Involvement of ethylene signaling in zinc oxide nanoparticle-mediated biochemical changes in Arabidopsis thaliana leaves

Abstract

The growing use of metallic nanoparticles in industry has resulted in their accumulation in agricultural land, which poses a serious threat to the yield and quality of crops worldwide. Various reports showed that plants face environmental stress through hormones; however, the regulation of ethylene under ZnO NP stress is still unknown. In this study, ethylene-signaling defective mutants (etr1-3 and ein2-1) were studied in comparison to Arabidopsis wild-type under ZnO NPs stress. We found that ZnO NPs significantly inhibited the growth of Arabidopsis and induced toxicity by regulating the expression of cell cycle-related genes. More importantly our results showed the involvement of ethylene signaling and biosynthesis in this process. The ZnO NPs affected the biomass, and chlorophyll and sugar contents in both the ethylene-insensitive mutants and wild-type plants. The higher ROS accumulation and increased lipid peroxidation showed that the ZnO NPs induced toxicity in Arabidopsis. The observed high antioxidant enzyme activities and mRNA transcript levels of their corresponding genes in the mutant plants clearly showed the tolerance of the ethylene-insensitive mutants compared to the wild-type plant against oxidative damage caused by ZnO NP stress. Overall, our results showed that the ethylene-insensitive mutants tolerated ZnO NP-induced stress more efficiently than WT. These results suggest that ethylene induces oxidative damage in plants under ZnO NP stress.

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Environmental significance

Zinc oxide nanoparticles (ZnO NPs) are significant metallic oxide nanoparticle due to their special physico-chemical properties. The utilization of these nano-particles is increasing in various fields. This study has significance since the number of investigations including phytohormone interaction with ZnO NPs are limited. Our findings showed that ZnO NPs induced toxicity and ethylene is involved in this process, which was further confirmed by the increased activity of the EBS::GUS lines under high exposure to ZnO NPs. These findings reaffirm the involvement of ethylene in ZnO NP toxicity. Furthermore, the significance of this investigation is that it provides insight into the modification of phytohormone pathways, which can be used to enable crops to cope with the alarming toxicity of ZnO NPs in the environment.

1. Introduction

Nanoparticles (NPs) are particles with one dimension and diameter ranging from 1 to 100 nm.¹ Due to their unique properties and novel features, NPs are utilized in various aspects of daily life and energy production, such as cosmetics, catalysts, semiconductors, drug carriers and in tomato plants.^2–4 However, their extensive use may become a health risk when they are released into the atmosphere.⁵ The use of nanoparticles in fertilizers and pesticides is the main way they enter the soil.⁶

Zinc oxide nanoparticles (ZnO NPs) are one of the most important metallic nanomaterials, which are used in various fields due to their specific chemical and physical properties.^7,8 ZnO NPs are employed in the rubber industry because they can protect rubber composites and enhance the intensity and toughness performance of high polymers.^9,10 Furthermore, due to their higher UV absorption ability, ZnO NPs are widely used in personal care products, such as sunscreen and cosmetics.¹¹ These are also used in other industries, including the electronics, concrete production, photocatalysis, and electrotechnology industries.^9,12 Moreover, ZnO is the third most widely used metal in NPs, where their estimated global production in between 550 and 33 400 tons per year.^13–15 The environmental concentration of ZnO NPs were found to be in the range of 3.1–31 μg kg⁻¹ and 76–760 μg L⁻¹ in soil and water, respectively.^16,17 ZnO NPs are the fifth most widely used materials in consumer products, as identified by a nanotechnology consumer product inventory.¹⁸

According to transcriptomic studies, nanoparticles up-regulate the expression of stress (biotic and abiotic)-related genes.^19,20 In tomato plants, nanoparticles induced ROS generation, affected photosynthetic pigments, and finally resulted in cell death.²¹ Recently, a comparative study of silver nanoparticles (AgNPs) and Ag⁺ in Arabidopsis thaliana showed that AgNPs reduced its fresh weight and photosynthetic capacity. Besides, they also showed chronic effects on plant yield and flowering time of Arabidopsis thaliana.²² Copper oxide nanoparticles with different concentrations (2, 5, 10, 20, 50, and 100 mg L⁻¹) increased the accumulation of O₂ and H₂O₂ in the roots and leaves of Arabidopsis thaliana.²³ Aluminium oxide nanoparticles (Al₂O₃ NPs) induced toxicity in wheat roots by increasing H₂O₂ levels and lipid peroxidation.²⁴ Cerium oxide (CeO₂ NPs) and indium oxide nanoparticles (In₂O₃ NPs) altered the antioxidant defense system in Arabidopsis seedlings.²⁵ A life cycle study on peanuts (Arachis hypogaea L.) demonstrated their physiological changes with different metallic nanoparticles.²⁶ Cerium oxide nanoparticles induced oxidative stress by increasing lipid peroxidation in Asparagus lettuce.²⁷ Besides plants, nanoparticles also showed toxicity in other organisms, such as titanium dioxide nanoparticles (TiO₂ NPs) induced toxicity in zebrafish including organ pathology, malformations, delay metamorphogenesis and DNA damage.²⁸

The adverse influence of ZnO NPs on plant growth and development has been reported in several studies. One of the previous studies also reported that ZnO NPs with concentrations of 100 μM and 200 μM enhanced H₂O₂ and lipid peroxidation in wheat seedlings, respectively.²⁹ Beside toxicity, ZnO NPs at a lower concentration (10 mg kg⁻¹) enhanced the biomass and net photosynthetic rate in lettuce.³⁰ In maize plants, they altered the expression pattern of different genes in their roots and shoots.³¹ Also, ZnO NPs inhibited root growth in different species of plants (Zea mays L., Brassica napus L., Cucumis sativus L., Raphanus sativus L., Lactuca sativus L., and Lolium perene L.),³² and have the capability to prompt the generation of reactive oxygen species (ROS), which may lead to cell death.^3,33 A reduction in chlorophyll production was observed in Zea mays plants grown in soil modified with 800 mg kg⁻¹ of ZnO NPs.³⁴ ZnO NPs also induced oxidative stress and altered the antioxidant enzyme activities in rice seedlings.³⁵ Therefore it is very important to study the antioxidant enzyme activities of plants under ZnO NPs stress.

Plant hormones act as signaling molecules under biotic and abiotic stress.³⁶ Among them, ethylene, which is involved in various physiological, biochemical and molecular processes during the life cycle of plants, enhanced ROS accumulation.³⁷ It was found to enhance ROS accumulation under different abiotic stresses, such as cadmium (cd), drought, high salinity, and low temperature.^38–41 A previous report suggested that the effects of oxidative stress decreased by using an ethylene inhibitor or through mutation in ethylene genes.⁴² In another report it was also mentioned that exogenous use of ethylene could accelerate ROS generation in Arabidopsis plants.⁴³

Ethylene is produced from methionine via S-adenosyl L methionine and 1-aminocyclopropane-1-carboxylic acid (ACC), which is catalysed by (ACS) 1-aminocyclopropane-1-carboxylic acid synthase and (ACO) 1-aminocyclopropane-1-carboxylic acid oxidase.⁴⁴ Two basic components of ethylene biosynthesis are ACS and ACO. Under heavy metal stress, ethylene production increase due to increased transcript levels of genes encoding these two proteins.^45,46 Exposure to heavy metal stress, such as Cd, aluminium (Al) and BPA could regulate ethylene biosynthesis, which confirmed the involvement of ethylene-induced heavy metal toxicity in plants.^47–49 Meanwhile, ethylene also plays a key role in plants adapting to abiotic stress.⁵⁰ In Arabidopsis, ethylene enhanced the accumulation of soluble solutes under drought stress by decreasing oxidative stress.^51–53 The aim of the current study was to clarify the antagonistic or synergistic interaction existing between ethylene and ZnO NPs toxicity. Investigation of ethylene-insensitive mutants and WT under ZnO NPs stress at the biochemical and molecular levels is a very convenient approach to explore the interaction between ethylene and ZnO NPs stress.

2. Materials and method

2.1. Characterisation of ZnO NPs

ZnO NPs (purity 99.9%) were purchased from Cw-nano (www.cwnano.com). The ZnO NP suspension were prepared using DI water.

The morphology and particle size of the ZnO NPs were determined via transmission electron microscopy (TEM, FEI Tecnai F20 S-TWIN). The crystal form of the ZnO NPs was detected by X-ray diffraction. The XRD data was collected in the 2θ range of 10–80°. The X-ray diffraction pattern of the ZnO NPs is shown in Fig. S1.†

The size distribution of the ZnO NPs in media (water suspension) with different concentrations (50, 100, 200, and 300 mg L⁻¹) was determined using a Malvern Zetasizer (Nano ZS, Malvern, UK). To minimize agglomeration we sonicated the different concentration of ZnO NP suspensions for 1 h before use.

2.2. Plant material and ZnO nanoparticle treatment

In this study, Arabidopsis thaliana (Columbia ecotype) wild-type and the ethylene signaling defective mutants ein2-1 and etr1-3 together with the ethylene reporter EBS::GUS transgenic lines were used. Seeds of the ethylene-insensitive mutants (etr1-3 and ein2-1) were received from the Arabidopsis Biological Resource Centre Columbus, OH, USA, as described before.⁵⁴ Seeds of all genotypes were sterilized for 10 minutes in 5% sodium hypochlorite, then washed 5 times with autoclaved distilled water and stored in the dark at 4 °C for 3 days. For six days, the seeds were grown on ½ MS medium at pH 5.8. The seedlings were placed in a growth room under controlled growth conditions for Arabidopsis, as described previously.^55–58 After six days the seedlings were transferred from the MS medium to soil pots. After two weeks, the plants were watered twice daily with 30 mL suspensions of ZnO NPs with different concentrations (0, 50, 100, 200, and 300 mg L⁻¹) and supernatant of 300 mg L⁻¹ of ZnO NPs. To obtain the supernatant, 300 mg L⁻¹ of ZnO NPs were centrifuged at 5000 rpm for 15 min. For complete separation of the Zn²⁺ ions, further supernatant was filtered using 0.22 um filters. The supernatant containing Zn²⁺ ions released from the suspension with 300 mg L⁻¹ concentration of ZnO NPs was used to evaluate if it had any effect on plant growth.

2.3. Biomass measurement

The plants were removed from the soil pots after 8 days of ZnO NPs stress. To remove debris material from the plant surface, the plants were washed with tap and distilled water, respectively. Paper towels were used to remove extra water. A digital balance was used to calculate the fresh weight (FW) of the plants, and their dry weight (DW) was measured after they were dried in a drying oven at 80 °C for 15 minutes followed by incubation at 65 °C in an incubator until completely dry.

2.4. GUS staining

To investigate the effect of ZnO NPs on ethylene biosynthesis and distribution, the expression of the ethylene reporter construct EBS::GUS was tested. GUS staining was carried out using the protocol described previously.^54,59 Leaves of the EBS::GUS lines were immersed in GUS substrate supplemented with X-gluc solution overnight in the dark at 37 °C. The stained leaves were washed with following concentrations of ethanol: 30%, 50%, 70%, 95% and 100%, for 1 h at room temperature. GUS staining in the leaves was photographed with a Leica stereo microscope.

2.5. Measurement of chlorophyll and total soluble sugar contents

After 8 days of ZnO NPs treatment, the chlorophyll contents (Chla and Chlb) of the fresh leaves were determined. Pure acetone, pure ethanol and distilled water (4.5 : 4.5 : 1) were used to extract the chlorophyll contents (Chla and Chlb). The chlorophyll (a and b) contents were measured spectrophotometrically (SPECTROstar^Nano) at wavelengths of 663 and 645 nm, respectively, using the equations reported in ref. 60.

The fresh leaves of the Arabidopsis ethylene mutants and wild-type plants were weighed (0.05 mg) and then crushed in 5 mL 95% ethanol following the method in ref. 61. The homogenized samples were centrifuged at 12 000 rpm to obtain 1 mL supernatant. 4 mL anthrone was mixed with 1 mL supernatant and heated in a water bath for 10 min. After cooling the mixture, the sugar contents were measured using a UV-VIS spectrophotometer (UV-2450, SHIM ADZU) at a wavelength of 620 nm.

2.6. Measurement of anti-oxidant enzymes activity

To measure the anti-oxidant enzymes activity, 0.3 g fresh sample was homogenized in potassium phosphate buffer (6 mL of 50 mM with pH 7.8). The homogenized samples were centrifuged at 4 °C for 15 min at 14 000 rpm and the extracted supernatant was used to further measure enzyme activities using a UV-VIS spectrophotometer (UV-2450, SHIM ADZU).

The activity of SOD was measured using the nitroblue tetrazolium (NBT) photochemical assay and protocol in ref. 62. The activity was measured spectrophotometrically at a wavelength of 560 nm.

POD activity was assessed following the protocol described in ref. 63.

The ascorbate activity (APX) was determined at a wavelength of 290 nm by following the protocol in ref. 64.

The activity of catalase (CAT) was assessed by following the method in ref. 65. The activity was measured at a wavelength of 240 nm.

2.7. Malondialdehyde analysis (MDA)

According to the method in ref. 66, 0.2 g leave sample was homogenized in 1.6 mL 10% trichloroacetic acid (TCA), centrifuged at 12 000 × g for 10 min, then the resulting supernatant was mixed with an equal volume of 0.67% 2-thiobarbituric acid (TBA), then boiled at 100 °C for 30 min and finally cooled in an ice bath. The MDA contents were measured at an OD of 532 nm and 600 nm.

2.8. Histochemical analysis of H₂O₂ and O₂ by DAB and NBT in leaves of Arabidopsis

According to the protocol in ref. 67, accumulation of H₂O₂ was determined by staining the leaves of control plants and the plants treated with 300 mg L⁻¹ of ZnO NPs with 3,3-diaminobenzidine (DAB) solution. The accumulation of O₂ was identified by the staining leaves with nitroblue tetrazolium (NBT) solution according to the method described in ref. 49.

2.9. Quantitative real-time PCR

TRIzol reagent was used to extract total RNA from the leaves of all genotypes. Quality and concentration of RNA were determined with a Nanodrop 2000 UV-vis spectrophotometer. RNA was converted into cDNA using oligo dT 18 primers and M-MLV transcriptase (Promega). The Roche LightCycler 96 quantitative PCR system was used to conduct the gene expression experiment using 96-well plates. Actin2 was used as an internal reference gene to normalize the expression of all genes. The primers used in this study are listed in Table S1.†^41,54 The mRNA transcript level of all genes were examined following the method described in ref. 57 and 68_.

2.10. Statistical analysis

The data was evaluated via two-way analysis of variance (ANOVA) using the SPSS statistical software (Chicago, USA). Tukey's HSD test at P < 0.05 was used to compare the means. The standard error was illustrated in the figures. The experiments were performed with three replicates with similar results.

3. Results and discussion

3.1. Characterization of ZnO NPs

The TEM images show that the ZnO NPs have a hexagonal shape with an average size of 30 nm (Fig. 1A and B). XRD was also used for the characterization of the ZnO NPs. The peaks in the XRD pattern of the ZnO NPs clearly show their crystalline nature. The sample exhibited diffraction peaks corresponding to the pure ZnO NPs planes of (100), (002), (101), (102), (110), (103) and (112), as shown in Fig. S1.† These diffraction peaks are consistent with the standard peaks of bulk ZnO NPs, which have a hexagonal wurtzite structure.

Fig. 1 Characterization of the ZnO NPs. (A and B) TEM images of the ZnO NPs.

Based on the particle size distribution in the exposure medium (water suspension), the hydrodynamic size and ζ potential (mV) of the ZnO NPs were investigated using a Malvern Zetasizer (Nano ZS, Malvern, UK). The hydrodynamic particle size and ζ potential at different concentrations of ZnO NPs (50, 100, 200, and 300 mg L⁻¹) are given in Table 1. The hydrodynamic particle size increased in the exposure medium compared to the solid state. Hydration layers capped on the surface of particles can increase their particle size in water. Therefore, the hydrodynamic size of the ZnO NPs was larger in water than in the solid state (TEM size). A change in particle size and agglomeration of ENPs in the exposure medium were previously reported.^69–71 Another report also indicated that larger particles formed in the exposure medium, which gravitated and left behind assemblies of smaller particles.⁷² The ZnO NPs were dispersed in water suspension and had positive ζ potential at all concentrations of ZnO NPs, as shown in (Table 1). However, the overall hydrodynamic size of the ZnO NPs decreased at higher testing concentrations (100, 200, and 300 mg L⁻¹), but their zeta potential slightly increased in comparison to that at a low concentration (50 mg L⁻¹). Similarly, as previously reported, the size of the agglomerated ZnO NPs decreased with an increase in exposure concentration, but their zeta potential did not significantly increase at higher testing concentrations (125, 250, and 500 mg L⁻¹) of ZnO NPs.⁷² These observations are consistent with our findings. Moreover, in our study, the testing concentrations (100, 200 and 300 mg L⁻¹), with a higher zeta potential proved more toxic to the plants than the lower concentration (50 mg L⁻¹), with a lower zeta potential. Nanoparticles with a positive surface charge show higher cytotoxicity compared to negatively charged nanoparticles. In this case, the uptake and translocation of positive ζ potential nanoparticles are easier compared to negative ζ potential particles.⁷³

Table 1 Size and ζ potential (mV) of the different concentration ZnO NP suspensions in water. Each data value is the mean of triplicate measurements (mean ± SE)

30 nm ZnO NPs in distilled water	Size (nm)	Zeta potential (mV)
50 mg L^−1	380.4 ± 8.7	11.2 ± 0.3
100 mg L^−1	305.06 ± 4.2	19.9 ± 0.2
200 mg L^−1	319.1 ± 3.7	19.3 ± 0.6
300 mg L^−1	296.6 ± 2.2	16.6 ± 0.3

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3.2. Physiological responses of Arabidopsis under ZnO NPs stress

To illustrate the effect of ZnO NPs on Arabidopsis plants, 22 day-old plants were subjected to different concentrations of ZnO NPs (0, 50, 100, 200 and 300 mg L⁻¹) and supernatant of 300 mg L⁻¹ of ZnO NPs in soil pots for 8 days. The results clearly showed that the rosette size and weight (FW and DW) of the wild-type plants decreased significantly at higher ZnO NPs concentration (Fig. 2A–C). The supernatant (S300) suspension of 300 mg L⁻¹ of ZnO NPs had a negligible effect on the rosette size and weight (FW, DW) of the plants. Therefore, in our further study, we selected different concentrations of ZnO NPs (0, 50, 100, 200 and 300) mg L⁻¹ without supernatant suspension. More importantly, our results revealed that the ZnO NPs significantly down-regulated the expressions of cell cycle-related genes (CYCA2-1, CYCB1-1, CYCD1-1, CYCD2-1, and E2FB) in the leaves of the WT Arabidopsis plants, as shown in Fig. 2D. The mRNA transcript level of CYCA2-1, CYCB1-1, CYCD1-1, CYCD2-1, and E2FB decreased by 8.3, 2.5, 4.16, 3.33, and 2.6 fold in the leaves of the WT seedlings in comparison to that in the control, respectively. These results suggest that ZnO NPs reduce plant growth by down-regulating the expressions of cell cycle-responsive genes.

Fig. 2 (A) Effect of the ZnO NPs on the Arabidopsis phenotype. (B) Fresh weight and (C) dry weight of the wild-type Arabidopsis plants subjected to 0, 50, 100, 200 and 300 mg L⁻¹ of ZnO NPs and supernatant of the ZnO NP suspension. (E) Relative mRNA expression of the cell cycle-responsive genes in the wild-type plants subjected to 0 and 300 mg L⁻¹ of ZnO NPs. Error bars illustrate mean ± SE. In the figure, the different letters show the significant difference by applying one way ANOVA with Tukey's HSD test at a probability of P < 0.05. One way ANOVA with LSD test was used at the probability of ** P > 0.01 and *** P > 0.001.

Nanoparticles have a negative effect on plant growth and development. They reduce the total biomass, elongation of roots and seed germination.⁷⁴ It has already been reported that ZnO NPs reduce biomass accumulation and accelerate ROS generation, which result in cell death in plants.^3,33 Although previous studies indicated the toxic effects of ZnO NPs on different plant species (lettuce, cucumber, ryegrass, radish and rape),³² to date, the involvement of ethylene in ZnO NP-induced toxicity in plants has not been investigated. According to previous studies, Zn²⁺ ions released from the supernatant of ZnO NPs did not cause any toxic effect on different plants (radish, rape, and ryegrass).³² Another report also demonstrated that the toxicity induced by ZnO NPs in rice and maize was caused by the nanoparticles themselves, not the Zn²⁺ ions released from the supernatant of the ZnO NPs.⁷⁵ Hence, we believe that in this study, the toxic effects in Arabidopsis leaves are due to ZnO NP exposure. Therefore, in our further study we did not use the supernatant of the 300 mg L⁻¹ of ZnO NP suspension.

Plants respond to harsh environments, which are most often responsible for their retarded growth and suppressed development by inhibition of mitosis.^76,77 Abiotic stresses inhibit cell progression by changing the transcript levels of cell cycle-related genes.⁷⁸ Our results demonstrated that the reduction in Arabidopsis growth and biomass could be due to the change in the mRNA transcript level of the genes related to the cell cycle under ZnO NP stress. The ZnO NPs significantly downregulated the expressions of the cell cycle genes CYCA2-1, CYCB1-1, CYCD1-1, CYCD2-1 and E2Fb, as shown in Fig. 2D. The plant hormone ethylene negatively regulates cell development, and it is also reported that ethylene signaling is involved in aluminum, alkaline and chromium Cr(VI) stress in Arabidopsis roots.^47,54,79 Recently, in another report, it was highlighted that chromium Cr(VI) stress down-regulates the mRNA expressions of cell cycle-responsive genes and also induces higher ethylene levels in Arabidopsis leaves.⁸⁰ These findings are consistent with our present study that ZnO NPs induce higher levels of ethylene in Arabidopsis leaves (Fig. 3A–C). From the results, this inhibits the cell division process in the leaves by down-regulating the expressions of cell cycle-responsive genes (Fig. 2D). Thus, it can be considered that ethylene may inhibit plant growth and development by repressing cell cycle-responsive genes expressions under ZnO NP stress. Collectively, it can be concluded that ethylene may inhibit plant growth and development by repressing cell cycle responsive genes.

Fig. 3 Involvement of ethylene signaling and biosynthesis in ZnO NP-induced toxicity and growth inhibition. (A) mRNA transcript level of the ethylene signaling genes in the wild-type leaves under 0 and 300 mg L⁻¹ of ZnO NPs, (B) mRNA transcript level of the ethylene biosynthesis genes in the wild-type leaves under 0 and 300 mg L⁻¹ of ZnO NPs, and (C) effect of different concentrations (0, 100, 200 and 300 mg L⁻¹) of ZnO NPs on the accumulation of ethylene in the leaves of the ethylene reporter EBS::GUS transgenic lines. One way ANOVA with LSD test was used at the probability of ** P > 0.01 and *** P > 0.001.

3.3. Biosynthesis and signaling of ethylene is responsible for ZnO NP-induced growth regression

The relative expression of the ethylene signaling genes (ETR1, EIN2, and EIN3) was examined under 0 and 300 mg L⁻¹ of ZnO NPs stress. The expression levels of ETR1, EIN2 and EIN3 were significantly up-regulated under 300 mg L⁻¹ of ZnO NP stress (Fig. 3A). Moreover to confirm our hypothesis, we used the EBS::GUS transgenic lines and subjected them to 0, 100, 200 and 300 mg L⁻¹ of ZnO NPs. A noticeable increase in the activity of EBS::GUS was observed, especially at a higher exposure (200 and 300 mg L⁻¹) of ZnO NPs (Fig. 3C). These findings suggest that ethylene signaling is involved in ZnO NP-induced toxicity in Arabidopsis leaves. This increased ethylene signaling-induced inhibition of plant growth under ZnO NPs stress may be due to variations in the biosynthesis of ethylene. For further confirmation of our hypothesis that the overproduction of ethylene may be responsible for growth regression in Arabidopsis under ZnO NPs stress, we examined the mRNA expression levels of the genes (ACS2, ACS5, ACS6, ACS8, ACO1 and ACO2) responsible for ethylene production under 0 and 300 mg L⁻¹ of ZnO NPs stress. All of these ethylene biosynthesis genes were significantly up-regulated at 300 mg L⁻¹ of ZnO NPs stress (Fig. 3B). Overall, these results suggest that ethylene induces ZnO NP inhibition of plant growth.

In previous reports, it was confirmed that aluminum stress increases the ethylene signaling and biosynthesis in Arabidopsis roots and inhibits plant growth.⁴⁷ However, the involvement of ethylene in ZnO NP-induced toxicity and growth inhibition in Arabidopsis leaves has not been investigated before. Copper stress significantly enhanced the expression pattern of the ACS genes in Arabidopsis thaliana.⁸¹ Copper also increased the mRNA expression levels of the ACS and ACO genes in B. oleracea.⁸² Our results here showed the up-regulation of the mRNA expression level of the ethylene signaling and biosynthesis of genes under 300 mg L⁻¹ ZnO NPs. The up-regulation of these genes in Arabidopsis leaves accounts for the greater production of ethylene under ZnO NPs stress and decreased plant growth (Fig. 3A and B, respectively). An endogenous increase in ethylene production under ZnO NPs stress may cause deleterious effects on the growth and development of Arabidopsis.⁴⁵ These findings are also in agreement with the previous report by Li et al.,⁷⁹ who suggested that alkaline stress increased the biosynthesis and signaling of ethylene in Arabidopsis roots and reduced plant growth. Our hypothesis was further confirmed by the increase in activity of the EBS::GUS transgenic line under higher ZnO NPs concentrations (Fig. 3C). This clearly indicates the involvement of ethylene in ZnO NP-induced toxicity and growth inhibition in Arabidopsis.

3.4. Ethylene mutants alleviate ZnO NP-reduced rosette biomass accumulation in Arabidopsis thaliana

To further evaluate the involvement of ethylene in plant biomass reduction under ZnO NP stress, we used ethylene-insensitive mutants for comparison with the wild-type plants. ZnO NPs at lower concentrations did not have a significant effect on the FW and DW of all three genotypes. At (200 and 300 mg L⁻¹) of ZnO NP exposure, the FW and DW of the seedlings were higher in both ethylene-insensitive mutants (etr1-3 and ein2-1) than the WT (Fig. S1†). These results clearly indicate that ethylene is involved in ZnO NP-induced rosette biomass reduction in Arabidopsis.

ZnO NPs at concentration of 2000 mg L⁻¹ decreased the biomass in different plants (lettuce, cucumber, ryegrass, radish and rape).³² Our findings were supported by the role of ethylene in plant leaf growth inhibition.⁸³ Further, another study also reported that ethylene-insensitive mutants in Arabidopsis have a higher rate of rosette biomass than the WT under high BPA stress.⁴⁹ These findings support our data that ethylene may be involved in Arabidopsis growth inhibition under ZnO NP stress.

3.5. ZnO NPs caused reduction of chlorophyll and sugar contents in WT

Leaves of the WT and ethylene-insensitive mutants (etr1-3 and ein2-1) were used to measure the chlorophyll and sugar contents. No significant chlorophyll reduction was observed in the leaves at lower concentrations of ZnO NPs. Higher doses (200 and 300 mg L⁻¹) of ZnO NPs significantly degraded the chlorophyll contents in all genotypes. This chlorophyll damage at a concentration of 300 mg L⁻¹ of ZnO NPs was observed as a reduction in chl a of 60%, 41% and 29% and chl b of 80%, 61% and 37% in WT, etr1-3 and senescence ein2-1 (Fig. 4A and B), respectively. The total chlorophyll contents were significantly reduced in the WT compared to both ethylene-insensitive mutants (Fig. 4C). Furthermore, in response to ZnO NPs, the total soluble sugar content reduction was more pronounced in WT than both ethylene-insensitive mutants (Fig. 4D). At 300 mg L⁻¹ of ZnO NPs, 38%, 35% and 25% reduction was observed in WT, etr1-3 and ein2-1, respectively. Overall, these results indicate that the ethylene mutants showed more resistance against chlorophyll degradation and sugar content reduction induced by ZnO NPs stress than the wild-type plants.

Fig. 4 Effect of ZnO NPs on the light-harvesting pigments and total soluble sugar contents. (A) Chlorophyll a, (B) chlorophyll b, (C) total chlorophyll contents, and (D) total soluble sugar contents in the Arabidopsis ethylene mutants and wild-type plants subjected to (0, 50, 100, 200 and 300 mg L⁻¹) of ZnO NPs. Error bars illustrate ± SE. In the figure, the different letters show the statistically significant difference. Two way ANOVA was used at the probability of P < 0.05.

Various reports investigated the involvement of nanoparticles in chlorophyll degradation.^84,85 In Arabidopsis, 0.5 mg L⁻¹ concentration of AgNPs was reported to inhibit chlorophyll synthesis and plant growth.⁸⁶ In another report, it was also mentioned that AgNPs with different concentrations (0.2, 0.5, and 3 mg L⁻¹) induced chlorophyll degradation.⁸⁷ A significant decrease in chlorophyll content under copper oxide nanoparticles (CuO NPs) was also reported in Arabidopsis.²³ This was the same for titanium dioxide nanoparticles (TiO₂ NPs). Titanium dioxide nanoparticles (TiO₂ NPs) reduced growth by reducing the chlorophyll pigments in Arabidopsis.⁸⁸ Furthermore, the application of cerium dioxide nanoparticles with different concentrations (1000 and 2000 ppm) resulted in almost 60% and 85% chlorophyll reduction, respectively, in Arabidopsis.⁸⁹ Senescence of plants naturally results in chlorophyll degradation,⁹⁰ and ethylene is considered as the key modulator of senescence.^91,92 This perspective of ethylene is quite consistent with our report that under ZnO NPs stress, both ethylene mutants had a higher chlorophyll content in comparison to their WT. This indicates that ethylene may enhance chlorophyll degradation in WT plants under ZnO NP stress. These findings are also consistent with an earlier report, which showed the involvement of ethylene-induced chlorophyll degradation under bisphenol A (BPA) stress.⁴⁹ In conclusion, the lower chlorophyll contents in the WT plants strongly indicate the involvement of ethylene signaling-induced chlorophyll degradation under ZnO NP stress.

3.6. Involvement of ethylene in ZnO NP-induced ROS and MDA content accumulation

ROS accumulation and lipid peroxidation are the parameters of oxidative stress commonly used as cell damage indicators under environmental stress conditions. NBT and DAB staining were used to measure the reactive oxygen species (O₂ and H₂O) accumulation in the leaves of the ethylene mutants and WT plants, respectively. After exposure to 300 mg L⁻¹ of ZnO NPs, all the genotypes had higher accumulation of H₂O₂ and O₂ compared to their untreated control plants. Moreover, the accumulation was more pronounced in the leaves of the WT compared to the ethylene mutants (Fig. 5A).

Fig. 5 Involvement of ethylene in oxidative damage under ZnO NP stress. (A) DAB and NBT assays under 0 and 300 mg L⁻¹ of ZnO NPs, (B) MDA content accumulation under different concentrations of ZnO NPs (0, 50, 100, 200 and 300 mg L⁻¹) in the leaves of the ethylene mutants and WT plants. Error bars illustrate ± SE. In the figure, the different letters show the statistically significant difference at the probability of P < 0.05.

The production of MDA contents in the tissues of plants indicates ROS formation. Our results showed that at lower ZnO NPs concentrations (50 and 100) mg L⁻¹, the MDA contents remained almost the same as that in the control plants, but significantly increased at higher doses (200 and 300 mg L⁻¹) of ZnO NPs stress in all genotypes. This increase was 244%, 165% and 115% in the leaves of the WT, etr1-3 and ein2-1 plants, respectively (Fig. 4B). Thus, the reduced MDA contents in the ethylene mutants under 300 mg L⁻¹ of ZnO NPs compared to the WT indicate that ethylene is involved in ZnO NP-induced toxicity in Arabidopsis (Fig. 5B).

Small-sized zinc oxide nanoparticles have the ability to enter and diffuse into plant cells and accelerate ROS production in plants.³ Similarly, in this study, the ZnO NPs accelerated ROS production in the Arabidopsis leaves (Fig. 5A and B). The higher rate of ROS cause lipid, protein and DNA damage, which led to cell death.⁹³ Thus, the accumulation of ROS in Arabidopsis leaves under ZnO NP stress proves to be an important factor for plant growth inhibition. Therefore, it is very important to balance the ROS in plant cells. ROS are closely associated with the signaling network of hormones.^94,95 Various studies illustrated the role of ethylene in the induction of oxidative burst.⁹⁶ In the present study, MDA accumulation in the Arabidopsis leaves proved the toxicity of the ZnO NPs (Fig. 5B). A higher MDA content was recorded in the wild-type plants than both ethylene mutants, and previous reports also illustrated the involvement of ethylene in lipid peroxidation.^97–99 It is considered that enhanced MDA contents in wild-type leaves may be toxic to cell organelles.¹⁰⁰ These results suggested that ethylene may be involved in the induction of oxidative damage and increased lipid peroxidation under ZnO NPs stress. The results of the present study are in full agreement with the previous reports about the role of ethylene in regulating MDA content accumulation under aluminium stress.¹⁰¹ Similarly, histochemical staining clearly indicated that O₂ and H₂O₂ were more accumulated in the leaves of the WT plants than both ethylene mutants (especially ein2-1) under 300 mg L⁻¹ of ZnO NP stress (Fig. 5A). The generation of ROS in response to different stress situations is common in plants,¹⁰² which is deleterious for different types of biological molecules.¹⁰³ Thus, herein, the higher accumulation of ROS in the wild-type leaves may result in toxicity to biological molecules and reduced plant growth under ZnO NP stress. These findings are consistent with the study of Cao et al.,⁴² who reported that ein2-1 mutants showed lower H₂O₂ and O₂ accumulation under paraquat. The higher accumulation of ROS in the WT leaves revealed that ethylene amplifies oxidative damage under ZnO NPs.

3.7. Ethylene-insensitive mutants tolerate ZnO NP-toxicity more efficiently

To examine the possible role of antioxidant enzymes against ZnO NP-induced ROS accumulation, the SOD, POD, APX and CAT activities were analyzed in the leaves of etr1-3 and ein2-1 and compared with that in the WT plants. The ZnO NP stress altered the SOD, POD, CAT and APX activities in all the genotypes. These activities were found to be significantly higher in the ethylene signaling-defective mutants than the WT plants. This indicated that ethylene participates in ZnO NP-induced ROS accumulation in Arabidopsis. Under ZnO NPs stress, an increasing trend was observed in SOD activity except at 100 mg L⁻¹ in both mutants and wild-type plants. However, as the dose of ZnO NPs increased (200 and 300 mg L⁻¹), it was significantly higher in both ethylene mutants than in the WT plants (Fig. 6A). Higher POD activity was observed in the etr1-3 mutant than both the wild type and ein2-1 mutant at all ZnO NPs doses. However, at 50 mg L⁻¹ of ZnO NP exposure, higher POD activity was recorded in WT than in the ein2-1 plants (Fig. 6B). In the control and at 50 mg L⁻¹ of ZnO NP exposure, the APX activity remained higher in WT than etr1-3, but at higher exposure, it significantly declined in the WT plants compared to that in both mutants (Fig. 6C). In comparison to both ethylene mutants, the CAT activity in the wild-type was higher at exposure less than 100 mg L⁻¹ ZnO NPs. However at 100, 200 and 300 mg L⁻¹ of ZnO NPs, the CAT activity was significantly higher in both mutants than in the WT plants (Fig. 6D). Overall, our results showed higher antioxidant enzyme activities in the ethylene-insensitive mutants under 300 mg L⁻¹ of ZnO NPs than that in the WT. This indicates that ethylene mutants can withstand ZnO NP-induced oxidative stress more efficiently than WT.

Fig. 6 Effect of the ZnO NPs on enzyme activity. (A) SOD, (B) POD, (C) APX, and (D) CAT activities in the leaves of the Arabidopsis ethylene mutants and wild-type plants subjected to 0, 50, 100, 200 and 300 mg L⁻¹ of ZnO NPs. Error bars illustrate ± SE. In the figure, the different letters show the statistically significant difference at the probability of P < 0.05.

For the conformation of the antioxidant enzymes response under ZnO NP stress at the molecular level, we examined the mRNA transcript levels of the genes related to the SOD, POD, APX, and CAT enzymes in the leaves of the wild-type and both ethylene mutants (Fig. 7). In all the genotypes, the relative mRNA expression level of SOD-related genes (MSD1 and CSD2) was maximum at high doses of ZnO NP stress (Fig. 7A and B). However, in comparison with WT, the transcript level of these genes remained significantly higher in both mutants. The mRNA transcript level of the POD3 gene was more up-regulated in the mutant plants exposed to ZnO NPs (especially at 200 mg L⁻¹), while the WT showed a lower transcript level of POD3 gene at high doses of ZnO NPs (Fig. 7C). In case of the ethylene mutants, the transcript level of CAT2 increased in a dose-dependent manner, while the trend of the CAT2 gene showed a decline at 300 mg L⁻¹ of ZnO NPs in all the genotypes, but it was significantly reduced in the WT than ethylene mutants (Fig. 7D). The mRNA expression level of the APX1 gene showed no significant changes in the WT at all doses of ZnO NP exposure. However, at 200 mg L⁻¹ of ZnO NP exposure, significant up-regulation of the APX1 gene was observed for ein2-1 than etr1-3 and WT (Fig. 7E). Moreover, at a higher concentration (300 mg L⁻¹) of ZnO NPs, a decrease in the expression pattern of the POD3, CAT2, and APX1 genes was observed. Taken together, our results demonstrated that WT had a lower transcript level of antioxidant enzyme-related genes than the ethylene mutants under ZnO NP stress.

Fig. 7 Effect of ZnO NPs on the transcript level of antioxidant enzyme synthesis genes. (A) MSD, (B) CSD2, (C) POD3, (D) CAT2, and (E) APX genes expression under different concentrations (0, 50, 100, 200 and 300 mg L⁻¹) of ZnO NPs in the leaves of the Arabidopsis ethylene mutants and wild-type plants. Error bars illustrate ± SE. In the figure, the different letters show the statistically significant difference at the probability of P < 0.05.

ZnO NPs enhance the production of ROS in plants.³ Plants have a defense system in the form of antioxidant enzymes, which give protection against oxidative damage.¹⁰⁴ This is similar to our data that ZnO NP exposure altered the antioxidant enzymes activities in Arabidopsis leaves. Previous reports illustrated that the coordination of CAT and SOD activities plays a protective role against oxidative stress induced by ROS (O₂ and H₂O₂).¹⁰⁵ The POD enzyme has the ability to detoxify H₂O₂.¹⁰⁶ Our data showed that the activities of this antioxidant enzyme remained significantly higher in the ethylene mutants than in the wild-type. This clearly indicated the tolerance of the ethylene mutants to oxidative damage caused by ZnO NP stress (Fig. 5A–D). The increment in these antioxidant enzyme activities is supported by the greater up-regulation of their corresponding genes in the mutant plants (Fig. 7A–E). A previous report also suggested that the ein2-1 mutant had a higher mRNA transcript level of SOD- and CAT-responsive genes under paraquat stress.⁴² The decrease in the expression patterns of the POD3, CAT2, and APX1 genes at higher exposure (300 mg L⁻¹) of ZnO NPs may be due to the higher rate of ROS accumulation in the Arabidopsis leaves. The expression patterns of some antioxidant enzyme-related genes have also been reported to be decreased under high chromium Cr(VI) and BPA stress in Arabidopsis seedlings.^49,80 The increased antioxidant activities in ethylene mutants can decrease the ROS level by converting them into oxygen and water.¹⁰⁷ Thus, the increased activation of the antioxidant defense system in the mutant plants revealed that the ethylene mutants can withstand ZnO NP toxicity more efficiently than WT.

4. Conclusions

ZnO NPs are generally toxic to plants because they induce biochemical changes, as observed by the alternations in sugar contents and chlorophyll contents, DAB and NBT staining and anti-oxidant defense system in all the genotypes. However, the WT Arabidopsis plants were more sensitive to ZnO NP toxicity compared to the ethylene-insensitive mutants. Ethylene is responsible for the down-regulation of cell cycle-related genes and reduced Arabidopsis growth under ZnO NP stress. This was confirmed by the up-regulation of the ethylene biosynthesis and signaling genes under ZnO NP exposure in the WT leaves, which was further supported by the increased activity of the EBS::GUS transgenic lines exposed to ZnO NPs. These results demonstrated the novel role of ethylene in response to ZnO NP stress. Moreover, further research on the molecular mechanisms related to the interaction between ethylene and ZnO NPs needs to be conducted.

Conflicts of interest

There are no conflicts of interest.

Acknowledgements

We are thankful to Dr. Javed Iqbal from College of Life Sciences and Oceanography, Shenzhen University, Shenzhen, P. R. China for reviewing this manuscript. The research was funded by the National Key R & D Program of China (2016YFD0100701); Major State Basic Research Development Program (973 Program, grant no. 2015CB150200); Zhejiang Provincial Natural Science Foundation of China (Grant No. LZ15C020001) and National Natural Science Foundation of China (Grant No. 31570183; 31529001; 31661143004) and China Postdoctoral Science Foundation (Grant No. 2018M632476).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c8en00971f

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