Negatively but not positively charged nanoceria promoted lateral root growth via modulating the distribution of reactive oxygen species rather than auxin

Negatively but not positively charged nanoceria promoted lateral root growth via modulating the distribution of reactive oxygen species rather than auxin | 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 Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed.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. 27 March 2025 V1 Latest version Share on Negatively but not positively charged nanoceria promoted lateral root growth via modulating the distribution of reactive oxygen species rather than auxin Authors : Guangjing Li , Jie Qi , Wenying Xu , Linlin Chen , Ashadu Nyande , Zhouli Xie , Jiangjiang Gu , Zhaohu Li , and Honghong Honghong 0000-0001-6629-0280 [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.174306802.27429747/v1 Published Global Challenges Version of record Peer review timeline 240 views 109 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Lateral root (LR) formation is critical for plant growth. ROS (reactive oxygen species), especially hydrogen peroxide, play important role in LR formation. While the role of superoxide anion in primordia in LR formation is still unclear. Cerium oxide nanoparticles (nanoceria), as a potent ROS scavenger, are widely used in plants in terms of maintaining ROS homeostasis to improve stress tolerance. Whether ROS scavenging nanomaterials can promote LR formation or not and how to use ROS scavenging nanomaterials to study the role of ROS in LR formation remain unclear. In this study, we investigated the effects of three types of nanoceria—poly (acrylic acid) nanoceria (PNC, 6.5 nm, -36 mV), aminated nanoceria (ANC, 6.9 nm, 30 mV), and bulk nanoceria (BNC, 84.9 nm, -5.5 mV)—on LR development in Arabidopsis. Only PNC promoted LR formation, increasing LR numbers by 73.5%. Compared to control plants, PNC-treated Arabidopsis showed reduction in root H 2 O 2 levels (up to 90.44%), alongside decreasing in superoxide anions (O 2 .─ ) and the changes of O 2 .─ distribution in LR primordia (LRP). Furthermore, DPI (diphenyleneiodonium, O 2 .─ inhibitor) treatment decreased LR numbers by 18.9%, while PNC treatment reversed this inhibition (12.25 ± 0.53 vs 8.38 ± 0.52). Transcriptome analysis revealed that PNC promoted LR development in Arabidopsis by modulating ROS metabolism and homeostasis, primarily through the regulation of ROS-related genes such as peroxiredoxins, peroxidases, and glutathione transferases. Interestingly, PNC treatment did not affect auxin distribution in Arabidopsis roots, as confirmed by DR5pro::GFP transgenic lines. Additionally, PNC did not alleviate the inhibition of LR formation caused by NPA (N-1-naphthylphthalamic acid, an auxin transport inhibitor). These findings suggest that PNC enhances LR formation through ROS modulation rather than auxin signaling. Negatively but not positively charged nanoceria promoted lateral root growth via modulating the distribution of reactive oxygen species rather than auxin Guangjing Li 1,2 , Jie Qi 1,2 , Wenying Xu 1,2 , Linlin Chen 1,2 , Ashadu Nyande 1,2 , Zhouli Xie 1,2 , Jiangjiang Gu 2,3 , Zhaohu Li 1,2 , Honghong Wu 1,2,4,5* 1 National Key Laboratory for Germplasm Innovation & Utilization of Horticultural Crops, The Center of Crop Nanobiotechnology, College of Plant Science & Technology, Huazhong Agricultural University, Wuhan, China, 430070 2 Hubei Hongshan Laboratory, Wuhan, China, 430070 3 College of Chemistry, Huazhong Agricultural University, Wuhan, China, 430070 4 Shenzhen Institute of Nutrition and Health, Huazhong Agricultural University, Shenzhen, China, 511464 5 Shenzhen Branch, Guangdong Laboratory for Lingnan Modern Agriculture, Genome Analysis Laboratory of the Ministry of Agriculture, Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen, China, 511464 * Corresponding author: [email protected] Abstract Lateral root (LR) formation is critical for plant growth. ROS (reactive oxygen species), especially hydrogen peroxide, play important role in LR formation. While the role of superoxide anion in primordia in LR formation is still unclear. Cerium oxide nanoparticles (nanoceria), as a potent ROS scavenger, are widely used in plants in terms of maintaining ROS homeostasis to improve stress tolerance. Whether ROS scavenging nanomaterials can promote LR formation or not and how to use ROS scavenging nanomaterials to study the role of ROS in LR formation remain unclear. In this study, we investigated the effects of three types of nanoceria—poly (acrylic acid) nanoceria (PNC, 6.5 nm, -36 mV), aminated nanoceria (ANC, 6.9 nm, 30 mV), and bulk nanoceria (BNC, 84.9 nm, -5.5 mV)—on LR development in Arabidopsis. Only PNC promoted LR formation, increasing LR numbers by 73.5%. Compared to control plants, PNC-treated Arabidopsis showed reduction in root H 2 O 2 levels (up to 90.44%), alongside decreasing in superoxide anions (O 2 .─ ) and the changes of O 2 .─ distribution in LR primordia (LRP). Furthermore, DPI (diphenyleneiodonium, O 2 .─ inhibitor) treatment decreased LR numbers by 18.9%, while PNC treatment reversed this inhibition (12.25 ± 0.53 vs 8.38 ± 0.52). Transcriptome analysis revealed that PNC promoted LR development in Arabidopsis by modulating ROS metabolism and homeostasis, primarily through the regulation of ROS-related genes such as peroxiredoxins, peroxidases, and glutathione transferases. Interestingly, PNC treatment did not affect auxin distribution in Arabidopsis roots, as confirmed by DR5pro::GFP transgenic lines. Additionally, PNC did not alleviate the inhibition of LR formation caused by NPA (N-1-naphthylphthalamic acid, an auxin transport inhibitor). These findings suggest that PNC enhances LR formation through ROS modulation rather than auxin signaling. Keywords: nanoceria, lateral root formation, ROS, auxin, transgenic lines Introduction Lateral roots (LRs) play a crucial role in plant fixation, plant water and nutrient absorption [1]. The development of LRs enhances the absorptive capacity and surface area of the root system [2]. Plants with larger LR systems always show better growth performance than those with smaller ones [3]. Therefore, promoting LR formation in plants is of importance for efficient crop production and the sustainable agriculture [4]. Lateral root (LR) development is a complex process regulated by various substances, including hormones[5], signaling molecules, proteins, and organic compounds[6]. Auxin[7] and reactive oxygen species (ROS) [8] play crucial roles in orchestrating this process. Auxin forms a gradient along the primary root, promoting pericycle cell division to initiate lateral root primordia (LRP) [9]. This gradient is maintained by auxin transporters, facilitating cell elongation and differentiation within the primordium [10, 11, 12]. ROS also contribute to LR formation by increasing LR density and supporting auxin-mediated cell wall remodeling in cortical cells. ROS accumulate in the apoplast of epidermal cells, where they are induced by respiratory burst oxidase homolog (RBOH) [8]. Together, auxin and ROS regulate key aspects of LR formation, including cell division, elongation, and vascular tissue patterning, ensuring proper root architecture. Exogenous auxin treatments [13], genetic engineering [1], biochemical agents (such as auxin analogs) [14], nutrient patches [15], and soil microbial interactions [16] are established methods for stimulating lateral root (LR) formation. However, these approaches face challenges in practical applications due to issues such as environmental sensitivity [17], non-specific physiological effects [18], high costs, and potential ecological risks [19]. In contrast, nanobiotechnology, particularly the use of nanomaterials, offers a promising alternative due to its precision, stability, environmental compatibility, and sustainability. Nanomaterials have been shown to enhance plant growth [20], improve nutritional quality, and increase growth success rates [21]. Despite the promising potential of nanomaterials, the mechanisms underlying their impact on LR formation remain poorly understood. Recent studies suggest that reactive oxygen species (ROS) and auxin (IAA) are key signaling components in nanomaterial-mediated root development. For example, bismuth vanadate nanoparticles promote primary root and LR growth in Arabidopsis by modulating ROS levels and hormone-related gene expression [22]. Graphene oxide (GO) has been shown to affect IAA content and regulate rice root growth. While cerium oxide nanoparticles (nanoceria) are widely known to enhance plant stress tolerance, their effect on LR formation remains inconclusive [23, 24]. Previous studies have shown that nanoceria with a neutral charge and a particle size of 25 nm have little effect on root development and lateral root (LR) formation in plants [25]. In contrast, our prior research demonstrated that negatively charged, smaller-sized PNCs can stimulate LR formation, alongside a reduction in ROS levels and Ca²⁺ concentrations in roots [26]. However, the mechanisms underlying these effects remain unclear. Specifically, it is still uncertain how a decrease in ROS levels can promote LR formation and whether auxin (IAA) plays a critical role in this process. Further investigation is needed to elucidate the interactions between nanomaterials, ROS, and IAA in promoting LR formation, which could lead to more targeted and effective strategies for improving root growth. In this work, we synthesized nanoceria with different size and charge and investigated its biological role on the promoting LR formation in Arabidopsis thaliana . By using laser confocal imaging technique, we investigated the distribution of PNC and the level and distribution of ROS and auxin in roots. We further confirmed the role of PNC in stimulating LR formation by using DPI. Transcriptomic analysis was conducted to investigate the possible key genes/pathways involved in PNC improved lateral root formation. The auxin distribution in Arabidopsis root treated with/without PNC was illustrated with DR5pro::GFP lines. Moreover, IAA inhibitor NPA was used to assess the role of auxin in PNC improvement of LR formation. Materials and Methods not-yet-known not-yet-known not-yet-known unknown 2.1 Preparation of PNC, ANC and BNC The poly (acrylic acid) nanoceria (PNC) were synthesized employing a previously described methodology by Wu et al. with slight modifications [27]. In brief, 1.08 g cerium nitrate (Sigma-Aldrich, 99%) was dissolved in 2.5 mL of deionized water (solution A); 4.5 g of polyacrylic acid (1800 MW, Sigma-Aldrich) was dissolved in 5.0 mL of deionized water (solution B). Mix the two solutions A and B well at 2000 rpm for 15 min using a vortexer, and add the mixture dropwise into a 50 mL beaker containing 15 ml of 30% ammonium hydroxide solution. Stir the mixture at room temperature at 500 rpm for 24 hours. Divide it into 1.5 mL centrifuge tubes and centrifuge at 4500 r for 1 hour. Discard about 100 μL of liquid at the bottom of the centrifuge tube. Aspirate the supernatant into an ultrafiltration tube (MWCO 30K, Millipore Inc.), then centrifuge it at 4,500 rpm for 6 cycles (one cycle every 45 minutes). After the cycle is over, collect the supernatant which is the PNC solution. Measure the absorption peak of the collected solution with a spectrophotometer (UV-1800, AOE). Then, calculate PNC concentration according to the Beer-Lambert law (see below for details). Finally, store it in a refrigerator at 4 °C. The synthesis of amino nanoceria (ANC) followed the methods outlined by Asati et al. with some modifications [28]. Simply, Mix 3.5 mL of 5 mM PNC with 1.5 mL of ultrapure water at 500 rpm for 2 min at room temperature. Then, 306.8 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (Sigma-Aldrich) (EDC) was dissolved in 0.5 mL of MES buffer (100 mM, pH 6.0), which was added dropwise to the mixture and stirred for 4 minutes. Then, 800 mM (6 mL) ethylenediamine (EDA, 99%, Sigma-Aldrich) (adjusted to pH 6.8 with HCl) was added dropwise to the final reaction mixture under constant stirring at 500 rpm, and stirring was continued for 3 h. Transfer the resulting solution to a 1.5 mL centrifuge tube and centrifuge at 4,500 rpm for 15 min to remove any debris and large clumps. Supernatants were purified by removing excess EDA and other reagents by centrifugation at 4,500 rpm (Allegra X30, Beckman) for 5 cycles (15 min per each cycle) using ultrafiltration tubes (MWCO 30K, Millipore Inc.). After the cycle was over, the absorption peak of the solution was measured with a spectrophotometer, the concentration was calculated according to the Beer-Lambert law, and finally stored in a refrigerator at 4 °C for future use. Bulk nanoceria (BNC) were commercial cerium oxide nanoparticles purchased from the company (Sigma-Aldrich). The marked particle size was <25 nm, and the measured particle size was about 60-70 nm, with a weak negative charge. 2.2 Preparation of DiI-PNC Label PNCs with DiI (1,1’-dioctadecyl-3,3,3’,3’tetramethylindocarbocyanine, 1,1’-dioctacyl-3,3,3’,3’tetramethylindocarbocyanine, a fluorescent protocol). Put 4 mL, 0.5 mM PNC and 200 µL, 0.3 mg/mL DiI (dissolved in DMSO) into a 20 mL glass vial and mix at 1000 rpm for 1 min. Purify the resulting mixture using a 10 kDa filter (4,500 rpm, every 5 min, at least five times) to remove free chemicals. Finally, the marked DiI-PNC was obtained, and the characteristic peak of DiI-PNC was detected with a UV spectrophotometer to determine the successful labeling of DiI. 2.3 Characterization of PNC, ANC, BNC and DiI-PNC The absorbance of the final PNC, ANC, and DiI-PNC solutions was measured with an ultraviolet-visible spectrophotometer (UV-1800, AOE). The concentration of PNC, ANC, and DiI-PNC was analyzed via the Beer-Lambert law (A = εCL). Here, A represents the absorbance at 271 nm (for PNC and DiI-PNC) and 264 nm (for ANC), ε denotes the molar absorption coefficient of PNC (3 cm −1 mM −1 ), L signifies the optical path length (cuvette width, 1 cm), and C stands for the molar concentration of the measured PNC, ANC, and DiI-PNC. The hydrodynamic diameter (DLS size) and zeta potential were evaluated using a 90 Plus PALS instrument (Brookhaven Instruments Corporation, USA). For TEM imaging, 20 μL of PNC and BNC were applied onto a holey carbon-coated copper grid, followed by imaging using a FEI Talos microscope operating at 300 kV. 2.4 Cultivation and treatment of Arabidopsis, rapeseed and rice The Arabidopsis thaliana in this study was of type Col-0. Arabidopsis seeds were soaked in 70% ethanol for 10 seconds, washed twice in sterile water, soaked in 5% NaClO for 10 minutes, and washed three times in sterile water. The seeds were evenly sprinkled into the MS square medium, dried on the surface, vernalized at 4 °C in a dark environment for 3 days, and then placed in the light (23 °C, 14/10h, 200 μmol m −2 s -1 ) for 5 days. After the Arabidopsis seedlings grew in growth chamber (full spectrum 395−800 nm; Boante company, Wuhan, China) for 5 days, Arabidopsis seedlings with uniform growth status and approximately the same root length were moved to the experimental treatments (PNC, ANC, BNC, CeCl 3 , DiI-PNC, H 2 O 2 , DPI, NAA, NPA) and the control treatment (adding corresponding volume of solvent) MS solid medium. 6 seedlings per plate, and 4 plates per treatment. After 7 days, take images of Arabidopsis seedlings with a camera. The root length and number of Arabidopsis thaliana in each treatment were counted by Image J software. The dry and fresh weight were recorded accordingly. The specific method of PNC treated rapeseed and rice seedlings: After germinating in the germination box for 3 days, select the seedlings with uniform growth for hydroponic cultivation. Then, set up the PNC treatment and the control treatment respectively. After 6 days’ treatment, take out the seedlings to record the root morphology. 2.5 Imaging of ROS in Arabidopsis roots Imaging of ROS in Arabidopsis roots using confocal laser microscopy. Dihydroethidium (DHE) and 2′,7′-dichlorodihydrofluorescein diacetate (H 2 DCFDA) were used as fluorescent dyes for O 2 .─ and H 2 O 2 , respectively. Arabidopsis roots grown on MS, PNC (0.1 mM) ± MS medium for 7 days were soaked in 25 μM H 2 DCFDA or 10 μM DHE dye (diluted with 10 mM TES, pH=7.5), and incubated for 30 min in the dark. After the incubation, rinse with TES three times, put it into a slide (a drop of perfluoronaphthylamine (PFD) was added to the slide in advance to enhance the effect of fluorescence imaging), cover with a cover slip, and make sure that there is no bubble. The laser confocal microscope was set as follows: 40x objective lens, 488 nm excitation light; emission channel for DHE: 520-580 nm; emission channel for DCF: 500-550 nm, and emission channel for chloroplast: 700-800 nm. 4-6 repetitions were made, and the fluorescence intensity of DCF and DHE was calculated using Image J software. 2.6 RNA-seq and data analysis Total RNA from plant samples was extracted using the RNAprep Pure Plant Kit (Tiangen, Beijing, China), and RNA samples were subsequently sent to BioMarker (BMK, Beijing) for sequencing, with three biological replicates. Gene functional annotations were based on the following databases: Nr (NCBI non-redundant protein sequences), Pfam (Protein family), KOG/COG (Clusters of Orthologous Groups of proteins), Swiss-Prot (manually annotated and reviewed protein sequences), KO (KEGG Ortholog database), and GO (Gene Ontology). The fragments per kilobase of transcript per million mapped reads (FPKM) were calculated using the formula: \begin{equation} \text{FPKM}=\frac{\text{cDNA\ }f\text{ragments}}{\text{Mapped\ }f\text{ragments}\left(\text{Millions}\right)\times\text{\ \ }Transcript\ length(kb)\text{\ \ \ \ \ \ }}\nonumber \\ \end{equation} Differential expression analysis between two conditions/groups was performed using DESeq2, which employs a negative binomial distribution model to identify differentially expressed genes from digital gene expression data. P-values were adjusted for multiple testing using the Benjamini-Hochberg method to control the false discovery rate. Genes with an adjusted P-value < 0.01 and a fold change ≥ 2 were considered differentially expressed. Gene Ontology (GO) enrichment analysis of differentially expressed genes (DEGs) was conducted using the clusterProfiler package, which implements the Wallenius non-central hypergeometric distribution to account for gene length bias. 2.7 DR5pro::GFP Arabidopsis GFP fluorescence monitoring The 5-day-old DR5pro::GFP transgenic Arabidopsis seedlings were moved to the MS solid medium containing PNC, and the GFP fluorescence in the root system was monitored at 24 h, 36 h, and 48 h after culture, respectively. Fluorescence monitoring was performed using a Leica laser confocal SP8 with excitation at 514 nm and emission at 550-615 nm. not-yet-known not-yet-known not-yet-known unknown 2.8 Statistical analysis All data were represented as mean ± SE (n = biological replicates) and analyzed using Excel 2019 and SPSS 23.0. The comparison was performed by an independent sample t-test (two-tailed) or one-way ANOVA based on Duncan’s multiple range test (two-tailed). * and ** represent P < 0.05 and P < 0.01, respectively. Different lowercase letters mean the significance at P < 0.05. 3. Results and discussion 3.1 PNC, ANC and BNC characterization The PNC and BNC were characterized by transmission electron microscopy (TEM). ANC are derived from PNC, sharing the same core. TEM images showed that PNC were well dispersed, while BNC were with irregular shape showing the mixing of diamond, rectangle, and spherical shapes (Fig. 1A). The hydrodynamic diameters of PNC and ANC, measured by using dynamic light scattering, were 6.5 ± 1.7 nm and 6.9 ± 0.6 nm, respectively (Fig. 1B). The size and shape of the synthesized PNC and ANC were similar with previous study [27]. Similar with previous study [27], BNC exhibit a larger hydrodynamic diameter of 84.9 ± 1.9 nm. ζ-potential results showed that PNC (-41.5 ± 3.0 mV) and ANC (28.3 ± 0.6 mV) are charged, compared with the neutral charge of BNC (-5.5 ± 0.3 mV) (Fig. 1C). The UV-Vis spectra showed that PNC and ANC had characteristic absorption peaks at 271 nm and 264 nm, respectively. To track the root distribution of PNC, PNC were labelled with the fluorescent dye 1,1’-dioctadecyl-3,3,3’,3-tetramethylindocarbocyanine perchlorate (DiI). DiI-PNC showed absorption peaks at 271 nm, 520 nm, and 557 nm, indicating successful coating of the DiI to the PNC (Fig. 1D). Figure 1. Characterization of cerium oxide nanoparticles. A. TEM images of PNC and BNC. B and C. Measurement of hydrodynamic size and ζ-potential for PNC, ANC, and BNC. D. The absorbance spectra of PNC, DiI-PNC and ANC by UV-Vis spectrophotometry. Scale bar, 100 nm. Mean ± SE (n = 3–6). 3.2 Negatively charged PNC increased the number of LRs Previous studies showed that nanoparticles’ size and charge could affect its biological role in plants. For example, negatively charged cerium oxide nanoparticles (about 10 nm) enhanced plant tolerance to high light, heat, cold, and salinity across various plant species such as cotton, rapeseed, cucumber, and Arabidopsis [27]. In contrast, particles sized around 99 nm with a charge of 47 mV were found to be toxic to asparagus lettuce plants, leading to reduced root length [29]. While, these studies are always focused on whole plant response or are done with foliar application. How nanoparticles’ properties such as size and charge affect their biological role in root development especially LR formation is still obscure. In this study, our results demonstrated how variations in the size and surface charge of cerium oxide nanoparticles affect LR formation of Arabidopsis . Compared with the control treatment, the application of 0.1 mM PNC with a smaller particle size led to a significant increase in the number of LRs (17.04 ± 0.43 vs 9.83 ± 0.46, Fig. 2A and B), along with a 12.4% increase in root fresh weight (0.23 ± 0.007 vs 0.21 ± 0.004 g) and a 9.5% increase in dry weight (0.018 ± 10^-5 vs 0.016 ± 7*10^-5 g, Fig. 2C). No significant difference was observed between BNC (with large particle size) and the control group in stimulating LR formation in Arabidopsis (9.32 ± 0.42 vs 9.83 ± 0.46, Fig. 2B). Together, these results suggest that size of nanoparticles is a factor affecting its role in LR formation, at least in Arabidopsis . It is known that plant cell wall porosity is around 13 nm. Indeed, nanomaterials with smaller particle sizes typically exhibit increased mobility and enhanced uptake into plant roots compared to larger particles [30, 31]. Larger particles often face significant barriers during root penetration and uptake [31]. The size-dependent uptake of nanomaterials by plant roots emphasizes the necessity to consider particle size during its applications in plants. Figure 2. Arabidopsis lateral root formation and distribution of cerium oxide nanoparticles (PNC) in roots. (A) Phenotype of Arabidopsis seedlings treated with various cerium oxide nanoparticles (0.1 mM) for 7 days. (B) Number of lateral roots in Arabidopsis seedlings after 7 days of treatment. (C) Fresh and dry weight of roots of Arabidopsis after 7 days of treatment. Scale bar = 1 cm. Mean ± SE (n = 24). (D and E) Representative confocal images showing the distribution of PNC in Arabidopsis roots at 24 h (D) and 48 h (E) post-treatment. PNC were labeled with DiI fluorescent dye. White arrows indicate the root apical meristem (RAM) and lateral root primordia (LRP), respectively. Scale bar = 50 μm. n = 6. More and more evidences show that negatively charged nanoparticles are more favored by plants. For example, PNC (10.3 nm, -16.9 mV) exhibited a significantly higher colocalization rate (46%) with chloroplasts in leaf mesophyll cells compared to similarly sized ANC (12.6 nm, 9.7 mV), which showed a colocalization rate of 27% [27]. Also, negatively charged carbon dots preferentially entered mesophyll cells in cucumber and Arabidopsis , rather than accumulating in epidermal cells. Conversely, positively charged carbon dots exhibited an opposite distribution pattern within the leaves [32]. Indeed, nanoparticles with positive or negative surface charges demonstrate distinct uptake kinetics and translocation patterns within plants [33]. Parkinson et al. demonstrated that positively charged particles accumulated near the root surface but were not absorbed. In contrast, negatively charged nanoparticles progressively accumulated and, over time, became visible within the xylem of roots [30]. This is the similar case in this study, showing negatively but not positively charged nanoceria improved the LR formation in Arabidopsis . Notably, ANC induced an effect diametrically opposed to that of PNC on the formation of LRs in Arabidopsis , resulting in a significant reduction of 25.7% in the number of LRs (7.30 ± 0.47 vs 9.83 ± 0.46, Fig. 2A and 2B). CeCl 3 treatment did not significantly affect LR numbers in Arabidopsis compared to the control group (11.27 ± 0.40 vs 9.83 ± 0.46, Fig. 2A and 2B). This suggested that the promotion of LR formation by nanoceria was not related to cerium ions. Furthermore, our results showed PNC could also improve LR formation in rapeseed and rice (Fig. S1), showing the broad application potential of applying PNC to promote LR formation. Confocal imaging results showed a predominant accumulation of DiI-PNC in the root apical meristem (RAM) area after 24 h treatment (Fig. 2D). The RAM consists of actively dividing cells characterized by thin and flexible cell walls, which exhibited increased permeability [34]. After 48 h treatment, in comparison to 24 h, we observed intensified fluorescence signals on the outer epidermis of the root tip, with fluorescence accumulation notably in cells within the mature zone, particularly in the LRP (Fig. 2E). The initiation of LRs takes place within the inner layers of the PR [35]. Subsequently, the initiation of LRP requires cellular separation within the surrounding tissue, leading to intercellular spaces between epidermal cells at the point of LR emergence [36]. Li et al observed a similar pattern showing the entrance of submicron plastics into crop roots through LRP [37]. 3.3 PNC modulate ROS level in Arabidopsis root tip and LR primordia ROS play crucial roles in root development by modulating fundamental cellular processes such as cell division, elongation, and differentiation [38]. In this study (Fig. 3), to better illustrate ROS levels in PNC treated LR and LRP, DCFH-DA (indicating H 2 O 2 ), DHE (indicating O 2 .─ ) and FM 4-64 (staining membrane) fluorescent dyes were used. The fluorescent dye, 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA), can be oxidized by H 2 O 2 to produce the fluorescent derivative dichlorofluorescein (DCF), characterized by green fluorescence within cells [39]. The DCF fluorescence intensity correlates with intracellular H 2 O 2 levels [40] . Dihydroethidium (DHE) fluorescent dye undergoes dehydrogenation facilitated by intracellular O 2 .─ , leading to the production of ethidium bromide, which can interact with RNA or DNA, resulting in fluorescence emission [41]. Consequently, the fluorescence intensity of DHE serves as a quantitative measure of intracellular O 2 .─ levels, with heightened fluorescence corresponding to increased concentrations [42]. Additionally, FM 4-64 fluorescent dye, which stains cell membranes, was employed to label the plasma membrane, helping to identify the developmental stages of LRP in Arabidopsis [43] . Figure 3. ROS content in Arabidopsis roots treated with PNC for 7 days. A and B. Confocal images showing the fluorescence of DCF (A) and DHE (B) in Arabidopsis roots after PNC treatment for 7 days. C and D. Calculated intensity of DCF and DHE in different developmental stages of LRP. YLR: young lateral root. Scale bar, 100 μm. Mean ± SE (n = 8-10). Confocal imaging results revealed a significant reduction in DCF fluorescence intensity within PNC-treated Arabidopsis roots as compared to the control group. This reduction was evident in the root tips (1.44 ± 0.50 vs 15.09 ± 3.72, 90.44%), young LRs (1.47 ± 1.25 vs 3.50 ± 1.56, 57.9%), stages I-IV of LRP (4.80 ± 1.08 vs 11.53 ± 2.07, 58.3%), and stages V-VIII of LRP (2.05 ± 0.73 vs 13.92 ± 2.82, 85.3%) (Fig. 3A and 3C). This is different with previous studies showing that exogenously applied H 2 O 2 can promote LR formation in plants [8, 44]. This might be due to the stimuli effect of PNC in plants. For example, PNC can stimulate the antioxidant enzyme activities in plants. Previous study showed that upon the application of PNC on cucumber plants, root applied PNC increased the DAB intensity in root apex [45]. These results showed that the effect of PNC on H 2 O 2 level is different in plant species. Many factors such as root exudates, root anatomy, and uptake efficiency etc. could contribute to this phenomenon. Similar to DCF intensity results, confocal imaging analysis showed that PNC treatment reduced the DHE intensity in root tips, compared to the control group (5.55 ± 0.65 vs 7.62 ± 0.63, a decrease of 27.2%, Fig. 3B and 3D). While PNC-treated Arabidopsis roots exhibited a significant increase in DHE intensity during early stages I-IV of LRP development (13.42 ± 0.53 vs 11.50 ± 0.50, an increase of 16.7%) and in young LR (15.2 ± 2.48 vs 9.69 ± 1.01, a rise of 57.9%) (Fig. 3B and 3D). However, in the later stages of LR development (stage V-VIII), no significant changes in DHE intensity were detected between the PNC-treated plants and control plants (14.92 ± 0.83 vs 17.47 ± 1.65) (Fig. 3B and 3D). Not surprisingly, O 2 .─ inhibitor DPI (1 μM, diphenylene iodonium) decreased the LR numbers by 18.9% (8.38 ± 0.52 vs 10.33 ± 0.5, Fig. S2A and S2B). While, co-application of PNC and DPI completely alleviated the inhibitory effect observed with DPI treatment alone on LR numbers in Arabidopsis (12.25 ± 0.53 vs 8.38 ± 0.52, Fig. S2A and S2B). Furthermore, this combined treatment exhibited a root-promoting effect comparable to that of PNC alone (12.25 ± 0.53 vs 13.17 ± 0.54, Fig. S2A and S2B). These results further confirmed the important role of O 2 .─ in PNC improvement of LR numbers in Arabidopsis . Previous studies showed that the distribution of H 2 O 2 and O 2 .─ within LRs is not uniform, highlighting their specialized roles in distinct developmental stages [46]. H 2 O 2 and O 2 .─ mainly accumulate in dividing and expanding cells in the elongation and meristem zones [47, 48], respectively, and overlap within the “transition zone”. For example, the balance between H 2 O 2 and O 2 .─ controls the transition between root cell differentiation and proliferation [49]. H 2 O 2 and O 2 .─ are present throughout all stages of LRP development, and the accumulation amount is higher in the later stage when LR appears [50]. Herein, we found that H 2 O 2 is not the key player for nanoceria promoted LR formation in Arabidopsis . While the distribution of O 2 .─ during the early stages of LRP matters for nanoceria promoted LR formation in Arabidopsis . Our work suggests that the role of ROS in the improvement of nanoceria on LR formation is different with other non-nano approaches. 3.4 PNC alter ROS distribution via regulation of metabolism-related genes Transcriptome datasets for control and PNC-treated samples were highly correlated (Pearson coefficient > 0.9), indicating good reproducibility across biological replicates (Fig. 4A). Gene Ontology (GO) enrichment analysis revealed significant associations between differentially expressed genes (DEGs) and oxidoreductase and membrane transporter activities, suggesting these biological processes in Arabidopsis root are primarily affected by PNC treatment (Fig. 4B). A total of 1,002 DEGs were identified (FDR < 0.05, fold change ≥ 2), with 532 upregulated genes and 470 downregulated genes (Fig. 4C). Notably, genes involved in ROS metabolism, including peroxiredoxin (PRX) and peroxidase (PER) which regulate ROS levels by catalyzing the breakdown or conversion of hydrogen peroxide [51, 52], showed significant changes of the expression level, showing upregulation of PRX56, PRX37, PER28, and PER39, and downregulation of PRX62, PRX25, and PRX4 (Fig. 4D). Previous work showed that the upregulation of PRX56 and PRX37 likely promotes the scavenging of hydrogen peroxide, reducing its accumulation in the root system [53, 54]. Additionally, the expression level of glutathione transferase (GST) genes which are involved in plant oxidative stress response [55, 56] was altered, showing upregulation of GSTU7 and GST16, and downregulation of GST20 and GST13 after PNC treatment (Fig. 4D). During lateral root development, ROS balance, influenced by GST activity, is crucial for maintaining optimal ROS levels to support root growth. Previous studies showed that the regulation of GST genes may enhance the root system’s antioxidant capacity, preventing excessive ROS accumulation and potential cellular damage [57, 58]. These results indicated potential regulation of PNC on ROS signaling through the glutathione metabolic pathway. Moreover, PNC may indirectly stimulate superoxide anion accumulation in lateral root primordia through modulation of ROS metabolism, contributing to lateral root formation [59]. Overall, PNC treatment can promote lateral root development by modulating ROS distribution and homeostasis, primarily through the regulation of ROS homeostasis-related genes such as peroxiredoxins, peroxidases, and glutathione transferases. Figure 4. Comprehensive analysis of differential gene expression and functional enrichment in Arabidopsis roots under PNC treatment. A, Correlation heatmap of samples. B, Chord diagram of GO enrichment analysis for DEGs in Arabidopsis roots under PNC treatments. C, Volcano plot of DEGs between control and PNC treatment. Red dots represent upregulated genes, blue dots represent downregulated genes (FC ≥ 2; FDR < 0.01). D, Differential expression of oxidoreductase genes in Arabidopsis roots comparing control and PNC treatment groups. Mean ± SE (n = 3). 3.5 PNC promoted LR formation bypassed the auxin pathway Auxin plays a crucial role in regulating various aspects of plant growth and development [59]. To assess the effect of PNC treatment on auxin distribution in Arabidopsis roots, we utilized DR5 transgenic Arabidopsis seedlings equipped with GFP fluorescent markers to visualize auxin distribution at 24, 36, and 48 hours post-PNC application (Fig. 5A). Interestingly, no significant changes in auxin distribution were detected following 24, 36, or 48 hours of PNC treatment when compared to the control group (Fig. 5A). This is different with the results in previous studies showing that auxin plays an important role in promoting LR numbers in plants [60]. not-yet-known not-yet-known not-yet-known unknown Figure 5. Effect of PNC treatment on auxin distribution and lateral root formation in Arabidopsis. (A) GFP fluorescence imaging of DR5pro::GFP Arabidopsis roots exposed to PNC for 24h, 36h, and 48h, showing consistent auxin distribution patterns across all time points. Scale bar = 100 μm. n = 12. PR: primary root, LRP: lateral root primordium. (B) Phenotype of Arabidopsis seedlings treated with PNC, NAA, and NPA for 7 days. (C) Number of lateral roots in Arabidopsis after 7 days of treatment with PNC, NAA, and NPA. Scale bar = 1 cm. Mean ± SE (n = 6). To explore whether PNC promote LR development in Arabidopsis through the auxin pathway, Arabidopsis seedlings were treated in vitro with the synthetic auxin naphthaleneacetic acid (NAA) and the auxin polar transport inhibitor N-1-naphthylphthalamic acid (NPA). Similar to previous studies [61], NAA treatment alone led to a significant increase of 54.3% in LR number in Arabidopsis compared to the control group (15.94 ± 0.48 vs 10.33 ± 0.50, Fig. 5B and 5C). However, co-application of NAA and PNC did not show higher LR numbers than NAA treatment alone (14.63 ± 0.45 vs 15.94 ± 0.48, Fig. 5C). NPA inhibits the polar transport of auxin, leading to a decreased ability of the Arabidopsis roots to grow vertically, and subsequently, a reduction in LR formation [60]. Following NPA treatment, the number of LRs in Arabidopsis decreased markedly by 31.8% of the control group (3.29 ± 0.49 vs 10.33 ± 0.50, Fig. 5C). This is similar to previous studies showing NPA application can reduce the LR numbers [60]. In the presence of NPA, the addition of PNC can partially restore the degree of root curvature of Arabidopsis, but it does not significantly increase the number of LRs (3.92 ± 0.32 vs 3.29 ± 0.49) (Fig. 5B and 5C). Recent evidence indicates that the auxin-independent transcription factor ZINC FINGER OF ARABIDOPSIS THALIANA 6 (ZAT6) plays a pivotal role in regulating lateral root development under salt stress [62]. Together, it further confirmed that the improvement of Arabidopsis LR numbers by PNC may bypass the auxin pathway. Conclusion In this study, we examined the effects of CeO2 nanoparticles on ROS and auxin distribution, focusing on their role in promoting lateral root (LR) formation in Arabidopsis thaliana. Three types of nanomaterials were tested: poly(acrylic) acid coated cerium oxide nanoparticles (PNC, 6.5 ± 1.7 nm, -41.5 ± 3.0 mV), aminated PNC (ANC, 6.9 ± 0.6 nm, 28.3 ± 0.6 mV), and bulk cerium oxide nanoparticles (BNC, 84.9 ± 1.9 nm, -5.5 ± 0.3 mV). Only PNC significantly promoted LR formation, which was associated with modulation of ROS, particularly O₂⁻ in LRP, and was independent of auxin signaling. Transcriptomic analysis indicated that PNC treatment enhances LR development by regulating genes involved in ROS homeostasis. These findings suggest that nanoceria-based approaches to stimulate LR formation differ from conventional methods and highlight the potential of PNC in enhancing root growth for agricultural applications. not-yet-known not-yet-known not-yet-known unknown Supplementary Information The supplementary materials are available at the online version. Acknowledgements We thank A/Prof. Mr. Jianbo Cao, and Ms. Limin He for their help in TEM imaging at Public Laboratory of Electron Microscopy, Huazhong Agricultural University. We thank Prof. Honghong Hu from Huazhong Agricultural University for providing the seeds of Arabidopsis DR5 lines. Authors’ contributions Guangjing Li: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing – original draft, Writing – review & editing. Jie Qi: Investigation, Validation. Wenying Xu: Investigation, Data curation. Linlin Chen: Resources. Ashadu Nyande: Writing – review & editing. Zhouli Xie: Writing –review & editing. Jiangjiang Gu: Writing –review & editing. Honghong Wu: Supervision, Conceptualization, Funding acquisition, Writing – review & editing. All authors edited the manuscript and approved the final version. Funding This work was supported by the Key Research and Development Projects of Hubei Province (2024BBB065), the National Key Research and Development Program of China (2023YFD19101700-3), Fundamental Research Funds for the Central Universities (2662023ZKPY002), the HZAU-AGIS Cooperation Fund (SZYJY2021008), the Key Research and Development Projects of Henan Province (231111113000), and the Hubei Agricultural Science and Technology Innovation Center Program (2021-620-000-001-032) to H.W. Data availability All data generated or analyzed during this study are included in this published article. Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare no competing financial interests. Reference [1] Nibau C, Gibbs DJ and Coates JC. Branching out in new directions: the control of root architecture by lateral root formation. New Phytol 2008; 179: 595-614.[2] Lynch J. Root Architecture and Plant Productivity. 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Keywords auxin growth lateral root formation nanoceria ros Authors Affiliations Guangjing Li Huazhong Agricultural University College of Plant Science and Technology View all articles by this author Jie Qi Huazhong Agricultural University College of Plant Science and Technology View all articles by this author Wenying Xu Huazhong Agricultural University College of Plant Science and Technology View all articles by this author Linlin Chen Huazhong Agricultural University College of Plant Science and Technology View all articles by this author Ashadu Nyande Huazhong Agricultural University College of Plant Science and Technology View all articles by this author Zhouli Xie Huazhong Agricultural University College of Plant Science and Technology View all articles by this author Jiangjiang Gu Huazhong Agricultural University View all articles by this author Zhaohu Li Huazhong Agricultural University College of Plant Science and Technology View all articles by this author Honghong Honghong 0000-0001-6629-0280 [email protected] Huazhong Agricultural University College of Plant Science and Technology View all articles by this author Metrics & Citations Metrics Article Usage 240 views 109 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Guangjing Li, Jie Qi, Wenying Xu, et al. Negatively but not positively charged nanoceria promoted lateral root growth via modulating the distribution of reactive oxygen species rather than auxin. Authorea . 27 March 2025. DOI: https://doi.org/10.22541/au.174306802.27429747/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu . 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