Cerium oxide nanoparticles promoted lateral root formation in Arabidopsis by modulating reactive oxygen species and Ca²⁺ level

Plant growth

Arabidopsis thaliana L. (Col-0) seeds were sterilised with 70% ethanol for 10 s, followed by two rinses in sterile water, treated with 5% NaClO for 10 min, and washed three times with sterile water. The seeds were then plated on Murashige and Skoog (MS) medium, vernalised at 4°C in darkness for 3 days, and subsequently grown under light conditions (23°C, 14/10 h light/dark cycle, 200 μmol m⁻² s⁻¹) for 5 days. Seedlings with uniform growth and root lengths were selected and transferred to either poly (acrylic) acid coated nanoceria (PNC)-treated or control MS medium (six seedlings per plate, four plates per treatment). After 7 days of treatment, seedlings were photographed, and root parameters were quantified using Image J software (National Institutes of Health, Bethesda, MD, USA).

Synthesis and characterisation of PNC

PNC were synthesised following a methodology adapted from previous publications (Wu et al. 2017). Cerium nitrate (1.08 g, 99%, Sigma-Aldrich) was dissolved in 2.5 mL of deionised water (solution A), while poly acrylic acid (4.5 g, 1800 MW, Sigma-Aldrich) was dissolved in 5.0 mL of deionised water (solution B). Solutions A and B were thoroughly mixed at 224g for 15 min using a vortexer and then added dropwise into a 50 mL beaker containing 15 mL of a 30% ammonium hydroxide solution. The mixture was stirred at room temperature (500 rpm) for 24 h. Subsequently, it was aliquoted into 1.5 mL centrifuge tubes and spun at 1920g for 1 h, with approximately 100 μL of liquid discarded from the bottom of each tube. The supernatant was transferred to an ultrafiltration tube (MWCO 30K, Millipore Inc.) and centrifuged at 1920g for six cycles (one cycle every 45 min). The resulting supernatant constituted the PNC solution, which was stored at 4°C.

The absorbance of the PNC solution was measured using an ultraviolet-visible spectrophotometer (UV-1800, AOE). PNC concentration was determined using the Beer-Lambert law (A = εCL), where A is the absorbance at 271 nm, ε is the molar absorption coefficient of PNC (3 cm⁻¹ mM⁻¹), L is the optical path length (cuvette width, 1 cm), and C is the molar concentration of PNC. The zeta (ζ) potential was evaluated using a 90 Plus PALS instrument (Brookhaven Instruments Corporation, USA). For transmission electron microscopy (TEM) imaging, 20 μL of PNC solution was deposited onto a holey carbon-coated copper grid and imaged using a FEI Talos microscope operating at 300 kV. The size of PNC in TEM images was measured using Image J software.

H₂O₂ and O2·− content

Quantification of H₂O₂ and O2·− levels was performed using commercial kits. H₂O₂ levels were measured with a kit from Nanjing Jiancheng Bioengineering Institute (Product No. A064-1-1). O2·− levels were determined using a kit from Solarbio Life Sciences (Product No. BC1295).

Diaminobenzidine (DAB) and nitro blue tetrazolium (NBT) staining

DAB and NBT staining followed the method described in a previous study (Kumar et al. 2013). Arabidopsis roots were stained with 1 mg mL⁻¹ DAB (pH 5.5, 50 mM Tris-HCl) at 37°C for 6 h and 0.5 mg mL⁻¹ NBT (pH 7.8, 50 mM phosphoric acid buffer) at 37°C for 1 h. Seedlings were subsequently washed rapidly with 70% ethanol and observed under an Olympus IX71 microscope. Quantitative analysis of staining intensity was conducted using Image J software.

Antioxidant enzyme activity assay

The activity levels of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) were quantified using assay kits from Solarbio Life Sciences. Specifically, SOD activity was measured with Product No. BC5165, POD activity with Product No. BC0090, and CAT activity with Product No. BC0205. Each experiment included six replicates.

Imaging of Ca²⁺ distribution in roots

Fluo-3 AM (Thermo Fisher Scientific) and FM 4–64 (Thermo Fisher Scientific) were used as fluorescent dyes for Ca²⁺ and plasma membrane staining, respectively. Fluo-3 AM and FM 4–64 dyes were dissolved in pure dimethyl sulfoxide (DMSO) and diluted to 20 μM with TES buffer (10 mmol L⁻¹, pH 6.1). For root staining, 5-day-old Arabidopsis plants were further root treated with PNC for 7 days. The roots were then stained with a mixture of 20 μM Fluo-3 AM and 20 μM FM 4–64, incubated in the dark for 2 h, and subsequently washed three to five times with TES buffer. Fluorescent signals from the roots were captured using a confocal laser scanning microscope (SP8, Leica, Germany) with the following settings: 40× objective; 488 nm excitation light; PMT1, 510−580 nm (Ca²⁺ fluorescence); and PMT2, 610−630 nm (plasma membrane fluorescence). Each experiment included 8–10 replicates, and Ca²⁺ fluorescence intensities were analysed using LAS AF Lite software as described in our previous studies (Wu et al. 2017; Liu et al. 2021).

Real time quantitative PCR

Total RNA was extracted using the EASYspin Plus Complex Plant RNA kit (RN53, Aidlab, Beijing, China), and 1 μg of total RNA was reverse transcribed into complementary DNA (cDNA) using the TRUEscript RT MasterMix kit (PC7002, Aidlab). Real time quantatitive PCR (qPCR) was performed using a 25-μL reaction mixture containing SYBR Green qPCR Mix (PC3302, Aidlab) following the manufacturer’s protocol. The qPCR analysis was conducted on a Bio-Rad CFX Connect Real-Time PCR System (Bio-Rad, CA, USA). Each gene was analysed with four biological replicates and three technical replicates. Relative gene expression was calculated using the 2^-ΔΔCt method as mentioned elsewhere. For a list of primers used for qPCR, see Supplementary Table S1.

Statistical analysis

The statistical analysis was conducted using Excel 2019 and SPSS 23.0. Data are presented as mean ± s.e. (n = biological replicates). Comparisons were performed using an independent sample t-test (two-tailed) or one-way ANOVA followed by Duncan’s multiple range test (two-tailed). Statistical significance was denoted as: *P < 0.05; **P < 0.01; and ***P < 0.001. Different lowercase letters indicate significant differences.

PNC characterisation

TEM analysis revealed that PNC exhibited good dispersion with an average particle size of 10.88 nm (Fig. 1a), consistent with previous studies (Wu et al. 2017). UV-Vis spectra showed absorption peaks of PNC at 271 nm (Fig. 1b). Zeta (ζ)-potential measurements showed that PNC have a negative charge of −41.7 ± 0.2 mV (Fig. 1c). These results are similar to previous studies (Wu et al. 2017; Liu et al. 2021; Li et al. 2022).

Fig. 1.

PNC characterisation by (a) TEM, (b) absorbance spectrum by UV-Vis spectrophotometry, and (c) measurement of zeta (ζ)-potential. Scale bar, 50 nm. Mean ± s.e. (n = 3).

Screening of application concentration of PNC for LR formation

Previous studies showed that engineered nanomaterials promoted early root growth (Andersen et al. 2016). Here, we conducted a screening experiment to determine the optimal concentration of PNC applied to Arabidopsis roots. Arabidopsis seedlings (5 days old) were exposed to varying concentrations of PNC (0.05, 0.07, 0.1, 0.15, 0.2 mM), and the subsequent effects on LR development were investigated after 7 days (Fig. 2a). Increasing concentrations of PNC resulted in a gradual increase in LR density (LRD) in Arabidopsis, with enhancements of 3.5%, 22.9%, 17.9%, 27.5%, and 61.1% observed from the lowest to highest concentrations of PNC, respectively (Fig. 2b). However, at 0.15 mM, PNC significantly reduced the length of the primary root from 7.1 ± 0.05 cm to 6.68 ± 0.06 cm (Fig. 2c). Similarly, previous studies demonstrated that elevated concentrations of nanoceria decreased pea (Pisum sativum) root elongation while increasing LRD (Skiba et al. 2020). This suggests a general trend where higher concentrations of nanomaterials may inhibit PR growth while stimulating LR formation and altering root morphology. Determining the optimal concentration of nanomaterials that balances main root growth with LR development is crucial for their practical application in agriculture. In this study, to minimise potential interference from high nanoparticle concentrations in investigating LR development mechanisms, we identified 0.1 mM PNC as the optimal concentration for promoting LR formation in Arabidopsis without reducing the length of the primary root.

Fig. 2.

PNC concentration screening on Arabidopsis roots. (a) Phenotype, (b) average emerged lateral root density, and (c) root length of Arabidopsis seedlings where LR length is the total length of lateral roots for each Arabidopsis plant. Scale bar, 1 cm. Mean ± s.e. (n = 24). Different lowercase letters indicate the significance difference at P < 0.05.

PNC promotes LR formation in Arabidopsis

Previous studies have extensively demonstrated the beneficial effects of nanoparticles on plant growth and development (Li et al. 2023; Huang et al. 2024). However, the less known is whether nanoparticles can promote LR formation, and the underlying mechanisms. In this study, we found a significant increase in the number of LRs in PNC-treated (0.1 mM) plants compared to the control (15.04 ± 0.46 vs 11.92 ± 0.37). Additionally, the LRD in PNC-treated plants also showed a significant increase (2.17 ± 0.05 vs 1.72 ± 0.05) than control plants, indicating enhanced LR formation. The length of both primary (6.91 ± 0.08 vs 6.93 ± 0.08) and lateral roots (8.05 ± 0.43 vs 8.41 ± 0.43) did not exhibit significant changes (Fig. 3a, b). Previous studies showed that PNC can enhance growth and salt tolerance in cucumbers by stimulating early antioxidant responses (Chen et al. 2022). Additionally, PNC has been shown to elevate salicylic acid levels and enhance ROS scavenging during seed germination, thereby improving salt tolerance in rapeseed (Brassica napus) (Khan et al. 2022). PNC also mitigates membrane oxidation damage and enhances salt tolerance in rapeseed through modulation of Cu-Zn SOD and lipid oxidase-IV isoenzyme activities (Li et al. 2022), and maintains cytoplasmic K⁺/Na⁺ ratios, thereby improving salt tolerance in cotton (Liu et al. 2021). In this work, we showed that PNC can improve LR formation in Arabidopsis, underscoring its potential in agricultural applications.

Fig. 3.

Lateral root initiation in Arabidopsis after 7 days of treatment with PNC. (a) Root phenotype, and (b) root length, lateral root (LR) number, LR length, and LR density. Scale bar, 1 cm. Mean ± s.e. (n = 6). *P < 0.05; **P < 0.01; ***P < 0.001.

PNC reduce ROS levels in primary and lateral roots

ROS play crucial roles in root growth and the initiation of LRs (Orman-Ligeza et al. 2016; Pasternak et al. 2023). During LR emergence, both H₂O₂ and O2·− accumulate in the vascular zone and epidermis/cortex of the mature root zone (Pasternak et al. 2023). H₂O₂ regulates auxin distribution by modulating polar auxin transport, thereby promoting LR formation and influencing root system architecture (Su et al. 2016). In this study, we assessed the impact of PNC application on Arabidopsis root ROS content (Fig. 4a, b). Our results show that roots treated with PNC exhibited reduced levels of H₂O₂ and O2·−, compared to control plants. Specifically, H₂O₂ levels decreased in both PRs (0.43 ± 0.04 vs 0.53 ± 0.02) and LRs (0.41 ± 0.05 vs 0.64 ± 0.10) after PNC treatment (Fig. 4a). Furthermore, O2·− content decreased by 47.7% in PRs (1.74 ± 0.21 vs 3.65 ± 0.15) and by 88.5% in LRs (0.12 ± 0.01 vs 1.04 ± 0.20) after PNC treatment (Fig. 4b).

Fig. 4.

Contents of (a) H₂O₂ and (b) O2 · − in Arabidopsis primary root (PR) and lateral root (LR) after 7 days of treatment with PNC. Mean ± s.e. (n = 8–10). *P < 0.05; **P < 0.01; ***P < 0.001.

To confirm the ROS findings, in situ histochemical staining for H₂O₂ (Fig. 5a) and O2·− (Fig. 5b) was conducted within the root system. Roots of control plants exhibited strong intensity with 3,3′-DAB (dark brown) and NBT (purple-blue). In contrast, Arabidopsis roots treated with PNC showed reduced staining intensity for both H₂O₂ and O2·− compared to control plants. Specifically, DAB staining intensity decreased by 3.3% in the PRs (201.14 ± 1.15 vs 208.73 ± 1.28) and by 7.2% in LRs (165.59 ± 6.36 vs 178.43 ± 1.92) (Fig. 5c). NBT staining intensity decreased by 5.8% in the PRs (218.07 ± 3.09 vs 231.61 ± 3.30) and by 10.1% in LRs (150.95 ± 3.47 vs 167.91 ± 2.67) (Fig. 5d). These results demonstrate that PNC reduces ROS levels in both primary and lateral roots during the promotion of LR formation in Arabidopsis. Interestingly, a previous study on cucumber plants indicated that PNC application increased NBT intensity in the root apex (Chen et al. 2022), suggesting species-specific variations in ROS responses to PNC. Factors such as root exudates, root anatomy, and uptake efficiency may contribute to these observed differences. These findings underscore the intricate relationship between ROS regulation and PNC effects on root physiology, providing insights into potential mechanisms underlying root development modulation. Mechanistic insights gained from this study highlight how PNC affects root development through ROS modulation. Specifically, the PNC-induced reduction in ROS levels likely influences auxin distribution and transport, which are critical for LR initiation and elongation (Su et al. 2016). Further investigation into the specific pathways that PNC interacts with ROS signalling cascades could provide deeper insights into its mode of action in root development. These findings underscore potential applications of PNC and similar compounds in agriculture. By manipulating ROS levels through chemical treatments, researchers and farmers could potentially enhance root growth, nutrient uptake efficiency, and stress tolerance in crops.

Fig. 5.

Staining of Arabidopsis roots with (a, c) DAB and (b, d) NBT. Scale bar, 100 μm. Mean ± s.e. (n = 8–10). *P < 0.05; **P < 0.01; ***P < 0.001.

Antioxidant enzymes such as POD, SOD, and CAT, play a crucial role in degrading ROS, thereby reducing their accumulation in cells (Dumanović et al. 2021). The observed decrease in H₂O₂ and O2·− in the PR and LR of Arabidopsis may be associated with increased antioxidant enzyme activity. When compared to the control group after 7 days of PNC treatment, POD and SOD activities in the roots were significantly enhanced by 29.4% (100,124.29 ± 6681.17 vs 77,374.16 ± 3023.43) and 25.5% (289.91 ± 3.52 vs 230.36 ± 23.67), respectively (Fig. 6a, b). No significant difference in CAT activity was observed between the control and PNC treatment groups (Fig. 6c). The increased POD and SOD activities effectively neutralised H₂O₂ and O2·−, which contributed to their reduced levels in the root system.

Fig. 6.

Antioxidant enzyme activities in Arabidopsis roots after 7 days treatment with PNC. (a) POD activity, (b) SOD activity, and (c) CAT activity. Mean ± s.e. (n = 6). **P < 0.01.

PNC reduces Ca²⁺ levels in Arabidopsis roots

Previous studies showed that Ca²⁺ is a crucial signalling molecule in the process of LR formation in plants (Zhang et al. 2022). To investigate the impact of PNC on Ca²⁺ signalling during LR development, we utilised the Ca²⁺-specific fluorescent dye Fluo-3AM to monitor Ca²⁺ levels in Arabidopsis roots. Ca²⁺ fluorescence signals were observed in both the PR tip and LRs of Arabidopsis plants (Fig. 7a). Our results reveal that treatment with PNC resulted in reduced Ca²⁺ fluorescence levels in both the primary and lateral roots. Specifically, compared to the control group, Ca²⁺ fluorescence intensity decreased by 35.7% in the primary root (13.75 vs 21.40) and by 22.7% in the lateral roots (10.40 vs 13.46) (Fig. 7b). Changes in Ca²⁺ dynamics can significantly affect various cellular processes, including root growth and development. The reduced Ca²⁺ fluorescence intensity observed in both PR and LR suggests that PNC may disrupt processes such as Ca²⁺ influx, efflux, or intracellular compartmentalisation. Such disruptions could alter the activation of Ca²⁺-dependent proteins and downstream signalling cascades critical for LR initiation and elongation. These findings are consistent with previous research highlighting the regulatory role of Ca²+ in root architecture. For example, fluctuations in Ca²⁺ levels have been associated with changes in root meristem activity, cell differentiation, and responses to environmental stimuli (Zhang et al. 2022). Our study suggested the important role of Ca²⁺ in mediating PNC effects on LR development in Arabidopsis.

Fig. 7.

Ca²⁺ distribution in Arabidopsis roots with (a) confocal images of Fluo-3/AM fluorescence showing Ca²⁺ accumulation, and (b) fluorescence intensity of Fluo-3/AM. Scale bar, 100 μm. Mean ± s.e. (n = 8–10). **P < 0.01; ***P < 0.001.

The ACA gene family (autoinhibited Ca²⁺-ATPase) is a critical component of calcium regulation in plants, and is responsible for maintaining intracellular calcium balance through the active transport of Ca²⁺. This family of calcium pumps plays essential roles in various physiological processes, including cell signalling, environmental stress responses, root development, and overall plant growth (Rahmati Ishka et al. 2021). In our study, Arabidopsis roots treated with PNC exhibited a decrease in intracellular calcium concentration. Concurrently, the expression levels of ACA1 and ACA2 genes were significantly upregulated, whereas ACA8 and ACA10 showed no substantial changes in expression (Fig. 8). These observations suggest that ACA1 and ACA2 are predominantly involved in regulating calcium distribution under PNC treatment. The observed upregulation of ACA1 and ACA2 likely enhances the activity of these calcium pumps, facilitating increased calcium ion transport or excretion, thereby contributing to the reduction of intracellular calcium levels. Conversely, the stable expression of ACA8 and ACA10 indicates that these genes have a minimal impact on calcium concentration regulation in this specific context. This differential expression pattern underscores the distinct roles and regulatory mechanisms of various ACA family members in response to PNC treatment, highlighting the specialised functions of ACA1 and ACA2 in managing calcium dynamics in plant roots.

Fig. 8.

Expression levels of the ACA gene in Arabidopsis roots after 7 days of treatment with PNC. Mean ± s.e. (n = 6). *P < 0.05.