysis, and used for data normalization. Blank samples (75%ACN in water) and QC samples were injected every six samples during acquisition. Data preprocessing and filtering The raw MS data were processed using MS-DIAL for peak alignment, retention time correction and peak area extraction. The metabolites were identified by accuracy mass (mass tolerance < 0.01Da) and MS/MS data (mass tolerance < 0.02Da) which were matched with HMDB, massbank and other public databases and our self-built metabolite standard library. In the extracted-ion features, only the variables having more than 50% of the nonzero measurement values in at least one group were kept. KEGG enrichment analysis To identify the perturbed biological pathways, the differential metabolite data were performed KEGG pathway analysis using KEGG database. KEGG enrichment analyses were carried out with the Fisher’s exact test, and FDR correction for multiple testing was performed. Enriched KEGG pathways were nominally statistically significant at the p < 0.05 level. Data processing The data were organized via Microsoft Excel and statistically analyzed via SPSS 26.0, and multiple comparisons were made via the least significant difference (LSD) method. Differences were considered significant at P < 0.05, and all data are presented as the means (± standard errors (SEs)) of three replicates. SigmaPlot 14.0 was used for plotting. Results Effects of copper at different concentrations and AMF inoculation on the growth and root characteristics of Vetiveria zizanioides After 30 days of copper stress, the leaves of vetiver showed yellowing and gradually wilted with increasing copper concentration. Under 400 mg/kg Cu stress, the vetiver grass stopped growing. As shown in Fig. [74]1, the height of the plants in the inoculated AMF treatment group first increased but then decreased with increasing Cu concentration. Compared with those in the uninoculated treatment group, the plant height in the AMF-inoculated treatment groups increased by 19.50%, 38.21%, 0.11% and 0.01%, respectively, at 0 mg/kg, 100 mg/kg, 200 mg/kg and 400 mg/kg Cu. The biomass of comfrey decreased with increasing Cu concentration. The biomass of the inoculated group was 0.04%, 32.14%, 31.25% and 28.74% greater than that of the uninoculated group at 0 mg/kg, 100 mg/kg, 200 mg/kg and 400 mg/kg Cu, respectively. Fig. 1. [75]Fig. 1 [76]Open in a new tab The effects of AMF inoculation and different copper concentrations on the height (a) and biomass (b) of Vetiveria zizanioide. Note: Different letters at the top of the bar graphs indicate significant differences (P < 0.05) at different levels of copper addition and with or without AMF inoculation treatment. NM and M are for plants not inoculated with AMF treatment and plants inoculated with AMF treatment. The effects of AMF inoculation and copper at varying concentrations on the growth of Vetiveria zizanioides roots are presented in Table [77]4. The total root length, total surface area, total root volume, and total number of root tips of the Vetiveria zizanioides root system initially tended to increase but then decreased with increasing copper concentration, peaking at a copper concentration of 200 mg/kg. Specifically, under 200 mg/kg copper stress, the total root area, total root volume, and total number of root tips increased by 51.01%, 42.55%, and 76.97%, respectively, compared with those of noninoculated plants (p < 0.05). However, no significant differences (p < 0.05) were detected in the mean root diameter or total root length, regardless of the presence of AMF or the level of Cu stress. Table 4. Effects of AMF inoculation and different cu concentration on root growth of V. zizanioides. Treatment (mg/kg) Total root length (cm) Total surface area (cm^2) Total root volume (cm^3) Avreage diameter (mm) Total root tip amount (N.plant^−1) 0 NM 384.06 ± 13.59^a 44.52 ± 2.05^c 0.41 ± 0.02^c 0.42 ± 0.02^a 2326.33 ± 390.34^bc M 425.72 ± 17.75^a 50.33 ± 1.99^c 0.59 ± 0.04^ab 0.42 ± 0.03^a 3079.67 ± 383.66^ab 100 NM 395.62 ± 48.6^a 47.3 ± 2.24^c 0.47 ± 0.04^bc 0.4 ± 0.03^a 2354.33 ± 142.38^bc M 451.47 ± 27.78^a 52.1 ± 1.96^bc 0.66 ± 0.04^a 0.44 ± 0.01^a 3745.67 ± 435.51^a 200 NM 452.27 ± 67.54^a 60.88 ± 4.77^b 0.57 ± 0.05^abc 0.41 ± 0.01^a 3590.67 ± 268.37^a M 460.54 ± 36.98^a 71.43 ± 4.14^a 0.67 ± 0.10^a 0.46 ± 0.02^a 4165.67 ± 698.75^a 400 NM 348.76 ± 31.6^a 43.75 ± 5.02^c 0.47 ± 0.06^bc 0.43 ± 0.08^a 1822.33 ± 200.96^c M 412.88 ± 14.76^a 44.49 ± 2.41^c 0.45 ± 0.04^bc 0.4 ± 0.02^a 2218.33 ± 160.58^bc [78]Open in a new tab Different letters in the same column indicate significant differences (p < 0.05). NM and M are for plants not inoculated with AMF treatment and plants inoculated with AMF treatment. Effects of copper at different concentrations and AMF inoculation on the copper content in Vetiveria zizanioides The variations in the Cu content and transfer coefficient of Vetiveria zizanioides among the distinct groups are shown in Table [79]5. Regardless of AMF inoculation, the results revealed that both the aboveground and belowground Cu contents of Vetiveria zizanioides increased with increasing Cu concentration. The Cu content in the shoots and root system of Vetiveria zizanioides inoculated with AMF was greater than that in the uninoculated group at Cu concentrations of 0 mg/kg and 100 mg/kg. Conversely, compared with that in the control group, the Cu content in the leaves of AMF-inoculated Vetiveria zizanioides decreased by 23.71% and 111.16% at Cu concentrations of 200 mg/kg and 400 mg/kg, respectively; similarly, the Cu content in the root system decreased by 81.01% and 21.08%, respectively. The translocation factor of Cu was highest at 0 mg/kg Cu and decreased sharply with increasing Cu concentration. Table 5. Changes of AMF inoculation and different Cu concentration on Cu content in leaves and roots of V. zizanioides. Treatments (mg/kg) Cu content (mg/kg) Translocation factor Shoot Root 0 NM 12.88 ± 0.13^g 36.84 ± 1.58^g 0.35 ± 0.01^a M 16.61 ± 0.20^f 70.20 ± 0.74^f 0.28 ± ± 0.03^b 100 NM 23.49 ± 0.42^e 385.53 ± 1.44^e 0.06 ± 0.00^d M 25.05 ± 0.18^e 394.69 ± 1.29^e 0.06 ± 0.01^d 200 NM 57.02 ± 0.69^c 771.39 ± 2.04^c 0.08 ± 0.01^cd M 46.09 ± 1.00^d 426.17 ± 2.16^d 0.11 ± 0.00^c 400 NM 135.31 ± 2.41^a 1196.96 ± 5.49^a 0.12 ± 0.01^c M 64.08 ± 0.48^b 988.61 ± 2.62^b 0.07 ± 0.01^d [80]Open in a new tab Different letters in the same column indicate significantly different (p < 0.05) under different copper concentrations and whether or not they were inoculated with AMF treatment. NM and M are for plants not inoculated with AMF treatment and plants inoculated with AMF treatment. Effects of copper at different concentrations and AMF inoculation on the osmoregulatory substances in Vetiveria zizanioidess Figure [81]2 shows the changes in soluble sugar, soluble protein, and proline contents in the upper parts of Vetiveria zizanioides under different copper concentrations after AMF inoculation. The soluble sugar content of Vetiveria zizanioides (both AMF-inoculated and noninoculated) first increased but then decreased with increasing copper concentration, and the soluble sugar content of both the inoculated and noninoculated groups under different copper concentrations was significantly greater than that of the 0 mg/kg Cu treatment group (P < 0.05). On the other hand, the soluble protein content increased with increasing copper concentration. Specifically, compared with the 0 mg/kg Cu treatment, the soluble protein content in AMF-inoculated Vetiveria zizanioides at 100 mg/kg, 200 mg/kg, and 400 mg/kg Cu increased by 46.02%, 51.22%, and 54.76%, respectively. Furthermore, compared with that of the noninoculated group, the proline content of the inoculated group increased by 0.05%, 32.38%, and 51.53% at 0 mg/kg, 200 mg/kg, and 400 mg/kg Cu, respectively, with the proline content of the AMF-inoculated group being significantly greater than that of the noninoculated group at 200 mg/kg and 400 mg/kg Cu, respectively (P < 0.05). Fig. 2. [82]Fig. 2 [83]Open in a new tab Changes of soluble sugar (a), soluble protein (b) and proline (c) contents in Vetiveria zizanioides leaves after AMF inoculation and different Cu concentration treatment. Note: Different letters at the top of the bar graphs indicate significant differences (P < 0.05) at different levels of copper addition and with or without AMF inoculation treatment. NM and M are for plants not inoculated with AMF treatment and plants inoculated with AMF treatment. Effects of copper at different concentrations and AMF inoculation on the antioxidant activity and malondialdehyde content of Vetiveria zizanioides Figure [84]3 shows the alterations in the CAT, SOD, and POD activities and the MDA content in the upper portion of Vetiveria zizanioides subjected to various levels of Cu stress following AMF inoculation. The CAT and POD activities in AMF-inoculated Vetiveria zizanioides gradually increased with increasing Cu concentration, indicating a similar trend. In contrast, the CAT and MDA activities in the uninoculated group initially increased but then decreased. At Cu concentrations of 0 mg/kg and 100 mg/kg, the CAT activity in the inoculated group was lower than that in the uninoculated group (P < 0.05). However, at the 200 mg/kg and 400 mg/kg Cu concentrations, the CAT activity in the inoculated group was significantly greater than that in the uninoculated group (P < 0.05). At the 0 mg/kg Cu concentration, the POD activity in the inoculated group was only 49.56% of that in the uninoculated group, but at all other concentrations, the inoculated group presented greater POD activity. SOD activity decreased with increasing Cu concentration; specifically, at 0 mg/kg, 100 mg/kg, 200 mg/kg, and 400 mg/kg Cu, the SOD activity in the inoculated group was 3.36%, 82.94%, 74.63%, and 57.67% higher than that in the uninoculated group, respectively. Conversely, the MDA content decreased by 0.04%, 25.15%, 138.32%, and 106.42%, respectively. The highest MDA content (57.84 nmol/g) was detected in the uninoculated group under 200 mg/kg Cu. Fig. 3. [85]Fig. 3 [86]Open in a new tab Changes of MDA (a), CAT (b), SOD (c) and POD (d) content in Vetiveria zizanioides leaves under AMF inoculation and different Cu concentrations. Note: Different letters at the top of the bar graphs indicate significant differences (P < 0.05) at different levels of copper addition and with or without AMF inoculation treatment. NM and M are for plants not inoculated with AMF treatment and plants inoculated with AMF treatment. Metabonomics analysis Quality control In the sample cohort, a pool of samples were designated quality control (QC) samples to evaluate the stability and reproducibility of the system. MSDIAL software was employed for the extraction of metabolite ion peaks, and the peaks extracted from all samples and the QC sample were subjected to unitvariancescaling (UV) treatment before PCA was performed. Under positive ion mode (Fig. [87]4 left), PC1 and PC2 accounted for 37.59% and 19.18% of the variance, respectively, whereas under negative ion mode (Fig. [88]4 right), PC1 and PC2 accounted for 35.55% and 21.71% of the variance, respectively. These results indicate a clear separation between the different treatment samples and the control group, with significant differences observed, and the tight clustering of the QC samples suggests good experimental reproducibility. Subsequently, orthogonal partial least squares discriminant analysis (OPLS-DA) was performed on the M-Cu vs. Cu groups (Fig. [89]5). The results demonstrated distinct differences between the various treatments, with one principal component and one orthogonal principal component obtained under both positive and negative ion modes. The Table [90]6 indicate the evaluation parameters in positive ion mode were R2X = 0.527, R2Y = 0.999, and Q2 = 0.816, whereas those in negative ion mode were R2X = 0.529, R2Y = 0.999, and Q2 = 0.834. The fact that R2 is close to 1 and that Q2 > 0.30 suggests that the OPLS-DA models were robust and not overfitted, making them reliable for further analysis. Thus, the next step in the analysis could be taken. Fig. 4. [91]Fig. 4 [92]Open in a new tab PCA diagram of each treatment group in positive (left) and negative (right) ion mode. Fig. 5. [93]Fig. 5 [94]Open in a new tab OPLS-DA diagram of M-Cu vs. Cu group in positive (left) and negative (right) ion mode. Table 6. OPLS-DA parameters of M-Cu vs. Cu group in positive and negative ion modes. Sample comparison group A R2X (cum) R2Y (cum) Q2 (cum) POS 1 + 1 + 0 0.527 0.999 0.816 NEG 1 + 1 + 0 0.529 0.999 0.834 [95]Open in a new tab Screening of differential metabolites Compound identification was performed on the basis of the precise mass number, secondary fragmentation and isotopic distribution, and qualitative analyses of the M-Cu and Cu groups were performed via databases such as the Human Metabolome Database (HMDB) and MassBank (Fig. [96]6)^[97]35–[98]37. A total of 441 metabolites were identified, and those showing variable importance (VIP) values greater than 1 in multivariate statistical analyses and P values less than 0.05 in univariate statistical analyses were subsequently selected. These metabolites were considered differentially expressed metabolites (DEMs) with significant differences. A total of 154 DEMs were identified during the screening of the M-Cu and Cu treatments. Seventy-six DEMs were upregulated, and 78 were downregulated. These findings suggest that M-Cu treatment resulted in significant changes in the metabolic profiles, with the levels of some metabolites decreasing and those of others increasing compared with those in the Cu-treated group. Fig. 6. [99]Fig. 6 [100]Open in a new tab The overall cluster diagram of the sample. Note: the horizontal direction is the sample name, the vertical direction is the metabolite information, the Group is the grouping, and the different colors are the colors filled with different values obtained by standardizing different relative contents (red represents high content and blue represents low content). Comparative analysis of the differentially expressed metabolites between the AMF-inoculated and noninoculated plants under copper stress revealed significant changes in their metabolic profiles. Among the top twenty significantly upregulated metabolites (Fig. [101]7), we observed notable increases in PFCA-Cl (17.88-fold), hexose + C10H19O2 (8.66-fold), stictic acid (8.11-fold), PFSA-unsaturated (5.93-fold), quinic acid (5.57-fold), 3,5-dichloro-2-O-methylanziaic acid (5.54-fold), and several other metabolites. These findings suggest that AMF treatment may increase the production of these metabolites, which could contribute to the alleviation of copper stress in vetiver grass. Fig. 7. [102]Fig. 7 [103]Open in a new tab M-Cu VS Cu difference multiple bar chart. Note: the abscissa is log[2]FC of the differential metabolite, that is, the differential multiple of the differential metabolite is based on 2, and the ordinate is the differential metabolite. Red indicates that the metabolite content is up-regulated, and blue indicates that the metabolite content is down-regulated. Conversely, significantly downregulated metabolites included hispanolone (-5.63-fold), tolytoxin (-4.67-fold), tunaxanthin (-4.11-fold), and others, indicating that AMF treatment may reduce the levels of these compounds, which may be detrimental under copper stress conditions. The utilization of Pearson’s correlation coefficients to assess the consistency of metabolite changes across the metabolic landscape is a powerful approach for understanding the complex relationships existing between different metabolites. By constructing a correlation clustering heatmap, we can visualize the metabolic closeness among significantly differentially expressed metabolites (DEMs), thereby gaining insights into the mutual regulation of these metabolites during biological state transitions. Figure [104]8 indicate that stictic acid, benzyl benzoate, canarione, and quinic acid are strongly positively correlated with each other, with correlation coefficients exceeding 0.99. This significant positive correlation suggests that the expression patterns of these metabolites are highly synchronized, indicating a concerted response to copper stress in vetiver grass under AMF inoculation. Fig. 8. [105]Fig. 8 [106]Open in a new tab Differential metabolite correlation matrix thermogram. Note: red represents positive correlation, blue represents negative correlation, the smaller the gap, the stronger the correlation coefficient, and vice versa. Specifically, the upregulation of quinic acid, along with stictic acid, benzyl benzoate, and canarione, under AMF-inoculated conditions during copper stress suggests that these metabolites may play important roles in the defense mechanisms of plants against copper toxicity. The coordinated upregulation of these metabolites could be a result of AMF-mediated metabolic reprogramming that enables plants to better cope with the adverse effects of copper stress. KEGG enrichment analysis of differentially abundant metabolites Upon conducting a comprehensive KEGG pathway enrichment analysis of the 154 differentially expressed metabolites (DEMs), we discovered a total of 69 metabolic pathways, 19 of which were significantly enriched. Figure [107]9; Table [108]7 reveal that under Cu stress conditions, AMF play a pivotal role in mitigating stress in Vetiveria zizanioides by modulating a diverse array of pathways. Among these, pathways related to alanine, aspartate, and glutamate metabolism; neuroactive ligand‒receptor interaction; taurine and hypotaurine metabolism; ABC transporters; vitamin B6 metabolism; taste transduction; phenylalanine, tyrosine, and tryptophan biosynthesis; the pentose phosphate pathway; bile secretion; the FoxO signaling pathway; and pathways related to neurological diseases such as prion disease, Huntington disease, and spinocerebellar ataxia stand out. Additionally, pathways linked to addiction phenomena, including cocaine addiction and nicotine addiction, glutamatergic synapses, aldosterone-regulated sodium reabsorption, and glyoxylate and dicarboxylate metabolism, also demonstrate the multifaceted regulatory mechanisms of AMF. Notably, the alanine, aspartate, and glutamate metabolism pathways remained significantly enriched even after rigorous statistical correction, underscoring their potential as key players in alleviating Cu stress in Vetiveria zizanioides. Fig. 9. Fig. 9 [109]Open in a new tab DEMS enrichment pathway diagram. Note: level 1 pathway classification: M-Metabolism, E-Environmental Information Processing, O-Organismal Systems. Table 7. Significant differences in metabolic pathways. Description KEGG ID Metabolites log2FC Alanine, aspartate and glutamate metabolism [110]C00025 L-Glutamate − 0.4790 [111]C12270 N-Acetylaspartylglutamate 1.1672 [112]C00352 D-Glucosamine-6-Phosphate 2.3235 Taurine and hypotaurine metabolism [113]C00025 L-Glutamate − 0.4790 [114]C00606 3-Sulfino-L-Alanine − 0.7569 Vitamin B6 metabolism [115]C00847 4-Pyridoxate − 0.5664 [116]C00279 D-Erythrose 4-phosphate − 0.9787 Phenylalanine, tyrosine and tryptophan biosynthesis [117]C00296 Quinic Acid 5.5746 [118]C00279 D-Erythrose 4-phosphate − 0.9787 Pentose phosphate pathway [119]C03752 Glucosaminate 0.9092 [120]C00279 D-Erythrose 4-phosphate − 0.9787 Glyoxylate and dicarboxylate metabolism [121]C00025 L-Glutamate − 0.4790 [122]C00898 Tartaric Acid 1.0630 ABC transporters [123]C00114 Choline 0.3153 [124]C00299 Uridine 1.1782 [125]C00025 L-Glutamate − 0.4790 [126]C00294 Inosine 0.7589 [127]Open in a new tab Discussion Plant height and fresh weight serve as reliable indicators of a plant’s overall growth status and vitality. Our study consistently revealed that when Vetiveria zizanioides plants were inoculated with arbuscular mycorrhizal fungi (AMF), their plant height and fresh weight surpassed those of their uninoculated counterparts. This finding aligns with the research of Wang et al.^[128]19, who reported a significant increase in alfalfa biomass following AMF inoculation under heavy metal stress. Similarly, Yang et al.^[129]38 reported that, under lead (Pb) stress, AMF-inoculated plants presented greater dry weights in their roots, stems, and leaves than nonmycorrhizal plants did, mirroring the results of our current study. These converging findings suggest that AMF inoculation not only promotes plant growth and development under heavy metal stress but also contributes to the development of metal tolerance, ultimately increasing plant biomass. This is further supported by the work of Riaz et al.^[130]39, who underscored the beneficial effects of AMF on plant resilience in contaminated environments. Roots, in addition to their vital function as the primary organ for absorbing water and minerals from the soil, are pivotal in perceiving and responding to external stimuli^[131]40,[132]41. This sensory role underscores their crucial role in facilitating plant adaptation to adverse conditions. When confronted with environmental stressors, plants tend to evolve adaptive mechanisms, including the reconfiguration of their root system architecture, to accommodate variations in nutrient availability and effectiveness^[133]42. Our study revealed that as the copper concentration increased from 0 to 200 mg/kg, the total root length, root surface area, root volume, and total number of root tips in the plant root system notably increased. This observation aligns with the findings of Xiong et al.^[134]43, who reported that under environmental stress, larger and more intricate root systems develop, enhancing water and nutrient uptake capabilities and ultimately contributing to increased plant biomass and resilience. Thus, the observed increase in root complexity in our experiment could be a consequence of copper stress, further validating the adaptive potential of plant roots in response to adverse environmental conditions. The accumulation of Cu ions within plant root cells can significantly impact root development through multiple mechanisms. It can modulate the proliferation rate of cells in the meristematic tissues of the root, thereby altering root growth dynamics. Furthermore, it can regulate the levels of key phytohormones, such as indole-3-acetic acid (IAA, also known as auxin) and cytokinin (CTK), which play pivotal roles in regulating plant growth and development^[135]44,[136]45. Interestingly, studies have shown that the inoculation of arbuscular mycorrhizal fungi (AMF) can influence the endogenous hormone levels in plants, thereby exerting a profound effect on root growth and development^[137]46,[138]47. This finding is consistent with the outcomes of our study, in which AMF-inoculated plants presented greater total root length, total root surface area, total root volume, and total root tip number than their noninoculated counterparts did. Moreover, other studies have shown that AMF inoculation increases IAA levels, which in turn stimulates root growth^[139]39. This positive influence on IAA production underscores the symbiotic relationship between AMF and host plants, highlighting the potential of AMF as a tool to promote robust root systems and improve plant resilience under varying environmental conditions. Our study revealed a noteworthy phenomenon: the copper transfer coefficient of vetiver was greater at a copper concentration of 0 mg/kg, while the copper content of both the above- and below-ground parts of plants inoculated with AMF was significantly greater than that of uninoculated plants. This might be because plants require a certain amount of copper to maintain their vital life processes and to ensure that material cycling, energy flow and information transfer within the plant proceed smoothly^[140]48. AMF, known for their heavy metal chelating ability, can accumulate large amounts of copper ions in the soil. These copper ions are actively taken up by plant cells and bind to metallothionein or specific soluble copper ion chaperone proteins. They are subsequently transported to various organelles, including vesicles, chloroplasts and mitochondria^[141]49. However, under relatively high copper stress levels (200 mg/kg and 400 mg/kg), a significant decrease in copper enrichment occurred in the aboveground parts of vetiver inoculated with AMF compared with that in the noninoculated group. This phenomenon can be attributed to the robust heavy metal sequestration ability of AMF, which effectively bind heavy metal ions in the soil through both surface adsorption and the secretion of compounds such as globocystin-related protein (GRSP). GRSP, in particular, enhances the adsorptive capacity of soil particles for heavy metal ions, reducing the levels of metals in the bioavailable state and bioavailability. This, in turn, restricts the translocation of heavy metals from the soil to plants, resulting in a lower accumulation of heavy metals in aboveground plant tissues^[142]50–[143]52. The accumulation of osmoregulatory substances, particularly carbohydrates, constitutes a pivotal mechanism in the plant arsenal against adverse stress^[144]53. Both soluble proteins and proline are important osmoregulators in plants^[145]54, and their accumulation is a defense response to abiotic stress^[146]55. Increasing the levels of osmoregulatory substances represents a strategic adaptation in plants to mitigate the adverse effects of stress. However, our current study reveals an intriguing deviation: inoculation with arbuscular mycorrhizal fungi (AMF) led to a reduction in soluble sugar content within plants. This phenomenon can be rationalized by considering two primary hypotheses. First, soluble sugars function as primary osmoregulators, bolstering the stability of plasma membranes and protoplasts while safeguarding enzymes against the detrimental effects of excess inorganic ions within plant cells^[147]56,[148]57. In the AMF-treated group, the reduced transfer of Cu ions to the aerial portions of balsamgrass resulted in a reduction in osmotic potential difference. Conversely, the uninoculated vetiver grass presented relatively high Cu ion concentrations in its upper sections, triggering a robust self-preservation mechanism in which elevated soluble sugar levels reduce the osmotic potential and increase cellular water uptake or retention capabilities. Second, the symbiotic relationship between vetiver and AMF may involve the consumption of soluble nutrients by plants. This process, as suggested by Gadkar et al.^[149]58, could contribute to the observed decrease in soluble sugar content following AMF inoculation. In the present study, we observed a significant increase in the proline content of vetiver leaves subjected to copper stress at concentrations of 200 mg/kg and 400 mg/kg; the proline content was significantly greater in the AMF-inoculated group than in the non-AMF-inoculated group, highlighting the key role of proline as an important regulatory agent in plants for coping with abiotic stress challenges. Furthermore, the symbiotic relationship of the plant with AMF significantly increased the tolerance of vetiver to Cu, mainly through the promotion of proline synthesis, thereby increasing the ability of the plant to recover from unfavorable conditions^[150]59. Heavy metal toxicity leads to the accumulation of reactive oxygen species (ROS) in plants, which results in oxidative stress and damage to proteins, lipids and DNA^[151]60. Excess copper catalyzes the formation of reactive oxygen species (ROS) through the Haber-Weis and Fenton reactions^[152]61. However, plants have gradually evolved antioxidant defense systems to help mitigate the harmful effects of ROS^[153]62. Plants can activate their antioxidant defense system in response to heavy metal-induced ROS accumulation. This system includes enzymes that help remove and neutralize ROS, such as superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD)^[154]63,[155]64. SOD, POD, and CAT can maintain the dynamic balance of free radical production and removal in plants under normal conditions, thereby eliminating the potential damage caused by free radicals to the plant cell membrane structure^[156]65. By assessing the activities of these antioxidant enzymes, we gain insights into the toxic impact of heavy metals on plant cells. Our study demonstrated that arbuscular mycorrhizal fungi (AMF) inoculation enhances antioxidant enzyme activities in plants under copper stress. Specifically, under copper concentrations of 100 mg/kg, 200 mg/kg, and 400 mg/kg, AMF-treated comfrey plants presented increased antioxidant enzyme activities compared with uninoculated plants. This finding underscores the ability of AMF to bolster plant resilience against heavy metal stress by upregulating the antioxidant enzyme system, thereby improving overall plant tolerance and survival under adverse conditions. Malondialdehyde (MDA), a reliable indicator of membrane lipid peroxidation, is often directly correlated with the intensity of oxidative stress within plants^[157]66. An elevated MDA content in plants signifies an increased degree of cell membrane lipid peroxidation, which compromises membrane structural integrity, as emphasized by González-Guerrero et al.^[158]67. Our study reinforces the notion that AMF inoculation triggers a cascade of beneficial effects. Specifically, AMF inoculation activates a plant’s antioxidant enzyme system, effectively neutralizing the reactive oxygen species (ROS) generated under abiotic stress conditions. This, in turn, mitigates peroxidative damage to cell membranes, alleviates the deleterious effects of copper toxicity, and ultimately enhances plant resilience and adaptability to adverse environments. Numerous studies have convincingly demonstrated that inoculation with arbuscular mycorrhizal fungi (AMF) significantly enhances plant resilience against metal stress^[159]16,[160]68–[161]70. When confronted with stress, plants experience disruptions in their redox balance, and plants respond adeptly under the AMF‒plant symbiotic relationship by activating relevant genes^[162]71. Phenylalanine metabolism and associated pathways play pivotal roles in conferring stress tolerance to plants^[163]72. The intricate metabolic network centered on phenylalanine is involved in the synthesis of crucial secondary metabolites such as anthocyanins, lignins, rutin, and chlorogenic acid^[164]73. Anthocyanins, renowned for their potent antioxidant properties, not only increase the aesthetic appeal of fruits but also safeguard plants against UV radiation, pests, and diseases^[165]74. Moreover, lignin content is intimately tied to plant biomass accumulation, underscoring its importance in plant growth and development^[166]75. Crucially, the biosynthesis of L-phenylalanine (L-Phe), the cornerstone of this metabolic pathway, requires two compounds, phosphoenolpyruvate (PEP) and erythrose 4-phosphate (E4P)^[167]76. This finding underscores the importance of D-erythrose 4-phosphate as an indispensable precursor for L-Phe production, thereby linking it to the entire network of stress-responsive metabolites in plants. Furthermore, AMF inoculation effectively mitigates the deleterious effects of copper on plants, thereby contributing to an increase in photosynthetic efficiency. In the photosynthetic apparatus, light energy is harnessed within leaf cells and stored in the form of nicotinamide adenine dinucleotide phosphate (NADPH). This energy is subsequently transformed into intermediates such as D-erythritol-4-phosphate and glyceraldehyde-3-phosphate, which are integral to various physiological processes, including glycolysis and amino acid synthesis, within plant cells. These metabolic intermediates, in turn, impact the hormonal balance in plants^[168]77, ultimately orchestrating the stress response mechanisms of plants at the macroscopic level. Our study revealed a decrease in D-erythrose 4-phosphate under copper stress, suggesting that AMF inoculation influences the L-phenylalanine (L-Phe) synthesis pathway as a strategic means to combat this stress. This adaptive response underscores the intricate interplay between AMF, plant metabolism, and stress tolerance mechanisms. Chlorogenic acid is a pivotal antioxidant in plants and is classified as a carboxyl phenolic acid that arises from the condensation of quinic acid and caffeic acid within the trans-cinnamic acid framework^[169]78,[170]79. Both quinic acid and chlorogenic acid occupy central stages in the phenylpropanoid metabolic pathway, serving as key intermediates^[171]80. These compounds can inhibit the lipid peroxidation resulting from metal stress, thereby safeguarding cellular integrity^[172]81. Oksana et al.^[173]82 demonstrated the antioxidant properties of chlorogenic acid, revealing its capacity to shield plants from oxidative damage caused by environmental stressors. In line with this, studies have reported a substantial increase in chlorogenic acid concentrations and a concomitant increase in antioxidant defenses in plants subjected to salt stress, suggesting a potential interplay between these variables^[174]83,[175]84. The inoculation of arbuscular mycorrhizal fungi (AMF) in Vetiveria zizanioides plants under copper (Cu) stress significantly increased the accumulation of quinic acid. This, in turn, promoted the production of additional chlorogenic acid, which acts as a robust defense agent against Cu stress, highlighting the symbiotic benefits of AMF in bolstering plant resilience. The results of this study indicate that inoculation with AMF under Cu stress leads to the formation of more phytochelatins (PCs) from glutamate. PCs are small cysteine-rich peptides that are able to bind metals (classes) through the -SH motif. Although the biosynthesis of PCs can be induced in vivo by a variety of metals (classes), PCs are involved mainly in the detoxification of cadmium and arsenic(III) as well as mercury, zinc, lead, and copper ions and are key components of plant defense against heavy metal stress^[176]85. In response to external heavy metal stress, peptides bind to heavy metals via sulfhydryl groups, forming low-molecular-weight complexes, which are sequestered in vesicles. In the vesicles, the PCN molecules further conjugate with other PCN molecules to effectively reduce the biological activity of the metal ions and produce high-molecular-weight compounds with minimal toxicity to plant cells, thus mitigating the damage caused by excess copper to plants^[177]86 (Fig. [178]10). Fig. 10. [179]Fig. 10 [180]Open in a new tab Stress resistance mechanism diagram of vetiver grass inoculated with AMF under copper stress. In this study, the metabolite changes of AMF inoculation on the stems and leaves of Vetiver under CU stress were studied from non-targeted metabolomics, but in order to further investigate the reasons, it is necessary to analyze the multi-omics such as genomics, transcriptomics and proteomics to form a more complete life activity process. Conclusions This comprehensive study investigated the impacts of AMF inoculation on the growth, physiological responses, and biochemical profiles of Vetiveria zizanioides under various levels of Cu stress. Additionally, we explored the molecular intricacies governing the enhanced copper tolerance of Vetiveria zizanioides via metabonomic analyses. Our findings indicate that vetiver grass inoculated with AMF has better growth performance than its noninoculated counterparts; AMF inoculation significantly broadens the tolerance range of vetiver grass to copper stress. AMF inoculation influences the metabolism of phenylalanine and related pathways in vetiver grass, leading to the synthesis of crucial secondary metabolites such as anthocyanins, lignin, rutin, and chlorogenic acid through a complex metabolic network centered on phenylalanine. These secondary metabolites bolster the resistance of vetiver grass to copper stress. Inoculation with AMF facilitates the increased formation of phytochelatins (PCs) from glutamate, thereby mitigating copper stress. Author contributions W.C, Y.G and C.C. designed research; Y.G, H.W, and Y.Y. performed research. Y.G. and H.C. analyzed data; Y.G. and W.C. wrote the manuscript; all authors critically reviewed and approved the final manuscript. Data availability The data that support the findings of this study are available from the corresponding author upon reasonable request. Declarations Competing interests The authors declare no competing interests. Footnotes Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. References