CeO2-NPs alleviates Cd toxicity in red fescue by enhancing antioxidant defense and metabolic regulation

CeO2-NPs alleviates Cd toxicity in red fescue by enhancing antioxidant defense and metabolic regulation | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 5 January 2026 V1 Latest version Share on CeO2-NPs alleviates Cd toxicity in red fescue by enhancing antioxidant defense and metabolic regulation Authors : Xueling Zheng , Along Chen , Mengyao Sang , Zhenjian Bai , Jinwei Zhang , Qianhan Zhao , Shasha Liu , Lu Zhang , Fuchun Xie , Jia Jiang 0009-0002-8959-461X , and Yajun Chen [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.176762182.28782902/v1 122 views 88 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Cadmium (Cd) contamination in soil severely threatens plant growth and ecological safety. Engineered nanomaterials, as a newly emerging form with characteristics of environmental protection and efficient absorption, have potential in mitigating response of crops to abiotic stresses, while it is only limited understanding role of engineered nanomaterials on alleviating Cd toxicity in plants used for ecological restoration. This study using morpho-physiological and multi-omics methods, it’s found that foliar application of 100-500 mg/L CeO 2 nanoparticles (NPs) significantly promoted plant growth, improved photosynthesis and antioxidant capacity, while reduced Cd uptake and translocation in two red fescue ( Festuca rubra L.) cultivars under Cd stress. Moreover, the concentration of 350 mg/L was most effective. Integrated transcriptomic and metabolomic analyses revealed that CeO 2 -NPs affect the key pathways including ‘flavone and flavonol biosynthesis’ and ‘D-amino acid metabolism’. Further screening revealed that rutin is the key metabolite regulated by CeO 2 -NPs, furthermore, functional verification showed that application of key metabolite rutin enhanced Cd tolerance by inhibiting the formation of hydrogen peroxide and oxygen free radicals, and activating the antioxidant defense system in red fescue. In summary, CeO 2 -NPs alleviates Cd toxicity in red fescue through regulating key metabolic substances to enhance antioxidant capacity and reducing Cd accumulation. This study provides novel insights into the nano-enabled strategy for the phytoremediation and safe utilization of Cd-contaminated soils. CeO 2 -NPs alleviates Cd toxicity in red fescue by enhancing antioxidant defense and metabolic regulation Xueling Zheng 1* , Along Chen 1* , Mengyao Sang 2* , Zhenjian Bai 2 , Jinwei Zhang 2 , Qianhan Zhao 2 , Shasha Liu 2 , Lu Zhang 3 , Fuchun Xie 2# , Jia Jiang 2# , Yajun Chen 1# 1 College of Horticulture , Northeast Agricultural University , Harbin 150030 , China 2 College of Animal Science and Technology , Northeast Agricultural University , Harbin 150030 , 3 College of Landscape and Architecture, Zhejiang A&F University # Correspondience Yajun Chen , E-mail: [email protected] ; Fuchun Xie , E-mail: [email protected] ; Jia Jiang, E-mai: [email protected] . * These authors contributed equally to this work. Abstract: Cadmium (Cd) contamination in soil severely threatens plant growth and ecological safety. Engineered nanomaterials, as a newly emerging form with characteristics of environmental protection and efficient absorption, have potential in mitigating response of crops to abiotic stresses, while it is only limited understanding role of engineered nanomaterials on alleviating Cd toxicity in plants used for ecological restoration. This study using morpho-physiological and multi-omics methods, it’s found that foliar application of 100-500 mg/L CeO 2 nanoparticles (NPs) significantly promoted plant growth, improved photosynthesis and antioxidant capacity, while reduced Cd uptake and translocation in two red fescue ( Festuca rubra L.) cultivars under Cd stress. Moreover, the concentration of 350 mg/L was most effective. Integrated transcriptomic and metabolomic analyses revealed that CeO 2 -NPs affect the key pathways including ‘flavone and flavonol biosynthesis’ and ‘D-amino acid metabolism’. Further screening revealed that rutin is the key metabolite regulated by CeO 2 -NPs, furthermore, functional verification showed that application of key metabolite rutin enhanced Cd tolerance by inhibiting the formation of hydrogen peroxide and oxygen free radicals, and activating the antioxidant defense system in red fescue. In summary, CeO 2 -NPs alleviates Cd toxicity in red fescue through regulating key metabolic substances to enhance antioxidant capacity and reducing Cd accumulation. This study provides novel insights into the nano-enabled strategy for the phytoremediation and safe utilization of Cd-contaminated soils. Keyword: CeO 2 -NPs, Festuca rubra L., Cd tolerance, Rutin, Antioxidant system, nanomaterial 1. Introduction With the rapid industrial development, the activities in electroplating, metallurgy, industrial emissions, and the overuse of fertilizers and pesticides have intensified heavy metal pollution of soil. Compared with other heavy metals, cadmium (Cd) has the characteristics of highly toxic, persistent, and highly mobile [1] , posing a significant threat to both human health and the ecological environment. Plants growing in Cd-contaminated environments experience various morphological, physiological, and biochemical changes, including slow growth, leaf chlorosis [2] , damage of photosynthetic tissues [3] , and abnormal cell proliferation [4] . These changes disrupt plant growth, and severely affect function of plant ecological restoration [5] . Moreover, plants absorb Cd from the soil through their roots, accumulating it in the xylem, which is then distributed throughout the plant. Cd can be transferred to humans via the food chain, ultimately threatening human health [6] . Therefore, it is crucial to explore effective, economical, and sustainable strategies to reduce cadmium damage to plants. Nanotechnology has shown great potential in agricultural production through the use of nanoparticles (NPs), addressing issues such as nutrient loss, low utilization, and secondary environmental pollution in soil remediation [7] . NPs are defined as particles smaller than 100 nm in at least one dimension [8] . Compared to traditional materials, NPs have a large specific surface area, high surface energy, and high stability, enabling them to effectively capture heavy metal ions and thus act as adsorbents and stabilizers for heavy metals [9-10] . In practice, NPs reduce heavy metal absorption and nutrient loss by hindering root transport of heavy metals, and significantly improved physiological metabolism and the growth of crops [11-14] . In rice, TiO 2 NPs(50, 100, 200, 400 mg/L) application alleviates the negative effects of Cd on photosynthesis and protein degradation [15] . Foliar application of 30 μM SeNPs reduced Cd absorption of roots and decreased Cd transfer to the aboveground parts in tomato ( Solanum lycopersicum L. ) [16] . Wheat ( Triticum aestivum L. ) treated with FeO and Se-NPs enhanced its Cd tolerance by reducing oxidative stress, increasing antioxidant content, and upregulating related genes, thereby reducing Cd absorption [17] . Clearly, engineered NPs significantly enhance crops resistance to Cd and hold great potential in Cd-contaminated soil remediation. Additionally, Cerium (Ce) is an abundant rare earth element, and CeO 2 -NPs also can improve crops stress resistance by inducing physiological and biochemical changes, and stimulating rhizosphere bacteria [18-21] . However, reports on the molecular mechanisms of engineered nanomaterials in enhancing plant stress resistance are limited, especially for CeO 2 -NPs, with studies mainly restricted to okra ( Abelmoschus esculentus L. ) [22] and fragrant rice ( Oryza sativa L.) [23] . It is worth noting that there is limited understanding of the effects of engineered CeO 2 -NPs on plants used for ecological remediation under Cd stress. Therefore, further research is needed to explore the metabolic and molecular mechanisms through CeO 2 -NPs enhancing Cd tolerance of ecological restoration plants. Red fescue ( Festuca rubra L.) has the characteristics of cold resistance, developed root system, strong tillering ability, and resistance to trampling, and is an excellent ecological restoration grass species as well as a dual-purpose species for both turfgrass and forage [24-25] . Red fescue is commonly used for soil stabilization and slope protection, urban environmental greening, and metal mine restoration to repair the ecological environment in high cold regions [26] . In addition, it plays a crucial role in recovering degraded grasslands and preventing desertification. Its growth environment is being poisoned by Cd from industrial waste gases, sewage irrigation, the accumulation of waste containing heavy metals and so on. As forage, the absorption of Cd by red fescue also presents a potential food safety risk. Therefore, strategies to mitigate Cd toxicity in red fescue need to be explored. This study employed foliar application of CeO 2 -NPs, combined with morphological, physiological, transcriptomic, and metabolomic analyses, to explore further mechanism of CeO 2 -NPs reducing the impact of Cd stress. 2. Materials and methods 2.1. Cultivation and treatment of experimental Material s The experiment was conducted in the greenhouse at the Horticultural Experiment Station of Northeast Agricultural University (E 126°14′; N 45°05′). The plant materials were red fescue cultivars ‘Frigg’ and ‘Reverent’, which were widely used in Northeast China. Seeds of uniform size, healthy, and fully developed were sown at a density of 25 g/cm² into pots containing 800 g of a mixture of nutrient soil and vermiculite (2:1), with dimensions of 16 cm (top diameter) × 14 cm (bottom diameter) × 14 cm (height). The plants were grown under controlled conditions, with a temperature of 25 ℃ ± 5 ℃, light intensity of 1000 ± 50 µmol·m -2 ·s -1 , a 16/8 hours light/dark photoperiod, and relative humidity of 75% ± 5%. After 35 days, once the plants had fully grown, treatments were applied. Based on the previous experiments, for each pot of plants add 100 mL of a solution with a concentration of 100 mg/kg of CdCl 2 , the percolate was returned to the pots. Additionally, different concentrations of CeO 2 -NPs were applied by foliar spraying. Six treatments were set up as follows: CK (no Cd stress + foliar spray of deionized water); Cd treatment (Cd + foliar spray of deionized water); Cd + 100 nCeO 2 (Cd + foliar spray of 100 mg/L CeO 2 -NPs); Cd + 200 nCeO 2 (Cd + foliar spray of 200 mg/L CeO 2 -NPs); Cd + 350 nCeO 2 (Cd + foliar spray of 350 mg/L CeO 2 -NPs); and Cd + 500 nCeO 2 (Cd + foliar spray of 500 mg/L CeO 2 -NPs). Each treatment was repeated three times. During spraying, aluminum foil was used to cover the surface of the soil to prevent the spray from contacting the soil. A volume of 20 mL was sprayed on each pot, and the application was repeated every 3 days, totaling three applications. Morphological observations and photosynthetic measurements were taken on day 12 after the Cd treatment, and samples were stored at -80°C for further analysis. The metabolite functional validation experiment included three treatments: no Cd stress with foliar spray of deionized water; Cd exposure with foliar spray of deionized water; Cd exposure with foliar spray of 5 mM rutin. The cultivation of plant materials, spraying methods, and sampling time followed the same procedures as in the exogenous CeO 2 -NPs application experiment. 2.2. Experimental methods 2.2.1. Characterization and observation of CeO 2 -NPs CeO 2 -NPs powder was purchased from Shanghai McLin Biochemical Technology Co., Ltd., with a specific surface area of 50 m² per gram. The morphology of the CeO 2 -NPs was evaluated using a Scanning Electron Microscope (SEM, SU8010, HITACHI, Japan). The crystal structure of the CeO 2 -NPs was analyzed using X-ray diffraction (XRD, D8 ADVANCE, Germany). 2.2.2. Biomass and root observation Red fescue plants were rinsed thoroughly with deionized water. A portion of the plants was scanned to assess root morphology and various parameters using a root scanner (LA-S, Wanshen, Zhejiang, China) [27] . The remaining plants were separated into above-ground and below-ground parts using scissors, and the fresh weights of the above-ground and below-ground parts were measured using an analytical balance (FA-C, Techcomp, Shanghai, China). Each measurement was conducted in triplicate for accuracy. 2.2.3. Cd content in root and leaf tissues Roots and leaves of red fescue from different treatments were collected, thoroughly washed with deionized water, dried to a constant weight, and ground into a fine powder. A 0.2 g sample of the powder was then pre-digested with a mixture of 12 mL of nitric acid (HNO 3 ) and hydrogen peroxide (H 2 O 2 ) in a 3:1 ratio for 8 hours. After a 24-hour incubation period, the mixture was placed in a microwave digestion system (MASTER-40, Shanghai, China) and digested at 150℃ for 30 minutes, 170℃ for 2 hours, and 190℃ for 1 hour. Once digestion was complete, the solution was heated at 150 ℃ to remove excess acid until the volume was reduced to 2 mL. The solution was then diluted to 50 mL with 2% HNO 3 . Cd concentrations were measured using Inductively Coupled Plasma Mass Spectrometry (ICP-MS), with the calculation method based on the procedure outlined by Wang et al. (2025) to determine the Cd content [28] . 2.2.4. Ultrastructural observation of roots and leaves Roots and leaves of red fescue from different treatments were collected and washed. A 3 mm segment of root or leaf was immersed in a 2.5% glutaraldehyde solution (pH = 6.8) for 24 hours to fix the tissues. After fixation, the samples were rinsed three times with 0.1 M phosphate-buffered solution. Leaf samples were then transferred to 1% osmium tetroxide solution for 2 hours of fixation, followed by three rinses with 0.1 M phosphate-buffered solution. The samples were dehydrated using ethanol-acetone and embedded in epoxy resin for 3 days. After embedding, ultra-thin sections (50-60 nm) were cut using an ultramicrotome and observed under a transmission electron microscope (HT7800, Hitachi, Japan). Root samples underwent dehydration using ethanol-acetone, followed by displace with ethanol-tertiary butanol, and were then dried using a freeze dryer (ES-2030 HITACHI). After drying, the samples were coated and observed and photographed using a scanning electron microscope (SU8010, Hitachi, Japan). 2.2.5. Measurement of photosynthetic-related parameters and chlorophyll content On clear days, between 9:00 and 11:00 a.m., the photosynthetic rate (Pn), transpiration rate (Tr), stomatal conductance (Gs), and intercellular CO 2 concentration (Ci) of red fescue were measured using a portable photosynthetic system (Li-6400 XT, LiCOR Inc.). Chlorophyll content was determined using the 95% ethanol extraction method. Each measurement was performed in triplicate for each treatment [29] . 2.2.6. Measurement of ROS, MDA, and osmotic regulating substances The superoxide anion (O 2 - ) content was measured using the hydroxylamine oxidation assay [30] . Hydrogen peroxide (H 2 O 2 ) content was quantified using a commercial assay kit (Mengxi Bio, Suzhou). DAB and NBT staining were performed using a commercial kit (Solarbio, Beijing). Malondialdehyde (MDA) content was determined by the thiobarbituric acid (TBA) method [31] . Soluble sugar (SS) content was measured using the anthrone colorimetric method [30] . Soluble protein (SP) content was determined by the Bradford method [32] . Free proline (Pro) content was assessed using the sulfosalicylic acid method [33] . Each measurement was performed in triplicate for each treatment. 2.2.7. Measurement of antioxidant defense system Superoxide dismutase (SOD) activity was measured using the NBT reduction assay [30] . Peroxidase (POD) activity was determined using the guaiacol assay method [30] . Catalase (CAT) activity was quantified by ultraviolet absorption at a specific wavelength [30] . Ascorbate peroxidase (APX) activity was determined according to the method described by Murshed et al. (2013) [34] . Reduced glutathione (GSH) content was measured by spectrophotometry [30] . 2.2.8 Transcriptomic and metabolomic analysis Based on the effects of different concentrations of CeO 2 -NPs on the morphological structure and physiological metabolic indicators of red fescue under Cd stress, leaves from the CK, Cd treatment, and Cd + 350 nCeO 2 group were selected for transcriptomic and metabolomic analyses. The transcriptomic analysis was performed with 3 replicates per treatment, totaling 18 samples from two cultivars. The metabolomic analysis was performed with 6 replicates per treatment, totaling 36 samples from two cultivars. Samples were sent to Shanghai Meiji Bio-Medical Technology Co., Ltd. for analysis. The detailed determination plan for the transcriptome is presented in Supplementary methods S(1). Differentially expressed genes were selected based on a |log2FC| ≧ 1 and FDR < 0.05 using DESeq2. Additionally, a Bonferroni-corrected FDR threshold of < 0.05 was applied, and Gene Ontology (GO) functional enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were conducted using Goatools and Python’s scipy software, respectively. Metabolite analysis was performed using an untargeted metabolomics approach, with the detailed protocol provided in Supplementary methods S(2). Data analysis was performed on the Meiji Bio Cloud Platform (https://cloud.majorbio.com). Missing values were removed using the 80% rule, and the data were normalized using sum normalization. Principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) were conducted using the R package. Student’s t-test and fold-change analysis were also applied. Variable importance in projection (VIP) values was calculated to assess the contribution of each variable, and metabolites with VIP > 1.0 and P < 0.05 (t-test) were considered differentially expressed metabolites (DEMs). 2.2.9. RT-qPCR validation A subset of genes was randomly selected for qPCR validation to verify the accuracy of the transcriptomic data. Gene IDs and primer sequences are provided in Table S1. RNA samples obtained from transcriptomic sequencing were used for quantitative reverse transcription PCR (qRT-PCR), following the protocol of the ChamQTM Universal SYBR® qPCR Master Mix kit (Novozan, Nanjing). Relative expression levels were calculated using the 2 -△△ct method. 2.3 Statistical analysis Data were analyzed using Microsoft Excel 2010 and IBM SPSS Statistics 26.0. One-way analysis of variance (ANOVA) followed by Duncan’s multiple range test was used to identify significant differences between sample groups. P < 0.05 was considered statistically significant, and different letters were used to denote significant differences between treatment groups. All figures were generated using GraphPad Prism v8 (GraphPad Software, La Jolla, CA, USA) and Origin v.2024. 3. Results 3.1. Characterization of CeO 2 -NPs SEM images revealed that CeO 2 -NPs have an irregular polygonal shape with a rough surface and sharp edges. The average particle size of CeO 2 -NPs, determined from 100 randomly selected nanoparticles, was 46.6 ± 9.3 nm. The absorption spectrum of CeO 2 -NPs shows a sharp peak with no significant impurity peaks, and the results correspond well with the characteristic peaks of the standard CeO 2 PDF card, confirming the presence of CeO 2 in the nanoparticles (Figure S1). 3.2. Effects of CeO 2 -NPs on growth and photosynthetic pigments of red fescue under Cd stress Cd significantly reduced the aboveground and underground biomass as well as root length in the two cultivars of red fescue (Figure 1). Under Cd stress, compared to the respective CK, the aboveground, underground biomass and root length of ‘Frigg’ decreased by 41.80%, 48.21%, and 34.27%, respectively, while in ‘Reverent’, the reductions were 45.54%, 50.67%, and 39.15%. Spraying of different concentrations of CeO 2 -NPs alleviated the Cd stress in both cultivars. The most significant effect was observed at a concentration of 350 mg/L. Compared with the Cd treatment, the aboveground, underground biomass and root length of ‘Frigg’ increased by 55.88%, 78.71% and 44.35% under the Cd + 350 nCeO 2 treatment, respectively, while those of ‘Reverent’ increased by 54.28%, 88.02% and 37.18%. (Figure 1C-E). Under Cd stress, photosynthetic pigments (Chla, Chlb, and total Chl) in red fescue were significantly reduced. Compared to the CK, ‘Frigg’ decreased by 52.50%, 43.44%, and 48.58%, respectively, while ‘Reverent’ decreased by 64.27%, 49.77%, and 57.97% (Figure S2). The photosynthetic parameters of both cultivars were also significantly affected, with ‘Reverent’ showing a greater decline in Pn, Tr, Gs, and Ci, which decreased by 33.19%, 51.87%, 12.10%, and 38.55% respectively, compared to CK. The application of CeO 2 -NPs effectively enhanced the chlorophyll content and photosynthetic capacity under Cd stress. The total chlorophyll content of ‘Frigg’ increased by 6.8%, 41.35%, 72.47% and 41.60% respectively when exposed to 100, 200, 350 and 500 mg/L CeO 2 -NPs compared to the Cd treatment, while ‘Reverent’ showed increases of 72.00%, 72.74%, 73.13%, and 23.80%, respectively. 3.3. Effects of CeO 2 -NPs on antioxidant system and osmotic regulation in red fescue under Cd stress Cd stress caused redox imbalance in both red fescue cultivars, significantly increasing the levels of H 2 O 2 , O 2 - , and MDA (Figure 2A-C). Under Cd stress, MDA content in ‘Reverent’ increased from 0.13 μmol/g to 0.55 μmol/g. Meanwhile, the levels of H 2 O 2 and O 2 - were 1.6times and 61.17% higher than those in CK. However, CeO 2 -NPs application significantly alleviated oxidative stress caused by Cd in red fescue. Compared to Cd treatment, the Cd+350 nCeO 2 treatment reduced H 2 O 2 accumulation in ‘Frigg’ and ‘Reverent’ by 22.62% and 32.61%, respectively, while the Cd + 500 nCeO 2 treatment resulted in the greatest decrease in O 2 - , with reductions of 18.79% and 24.27% in both cultivars (Figure 2C). Compared to the Cd treatment, the MDA content of ‘Frigg’ and ‘Reverent’ decreased most significantly at CeO 2 -NPs concentrations of 500 mg/L and 350 mg/L, reducing by 56.17% and 23.37%, respectively (Figure 2A). Cd stress reduced antioxidant enzyme activity in both red fescue cultivars (Figure 2E-I). In ‘Reverent’, the activity of SOD, POD, CAT, GSH, and APX decreased by 36.54%, 41.11%, 61.61%, 21.09%, and 65.84% respectively, compared to CK (Figure 2D-H). However, CeO 2 -NPs application significantly increased antioxidant enzyme activity in both cultivars. Among all treatments, the Cd + 350 nCeO 2 treatment had the most significant effect on SOD and APX activities. Compared to Cd treatment, ‘Frigg’ increased by 38.98% and 2.01 times, while ‘Reverent’ increased by 66.58% and 3.42 times (Figure 2E, G), and POD activity increased significantly under CeO 2 -NPs treatments at 500 mg/L, with increases of 1.24times in ‘Frigg’ and 1.43 times in ‘Reverent’ (Figure 2F). The activities of CAT and GSH were significantly enhanced under the treatment with CeO 2 -NPs (Figure 2D, H). Cd stress lowered soluble sugars in ‘Reverent’, but no significant changes were observed in ‘Frigg’. Application of CeO 2 -NPs increased the proline and soluble sugar content in red fescue. CeO 2 -NPs at 200, 350, and 500 mg/L significantly increased proline content with ‘Frigg’ increasing by 15.94%, 16.31%, and 17.44%, respectively, compared to the Cd treatment. In ‘Reverent’, proline content increased by 19.11%, 20.63%, and 17.27% respectively. Among all treatments, the Cd+350 nCeO 2 treatment had the most significant effect on soluble sugar content, with increases of 13.15% and 16.07% in ‘Frigg’ and ‘Reverent’ respectively (Figure S3). 3.4. Effects of CeO 2 -NPs on Cd accumulation in red fescue under Cd stress Under Cd stress, both ‘Frigg’ and ‘Reverent’ significantly accumulated Cd in their roots and leaves (Figure S3). Cd accumulation in the leaves and roots of ‘Reverent’ was 19.49% and 12.41% higher than in ‘Frigg’. Additionally, the Cd transport from roots to leaves was higher in ‘Reverent’ than in ‘Frigg’. The application of CeO 2 -NPs significantly reduced Cd accumulation in both roots and leaves, as well as the Cd transport factor (TF) (Figure 2I). With CeO 2 -NPs concentrations of 100, 200, 350, and 500 mg/L, the TF values of ‘Frigg’ decreased by 6.00%, 17.30%, 24.28%, and 8.27% respectively, compared to the Cd treatment. In ‘Reverent’, the TF values decreased by 3.90%, 10.25%, 21.72%, and 19.16%. 3.5. Optimization of the optimal exogenous concentration of CeO 2 -NPs The effects of different concentrations of CeO 2 -NPs on the physiological indicators of red fescue under Cd stress were analyzed based on the membership function. The results showed that the treatment of Cd +350 nCeO 2 had the best ameliorative effect on cadmium stress (Table S2). Subsequent experiments on the anatomical structure, transcriptome, and metabolome of red fescue were all conducted using the selected concentration of 350 mg/L. 3.6. Effects of CeO 2 -NPs on the anatomical structure of red fescue under Cd stress Changes in the chloroplast ultrastructure of plant leaves and the root epidermal cells were observed (Figure 3, Figure S4). Under CK treatment, the chloroplast structure was intact, with well-organized grana and stroma lamellae. The chloroplasts were abundant, arranged in a single row, and tightly aligned with the cell wall. Various organelles, including mitochondria and starch granules, were clearly visible and exhibited normal characteristics. The root epidermal cells had smooth surfaces and intact shapes under CK treatment. Under Cd stress, the mesophyll cell walls became blurry, thickened, and showed plasmolysis. Organelles displayed severe abnormalities, including enlarged starch granules, vacuolization of mitochondria, and distorted thylakoid stroma. The surface of the root epidermal cells was rough and damaged. The application of CeO 2 -NPs alleviated Cd-induced damage to the mesophyll cells and root structure. This was reflected in a slight reduction in cell wall thickness, less plasmolysis, and reduced chloroplast damage, Moreover, the number of starch granules decreased. The damage to the root epidermal cells was reduced, resulting in a smoother surface. 3.7. Metabolomic changes of red fescue induced by CeO 2 -NPs and Cd To explore how exogenous CeO 2 -NPs alleviate Cd toxicity in red fescue growth, an untargeted metabolomic analysis was performed. PCA showed clear separation between the treatments, indicating that the data were stable and reliable (Figure 4A). A total of 685 metabolites were identified, including flavonoids, phenolic acids, amino acids, and their derivatives (Figure 4B). In the CK vs Cd treatment group, 274 and 241 DEMs were identified in ‘Frigg’ and ‘Reverent’, respectively. Under Cd stress, 270 DEMs were identified in the ‘Frigg’ vs ‘Reverent’ comparison. In the Cd + 350 nCeO 2 vs Cd group, 343 DEMs were found in ‘Frigg’, with 159 upregulated and 184 downregulated; while in ‘Reverent’, 276 DEMs were identified, with 85 upregulated and 191 downregulated. After CeO 2 -NPs treatment, 267 DEMs were identified in the ‘Frigg’ vs ‘Reverent’ comparison, with 119 upregulated and 148 downregulated (Figure 4C). UpSet plot analysis revealed significant differences in metabolite accumulation patterns between cultivars (Figure 4D). K-means analysis grouped 685 metabolites into 12 categories (Figure 4E, Table S3). In both red fescue cultivars (Sub class 7, Sub class 9), metabolites showed significant differences after Cd treatment, which were reversed to normal levels after exogenous spraying of CeO 2 -NPs. In Sub class 7, metabolites significantly decreased under Cd treatment, mainly composed of steroids and flavonoids (Figure 4E). These metabolites may play a crucial role in red fescue’s response to Cd stress. In the Cd + 350 nCeO 2 vs Cd group, both ‘Frigg’ and ‘Reverent’ showed significant enrichment in pathways such as stilbenoid, diarylheptanoid, and gingerol biosynthesis, as well as unsaturated fatty acid biosynthesis. Additionally, compared to ‘Reverent’, ‘Frigg’ showed specific and significant enrichment of DEMs in monoterpenoid biosynthesis, phenylpropanoid biosynthesis, and diterpenoid biosynthesis pathways (Figure S5). To explore the relationship between the Cd TF, physiological indices, and differential metabolites, Mantel test and Spearman correlation models were used for combined analysis. The results showed that antioxidant indices SOD, POD, CAT, AsA, and GSH were significantly negatively correlated with the Cd transport factor (r ≤ -0.8, P < 0.05), while MDA, H 2 O 2 , and O 2 - were significantly positively correlated with the Cd transport factor (Figure 5A).To further understand the relationship between metabolites and physiological regulatory networks, correlation analysis between the 12 metabolite categories identified by K-means clustering and physiological indices was conducted. Metabolites in Class 7 were significantly positively correlated with antioxidant indices (SOD, POD, CAT, AsA, and GSH), and metabolites in Class 3 were also positively correlated with these indices (Figure 5B). KEGG enrichment analysis showed that Class 7 metabolites were significantly enriched in pathways such as Flavone and flavonol biosynthesis, and Stilbenoid biosynthesis. Interestingly, this result was consistent with the KEGG enrichment analysis of ‘Frigg’ and ‘Reverent’ in the Cd + 350 nCeO 2 vs Cd group. Sub class 3 metabolites were enriched in amino acid metabolism pathways, including Histidine metabolism and Cyanoamino acid metabolism (Figure 5C-F). 3.8. Transcriptomic changes of red fescue induced by CeO 2 -NPs and Cd stress We performed transcriptomic sequencing of red fescue leaves from different cultivars and treatment groups to investigate the transcriptional changes in response to CeO 2 -NPs under Cd stress. The sequencing statistics from PacBio and Illumina Hi-seq are provided in Table S4. Both cultivars had Q2 and Q3 values greater than 99% and 95%, respectively, and GC content above 52%. PCA results showed clear separation between the treatment groups (Figure 6A). RT-qPCR validated the expression of 15 randomly selected genes, and the results showed a high correlation with RNA-seq data (r 2 = 0.9811), confirming the reliability of the RNA-seq data (Figure S6). Differential gene analysis showed that in the Cd vs CK group, ‘Frigg’ had 3933 DEGs (2754 upregulated, 1179 downregulated), while ‘Reverent’ had 4264 DEGs (3329 upregulated, 935 downregulated), and ‘Frigg’ and ‘Reverent’ had 900 and 1065 genes specifically expressed, respectively (Figure 6B). After CeO 2 -NPs treatment, 37917 DEGs were identified, with 20507 upregulated and 17410 downregulated. In the Cd +350 nCeO 2 vs Cd group, ‘Frigg’ had 3933 DEGs (2429 upregulated, 622 downregulated), while ‘Reverent’ had 3714 DEGs (746 upregulated, 2948 downregulated). There were 443 and 737 specifically expressed genes in the two cultivars respectively (Figure 6B-C). This indicates that CeO 2 -NPs treatment triggered the expression of specific genes in red fescue under Cd stress. GO enrichment analysis showed that in the Cd vs CK group, ribonucleoprotein complex, translation, and amide metabolic process were significantly enriched only in ‘Frigg’. After CeO 2 -NPs treatment, DEGs from both ‘Frigg’ and ‘Reverent’ were mainly enriched in oxidoreductase activity (Figure S7). Furthermore, both cultivars showed significant enrichment in oxidative phosphorylation, pentose and glucuronate interconversions, tyrosine metabolism, and fatty acid degradation pathways in response to CeO 2 -NPs (Figure 6D-E). These pathways may play important roles in regulating Cd tolerance for red fescue under CeO 2 -NPs treatment. 3.9. Integrated transcriptomic and metabolomic analysis To investigate the regulatory mechanism of exogenous CeO 2 -NPs in enhancing Cd tolerance in red fescue, we used WGCNA (Weighted Gene Co-expression Network Analysis) to identify four co-expression modules based on gene expression similarity (Figure S8). These modules exhibited significant differences in expression patterns across the treatments, indicating their potential role in plant responses to Cd stress and CeO 2 -NPs-mediated alleviation. In the blue module, gene expression was significantly higher in ‘Frigg’ than in ‘Reverent’. In contrast, the turquoise module exhibited an opposite pattern. The yellow module showed a significant decrease in expression under Cd stress in both cultivars, but increased significantly after CeO 2 -NPs treatment. This was highly correlated with the expression trend of metabolites in Class 7, suggesting a positive regulatory role in CeO 2 -NPs-mediated alleviation of Cd stress (Figure S8, Figure 4E). Further correlation analysis showed that the yellow module showed the highest correlation with rutin (r = 0.84, p < 0.0001) (Figure 5F, Table S5). A scatter plot analysis of module membership (MM) and gene significance (GS) was performed. Based on MM and GS values, the top 5 genes in the yellow modules were selected for clustering analysis based on their expression levels. These genes exhibited expression patterns consistent with those of rutin under different treatments (Figure 6G). This indicates that genes such as TRINITY DN50451_cl_gl, TRINITY_DN54597_c0_g1 are significantly positively correlated with rutin (r > 0.8). In conclusion, this highlights a close relationship between metabolite production and gene expression changes. 3.10. Rutin enhance Cd tolerance by increasing antioxidant enzyme activity Based on the screening of differentially expressed metabolites and genes before and after the application of CeO 2 -NPs, rutin was selected as the key metabolite mediating CeO 2 -NPs induced cadmium tolerance. WGCNA analysis and the correlation between Cd transport factor and physiological indices indicate that SOD, APX, POD, CAT and GSH activities were the most positively correlated with Cd tolerance enhancement in red fescue, then we selected these indices to evaluate effectiveness of rutin in enhancing cadmium resistance. The results showed that application of rutin significantly improved red fescue growth, reduced ROS accumulation, and enhanced antioxidant activity under Cd stress. Rutin treatments displayed more Cd-tolerant phenotypes compared to the Cd treatment group, with better and denser growth (Figure 7A-B). Exogenous rutin treatment significantly enhanced antioxidant enzyme activities in ‘Frigg’ and ‘Reverent’. Compared to Cd treatment group, rutin-pretreated ‘Frigg’ had increases of 1.28, 1.15 times, 20.54%, 88.77%, 26.88% in SOD, APX, POD, CAT and GSH, respectively, while ‘Reverent’ showed increases of 1.16, 1.94times 88.87%, 99.79%, 41.48% (Figure 7G-K). Furthermore, the application of rutin resulted in lower H 2 O 2 and O 2 - accumulation in red fescue compared with the Cd treatment group. DAB and NBT staining results further confirmed these findings, showing that staining intensity in leaves of spraying rutin was nearly to the CK level compared to the Cd treatment (Figure 7C-F). These results indicate that the key metabolite rutin primarily contribute to red fescue’s response to Cd stress through the antioxidant defense system. 4. Discussion 4.1. Mitigating effect of CeO 2 -NPs on Cd-induced growth inhibition Excessive accumulation of Cd in soil severely disrupts normal plant growth and development, leading to stunted shoots, shortened roots, reduced growth rate, and reductive biomass. Growth inhibition has been reported in tomato ( Solanum lycopersicum ) [35] and kenaf ( Hibiscus cannabinus L.) [36] . Consistently, our results showed that Cd toxicity markedly suppressed shoot growth and altered root structural traits of red fescue (Figure 2). This inhibitory effect may result from the high Cd accumulation in roots (Figure S2). Cd stress also damages the first defensive barrier of the plant, the root epidermal cell wall. Moreover, Cd 2+ competes with essential nutrients such as K, Ca, and P for uptake sites [37] , and the resulting root damage further limits water and nutrient absorption by other plant tissues [38-39] , ultimately impairing overall plant development. In contrast, the application of exogenous CeO 2 -NPs significantly mitigated Cd toxicity in red fescue , which may be attributed to the nutritional role of Ce combinating with other elements to promote plant metabolism and improve Cd-induced nutrient imbalance [40] . These findings are consistent with previous reports showing that foliar application of CeO 2 -NPs enhanced growth performance in fragrant rice ( Oryza sativa L.) under Cd stress [23] . 4.2. Regulation of antioxidant defense system by CeO 2 -NPs under Cd stress High Cd concentrations induce excessive accumulation of ROS in plants, leading to elevated MDA levels and disruption of normal metabolic processes [41-42] . In this study, contents of MDA, H 2 O 2 , and O 2 - significantly increased in both cultivars of red fescue under Cd stress (Figure 2B-D), which consistent with findings in cotton ( Gossypium hirsutum L.) [39] . Abiotic stresses commonly activate plant defense systems by altering the activities of antioxidant enzymes such as SOD, POD, CAT, APX, and GSH [43] . Among them, SOD, POD, and CAT mainly target O₂ - , thereby reducing its conversion to H 2 O 2 [44] . GSH plays a crucial role in ROS detoxification, and its reaction products can serve as substrates for other antioxidant enzymes to scavenge ROS [45] . Under cadmium stress, the antioxidant activity of red fescue decreases, and the toxicity of Cd interferes with the normal functioning of these antioxidants within the plant [46] . Application of exogenous agents can mitigate oxidative stress by lowering ROS accumulation and enhancing antioxidant enzyme activities, thereby compensating for stress-induced damage [47] . In the present study, CeO 2 -NPs effectively alleviated Cd-induced oxidative stress by modulating the activities of key antioxidant enzymes (Figure 2E–I). Similar protective effects of CeO 2 -NPs have been reported in Cilantro ( Coriandrum sativum L.) [48] , kidney bean ( Phaseolus vulgaris var. Red Hawk) [49] , and Radish [50] . Furthermore, GO enrichment analysis of the transcriptome revealed that the ‘oxidoreductase activity’ pathway was significantly enriched in red fescue treated with CeO 2 -NPs under Cd stress (Figure S7). Therefore, it can be inferred that CeO 2 -NPs enhance Cd tolerance in red fescue by upregulating enzymatic and non-enzymatic antioxidants, including POD, CAT, and GSH. 4.3. CeO 2 -NPs enhance photosynthetic performance under Cd stress High Cd concentrations disrupt photosynthetic functional proteins, inhibit pigment synthesis, and damage essential organelles such as chloroplasts, ultimately impairing photosynthetic performance in plants [51] . In our study, Cd stress significantly reduced chlorophyll content and photosynthetic parameters in red fescue . This trend is consistent with findings in wheat ( Triticum aestivum L.), where tolerant cultivars maintain smaller reductions in stomatal conductance (Gs) under stress through regulated stomatal closure [52] . Similarly, tolerant Brassica napus cultivars exhibit higher Gs and intercellular CO 2 concentrations (Ci) under stress, improving gas exchange and photosynthetic performance [50] . Application of CeO 2 -NPs effectively alleviated Cd-induced decreases in photosynthetic pigment content and photosynthetic activity in red fescue . Previous studies also reported that CeO 2 -NPs significantly increased chlorophyll a (Chl a) content in okra ( Abelmoschus esculentus L.) leaves [23] . Furthermore, the GO enrichment analysis revealed that foliar application of 350 mg/L CeO 2 -NPs significantly enriched differentially expressed genes in the carbohydrate metabolic process pathway (Figure S7). Since photosynthesis represents a major carbohydrate metabolism pathway in plants [53] , we infer that CeO 2 -NPs enhance photosynthetic capacity under Cd stress by increasing chlorophyll content and modulating carbohydrate metabolism, thereby improving the plant’s tolerance to Cd-induced damage. 4.4. Protective role of CeO 2 -NPs in preserving cellular ultrastructure and reducing Cd uptake Ultrastructural observations provide valuable insights into the physiological basis underlying these differences. Cd accumulation damages mesophyll cells, leading to impaired photosynthetic performance [54-55] . In this study, Cd stress caused visible structural injuries to chloroplasts, thylakoid membranes, and cell walls in both red fescue cultivars, which likely contributed to the observed decline in photosynthetic capacity (Figure 3). Similarly, Cd exposure in foxtail millet ( Setaria italica ) resulted in cell wall thickening across multiple tissues, consistent with our findings [56] . Excessive Cd accumulation has been widely reported to damage cellular organelles, reduce photosynthetic activity, and limit biomass production [57] . However, exogenous application of CeO 2 -NPs alleviated these structural damages in red fescue , restoring cell wall thickness to near-control levels, this might be associated with decreased Cd accumulation in the leaves (Figure 3). Previous studies demonstrated that CeO 2 -NPs strongly adsorb Cd, thereby reducing bioavailability and root uptake in corn ( Zea mays L.) [58] . We hypothesize that foliar-applied CeO 2 -NPs may undergo basipetal translocation within red fescue , reaching the roots and subsequently influencing Cd absorption from the soil [53] . Moreover, CeO 2 -NPs appear to regulate gene expression across plant tissues, particularly those involved in metal transport. Transcriptome data analysis revealed significant enrichment in the ‘transmembrane transporter activity pathway’ for both ‘Frigg’ and ‘Reverent’, while ‘Frigg’ uniquely exhibited enrichment in ‘active transmembrane transporter activity’ . In fragrant rice (Oryza sativa L.), CeO 2 -NPs were shown to downregulate HMA3 and NRAMP1 , key transporters mediating Cd and other metal ion translocation [41] . Collectively, these findings suggest that CeO 2 -NPs mitigate Cd toxicity in red fescue by reducing Cd uptake, thereby protecting chloroplasts, starch granules, and other organelles in mesophyll cells. This protective mechanism enhances photosynthetic efficiency, promotes biomass accumulation, and ultimately contributes to improved Cd tolerance in the plant. 4.5. Involvement of key metabolite in plant Cd tolerance enhancement Rutin, a naturally occurring flavonol abundant in plants, whose synthesis and medicinal function have been intensively studied in animals [59-60] . Additionally, research in plants has shown that rutin is involved in the defense responses against pathogens [61-62] and salt stress [63-64] . However, its role in plant tolerance to Cd stress has not been well or clearly documented, and remains particularly poorly understood in red fescue. In this study, rutin was identified as the key metabolite mediating exogenous CeO 2 -NPs-induced Cd stress tolerance in red fescue. It is noteworthy that exogenous rutin application partially alleviated the toxicity caused by Cd treatment in red fescue, reducing ROS levels and restoring growth (Figure 7). These results provide strong evidence for rutin’s role in regulating plant Cd tolerance. Moreover, they further demonstrate the effectiveness of exogenous CeO 2 -NPs in enhancing plant cadmium tolerance. Research has shown that Cd tolerance in plants is closely related to the mechanisms of ROS production and scavenging, including various antioxidants [65-66] . There is ample evidence indicating that exogenous rutin can directly trigger the synthesis of antioxidant secondary metabolites and activate antioxidant enzyme activities to enhance ROS clearance [67-68] . In our experiment, the provision of exogenous rutin significantly increased the levels of antioxidant enzymes and antioxidants in red fescue subjected to Cd stress and significantly reduced ROS content (Figure 2B-H). These findings are consistent with the previous results where exogenous CeO 2 -NPs improved cadmium toxicity in red fescue. Based on these results, we reasonably infer that exogenous CeO 2 -NPs enhance Cd tolerance by modulating rutin metabolism, activating both enzyme and non-enzyme antioxidants, and clearing accumulated ROS to improve the plant’s antioxidant defense system. Therefore, our work innovatively reveals the molecular mechanisms by which exogenous CeO 2 -NPs exert protective effects in plants suffering from cadmium toxicity. These findings hold great potential for enhancing plant Cd tolerance through the exogenous supply of specific metabolites. 5. Conclusion Exogenous application of CeO 2 -NPs at varying concentrations effectively enhanced the tolerance of two red fescue cultivars to Cd stress. CeO 2 -NPs improved photosynthetic efficiency, strengthened the antioxidant defense system, promoted osmolyte accumulation, maintained cellular structural integrity, and reduced Cd uptake, thereby mitigating Cd-induced toxicity. Additionally, a concentration of 350 mg/L of CeO 2 -NPs was most effective in improving the cadmium tolerance of red fescue. Integrated transcriptomic and metabolomic analyses revealed that CeO 2 -NPs regulated the main metabolic pathways of plants, and rutin was the most important metabolite. Independent treatments with rutin further confirmed that rutin, as a key metabolite regulating by CeO 2 -NPs, can enhance Cd tolerance by activating the antioxidant defense system of red fescue . Overall, this study provides novel insights into the mechanisms of CeO 2 -NPs effectively mitigating Cd-induced toxicity by reprograming transcriptomic and metabolome and regulating key metabolite to maintain the growth of red fescue . The finding offers theoretical guidance and experimental evidence for the application of engineered nanomaterials to reduce the Cd toxicity of plants used for ecological restoration and enhance its function in the ecological restoration process of cadmium contaminated environment. Supplementary Information The online version contains supplementary material available at Acknowledgements This research was supported by Natural Science Foundation of Heilongjiang Province (LH2023C01）and the China’s National Key R&D Program (2022YFD1600501-07). Funding This work is supported by Natural Science Foundation of Heilongjiang Province (LH2023C01), the China’s National Key R&D Program (2022YFD1600501-07) and National Natural Science Foundation of China (32171687). CRediT authorship contribution statement Zheng Xueling Writing-original draft, Software. Chen Along Formal analysis, Software, Data curation. Sang Mengyao Formal analysis, Data curation. Bai Zhenjian Investigation, Data curation. Zhang Jinwei Formal analysis, Data curation. Zhao Qianhan Investigation, Data curation. Liu Shasha Investigation, Formal analysis. Zhang Lu Investigation, Formal analysis. Xie Fuchun Writing-review & editing, Funding acquisition. Jiang Jia Project administration, Conceptualization. Chen Yajun Writing-review & editing, Project administration, Funding acquisition, Conceptualization. Data availability The authors declare that the data supporting the findings of this study are presented in the article and supplementary information files are available from the corresponding author upon request. The raw data of transcriptomic sequencing and metabolomic sequencing supporting the results of this study have been deposited in the China National GeneBank (CNGB) Sequence Archive (CNSA, accession code: CNP0008352). Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare no competing interests. References [1] Zhong, M., Yue, L., Qin, H., Wang, G., Xiao, L., Cheng, Q., Lei, B., et al., 2023. TGase-induced Cd tolerance by boosting polyamine, nitric oxide, cell wall composition and phytochelatin synthesis in tomato. Ecotoxicology and Environmental Safety 259, 115023. https://doi.org/10.1016/j.ecoenv.2023.115023. [2] Tanwir, K., Javed, M.T., Abbas, S., Shahid, M., Akram, M.S., Chaudhary, H.J., Iqbal, M., 2021. Serratia sp. CP-13 alleviates Cd toxicity by morpho-physio-biochemical improvements, antioxidative potential and diminished Cd uptake in Zea mays L. cultivars differing in Cd tolerance. 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Scientific Reports 5(1):24447. https://doi.org/10.1038/s41598-025-04235-6. Fig. 1 Effects of CeO 2 -NPs on the growth parameters of two red fescue cultivars under Cd Stress. (A) Phenotypic images of red fescue under different treatments. (B) Root images of red fescue under different treatments. (C) Root length. (D) Fresh leaf weight. (E) Fresh root weight.In Figures A and B, ”a” denotes the Cd + 100 mg/L nCeO 2 group, ”b” denotes the Cd + 200 mg/L nCeO 2 group, ”c” denotes the Cd + 350 mg/L nCeO 2 group, and ”d” denotes the Cd + 500 mg/L nCeO 2 group. In Figure C-E, the different lowercase and uppercase letters indicate significant differences between treatments for ‘Frigg’ and ‘Reverent’, respectively (Duncan’s test, P < 0.05) . Fig. 2 Effects of CeO 2 -NPs on lipid peroxidation, antioxidants and TF in two red fescue cultivars under Cd Stress. (A) MDA content; (B)O 2 - content; (C)H 2 O 2 content; (D)CAT activity;(E) SOD activity; (F)POD activity; (G)APX content; (H)GSH content, (I)TF. In the figure, different lowercase and uppercase letters indicate significant differences between treatments for ‘Frigg’ and ‘Reverent’ (Duncan’s test, P < 0.05) . Fig. 3 Ultrastructure of mesophyll cells observed by TEM. CW: Cell wall, TH: Thylakoid stroma, OG: Osmium granules, V: Vacuole, Mt: Mitochondria, SG: Starch granules, Chl: Chloroplast. Fig. 4 Metabolomic analysis of red fescue cultivars ‘Frigg’ and ‘Reverent’. (A) PCA analysis of DEMs in ‘Frigg’ and ‘Reverent’ cultivars;(B) Hierarchical clustering heatmap of DEMs in ‘Frigg’ and ‘Reverent’;(C) Statistics of upregulated and downregulated DEMs in ‘Frigg’ and ‘Reverent’;(D) UpSet plot showing the number of DEMs between different treatment comparisons in ‘Frigg’ and ‘Reverent’;(E) k-means classification of 12 categories of metabolites. The colored lines represent the dynamic expression of each metabolite across different groups. The black lines represent the representative expression of the clusters . Fig. 5 Relationship between Cd transport factor, physiological response, and metabolites in two red fescue cultivars. (A) Heatmap analysis of the correlation between physiological indices and Cd transport;(B) Heatmap analysis of the correlation between 12 Classes of metabolites and physiological indices;(C) KEGG enrichment analysis of Class 7 metabolites;(D) Metabolite expression analysis in significantly enriched KEGG pathways of Class 7 metabolites;(E) KEGG enrichment analysis of Class 3 metabolites;(F) Metabolite expression analysis in significantly enriched KEGG pathways of Class 3 metabolites. Fig. 6 Transcriptomic analysis of red fescue cultivars ‘Frigg’ and ‘Reverent’. (A) PCA analysis of DEGs in ‘Frigg’ and ‘Reverent’;(B) Venn diagram of DEGs in ‘Frigg’ and ‘Reverent’;(C) Statistics of upregulated and downregulated genes in the transcriptomes of ‘Frigg’ and ‘Reverent’;(D) KEGG enrichment analysis of DEGs in ‘Frigg’ and ‘Reverent’ under Cd vs CK treatment;(E) KEGG enrichment analysis of DEGs in ‘Frigg’ and ‘Reverent’ under Cd + nCeO 2 vs Cd treatment;(F) Heatmap showing module characteristic correlations with compounds. Each row represents a module, and each column corresponds to a compound. Green indicates a negative correlation, while yellow indicates a positive correlation;(G) Correlation analysis of module membership and gene significance in the yellow module. The x-axis represents gene membership in the module, and the y-axis represents the significance of the correlation with Rutin levels. Fig. 7 Effects of rutin on Cd tolerance in red fescue . (A) Phenotypic changes of ‘Frigg’ under different treatments;(B) Phenotypic changes of ‘Reverent’ under different treatments;(C) DAB and NBT staining of ‘Frigg’ leaves under different treatments;(D) DAB and NBT staining of ‘Reverent’ leaves under different treatments;(E) H 2 O 2 content;(F) O 2 - content;(G) SOD activity;(H) GSH activity;(I) CAT activity;(J) POD activity;(K) APX activity. Different letters indicate significant differences between different treatments (Duncan’s test, P < 0.05). Information & Authors Information Version history V1 Version 1 05 January 2026 Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords festuca rubra l antioxidant system cd tolerance ceo2-nps growth metabalome Authors Affiliations Xueling Zheng Northeast Agricultural University View all articles by this author Along Chen Northeast Agricultural University View all articles by this author Mengyao Sang Northeast Agricultural University View all articles by this author Zhenjian Bai Northeast Agricultural University View all articles by this author Jinwei Zhang Northeast Agricultural University View all articles by this author Qianhan Zhao Northeast Agricultural University View all articles by this author Shasha Liu Northeast Agricultural University View all articles by this author Lu Zhang Zhejiang A and F University Jiyang College View all articles by this author Fuchun Xie Northeast Agricultural University View all articles by this author Jia Jiang 0009-0002-8959-461X Northeast Agricultural University View all articles by this author Yajun Chen [email protected] Northeast Agricultural University View all articles by this author Metrics & Citations Metrics Article Usage 122 views 88 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Xueling Zheng, Along Chen, Mengyao Sang, et al. 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