Analytical grade CeO₂NPs (99.5%, 20–50 nm, CAS 1306-38-3) were purchased from Aladdin Reagent Company (www.aladdin-e.com, Shanghai, China). The seeds of the alfalfa variety ‘Huanghou’ were purchased from Jiangsu Qianhuabaimei Seed Industry Co., Ltd. (Suqian, China) in May 2022.

To determine the shape and size of CeO₂NPs, the CeO₂NPs were dissolved in distilled water, and a drop of CeO₂NP sample was placed on a Cu grid and dried under vacuum for analysis. The prepared samples were observed using a transmission electron microscope (TEM) (Hitachi High-Technologies Corporation, HT7800, Tokyo, Japan) at an accelerating voltage of 120 kV. To characterize the size and zeta potential of CeO₂NPs, the samples were made into a suspension and measured with a nanometer laser particle sizer (ZEN3600; Malvern Instruments Limited, Malvern, UK). The crystal properties of CeO₂NPs were determined by X-ray diffraction (XRD) analysis. The sample was also subjected to an X-ray diffractometer (Model D8 Advance A25; Bruker, Bremen, Germany) at 40 kV and 40 mA with Cu Kα1 radiation. The scan 2θ range was 10–80°.

To investigate whether CeO₂NPs have nanocatalytic activity against α-amylase, in-vitro experiments were conducted. The in-vitro experiment was adjusted according to the α-amylase activity measurement method. In short, 2 mL of 0, 50, 100, and 500 mg/L CeO₂NP solutions were mixed with 8 mL of α-amylase solution (3.7 U/mL) to form a mixed solution of 0, 10, 50, and 100 mg/L CeO₂NPs and incubated at 37°C for 2 h, and then, Kishorekumar’s (2007) method was used to determine the enzymatic activity of the mixed solution. In short, after incubation, the different amylase solutions were mixed with starch solutions; after sufficient reaction time, the absorbance value was measured using a spectrophotometer, and the enzyme activity was calculated using a standard curve for the glucose system. Each treatment was repeated three times.

Salt (NaCl) solutions with different concentrations (0, 50, 100, 150, 200, and 250 mM) were established to conduct germination experiments to characterize the effects of salt concentration. Referring to the processing method of Song et al. (2021), all seeds used for the germination experiment were disinfected with 75% alcohol for 30 s and washed five times with sterile water. For each treatment, seeds were germinated on double-layer filter paper in Petri dishes with 5 mL of treatment solution. Then, the seeds were put in a germination chamber maintained at 25°C ± 2°C, 85% relative humidity, and a 16/8-h photoperiod (light/dark) with 10,000 lx irradiance for 10 days; water was replenished daily to keep the filter paper and seeds wet. This was the setup used to determine the germination potential (GP, 4 days), germination rate (GR, 10 days), and root length (RL) and shoot length (SL) at the germination stage.

Univariate quadratic regression analyses were performed on the relative values of various test indicators and salt solution concentrations of alfalfa based on the following equation:

Here, a, b, and c are equation coefficients (Li et al., 2019).

The salt concentration at 50% lethality (S ₅₀) was calculated based on this regression equation, and the mean S ₅₀ values of each indicator were calculated to obtain the screening concentration.

CeO₂NP suspension solutions were used as nanopriming solutions. The particles were dispersed in deionized water by ultrasonic vibration (100 W, 40 kHz) with an ultrasonic cleaner (Ningbo Scientz Biotechnology Co., Ltd., Ningbo, China) for 30 min to prepare the nanopriming solutions of corresponding concentrations.

The CeO₂NPs were dispersed in deionized water to prepare nanopriming solutions with different concentrations (0, 10, 50, 100, 200, 500 mg/L). Six batches of seeds (500 seeds for each) with uniform size and full granules were placed into PE tubes with plugs, to which 20 mL of nanopriming solutions of various concentrations were added. After the seeds were soaked for 24 h (in the dark, 10°C), seeds were dried back at room temperature for 48 h and stored for later use. Control (CK, non-priming) and primed seeds were germinated under salt treatment (using the concentration identified in the previous section). The seed germination conditions were the same as those described in the previous section (2.2).

Referring to the method of Li et al. (2019), evaluations were conducted of the salt tolerance of each priming concentration by determining its salt tolerance membership function value.

Here, X_i is the membership function value of the salt tolerance index for treatment concentration i, X is the actual measured value of the salt tolerance index, and X _max and X _min are the maximum and minimum values of the salt tolerance index for each concentration treatment, respectively. First, the membership function values were calculated for each indicator and summed, and then, the average membership function values were calculated to evaluate salt tolerance under different concentration treatments. The average range was between 0 and 1, and the larger the average, the higher the salt tolerance. The optimal priming concentration was used for the subsequent experiments.

In order to better observe changes in the epidermal structure of seeds after priming treatment, verify whether these changes provide conditions for water absorption and nanomaterial infiltration, and further verify Ce penetrates the seed coat, we conducted ultrastructural observations of the seed coat and cross-section. Seeds were collected from different treatments [non-primed (CK), hydropriming (HP), CeO₂NP priming (Ce)], and their epidermises were observed by scanning electron microscopy (SEM; JSM 5600 LV; JEOL, Tokyo, Japan). Additionally, an energy-dispersive X-ray spectrometer (EDX; KEVE2; Hillsboro, USA) was used for the point analysis of epidermal elements. CeO₂NP-primed seeds were cut along the cotyledon–radicle axis, and the longitudinal section was observed under SEM, and elemental line scanning was conducted (on the cotyledon–radicle sample) to analyze the distribution of Ce.

Water absorption is an important factor affecting seed germination, and water can activate starch hydrolases to provide material reserves for germination. Therefore, seed water absorption and α-amylase activity were measured. Approximately 0.05 g of alfalfa seeds were tested for their seed water absorption rate for 24 h in a germination environment, with three replicates. Seeds were weighed at 0, 3, 6, 9, 16, and 24 h, respectively, and the seed water absorption rates were calculated for different treatments (i.e., CK, HP, and Ce). The activity of α-amylase was measured by the 3,5-dinitrosalicylic acid method (Kishorekumar et al., 2007). Specifically, samples (0.1 g) of alfalfa seeds that had germinated for 24 h under different treatments were homogenized and brought to a constant volume of 1.5 mL. Samples were extracted at room temperature for 15 min and then centrifuged at 6,000×g for 10 min, and the resulting supernatant was collected as the enzyme extract. Then, a spectrophotometer was used to measure the absorbance value, and thus, it was possible to calculate enzyme activity based on a standard curve for the glucose system.

Relative conductivity (REC) and malondialdehyde (MDA) content can indicate the degree of cellular damage. REC was measured according to the method described by Zou (2003). In detail, 0.1 g of germinated seeds (after 24 h) was washed three to five times with deionized water, and after the water was wiped away, 5 mL of deionized water was added. The electrical conductivity of the leachate when the seed was first added was measured with a conductivity meter (DDSJ – 318; Rex Electric Chemical, Jiashan, China) as the initial electrical conductivity (A1). After samples were placed under vacuum with a vacuum pump for 10 min, the conductivity (A2) of leachate after standing for 30 min was measured. After boiling the leachate in a 100°C water bath for 15 min and then cooling it to 25°C, the conductivity of the leachate was measured again as the absolute conductivity (A3). The formula for REC is as follows: REC (%) = (A2 − A1)/(A3 − A1) * 100. Malonaldehyde (MDA) content was determined following the approach of Heath and Packer (1968).

ROS can act as signaling molecules and also serve as an indicator of stress level. Seeds that had germinated for 24 h (0.2 g, n = 3) were placed into 3.0 mL of PBS (50 mM, pH 7.8) on ice and ground into a homogenate that was then centrifugated (12,000 r/min, 4°C) for 5 min, and the resulting supernatant was used as the sample extract solution for the determination of H₂O₂ and O₂ ^·− contents. The determination of H₂O₂ content was carried out using the method of Ferguson et al. (1983). The content of O₂ ^·− was determined by the hydroxylamine oxidation method (Ke et al., 2002).

Soluble proteins, amino acids, and soluble sugar content can be used as both osmotic regulators to alleviate osmotic stress and as nutrients for seed germination. Therefore, their contents should be measured when evaluating stress responses. The concentration of amino acid content in germinated seeds was measured by the ninhydrin method (Zou, 2003) and quantified using leucine as the standard. The supernatants containing the amino acids from germinated seeds were assayed for soluble protein content by the Bradford method (Bradford, 1976). The soluble sugar content of the germinated seeds was measured by the anthrone colorimetric method (Zou, 2003) using glucose as the standard.

Antioxidant enzymes play a crucial role in clearing harmful oxidative substances from cells. Superoxide dismutase (SOD) activity was calculated as the units of enzyme required for 50% inhibition of nitroblue tetrazolium per gram of fresh seeds per minute according to the approach of Beauchamp and Fridovich (1971). Catalase (CAT) activity was measured according to the approach of Aebi (1984). The reduction in absorbance owing to H₂O₂ degradation was measured at 240 nm (A ₂₄₀). A 0.1 decrease in A ₂₄₀ within 1 min was defined as one unit of CAT enzyme activity. Peroxidase (POD) activity was analyzed following the method of Hemeda and Klein (1990). An increase of 0.01 in A ₄₇₀ per minute was defined as one unit of POD enzyme activity.

The role of plant hormones as signaling molecules during seed germination cannot be ignored, and metabolic analysis can help to elucidate the underlying causes of germination differences. Alfa seeds (0.1 g) germinated under different treatments for 24 h were used as sample material for the measurement of hormone metabolism levels. Fresh seeds were harvested, immediately frozen in liquid nitrogen, ground into powder (30 Hz, 1 min), and stored at −80°C until subsequent analysis. Fifty-milligram samples of seeds were weighed into 2-mL plastic microtubes, frozen in liquid nitrogen, and dissolved in 1 mL of methanol/water/formic acid (15:4:1, V:V:V). Ten microliters of an internal standard mixed solution (100 ng/mL) was added into the extract as internal standards (IS) for quantitation. The mixture was vortexed for 10 min and then centrifuged for 5 min (12,000 r/min, 4°C), and the resulting supernatant was transferred to clean plastic microtubes, evaporated to dryness and dissolved in 100 μL of 80% methanol (V/V), and filtered through a 0.22-μM membrane filter for further LC-MS/MS analysis.

A one-way ANOVA was performed to determine the effects on the means of treatment samples across three replications. Data were analyzed using SPSS 26.0 (IBM Corp, Armonk, NY, USA). The significance levels of differences among the means of all the measured traits were determined by Duncan’s multiple range test at a 5% level. Values of P smaller than or equal to 0.05 were considered statistically significant.

Figure 1A shows the XRD pattern of cerium oxide (CeO₂) nanoparticles, with card ID 81-0792. The TEM image of typical CeO₂NPs in Figure 1B shows a loose cluster composed of uniformly sized nanoparticles. The hydrodynamic fluid size of CeO₂NPs is 1,414.9 ± 548.1 nm ( Figure 1C ), while the zeta potential analysis results are −8.98 ± 0.17 mV ( Figure 1D ). In the in-vitro experiments, as the concentration of CeO₂NPs increased, the amylase activity also increased, and the 100-mg/L treatment achieved the maximum increase in enzyme activity, an increase of 6.55% ( Figure 1E ).

As shown in Figure 2 , the seed vigor of alfalfa seeds decreased with increases in salt concentration. Specifically, the salt concentration of 100 mM significantly (P< 0.05) reduced the germination potential (29.63%), germination rate (23.78%), root length (53.69%), and seedling length (56.35%) of alfalfa during the germination period. S ₅₀ was calculated based on the salt damage membership function and estimated to be 202.1 mM ( Table 1 ); thus, subsequent experiments were based on the salt concentration of 200 mM.

Salt concentration screening identified the S ₅₀ value as 200 mM ( Figure 2 ; Table 1 ). On this basis, the effects of different concentrations of CeO₂NPs on the seed vigor of alfalfa under salt stress were studied ( Figure 3 ). Priming treatment was able to improve seed germination potential and germination rate, as well as the root and seedling length during seedling germination under salt stress. Different concentrations of CeO₂NPs had a significant (P< 0.05) promoting effect on alfalfa seeds under salt stress. The optimal effect was achieved at a concentration of 50 mg/L ( Table 2 ), with the germination potential increasing from 4.0% to 51.3% ( Figure 3A ) and the germination rate increasing from 10.0% to 62.7% ( Figure 3B ), significantly (P< 0.05) improving the growth of alfalfa seedlings during the germination period, increasing the root length from 8.3 cm to 23.1 cm ( Figure 3C ), and promoting the growth of seedlings from 9.8 cm to 13.7 cm ( Figure 3D ).

Through observing the alfalfa seeds under different treatments by SEM, it was found that the priming treatment had a significant impact on the seed coat structure ( Figure 4 ). The non-primed seeds were plump and smooth, with tightly arranged epidermal cells faintly visible, and the surface was covered with a waxy layer ( Figure 4A ). Under hydropriming, rough epidermis and cracks between epidermal cells appeared ( Figure 4B ). Under CeO₂NP priming, the epidermis of the seeds was rough, with the wax layer fully consumed; additionally, the epidermal cells were loose, and the gaps between the epidermal cells were obvious ( Figure 4C ). Thus, it could be seen that priming treatment promoted the erosion of the seed coat wax and loosened the epidermal cells. Accordingly, the effect of CeO₂NP priming was visibly better than that of hydropriming. Using EDX point scanning analysis, it was found that cerium deposition was present in the seed epidermis of seeds that had been primed by CeO₂NPs, with concentrations of 0.03% (atomic) and 0.3% (weight), while there was no cerium deposition in the hydroprimed and non-primed seed epidermis ( Figures 4A–C ; Table 3 ). Through EDX line-scanning analysis on the seed profile, it was determined that CeO₂NPs fully entered the radicle and cotyledon tissues of alfalfa seeds and were evenly distributed ( Figure 4D ). Compared with the non-primed control treatment, the seed water absorption curve ( Figure 4E ) indicated that priming treatments (including hydropriming and CeO₂NP priming) can significantly improve the water absorption of alfalfa seeds. In addition, the water absorption effect of seeds primed with CeO₂NPs was better than that of hydroprimed seeds.

As shown in Figure 5A , hydropriming and CeO₂NP priming treatments significantly (P< 0.05) increased the α-amylase activity of alfalfa seeds, and the promotion effect caused by CeO₂NP priming was better than that of hydropriming. Additionally, salt stress significantly (P< 0.05) decreased α-amylase activity. The hydropriming treatment significantly (P< 0.05) increased the soluble sugar content of germinating alfalfa seeds, but both hydropriming and CeO₂NP priming treatment significantly (P< 0.05) decreased the soluble sugar content compared with the control under salt stress ( Figure 5B ). Overall, salt stress increased the soluble protein content of germinated seeds. Figure 5C shows that the priming treatment significantly reduced the soluble protein content of germinated seeds, and the CeO₂NP priming treatment had the lowest content. However, CeO₂NP priming relatively increased the soluble protein content under salt stress. Compared with the control treatment, hydropriming reduced free amino acid content, and the free amino acid content under CeO₂NP priming was higher than that under hydropriming; salt stress reduced free amino acid content in general ( Figure 5D ).

Compared with the control and hydropriming treatments, CeO₂NP priming significantly (P< 0.05) increased the relative conductivity of germinated seeds under control conditions and significantly (P< 0.05) reduced conductivity under salt stress. Overall, salt stress increased the conductivity of germinated seeds ( Figure 6A ). Hydropriming and CeO₂NP priming significantly (P< 0.05) increased the MDA content of germinated seeds, while CeO₂NP-primed seeds had lower MDA content. Both hydropriming and CeO₂NP priming significantly (P< 0.05) reduced MDA content under salt stress ( Figure 6B ).

As shown in Figures 6C, D , under control conditions, the priming treatment had no significant effect on the ROS content of germinated seeds. However, under salt stress, both hydropriming and CeO₂NP priming reduced the H₂O₂ content of the seeds, while hydropriming increased the O₂ ^.− content of the seeds. CeO₂NP priming significantly (P< 0.05) reduced H₂O₂ and O₂ ^.− contents compared with hydropriming.

Hydropriming and CeO₂NP priming significantly (P< 0.05) reduced SOD enzyme activity, with the activity of hydropriming promoting the lowest level. Under salt stress, the SOD enzyme activity of primed seeds significantly decreased, with CeO₂NP priming promoting the lowest level ( Figure 6E ). Priming reduced the POD enzyme activity of germinated seeds, while only CeO₂NP priming significantly (P< 0.05) reduced the POD enzyme activity under salt stress ( Figure 6F ). The two priming treatments (HP and Ce) significantly (P< 0.05) increased the CAT enzyme activity of germinated seeds, while only CeO₂NP priming significantly (P< 0.05) increased the CAT enzyme activity under salt stress ( Figure 6G ).

ABA is mainly a hormone that promotes seed dormancy and inhibits germination (Guillaume et al., 2017), while jasmonic acid (JA) hormones are also important hormones that promote seed dormancy and may interact with ABA. Both under control and salt stress conditions, priming treatments significantly (P< 0.05) reduced the ABA content of germinated alfalfa seeds, and the ABA levels under hydropriming and CeO₂NP priming were similar ( Figure 6H ). Priming significantly (P< 0.05) reduced the JA content of alfalfa seeds under control and salt stress, and CeO₂NP priming promoted lower JA content compared with hydropriming ( Figure 6I ).

As shown in Figure 7 , hydropriming and CeO₂NP priming downregulated the metabolites of cytokinins, auxins, and GAs compared with non-primed seeds. Under salt stress, hydropriming and CeO₂NP priming downregulated JA, salicylic acid, GA, ABA, and strigolactones, as well as some metabolites related to auxins and cytokinins. The number of differential metabolites in the CK vs. HP comparison that were upregulated was 7 and that of metabolites that were downregulated was 24, which is 1 more downregulated metabolite compared with the CK vs. Ce comparison. The number of differential metabolites in the SCK vs. SHP comparison was 18, with 3 upregulated and 15 downregulated, which is 1 more upregulated and 8 fewer downregulated than in the SCK vs. SCe comparison ( Figure 8A ). CK vs. HP and CK vs. Ce comparisons had 7 and 9 unique differential metabolites, respectively, and 21 shared metabolites ( Figure 8B ). SCK vs. SHP and SCK vs. SCe comparisons had 2 and 9 unique differential metabolites, respectively, and 16 shared differential metabolites ( Figure 8C ).

The Kyoto Encyclopedia of Genes and Genomes (KEGG) classification ( Figure 9 ) and enrichment ( Figure 10 ) analyses were conducted on the differential metabolites of alfalfa seeds germinated under CK, HP, and Ce treatments with and without salt stress. The KEGG pathway analysis between CK and HP indicates that significantly different metabolites were mainly enriched in α-linolenic acid metabolism, diterpenoid biosynthesis, zeatin biosynthesis, tryptophan metabolism, plant hormone signal transduction, biosynthesis of secondary metabolites, metabolic pathways, and carotenoid biosynthesis pathways. The numbers of significantly different metabolites in these metabolic pathways were one, three, three, one, six, eight, seven, and one, respectively ( Figure 9A ), and among these, the differences associated with the metabolism of α-linolenic acid and the enrichment of plant hormone signal transduction were relatively significant ( Figure 10A ). The KEGG classification of differential metabolites between CK and Ce treatments in the same pathways identified in the CK vs. HP comparison involved one, two, four, one, eight, nine, seven, and two differential metabolic pathways ( Figure 9B ), and the enrichment ratio of differential metabolites in the carotenoid biosynthesis and plant hormone signal transduction pathways was significant ( Figure 10B ). The differential metabolites between HP and Ce treatments were mainly enriched in diterpenoid biosynthesis, zeatin biosynthesis, plant hormone signal transduction, biosynthesis of secondary metabolites, and carotenoid biosynthesis pathways. The KEGG analysis ( Figures 9C , 10C ) between seeds receiving no priming and hydropriming (SCK vs. SHP) under salt stress showed that there was a significant enrichment of one differential metabolite in the biosynthesis and carotenoid biosynthesis pathways of various alkaloids, with two differential metabolites enriched in the zeatin biosynthesis and tryptophan metabolism pathways and four differential metabolites among the plant hormone signal transduction, biosynthesis of secondary metabolites, and metabolic pathways. The KEGG analysis ( Figures 9D , 10D ) of seeds under Ce treatment compared with non-priming under salt stress (SCK vs. SCe) showed that differential metabolites were mainly significantly enriched in the biosynthesis of various alkaloids, α-linolenic acid metabolism, diterpenoid biosynthesis, zeatin biosynthesis, tryptophan metabolism, plant hormone signal transduction, biosynthesis of secondary metabolites, metabolic pathways, and carotenoid biosynthesis pathways, with the number of significant differences in metabolites being one, one, two, three, two, seven, nine, eight, and two, respectively. The enrichment of carotenoid biosynthesis and plant hormone signal transduction pathways was significant. In addition, the KEGG analysis showed that compared with SHP, SCe induced α-linolenic acid metabolism and diterpenoid biosynthesis pathway enrichment of differential metabolites, and there were quantitative differences in the enrichment of differential metabolites in the zeatin biosynthesis, plant hormone signal transduction, biosynthesis of secondary metabolites, metabolic pathways, and carotenoid biosynthesis pathways.