Study on the effect of sodium-nanoparticle in rice root development

ABSTRACT

Micro-nanoparticles can enter the root tissue of plant cells along with the multiple lanes, and then accumulate in the tissue. But the plant physiological effect is still less studied. In this study, rice seedlings at germination stage were treated with 100 µM NaBiF₄ and BiF₃. We found that exogenous application of NaBiF₄ treatment inhibited the elongation of rice roots and promoted the generation of adventitious roots, but treated BiF₃ did not mediate obvious phenotype. Further analysis of the peroxidase activity in related tissues showed that NaBiF₄ induced the activity of SOD and CAT decreased, and POD increased, while BiF₃ only induced the activity of SOD to reduced, but the activity of CAT and POD were no changed. Further analysis of the sodium element and potassium element concentration in tissues showed that only the NaBiF₄ treatment reduced content of potassium, but not sodium. Finally, stress-related genes OsMT1, OsMT2, OsOVP1, OsNIP2;1, and OsMT2b were analyzed by Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR). These results showed that NaBiF₄ induced the expression of OsMT2, OsOVP1, OsNIP2;1 decreased, and OsMT2b increased. However, BiF₃ only induced expression of OsMT1 increased. These results provide a physiological basis for further analysis of the effects of sodium salt-nanoparticles in crop plants.

As the development of science and technology, ENPs (engineering nanoparticles) were used in multiple fields.^1–7 However, there is a great controversy about its potential impact on the ecosystem, especially in plant development, such as germination, water absorption, root elongation, photosynthesis, nitrogen metabolism, and antioxidant stress.^8–13 At genetical level, the toxic mechanism of ENPs affects cell signaling pathways, and then nanoparticles caused phytotoxicity might due to the dissolution and release of toxic ions, mechanical damage and clogging, the production of excess ROS and so on, which might affect photocatalytic activity, cell surface adhesion, dissolution characteristics, surface characteristics, occurrence forms, solvent effect, and synergistic effect with other environmental pollutants are also possible toxic action mechanisms.¹²

As the plant development, there is a series of barriers for the uptake and transport of nanoparticles, such as cell wall, extracellular spaces of cell walls, cell membranes, plasmodesmata, casparian strip, vasculature, pit membranes, cuticles, stomata, hydathodes/lenticels, and cell wall junction.¹² During these barriers, size of 50–100 nm can be used analyze how nanoparticles enter the cell by membrane. We analyzed the effect of sodium nanoparticles (NaBiF₄) and no sodium nanoparticles (BiF₃) that size of 50–100 nm in rice development at seedling stage. Both NaBiF₄ and BiF₃ entered into the cell, sodium nanoparticles (NaBiF₄) inhibited root elongation at 100 µM, but no-sodium nanoparticles (BiF₃) did not inhibit root elongation at such concentration.^14,15 Here, we provided an addendum, how NaBiF₄ and BiF₃ mediate the peroxidase activity in related tissues.

Compared with the negative control, nanoparticle NaBiF₄ inhibited the root elongation both primary root and lateral roots, but increased the number of adventitious roots obviously (Figure 1ae). However, BiF₃ nanoparticles did not change the root elongation and adventitious roots number (Figure 1). These results were similar to previously reported data.^14,15 By analyzing the roles of NaBiF₄ and BiF₃, SOD enzyme activity were obviously decreased (Figure 2a). However, the activity of CAT enzyme decreased only in the NaBiF₄ treatment, but not BiF₃ treatment (Figure 2b). Meanwhile, the POD enzyme activity only increased significantly in the NaBiF₄ treatment, but not in the BiF₃ treatment (Figure 2c). So, both nanoparticles NaBiF₄ and BiF₃ have certain toxic effects on the root system of experimental rice.

Figure 1.

Effect of NaBiF₄ and BiF₃ on rice root development. (a) Phenotypes of nanoparticles treatment plants compared with WT at 7 DAG. (b) Primary roots length in 5 DAG; (c) lateral roots length at 5 DAG; (d) number of lateral roots at 5 DAG; (e) number of adventitious roots at 5 DAG. Each datapoint represents the mean (±SE) from at least four different plants. *P < .05. **P < .01.

Figure 2.

Activity of SOD, CAT, and POD in root tissues under exogenous application of NaBiF₄ and BiF_3. (a) SOD activity; (b) CAT activity; C:POD activity. Each datapoint represents the mean (±SE) from at least four different plants. *P < .05.**P < .01.

To further analyze the different roles between nanoparticles NaBiF₄ and BiF₃, 3 DAG of rice seedlings were treated by 100 uM NaBiF₄ and BiF₃. The content of sodium element in rice tissues did not change both under exogenous application of 100 µM NaBiF₄ and BiF₃ (Figure 3a). The content of potassium element was decreased in rice seedlings under the treatment of NaBiF₄, but not in BiF₃ (Figure 3b). These results showed that NaBiF₄ and BiF₃ had different roles in balance between sodium and potassium element.

Figure 3.

Sodium and potassium contents in tissues under exogenous application of NaBiF₄ and BiF_3. (a) Sodium element concentration per plant; (b) Potassium element concentration per plant. Each datapoint represents the mean (±SE) from at least four different plants. *P < .05.**P < .01.

Exogenous application of nanoparticles entered into the tissues and further affected the balance of sodium and potassium elements in rice root tissues, thus affecting the vitality of tissues. Previously, some genes including OsOVP1 (a vacuolar H⁺-translocating inorganic pyrophosphatase), nodulin 26-like intrinsic protein (OsNIP2;1), metallothionein (OsMT1, OsMT2), and metallothionein 2b (OsMT2b) were responded to ROS pathway signaling in rice.^15–17 We analyzed the related gene expression levels that affect the peroxidase activity level in root tissues. Under the NaBiF₄ nanoparticles treatment, the transcription expression level of OsOVP1, OsNIP2;1, and OsMT2 was decreased, OsMT2b increased, but OsMT1 was not changed. By treatment of BiF₃ nanoparticles, OsOVP1, OsNIP2;1, OsMT2, OsMT2b has no obvious change, while OsMT1 was enhanced. These results showed that both NaBiF₄ and BiF₃ nanoparticles affected the peroxidase activity of rice root tissues, although the root elongation was not obviously changed Figure 4.

Figure 4.

ROS-related genes expression level, relative to OsUBQ5, under exogenous application of NaBiF₄ and BiF₃ treatment. (a) Transcription level of OsOVP1; (b) transcription level of OsMT2; (c) transcription level of OsNIP2; 1; (d) transcription level of OsMT1; (e) transcription level of OsMT2b. Each datapoint represents the mean (±SE) from at least four different plants. *P < .05. **P < .01.

This topic mainly studied the effect of sodium salt-nanoparticles on rice root system, and preliminarily explored the physiological mechanism of rice root system in response to sodium salt-nanoparticles (NaBiF₄). By conducting a series of experiments, these results showed that the root elongation rate and the lateral root elongation were inhibited, and the number of adventitious roots increased significantly in rice treated with nanoparticles (NaBiF₄). By measuring the activity of rice root-related peroxidase activity, it was found that both nanoparticles had a certain degree of toxic effect on the experimental rice root system. These results indicated that the rice root system was subjected to a certain degree of oxidative stress, thus it can be seen that nanoparticles induced phytoxicity. In addition, the results of related genes expression show that the exogenous application of nanoparticles affected the stability of rice root tissues, further supporting the view that nanoparticles are harmful to plants. Compared with BiF3 nanoparticles, NaBiF4 has one more element of sodium. Interestingly, NaBiF₄ entered the rice root system, resulting in no obvious change in sodium content and forcing outflow of potassium element, which caused the abnormal balance of potassium and sodium content. These results, above, indicated that NaBiF4 nanoparticles resulted in rice root toxicity mainly in the NaBiF4 accumulation in root and output of potassium element. And BiF3 nanoparticles could also induce ROS signaling response only in BiF3 accumulation in root. However, how do nanoparticles enter into the cell is unknown, which might give more insights in further development.

Materials and methods

Plant materials and growth conditions

Rice (Oryza sativa L. japonica. cv. Nipponbare) plants were grown in controlled environment rooms. According to previous report, good quality seeds were emerged by NaClO with 1.5% of Cl₂ for 30 minus, washed by ddH₂O for 4–5 times, and then germinated on a Murashige and Skoog (MS) medium with or without 20 µM, and 50 µM nanoparticles in square Petri dishes. All the square Petri dishes were set vertically at the temperature of 28°C under continuous light exposure.

Determination of K+ and Na+ concentrations

The K⁺ and Na⁺ concentrations were measured as described previously.^18,19,20 Seven days after germination rice plant in MS0-medium were harvested. Briefly, the total seedling samples were oven-dried for 3 d at 80°C and then extracted in 100 mM acetic acid for 2 h at 90°C after weighing. The K⁺ and Na⁺ concentrations of the extracts were quantified using inductively coupled plasma-optical emission spectrometry (ICP-OES; Perkin-Elmer, Norwalk, CT, USA). For material of K⁺ and Na⁺ concentrations in water, it was directly used for quantification.

SOD, CAT, POD assay

The activities of SOD, CAT, and POD activity of rice root was measured as described previously.^21,22  Four days after germination, the seedling rice plants were moved to 50 µM NaBiF₄ and BiF₃ nanoparticles water solution for 3 d. About 100 mg of mixed material were harvested and ground in liquid nitrogen to a fine powder and then homogenized in 5 ml 10 mmol/L PBS (pH 7.0) containing 1% PVP (w/v), 1 mM PMSF, 0.1% Triton-X100 (w/v) and 0.1 mM EDTA. The extraction was performed at 4°C. After centrifugation at 12,000 g for 20 min, the supernatant solution was used as the preparation for individual enzyme activity. Then, SOD and CAT activity were measured by spectrophotometer at 560 nm and 240 nm, respectively. The adrenochrome formation in the next 3 min was recorded at 470 nm in a UV-Vis spectrophotometer.

RT-PCR analyses

Total RNA was isolated from root, 4 d after germination, the seedling rice plants were moved to 100 µM NaBiF₄and BiF₃ nanoparticles water solution for 3 d. The cDNAs were synthesized and quantitative real-time RT-PCR was performed as previously described. The internal control was rice UBQ5 (LOC_Os01g22490). All experiments were conducted at least three times, with three or more samples taken at each point. To ensure primer specificity, we performed the experiments when the melting curve showed a single sharp peak. The PCR products were sequenced to verify the specificity of the reaction. All primers used for studying gene expression were listed in our previous report.¹⁴

Statistical analysis

Students’ t-tests were performed to determine any statistically significant differences among values measured from control and treatment experiments.

Funding Statement

This study was supported by the Natural Science Foundation of Jiangsu Province [Grant BK20190889], the Natural Science Foundation of the Jiangsu Higher Education Institutions of China [Grant No. 19KJB180033], the Project funded by China Postdoctoral Science Foundation [2019M660130], “Lvyang Jinfeng” talents attracting plan, the Talent Support Program of Yangzhou University, and Yangzhou University Postdoctoral Science Foundation, a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).