Impact of nanometer hydroxyapatite on seed germination and root border cell characteristics

Abstract

Nanomaterials may have effects on health and environmental safety due to their unique physical and chemical properties. Nanometer hydroxyapatite (NHAP) is a commonly used passivator for fixing heavy metals in soil and remediating soil pollution. We evaluated the phytotoxicity of NHAP, its effects on the sprouting of plants, and the growth of root tip border cells by measuring the effects of NHAP on the germination rate of cucumber and the number and activity of root border cells in indoor cultivation and hanging cultivation by flow cytometry. The germination percentage and germination index of cucumber increased rapidly with increasing concentration of NHAP. When the concentration was greater than 1000 mg L⁻¹, both root growth and shoot growth were inhibited to varying degrees. The highest number and activity of root border cells were observed during germination of cucumber seeds at a root length of 20 mm. Cell survival decreased steadily with increasing root growth, reaching the lowest survival rate of 55.4% at 40 cm. Decrease in the number of root border cells was observed following treatment with NHAP, with the rapid decreases observed at NHAP concentrations greater than 500 mg L⁻¹. This result demonstrates that a high concentration of NHAP exerts a harmful or inhibitory effect on the growth of cucumber root. Pectin methylesterase (PME) activity increased with increasing NHAP concentration, therefore protecting the plant from the increased entry of metal ions into the cells.

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Introduction

Hydroxyapatite (HAP), the main inorganic component of bones and teeth, has favorable biological compatibility. Due to its large micro-interface and high microporosity, nanometer hydroxyapatite (NHAP) can perform photocatalytic adsorption, specific adsorption and surface adsorption when it is exposed to pollutants, therefore reducing the biological toxicity and mobility of these pollutants.¹ Compared to conventional passivators, NHAP can significantly reduce the addition of conditioner by several fold, thus reducing adverse environmental effects. In addition, NHAP plays an important role in remediating soil pollution, particularly for the in situ passivation remediation of heavy metal soil pollution.^2,3 However, the application of NHAP for soil pollution remediation has only recently been investigated. Researchers have focused more on remediation effects such as the use of NHAP to reduce the bioavailability of Cu and Cd⁴ in soil by increasing the soil pH and increasing the migration and dissociation of steady-state Cu²⁺.⁵ The application of NHAP can increase biological yield and germination rates as well as promote root growth. However, much of the literature has focused on the growth of individual plants and the response of soil biological activity. Few studies have examined the effects of NHAP on root tip growth and living cells. Such studies would not only enhance the systematic understanding of NHAP environmental remediation but also provide better data to support the use of NHAP.

NHAP has a particle size of approximately 1–100 nm. Its biological activity and chemical functions differ from those of micron- or millimeter-sized substances because NHAP, like other nanometer materials,⁶ can be adsorbed through the root tip surface of the plant or by the plant during growth. Similarly, NHAP released into the soil solution may serve as a source of ionic materials and can therefore affect the exposed root tip and growth environment of the root system, altering the biological properties of the plant and the accumulation and transmission pathways of the substance in the food chain.^7,8 Therefore, in addition to studying the remediation of environmental pollution by NHAP, it is imperative to conduct research on the biological effects of NHAP. Root border cells are specialized cells that occupy the surface of the root cap and surround the root.⁹ Root border cells synthesize and secrete a series of bioactive chemicals that induce and control the growth of rhizosphere microorganisms and counteract toxic substances,¹⁰ thus playing an important role in protecting the root tip from the influence of adverse factors. Adding NHAP will inevitably have an impact on soil biological activity and plant germination. Recent studies have investigated the response of the plant rhizosphere to NHAP. For example, Lin¹¹ observed that artificial nanozinc and nanozinc oxide inhibit the germination of ryegrass and maize, respectively. In addition, a cultivation study by Cui showed that NHAP has no significant effect on soil urease, catalase or acid phosphatase activities.¹² However, the corresponding mechanisms of the root system in response to NHAP, particularly in root border cells, the vanguard of the root system, have not been studied.^13,14 Therefore, studies of the protective function and adaptability of border cells in response to nanoparticles will help further elucidate the mechanisms underlying the resistance of border cells in the soil–plant system.

In our study, cucumber (Cucumis sativus L.) was used as the model system. We used indoor cultivation and hanging cultivation to study the effects of NHAP on cucumber germination and the growth characteristics and response mechanisms of root border cells. The results offer scientific support for the use of nanoparticles in heavy metal pollution remediation and support the biological safety of the nanoparticles.

Results

Effects of NHAP solutions of different concentrations on cucumber seed germination

NHAP solutions of different concentrations had different effects on cucumber seed germination and root tip growth. As shown in Fig. 1, the seed germination percentage and germination index increased rapidly with increasing NHAP concentration, reaching their maximum values at 1000 mg L⁻¹. The germination index and germination rate were significantly higher at an NHAP concentration of 1000 mg L⁻¹ than at 100, 200, 500 mg L⁻¹ and 2000 mg L⁻¹ (P < 0.05). As the NHAP concentration increased, the reduction in root length first decreased and then subsequently increased. Indeed, NHAP at concentrations of up to 1000 mg L⁻¹ stimulated root growth, as attested by the negative percentage of root growth observed (Fig. 1). On the other hand, 2000 mg L⁻¹ NHAP inhibited root growth by about 20%. However, when the NHAP concentration was 2000 mg L⁻¹, NHAP significantly inhibited root growth (P < 0.05). As shown in Fig. 1, NHAP did not affect sprout (seedling) growth, regardless of the concentration tested.

Fig. 1 Effects of NHAP at different concentrations on cucumber seeds (average ± standard error, LSD pairwise testing, *P < 0.05).

Number of cucumber root tip border cells and changes in survival rate

Fluorescence microscopy revealed that the root border cells were rod-shaped and appeared at approximately the same time as the root tip. Live cells dyed with FDA–PI exhibited a green color (Fig. 2(A)). At high concentrations of nanometer hydroxyapatite, dead cells became long and narrow and fused together, exhibiting red fluorescence (Fig. 2(B)). Flow cytometry analysis (Fig. 3) indicated that a high cell survival rate was achieved when roots up to 35 mm long were treated with NAHP, whereas the cells of 40 mm long roots exhibited cell survival rates as low as 55% under the same experimental conditions (P < 0.05). As the root tip grew, the number of cucumber root tip live border cells first increased and then decreased. There were fewer live cells when the root was 1 cm long, and the number of live border cells increased slowly as the root grew. The number of live cells reached a maximum at a root length of 2 cm. As the root continued to grow and the number of border cells continued to increase, PME activity decreased, and the cell survival rate dropped accordingly. Based on these results, in subsequent experiments, root border cells were collected when the cucumber root was 2 cm in length.

Fig. 2 Morphological changes in root border cells. (A) Living border cells dyed with FDA–PI, displaying green fluorescence; (B) dead border cells dyed with FDA–PI, displaying red fluorescence.

Fig. 3 Border cell numbers at different root lengths and changes in the cell survival rate (mean ± standard error). Different letters above the bars indicate significant differences in cell number between groups (P < 0.05); different numbers of * indicate a significant difference in the cell survival rate between groups (P < 0.05).

Effects of NHAP on the survival rate of cucumber root border cells

As shown in Fig. 4(A), at an NHAP concentration of less than 500 mg L⁻¹, living cells accounted for more than 80% of the border cells. In the range of 0–500 mg L⁻¹, increasing the NHAP concentration did not significantly alter the number of cells (P > 0.05). At NHAP concentrations greater than 500 mg L⁻¹, the border cells decreased slowly in number, whereas the percentage of dead cells increased rapidly, and the percentage of live cells decreased rapidly. At an NHAP concentration of 2000 mg L⁻¹, the percentage of live cells was only 31.7%. Thus, only some NHAP concentrations negatively affected border cell number and activity, and NHAP was safe to use on root tip border cells at concentrations less than 500 mg L⁻¹. Representative results of Annexin V-FITC/PI double-dye flow cytometry are presented in Fig. 4(B) and (C). The scatter diagram is divided into four quadrants, with upper left quadrant 1 (Annexin −, PI+) representing early cell death, upper right quadrant 2 (Annexin +, PI+) representing dead cells, lower left quadrant 3 (Annexin −, PI−) representing live cells, and lower right quadrant 4 (Annexin +, PI−) representing dead cells and late cell death. Comparative analysis of the data in Fig. 4(B) and (C) revealed that after treatment with 2000 mg L⁻¹ NHAP, the cell death rate of cucumber root border cells significantly increased compared to those treated without NHAP. High concentrations of NHAP accelerated the death of root border cells, in agreement with the results shown in Fig. 4(a).

Fig. 4 Changes in the number of live root tip cells due to treatment with different concentrations of NHAP. (a) Flow cytometry report for different concentrations of NHAP; (b) flow cytometry results for 0 mg L⁻¹ NHAP; (c) flow cytometry results for 2000 mg L⁻¹ NHAP.

Effects of NHAP on PME activity in the root tip

As shown in Fig. 5(A), as the root tip grew, the PME activity initially increased and then decreased. The PME activity in NHAP-treated roots was similar in samples of 5–25 mm length, averaging 11.13 μmol H⁺/h/mg total protein. This activity significantly decreased to 3.61 μmol H⁺/h/mg total protein, in average, in 30 mm or longer roots (Fig. 5; P > 0.05). Fig. 5(B) shows that the treatment with 100 mg L⁻¹ NHAP for 48 h caused an increase (P < 0.05) of PME activity in 2 cm long roots in comparison to untreated roots. Further increase in enzyme activity was observed after treatments with 200 to 1000 mg L⁻¹ NHAP, whereas a concentration of 2000 mg L⁻¹ NHAP restored PME activity to the levels found in the control roots. In fact, PME activity in 2000 mg L⁻¹ NHAP-treated roots was approximately 35% of that of roots treated with NHAP at concentrations in the range from 200 to 1000 mg L⁻¹ NHAP.

Fig. 5 Changes in the PME activity of cucumber root tips (mean ± standard error). (A) The PME activity of roots of different lengths. (B) The PME activity of root tips treated with NHAP at different concentrations at a root length of 2 cm. Different letters indicate significant differences (P < 0.05).

Discussion

Due to its unique crystalline properties, NHAP strongly adsorbs and fixes a variety of metal cations. Therefore, NHAP is widely used for heavy metal soil remediation. However, adding high concentrations of NHAP to the environment may have negative effects on plant growth by releasing –OH, changing the soil pH and allowing nanoparticles to enter the root tip cells. Adding NHAP increases soil catalase, urease and phosphatase activities to varying degrees;¹⁵ however, few studies have considered the effects of NHAP on plant germination and the growth and abscission of root border cells. Our experimental results show that increasing the NHAP concentration results in an initial increase and subsequent decrease in the cucumber germination percentage and germination index. In addition, the germination percentage and germination index were significantly higher at an NHAP concentration of 1000 mg L⁻¹ than at other treatment concentrations (P < 0.05). Increasing NHAP concentration significantly increases the inhibition of cucumber root growth. At 2000 mg L⁻¹, NHAP exerts a significant inhibitory effect (P < 0.05). Thus, NHAP may negatively affect cucumber growth.

Root border cells, which drop from the root tip, are an active group of cells with special physiological activity. Their growth is closely related to root growth. In the early period of root development, border cells develop rapidly and are highly active. For example, the root border cells of Glycine max are free from the start of development, nearly concurrent with the growth of the root. When the root grows to 15 mm, the border cells reach their maximum number of 5300.^16,17 When the roots of Hordeum vulgare grow to 20–25 mm, the root border cells reach their maximum number of 1400.¹⁸ Our study reveals that at an NHAP treatment concentration of 0 mg L⁻¹, border cells develop with the growth of the cucumber root tip, and live border cells are most abundant at root lengths shorter than 20 mm. The cell survival rate remains above 80% at 0 mg L⁻¹ NHAP, which is similar to previously reported findings. Recent studies have demonstrated that changes in the plant growth environment, such as changes in temperature and CO₂ concentration, may affect seed germination, sprout growth and root growth. Root border cells, as the messaging component of the rhizosphere, respond the most rapidly to environmental changes. The number and survival rate of root border cells change significantly, and extracellular signals play a greater role than intracellular signals in alterations of the development of root border cells.¹⁵ Currently, studies of border cell activity or the resistance reaction mainly focus on soil aluminum poisoning and heavy metal pollution. There has been little detailed research on the effects of NHAP on root cap border cells and enzymatic activity. In the studies of plant resistance to aluminum toxicity, root tip cells and extracellular mucus secretion are commonly used to reveal the effects of aluminum poisoning and the immune response of plants. Studies have also demonstrated antiviral mechanisms in Glycine max, corn, peas and beans.^19–21 We determined that NHAP exerts an effect when the cucumber root is 20 mm long and that the border cells respond rapidly (Fig. 4), maintaining a stable number of living cells to resist the adverse environment. Similar findings were also obtained in Zhao's study of the response of cucumber root border cells to CO₂.²² The process involves the regulation of root intrinsic signals and the adjustment of external environment signals, the mechanisms of which remain unclear. However, root border cell death increases under the influence of high NHAP concentration, which may be related to the decreases in root length and slow growth of the root.

PME can eliminate pectin methylation of the cell wall. Several researchers have speculated that the shedding of plant root border cells from the root system is mainly caused by PME demethylation. Studies have revealed that PME can cause pectin demethylation only when it is secreted in the form of zymogen outside the plant. PME can also enable the negatively charged group on the cell wall to gather more negative charges, therefore enhancing the adsorption capacity of metal cations, improving the structure of the cell wall and consequently strengthening crosslinking between cells. Furthermore, metal cations can substitute for Ca²⁺ in the cell walls of PME and change PME activity.²³ The impact of nanoparticles on root border cells has not been studied. The influence of NHAP on the environment and living organisms is primarily due to the dissociation of –OH and Ca²⁺. –OH may react with galacturonic acid polymer carboxylic acid, and an increase in Ca²⁺ stimulates the PME activity of the border cells. Consequently, the pectin of the cell wall undergoes demethylation to increase the number of cation binding sites, thus limiting the number of particles and cations that can enter the cells and damage the plant. Our study revealed that increasing the NHAP concentration steadily increased PME activity in the root. Highly active PME can cause the pectin of the root tip cell wall to undergo demethylation, thus increasing the number of binding sites of NHAP in the glue layer of border cells and reducing the risk of Ca²⁺ and nanoparticles entering the root tip cells. This process helps maintain the normal growth and development of the root tip. Observation of fluorescence revealed that under the influence of high NHAP concentration, the shape of the cucumber root tip border cells changed from stick-like to long and narrow, with a large number of cells gathering together (Fig. 2). A high NHAP concentration can exert a stress effect that increases the mucus layer of the cucumber root tip border cells. In conclusion, a change in the environment can stimulate the growth of border cells and increase PME activity. Stress in beans and peas can stimulate border cells to secrete a mucus layer, and this mucus layer can combine with aluminum ions to fix the aluminum. However, the mucus layer composition of the cucumber root tip border cells remains unknown, and its interaction with nanoparticles requires further study. Therefore, future research should determine the composition of the cucumber root tip border cells and the mechanism of the nanoparticles using methods such as GC-MS. Such efforts will provide a better understanding of the response mechanism of border cells and their mucus to nanoparticles and an accurate evaluation of the plant toxicity of nanoparticles.

Conclusions

Under water culture and hanging cultivation, the cells were greatest in number and most active when the root length reached 20 mm, and the lowest survival rate of 55.4% was observed at a root length of 40 mm. Although the effects of NHAP on the growth of cucumber sprouts were not significant, 2000 mg L⁻¹ NHAP significantly inhibited the growth of cucumber roots and PME activity. When PME activity reached its maximum values at 500 mg L⁻¹ NHAP, the germination index and germination rate were significantly higher at an NHAP concentration of 1000 mg L⁻¹ than at other NHAP concentrations (P < 0.05). When the NHAP concentration was less than 500 mg L⁻¹, living cells accounted for more than 80% of the border cells. The present study may serve as a useful reference for further research on the phytotoxicity of hydroxyapatite nanoparticles and may promote their application in environmental pollution remediation.

Materials and methods

Experiment materials

Huayan new no. 4 cucumber seeds were used (Xinhuayan Seed Firm, Ningyang County, Shandong Province), and the NHAP used in the experiment was prepared in the laboratory by sintering synthesis. NHAP was first synthesized using an EDTA : Ca : NH₄H₂PO₄ ratio of 2.67 : 2.67 : 1, with quantitative addition of Ca(NO₃)₂·4H₂O, (NH₄)₂–EDTA and NH₄H₂PO₄ to form a uniform solution. After slow evaporation of the solvent and drying at 80 °C, the dry matter was sintered for 4 h at 800 °C to prepare NHAP. The particle morphology distribution is shown in Fig. 6.

Fig. 6 Morphology distribution of NHAP visualized by scanning electron microscopy (a, magnified 30 000 times; b, magnified 10 000 times).

Border cell number and survival rate

Measurements of border cell number: four cucumber seeds were selected from each root length group (1, 1.5, 2, 2.5, 3, 3.5 and 4 cm) cultivated in distilled water. Their roots were cut and placed in an Eppendorf tube with 1 mL of ultrapure water. The border cells were shed in the water by oscillation on a vortex oscillator for 30 s and blown gently with a pipetting gun a few times. The border cells and their mucus were consequently dispersed fully and evenly in the water to form a suspension of border cells. Twenty microliters of the suspension were extracted from the middle tier of the Eppendorf tube with the pipetting gun and transferred to a flow cytometry tube. Then, the suspension was mixed with FDA (25 μg mL⁻¹) 1 : 1 in the dark and dyed for 20 min. The number of cells was counted by flow cytometry (Becton, Dickinson and Company, New Jersey, USA), and the measurement was repeated three times for each sample. The measurement results were averaged, and the number of root tip border cells in each root length group and the border cell survival rates were calculated.

Measurement of survival rate: a 20 μL aliquot of border cell suspension was mixed with FDA (25 μg mL⁻¹)–PI (10 μg mL⁻¹) in a 1 : 1 ratio to form 40 μL of dyed root border cell solution, and the mixture was incubated in the dark for 20 min. Ten microliters of the solution were spread evenly on a clean fluorescence microscope detection plate. At an excitation wavelength of 488 nm, healthy live cells exhibited green fluorescence, and dead cells did not exhibit any color. The remaining 30 μL of dyed solution was used to determine the change in fluorescence intensity within a certain range via flow cytometry, and the proportion of FDA-dyed cells was used to determine the number of cells and the survival rate.

Cultivation method

First, cucumber seeds were disinfected by soaking them in 10% NaClO for 15 min. After rinsing several times in distilled water, the seeds were placed in the incubator at 25 °C for 12 h to avoid light and accelerate germination. Then, 30 cucumber seeds that had initiated sprouting were selected from each group and placed in a petri dish with 200 mL of distilled water for hanging cultivation to a root length of 20 mm. For hanging cultivation, seeds were sown on gauze. A specific volume of water solution was added to the bottom layer of the gauze. Water evaporation allowed the humidity to be maintained during seed growth. The cultivation process included spraying culture solution to guarantee the healthy growth of the root system and to prevent the excessive shedding of cucumber root tip cells caused by the impact of water flow, which could affect the accuracy of the results. During cultivation, the different concentrations of NHAP solutions, such as 0, 100, 200, 500, 1000 and 2000 mg L⁻¹, were sprayed once every 2 h. The number of germinated seeds, root length and shoot length were recorded daily, and water was supplied to maintain a constant weight. After treating the root tip with NHAP for 48 h (root tips were 2 cm long), the root tips were collected, and the number and activity of border cells were measured. Then, the following indices were calculated:

Germination rate = the number of germinated seeds/the number of experimental seeds × 100%;

Germination index = ∑(the number of germinated seeds on the t-th day/the number of days), where t-th means day t;²⁴

Root length inhibition ratio = (1 − experimental root length/contrast root length) × 100%;

Bud length inhibition ratio = (1 − experimental bud length/contrast bud length) × 100%.

PME extraction and activity measurement

PME extraction. The shedding of border cells from the plant root tip is related to the expression of PME in the root tip.²⁵ Cucumber root tips of a specific length (3 mm) cultivated under different concentrations of NHAP solution were cut, placed in a mortar containing 500 μL of PME extraction solution (0.1 M citric acid, 0.2 M Na₂HPO₄, 1 M NaCl, and pH 5.8) and fully ground. To ensure the extraction concentration and the accuracy of the experimental results, for each treatment concentration, 3 root tips were selected to determine PME activity. The homogenate was placed in a 2 mL centrifuge tube and subjected to freezing for 1 h. Centrifugation was performed at 10 000 rpm and 4 °C for 10 min, and the resulting supernatant was stored at −20 °C.

PME activity testing. The specific steps for determining μmol H⁺/h/mg total protein were performed as described by Richard et al.:²⁶

(1) A standard curve was prepared by adding 40, 50, 60, 70, 80, 100 or 120 μL of 0.01 M HCl to 4 mL of substrate solution. The mixture was oscillated and shaken, and A₅₂₅ was obtained. A standard curve relating absorbance to μmol of H⁺ was developed, with A₅₂₅ as the abscissa and H⁺ μmol as the ordinate.

(2) Ten microliters of supernatant were mixed with 4 mL of substrate solution (0.5% (w/v) citrus pectin, 0.2 M NaCl, and 0.15% (w/v) methyl red, pH 6.8), then incubated in a water bath at 37 °C for 2 h.

(3) A₅₂₅ was obtained with a spectrophotometer. The standard curve regression equation was used to calculate PME activity. The process was repeated 3 times for each sample.

Statistical analysis. Each test was repeated 3 times. The derived test results were processed in Microsoft Excel to calculate the mean and standard error, and significant differences between treatments were examined with SPSS 11.5 software (LSD method, significant at P < 0.05).

Acknowledgements

This study was supported by the Project of International Cooperation of Hebei (14394204D) and the Postdoctoral Fund of Hebei University.

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