Journal of Hazardous Materials 297 (2015) 173–182

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Nitric oxide ameliorates zinc oxide nanoparticles-induced phytotoxicity in rice seedlings Juan Chen a,b,1 , Xiang Liu a,1 , Chao Wang b,1 , Shan-Shan Yin a , Xiu-Ling Li a , Wen-Jun Hu a,c , Martin Simon a , Zhi-Jun Shen a , Qiang Xiao d , Cheng-Cai Chu e , Xin-Xiang Peng f , Hai-Lei Zheng a,∗ a Key Laboratory for Subtropical Wetland Ecosystem Research of MOE, College of the Environment and Ecology, Xiamen University, Xiamen, Fujian 361005, China b State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, Nanjing 210008, China c Zhejiang Academy of Agricultural Sciences, Hangzhou, Zhejiang Province 310021, China d Laboratory of Biological Resources Protection and Utilization of Hubei Province, Hubei Institutes for Nationalities, Enshi, Hubei 445000, China e State Key Laboratory of Plant Genomics and National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China f College of Life Sciences, South China Agricultural University, Guangzhou 510642, China

h i g h l i g h t s • NO can alleviate ZnO NPs-induced growth inhibition of rice seedlings. • Zn concentration in ZnO NPs-treated rice seedlings was decreased by NO. • NO alleviates ZnO NPs-induced oxidative stress by mediating antioxidant system.

a r t i c l e

i n f o

Article history: Received 27 November 2014 Received in revised form 26 April 2015 Accepted 27 April 2015 Available online 29 April 2015 Keywords: Antioxidant Nitric oxide Phytotoxicity Rice Zinc oxide nanoparticles

a b s t r a c t Nitric oxide (NO) has been found to function in enhancing plant tolerance to various environmental stresses. However, role of NO in relieving zinc oxide nanoparticles (ZnO NPs)-induced phytotoxicity remains unknown. Here, sodium nitroprusside (SNP, a NO donor) was used to investigate the possible roles and the regulatory mechanisms of NO in counteracting ZnO NPs toxicity in rice seedlings. Our results showed that 10 ␮M SNP significantly inhibited the appearance of ZnO NP toxicity symptoms. SNP addition significantly reduced Zn accumulation, reactive oxygen species production and lipid peroxidation caused by ZnO NPs. The protective role of SNP in reducing ZnO NPs-induced oxidative damage is closely related to NO-mediated antioxidant system. A decrease in superoxide dismutase activity, as well as an increase in reduced glutathione content and peroxidase, catalase and ascorbate peroxidase activity was observed under SNP and ZnO NPs combined treatments, compared to ZnO NPs treatment alone. The relative transcript abundance of corresponding antioxidant genes exhibited a similar change. The role of NO in enhancing ZnO NPs tolerance was further confirmed by genetic analysis using a NO excess mutant (noe1) and an OsNOA1-silenced plant (noa1) of rice. Together, this study provides the first evidence indicating that NO functions in ameliorating ZnO NPs-induced phytotoxicity. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Nanoparticles (NPs) are ultrafine particles that typically have at least one dimension less than 100 nm in size [1]. With the indus-

∗ Corresponding author. Tel: +86 592 218 1005; fax: +86 592 218 5889. E-mail address: [email protected] (H.-L. Zheng). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.jhazmat.2015.04.077 0304-3894/© 2015 Elsevier B.V. All rights reserved.

trial development at the nanoscale, some metal oxide NPs such as zinc oxide (ZnO), titanium dioxide (TiO2 ), copper oxide (CuO) and cerium oxide (CeO2 ), are widely applied in market goods. Among them, due to unique electronic, optical, dermatological, and antibacterial properties, ZnO NPs are used in various commercial products including batteries, pigments, catalysts, semiconductors, cosmetics, drug carriers, etc. [2]. The production, use and disposal of a large number of ZnO NPs will inevitably increase their release

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into the environment and has become a serious threat to biological systems including plants [3]. Phytotoxicity of ZnO NPs on seed germination and root development has recently been studied in lettuce, radish, and cucumber [4]. ZnO NPs also seriously inhibited wheat growth under field conditions [5] and caused genotoxicity to broad bean [6]. A limited number of studies on ZnO NPs ecotoxicity suggested several mechanisms of action. First, the release of Zn2+ from ZnO NPs in exposure media may be a possible cause for phytotoxicity [3]. Second, ZnO NPs may directly disrupt membranes or DNA [2]. Most importantly, ZnO NPs promote the generation of reactive oxygen species (ROS), • that is superoxide radical (O2 − ) release and hydrogen peroxide (H2 O2 ) production, in the absence of photochemical energy [7]. Excessive generation of ROS can induce lipid membrane peroxidation and cellular damage, which has been suggested as one of the primary reasons contributing to nanotoxicity in general [3]. ZnO NPs-induced toxicity via ROS has been extensively demonstrated and clarified in the previous studies [8]. These studies expanded and deepened our knowledge on phytotoxicity of ZnO NPs. However, to date, no information concerning how to ameliorate ZnO NPs-induced phytotoxicity is available. Nitric oxide (NO), an important signaling molecule, mediates various plant physiological and developmental processes, including stomatal closure, flowering, root formation, etc. [9,10]. NO also plays a critical role in enhancing plant tolerance to environmental stresses such as salt, drought, chilling and heavy metals [11]. An important mechanism by which NO protects plants against environmental stresses is to eliminate excessive intracellular ROS by increasing the content of antioxidants, as well as regulating antioxidant enzyme activity [11,12]. For instance, Laspina et al. [13] reported that exogenous NO alleviated cadmium-induced oxidative stress in rice and sunflower leaves by increasing the contents of low-molecular-weight antioxidants including ascorbate and reduced glutathione (GSH). Our recent study also found that NO effectively activated antioxidant enzymes and mitigated oxidative damage caused by salt in a mangrove species, Aegiceras corniculatum [14]. Therefore, it is logical to hypothesize that NO could ameliorate phytotoxicity caused by ZnO NPs in plants. If this is the case, the detoxication mechanism of NO may be related to antioxidant system. In the light of these questions, we analyzed the effects of sodium nitroprusside (SNP, a widely used NO donor) on growth (e.g., root length, shoot height, biomass and total chlorophyll (Chl) content), Zn accumulation, ROS production, lipid peroxidation, antioxidant enzyme activity and gene transcript abundance in ZnO NPs-treated rice seedlings in the present study. By employing a NO excess mutant (noe1) and an OsNOA1-silenced plant (noa1) of rice, genetic evidences were provided to further confirm the role of NO in mediating ZnO NPs-induced phytotoxicity in rice. The objective of this study is to investigate whether and how NO functions in ameliorating phytotoxicity caused by ZnO NPs in plants. This study might provide novel and useful information to nanotoxicity studies in plants.

2. Materials and methods 2.1. Characterization of ZnO NPs ZnO NPs were purchased from DK Nano Technology Co., Ltd., Beijing, China, with a purity of 99.5%, particle size of 30 nm. The morphology of ZnO NPs was examined using scanning electron microscopy (SEM, S-4800, Hitachi, Ltd., Japan) and transmission electron microscopy (TEM, H-7650, Hitachi Ltd., Japan). The image and size distribution of ZnO NPs are shown in Fig. S1, with a size

ranging between 10–70 nm and a mean size of 28 ± 4 nm. The mean size was almost the same as that claimed (30 nm) by the producer. 2.2. Plant material and treatments Rice seeds (Oryza sativa L.) were first sterilized in 5% sodium hypochlorite solution for 15 min, followed by rinsing thoroughly with sterilized water and germinated on moist filter paper at 35 ◦ C for 48 h. The germinated seeds were transferred to a hydroponic culture containing Kimura B nutrient solution as described previously [15]. Seedlings were grown in an environmentally-controlled growth chamber with a 14 h/27 ◦ C day and a 10 h/25 ◦ C night regime, a light intensity of 400 ␮mol m−2 s−1 photosynthetically active radiation, and relative humidity of about 70%. Three-day-old seedlings of uniform size were selected and used in the following experiments. Rice (cv. Jiafuzhan) seedlings were divided into the following three experimental groups. In the first group, to study the phytotoxicity of ZnO NPs, the seedlings were transferred to ZnO NPs suspensions prepared in Kimura B nutrient solution as described previously [3]. In brief, the desired ZnO NPs mixtures with concentration of 0, 50, 100, 250, 500 and 1000 mg L−1 were stirred for 5 min and later sonicated by water bath ultrasonic treatment (25 ◦ C, 100 W, 40 kHz) for 1 h. In the second group, rice seedlings were transferred to ZnO NPs suspensions containing different concentrations of SNP (0, 5, 10, 25, 50 and 100 ␮M) to select an appropriate concentration of SNP. In the third group, 2-(4-carboxyphenyl)4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) was used as a NO scavenger and K3 Fe(CN)6 as additional control for SNP decomposition. The third group consisted of a control (CK, only Kimura B nutrient solution), 250 mg L−1 ZnO NPs (N), 10 ␮M SNP (S), 250 mg L−1 ZnO NPs + 10 ␮M SNP (N + S), 250 mg L−1 ZnO NPs + 10 ␮M K3 Fe(CN)6 (N + CN), 100 ␮M cPTIO (C), 250 mg L−1 ZnO NPs + 100 ␮M cPTIO (N + C), 250 mg L−1 ZnO NPs + 10 ␮M SNP + 100 ␮M cPTIO (N + S + C). To verify the role of NO in ZnO NPs tolerance at the genetic level, a NO excess mutant (noe1), an OsNOA1-silenced plant (noa1) of rice and their corresponding wild-type rice (cv. Nipponbare and Zhonghua 11) were exposed to 0 or 250 mg L−1 ZnO NPs. The NOE1 mutant obtained from a large T-DNA-tagged population by genetic screening accumulated more NO than its wild-type plants [16]. The noa1 mutant with defect in NO synthesis-associated protein1 (NOA1) was generated via RNA interference [15]. The reduced NO production has been observed in noa1 mutant plants [16,17]. All the treatment solutions were freshly prepared before each experiment and adjusted to pH 5.8. According to the method of Lin and Xing [3], the treatment solutions were stirred three times per day with an 8 h interval, and were renewed every day to maintain a constant ZnO NPs concentration. After various treatments for 3 days, fresh roots and shoots of rice seedlings were collected for further measurements. 2.3. Plant growth measurement Root length and shoot height were photographed and measured using Image J 1.4 software (Wayne Rasband National Institute of Health, Bethesda, MD, USA). Biomass was measured after drying at 70 ◦ C for 48 h and was recorded by weighing individual seedling. Total chlorophyll (Chl) content in rice shoot was determined according to the method of Yang et al. [15]. 2.4. TEM observation Fresh roots and shoots samples were cut into 0.5 × 1.0 mm pieces and prefixed in 2.5% glutaraldehyde for 4 h. After washing with phosphate buffer solution (PBS, 0.1 M, pH 7.0), the samples

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were post fixed in 1% OsO4 for 4 h. Then the samples were dehydrated in a series of acetone, embedded in Spurr’s resin, and finally, sectioned with an ultramicrotome (Leica EM UC6 and Reichert Ultracut S., Leica Microsystems GmbH, Wetzlar, Germany). A TEM (H-7650 Hitachi, Tokyo, Japan) was used for observing the ultrastructures of rice roots and shoots.

2.5. Zn concentration determination According to the method described previously [7], the roots and shoots were washed with HNO3 (10 mM) for 10 min, followed by rinsing with deionized water to remove ZnO NPs stuck on the surface. Subsequently, the washed roots and shoots were dried at 70 ◦ C for 48 h. Next, the dried samples (200 mg) were ashed for 12 h at 550 ◦ C in a muffle furnace and then dissolved in 1 ml concentrated HNO3 . After digestion, the acid solution was diluted to an appropriate volume with deionized water. Zn concentration in the solution was determined by atomic absorption spectrophotometry (AAS, Thermo Element MKII-M6, Thermo Electron, USA). Finally, Zn concentration in roots or shoots was expressed by mg per g of dry weight.

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2.9. Total RNA extraction and quantitative real-time PCR analysis Total RNA was extracted from root or shoot samples using RNA purification reagent (Invitrogen Inc. CA, USA) according to the manufacturer’s procedure. The extracted RNA was quantified using a ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) and RNA integrity was detected by 1% agarose gel electrophoresis. RNA was reverse transcribed to firststrand cDNAs using PrimescriptTM First-Strand cDNA Synthesis Kit (TaKaRa Bio Inc., Dalian, China). The cDNA was amplified using specific primers (Table S1). The quantitative real-time PCR (qRT-PCR) reaction was performed using an ABI Prism 7300 Sequence Detection System (Applied Biosystems, CA, USA). The qRT-PCR reaction included 1 ␮l of cDNA (equivalent to 10 ng of mRNA), 0.2 ␮M of forward and reverse primers, and 5 ␮l of Faststart Universal SYBR Green Master (ROX Molecular Biochemicals, Mannheim, Germany) in a final reaction volume of 10 ␮l. Amplifications were performed according to the following conditions: 2 min at 52 ◦ C, 5 min at 95 ◦ C, and 40 cycles of 30 s at 95 ◦ C, 30 s at 56 ◦ C and 30 s at 72 ◦ C. The actin gene of rice (acquired from NCBI, Genbank accession number AB047313.1) was selected as the internal standard. Relative transcript abundance of each target gene was expressed as value relative to corresponding control sample after normalization to actin using the Ct method [24].

2.6. NO content measurement 2.10. Statistical analysis NO content in the roots and shoots of rice seedlings was visualized using the highly specific NO fluorescent probe 4-amino5-aminomethyl-2 ,7 -difluorofluorescein diacetate (DAF-FM DA, Invitrogen, Carlsbad, CA, USA) as described previously [12]. The root or shoot slices were loaded with 10 ␮M DAF-FM DA in 20 mM HEPES-NaOH buffer (pH 7.5) for 30 min. Thereafter, the samples were washed four times in fresh buffer to wash off excess fluorophore. DAF-FM DA fluorescence was imaged using a laser confocal scanning microscope (LSM 510, Zeiss, Oberkochen, Germany) with 495 nm excitation and 515 nm emission. The images acquired from the microscope were analyzed using ImageJ 1.4 software as described above. At least six root or shoot slices were measured in each treatment and the NO content was expressed as the means of relative intensity of fluorescence over the control.

2.7. Measurement of lipid peroxidation, GSH and ROS production Level of lipid peroxidation was measured by estimating malondialdehyde (MDA) content using thiobarbituric acid reaction as described by Tewari et al. [18]. GSH content was determined using an assay kit purchased from Nanjing Jiancheng Bioengineering • Institute, China. O2 − and H2 O2 content was measured following the methods of Nair and Chung [19].

2.8. Assay of antioxidant enzyme activity The enzymes were extracted according to Chen et al. [14]. Superoxide dismutase (SOD) activity was determined according to Beyer and Fridovich [20]. The definition of one unit of SOD was the enzyme amount causing 50% decrease in the rate of nitrite formation from the oxidation of hydroxylamine by superoxide. Peroxidase (POD) was assayed as described by Dimkpa et al. [8]. Catalase (CAT) activity was determined spectrophotometrically by measuring the rate of H2 O2 decrease at 240 nm [21]. Ascorbate peroxidase (APX) activity was assayed as described by Nakano and Asada [22]. The activity of SOD, POD, CAT or APX was expressed as units per mg of protein. Protein concentration in the enzyme extract was determined using the method described previously [23].

At least 15 seedlings were randomly selected and used for root length, shoot height and biomass measurement. For other measurements, four replicates were conducted in each treatment. Values in figures and tables were expressed as means ± standard error (SE). All data were subjected to one-way analysis of variance (ANOVA) followed by Tukey–Kramer’s multiple comparison test subjected to the Bonferroni correction using SPSS 19.0 (Chicago, IL, USA). A probability of p < 0.05 was considered significant. 3. Results 3.1. Growth inhibition of rice seedlings caused by ZnO NPs Toxicity of ZnO NPs to rice seedlings was evident and increased with increasing concentration of ZnO NPs (Fig. 1A). The seedling growth was markedly inhibited by ZnO NPs, especially under higher than 250 mg L−1 ZnO NPs. After 3-day of 250 mg L−1 ZnO NPs treatment, root length, shoot height and biomass were 16.8, 35.6 and 12.3% lower, respectively, than those in ZnO NPs-free control (Fig. 1B–D). Similarly, ZnO NPs also decreased dramatically the total Chl content (Fig. 1E). Due to the obvious inhibition on seedling growth, 250 mg L−1 was used as the treatment concentration of ZnO NPs in the subsequent experiments. TEM images of rice seedlings show the presence of dark dots in the roots and shoots under ZnO NPs treatment (Fig. S2). NPs were observed to concentrate on the cell wall surface. Such dark dots were not observed in the seedlings without ZnO NPs treatment (Fig. S2A and C). Thus, it can be concluded that ZnO NPs have entered into the roots and shoots of rice seedlings. 3.2. NO ameliorates the growth inhibition caused by ZnO NPs in rice seedlings The inhibition on growth caused by ZnO NPs was markedly mitigated by low concentrations of SNP (1, 5 and 10 ␮M), especially 10 ␮M SNP (Figs. S4 and S5). However, high concentrations of SNP (25, 50 and 100 ␮M) inhibited seedling growth (Fig. S4). Therefore, 10 ␮M SNP was used to investigate the role of NO in amelioration of ZnO NPs toxicity in the subsequent experiments.

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Fig. 1. Symptoms (A), root length (B), shoot height (C), biomass (D) and Chl content (E) of rice seedlings after 3-day exposure to ZnO NPs at the different concentrations (0–1000 mg L−1 ).

As shown in Fig. 2B–D, root length, shoot height and biomass of rice seedlings grown under ZnO NPs and SNP treatment were higher by 12.8, 23.5 and 10.2%, respectively, than those under ZnO NPs treatment alone. SNP also significantly increased Chl content in ZnO NPs-stressed seedlings (Fig. 2E). However, no significant improvement was found after the addition of K3 Fe(CN)6 , which was as a control to SNP. The seedling growth was comparable between the control and cPTIO treatment alone, whereas, the application of cPTIO strongly aggravated ZnO NPs-induced growth inhibition. When cPTIO added together with SNP, the role of SNP in the alleviation of ZnO NPs-induced growth inhibition was blocked (Fig. 2). NO levels in roots and shoots with different treatments were detected by fluorescence imaging as shown in Fig. 3. Compared with the control (CK, only Kimura B nutrient solution), a slight increase of relative intensity of NO fluorescence was induced by ZnO NPs. As expected, compared with other treatments, more intense green fluorescence was observed in both roots and shoots of seedlings supplied with SNP (Fig. 3A). However, no significant increase of NO fluorescence was observed after K3 Fe(CN)6 addition, and cPTIO almost completely inhibited the appearance of fluorescence (Fig. 3). These results clearly suggest that NO specifically contributed to the effects of SNP on ZnO NPs tolerance response in rice. 3.3. Effects of NO on O2 accumulation

•−

, H2 O2 , MDA and GSH content and Zn

As shown in Fig. 4, ZnO NPs treatment stimulated the produc• tion of O2 − and H2 O2 in rice seedlings. For instance, H2 O2 content

in ZnO NPs-treated rice roots and shoots increased by 21.7 and 51.9%, respectively, compared with the control. However, ZnO NPs• induced increase of O2 − and H2 O2 content was reversed by SNP addition. Lipid peroxidation, which was measured by MDA content, showed a remarkable decrease in the roots exposed to ZnO NPs (Fig. 5A). By contrast, MDA content in ZnO NPs-stressed rice leaves increased by 25.3%, compared with the control, while this effect was reversed by SNP addition (Fig. 5B). As shown in Fig. 5C and D, ZnO NPs increased significantly the GSH content in rice seedlings. Interestingly, SNP addition substantially strengthened the increasing tendency of GSH in roots and shoots, being 10.0 and 15.1% higher, respectively, than those with ZnO NPs treatment alone (Fig. 5C and D). ZnO NPs dramatically increased Zn accumulation in roots and shoots, and this increase was effectively inhibited by SNP addition (Fig. 5E and F). 3.4. NO regulates activity and gene transcript abundance of antioxidant enzymes Under ZnO NPs treatment, SOD activity increased by 18.4 and 39.1% in roots and shoots, respectively, compared with the control (Table 1). However, the increases in SOD activity were reversed by SNP addition. In contrast to SOD, ZnO NPs resulted in decreases in CAT, APX and POD activity in roots and shoots of rice seedlings. It is noteworthy that SNP significantly increased CAT, APX and POD activity, compared with ZnO NPs treatment alone (Table 1). To better understand the molecular mechanism of NO-mediated antioxidant response in ZnO NPs-stressed rice seedlings, we inves-

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Fig. 2. Symptoms (A), root length (B), shoot height (C), biomass (D) and Chl content (E) of rice seedlings after 3 days of treatments including the control (CK), 10 ␮M SNP (S), 250 mg L−1 ZnO NPs (N), 250 mg L−1 ZnO NPs + 10 ␮M SNP (N + S), 250 mg L−1 ZnO NPs + 10 ␮M K3 Fe(CN)6 (N + CN), 100 ␮M cPTIO (C), 250 mg L−1 ZnO NPs + 100 ␮M cPTIO (N + C) and 250 mg L−1 ZnO NPs + 10 ␮M SNP + 100 ␮M cPTIO (N + S + C).

Fig. 3. Endogenous NO content in root and shoot of rice seedlings after 3 days of treatments including the control (CK), 10 ␮M SNP (S), 250 mg L−1 ZnO NPs (N), 250 mg L−1 ZnO NPs + 10 ␮M SNP (N + S), 250 mg L−1 ZnO NPs + 10 ␮M K3 Fe(CN)6 (N + CN), 100 ␮M cPTIO (C), 250 mg L−1 ZnO NPs + 100 ␮M cPTIO (N + C) and 250 mg L−1 ZnO NPs + 10 ␮M SNP + 100 ␮M cPTIO (N + S + C) (A). The relative NO fluorescence intensity in root (B) and shoot (C). Values are normalized to those of the control (r.u., relative unit).

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•

Fig. 4. O2 − content in root (A) and shoot (B) and H2 O2 content in root (C) and shoot (D) of rice seedlings treated with the control (CK), 10 ␮M SNP (S), 250 mg L−1 ZnO NPs (N) and 250 mg L−1 ZnO NPs + 10 ␮M SNP (N + S) for 3 days.

Fig. 5. MDA content in root (A) and shoot (B), GSH content in root (C) and shoot (D), and Zn concentration in root (E) and shoot (F) of rice seedlings exposed to the control (CK), 10 ␮M SNP (S), 250 mg L−1 of ZnO NPs (N) and 250 mg L−1 of ZnO NPs + 10 ␮M SNP (N + S) for 3 days. Table 1 Effects of 10 ␮M SNP on activity of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and peroxidase (POD) in roots and shoots of rice seedlings exposed to 250 mg L−1 ZnO nanoparticles (ZnO NPs) for 3 days. Treatment

Control SNP ZnO NPs ZnO NPs + SNP

SOD (U mg−1 protein)

CAT (U mg−1 protein)

APX (U mg−1 protein)

POD (U mg−1 protein)

Roots

Shoots

Roots

Shoots

Roots

Shoots

Roots

Shoots

3.8 ± 0.1b 3.9 ± 0.2b 4.5 ± 0.1a 4.1 ± 0.1b

9.2 ± 0.1c 9.1 ± 0.1c 12.8 ± 1.2a 11.1 ± 0.2b

48.2 ± 3.7a 42.0 ± 3.6b 26.5 ± 1.1c 40.1 ± 2.3b

213 ± 6.2b 258 ± 14.2a 124 ± 8.8c 193 ± 7.6b

20.4 ± 2.7a 21.2 ± 0.8a 10.3 ± 1.0b 23.7 ± 0.8a

322 ± 13.6a 330 ± 20.8a 283 ± 7.0b 341 ± 7.6a

89.7 ± 6.2a 85.6 ± 5.6a 44.8 ± 3.6c 65.9 ± 6.9b

1006 ± 51b 1371 ± 29a 945 ± 28c 1179 ± 62b

Values followed by the same letter in a column are not significantly different (p = 0.05) as described by one-way ANOVA.

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Fig. 6. Relative transcript abundance of Cu/Zn-SOD (A), Mn-SOD (B), CATa (C), CATb (D), APX (E) and POD (F) in root and shoot of rice seedlings exposed to the control, 10 ␮M SNP, 250 mg L−1 of ZnO NPs and 250 mg L−1 of ZnO NPs + 10 ␮M SNP for 3 days.

tigated the effect of SNP on the relative transcript abundance of corresponding antioxidant genes including Cu/Zn-SOD, Mn-SOD, CATa, CATb, APX and POD using qRT-PCR. As shown in Fig. 6, the relative transcript abundance of these genes was comparable between the control and SNP treatment alone. In addition, ZnO NPs treatment alone increased the relative transcript abundance of Cu/Zn-SOD and Mn-SOD gene, but decreased the gene expression of CATa, CATb, APX and POD in both roots and shoots of rice seedlings compared with the control. These increases in Cu/Zn-SOD and Mn-SOD gene or decreases in CATa, CATb, APX and POD gene were effectively inhibited or reversed by SNP addition (Fig. 6). To confirm the role of NO in mediating ZnO NPs-induced phytotoxicity in rice at the genetic level, two NO producing mutants of rice, noe1 and noa1, were used and treated with ZnO NPs. Our results showed that the noe1 mutant was more tolerant but the noa1 mutant was more vulnerable to ZnO NPs treatment compared with their corresponding wild-type plants, as measured by the responses of root length, shoot height, biomass and total Chl content (Fig. S6). We next examined the relative transcript abundance of genes encoding several antioxidant enzymes in roots and shoots of the mutants and wild-type plants. As shown in Fig. 7, upon ZnO NPs treatment, the relative transcript abundance of CATa, CATb, APX and POD gene was, with few exceptions, decreased in wild-type plants compared with their untreated controls, and this decrease was further strengthened in the noa1 mutant. In contrast, compared with the wild-type of noe1, significant increases in the expression of these antioxidant genes were observed in both roots and shoots of the noe1 mutant after exposure to ZnO NPs.

4. Discussion The accumulation, persistence and impact of NPs on plant growth depend on the size and applied concentration of NPs, as well as the plant species studied [25]. In the present study, 250 mg L−1 ZnO NPs significantly inhibited the growth of rice seedlings (Fig. 1). Similar to the results in this study, high ZnO NPs concentrations often lead to significant toxicity on plants. For example, Lin and Xing [4] reported that the suspensions of ZnO NPs at the concentration of 2000 mg L−1 practically terminated root elongation of six crops including radish, rape, ryegrass, lettuce, corn and cucumber. Significant retardation of root growth was also observed when soybean seedlings were exposed to 500 mg L−1 ZnO NPs [26]. Dissolved Zn2+ was reported to be a possible cause for the toxicity of ZnO NPs [27], because high level of Zn2+ significantly inhibit plant growth and development [28]. Many previous studies showed phytotoxic dose of Zn2+ ranging from 43 to 996 mg Zn L−1 to various plant species [29]. In this study, toxic symptoms were observed in rice seedlings exposed to Zn2+ with concentrations higher than 80 mg Zn L−1 by dissolving ZnSO4 ·7H2 O into the nutrient solution (Fig. S3). However, the soluble Zn2+ in all ZnO NPs treatment solution was less than 3 mg Zn L−1 (Table S2), which was far lower than the toxic threshold of Zn2+ to rice in this study. Thus, the phytotoxicity observed in this study is likely attributed to ZnO NPs rather than Zn2+ . In agreement with previous finding by Lin and Xing [3], TEM images showed that NPs were absorbed and agglomerated on the cell wall surface of rice root and shoot cells after ZnO NPs treatment (Fig. S2), implying that the phytotoxicity of ZnO NPs was likely due to the NPs per e.

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Fig. 7. Relative transcript abundance of CATa (A), CATb (B), APX (C) and POD (D) in root and shoot of the noe1, noa1 mutants, and their corresponding wild-type plants exposed to 0 and 250 mg L−1 ZnO NPs for 3 days. WT-noe1, the wild-type plant of noe1 mutant; WT-noa1, the wild-type plant of noa1 mutant.

NO has emerged as a crucial signaling molecule involved in multiple resistant responses to environmental stresses [11]. The methodology using exogenous NO donor (SNP) provides a useful tool to investigate the biological role of NO in plants [13,14]. Green fluorescence caused by NO was observed in rice roots and shoots (Fig. 3), suggesting that the increased endogenous NO was specially induced by SNP. To pinpoint whether NO functions in resisting ZnO NPs-induced phytotoxicity in rice, the effects of SNP concentrations (0–100 ␮M) on the growth of ZnO NPs-stressed rice seedlings were considered in this study. Our results clearly demonstrated that 10 ␮M SNP reversed ZnO NPs-induced plant growth inhibition (Fig. 2, Figs. S4 and S5). This finding was in agreement with NO-induced plant tolerance to various environmental stresses including salinity, heat, heavy metal, drought, etc. [14,17]. However, high concentrations of SNP (>50 ␮M) had the opposite effect (Fig. S3), indicating that the protective role of NO during ZnO NPs stress was in a dosage-dependent manner [30]. It has been clearly and extensively demonstrated that NPs can induce toxicity via ROS-mediated cellular damage [7]. ZnO NPs have the ability to generate ROS, due to their photocatalytic activity [2]. Thwala et al. [31] reported that ZnO NPs induced the overproduction of ROS in Spirodela punctuta, and similar result was also observed in ZnO NPs-treated rice seedlings in this study (Fig. 4). Excessive generation of ROS can induce cell membrane lipid peroxidation and/or facilitate accumulation and internalization of the NPs into cells, and eventually lead to cell death [2,25]. Thus, ZnO NPs-induced ROS accumulation appears to be an important cause of plant growth inhibition. MDA, a cytotoxic product of lipid peroxidation, has been considered as an indicator of oxidative damage induced by ROS [19]. Enhanced lipid peroxidation in plants exposed to various environmental stresses has been largely reported [14,18]. In the present study, an increase in the MDA content in ZnO NPs-treated rice shoots implied the oxidative damage of ZnO NPs (Fig. 5B). NO generation by SNP supplement effectively scavenged ROS in both roots and shoots, as well as decreased MDA level in rice shoots under ZnO NPs treatment (Figs. 4 and 5). These results indicated that exogenous NO reduced oxidative damage that caused by ZnO NPs and exhibited a protective effect on rice seedlings. However, MDA content was decreased in ZnO NPs-treated rice roots, and SNP supplement did not significantly affect MDA level (Fig. 5A). The decline of MDA content in roots may be a result of increasing

antioxidant defense capacity and/or the possible biomodification of NPs in the root cells [32,33]. The activation of antioxidant system is an important adaptive strategy to reduce ROS production and relieve oxidative damage in plant stress response [18,34]. Some low-molecular-weight antioxidants including GSH play important roles in the control of the cellular redox homeostasis and H2 O2 elimination [35]. In this study, we noticed that ZnO NPs treatment increased the GSH content (Fig. 5C and D), a similar trend has also been reported in the leaves of rice subjected to CuO NPs stress [1]. Interestingly, SNP induced even higher GSH level in ZnO NPsstressed rice roots and shoots (Fig. 5C and D), indicating that NO could reduce H2 O2 accumulation and alleviate oxidative damage by regulating GSH level in rice seedlings upon ZnO NPs exposure. Besides GSH, antioxidant enzymes including SOD, POD, CAT, and APX also play critical roles in reducing ROS accumulation and maintaining cellular redox steady state in plants under environmental stresses [8,31]. Effects of NPs on antioxidant enzymes are very complex and related to the properties of NPs, treatment time and concentration, plant species and genotypes [27]. Thwala et al. [31] reported that 100 mg L−1 ZnO NPs significantly increased SOD activity in Spirodela punctuta. However, CeO2 NPs at low concentrations (≤125 mg L−1 ) resulted in an obvious decrease in CAT activity in rice roots, but no significant change was observed in SOD, POD and APX activity [36]. In this study, after ZnO NPs treatment, rice roots and shoots had a significantly higher SOD activity compared with the control (Table 1), which might be attributed to the high • level of O2 − generated by ZnO NPs stress (Fig. 4A and B). SNP addition resulted in the reduced induction of SOD activity (Table 1), • which coincided with the lower H2 O2 and O2 − content in rice seedlings exposed to SNP and ZnO NPs combined treatment (Fig. 4). POD, CAT and APX are the principal H2 O2 scavenging enzymes in plants [34]. Unlike SOD, the activity of POD, CAT and APX was decreased by ZnO NPs, but this decrease was remarkably reversed by SNP supplement (Table 1), suggesting that NO was involved in reducing H2 O2 accumulation by activating POD, CAT and APX. Although the varying activity of antioxidant enzyme has been considered as a quick activation of defense response to combat NPs-induced oxidative damage [1], knowledge on the genetic and molecular mechanisms responsible for the tolerance of plants to nanotoxicity is still very limited. Several recent studies provided new insight into the transcriptomic responses of plants upon exposure of nanomaterials. Khodakovskaya et al. [37] investigated the

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Fig. 8. Proposed model for the role of NO in alleviating ZnO NPs-induced oxidative damage in rice seedlings. Increased component is marked by an upward arrow and red color, decreased component is marked by a downward arrow and blue color. CK, the control; N, ZnO NPs treatment alone; N + S, ZnO NPs + SNP treatment. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

effects of carbon nanotubes on gene expression in tomato roots by microarray analysis, and discovered a number of genes involved in stress-related processes. The transcript expression of genes encoding antioxidant enzymes including APX and SOD were up-regulated in Arabidopsis thaliana exposed to ZnO NPs [38]. On the contrary, silver NPs decreased the gene expression level of APX in rice roots [19]. In this study, significant transcript up-regulation of Cu/Zn-SOD and Mn-SOD gene, as well as down-regulation of CATa, CATb, APX and POD gene were observed in rice seedlings upon ZnO NPs exposure (Fig. 6). The different expression patterns of genes encoding antioxidant enzymes reflect a complex gene regulation pathway in plants in response to various types of NPs [19,38]. It is known that NO plays an important role in counteracting stress-induced cytotoxic processes in plants through mediating transcript changes of genes involved in ROS production and degradation [9,10]. Xie et al. [12] found that the SNP pretreatment could block the downregulation of antioxidant-related gene expressions in A. thaliana under salt stress. Similarly, in this study, SNP addition significantly up-regulated the expression of CATa, CATb, APX and POD gene (Fig. 6), which perfectly matched the changes in corresponding antioxidant enzyme activity (Table 1). These results, along with the lower ROS level and the alleviation on growth inhibition of rice seedlings, clearly indicate that NO generated by SNP can help to reestablish the redox balance and to protect against oxidative damage in rice seedling upon ZnO NPs stress. To further confirm the alleviating role of NO in ZnO NPs-induced phytotoxicity from the genetic viewpoint, two NO producing mutants of rice, noe1 and noa1, were used in this study. The noe1 mutant accumulated more NO than its wild-type plant [16]. On the contrary, the noa1 mutant with defect in NO synthesis-associated protein1 (NOA1) reduced NO biosynthesis [15]. Genetic mutants with altered endogenous NO levels provide a powerful tool for dissecting the physiological function of NO in plants [9,17]. For example, He et al. [9] reported NO repressed the floral transition

in A. thaliana by using a mutant overproducing NO (nox1). In rice, by using noe1 mutant, Lin et al. [16] demonstrated that NO was an important mediator in H2 O2 -induced leaf cell death. In this study, compared with the wild-type plant of rice, the inhibitions on growth and gene expression of CATa, CATb, APX and POD caused by ZnO NPs were more severe in the noa1 mutant (Figs. S6 and 7). On the contrary, due to more NO accumulation, the noe1 mutant was more tolerant to ZnO NPs stress and exhibited the increased expression of antioxidant genes than its wild-type plant (Figs. S6 and 7). These results support the conclusion that NO is involved in enhancing ZnO NPs tolerance in rice through modulating antioxidant gene expression. 5. Conclusion This study provides the first evidence, to our knowledge, that NO functions in alleviating ZnO NPs-induced growth inhibition and oxidative damage in rice. We proposed a schematic model to explain the regulatory mechanism of NO against ZnO NPs stress (Fig. 8). ZnO NPs stimulated ROS production and destroyed H2 O2 scavenging system in rice seedlings through inhibiting the activity and gene expression of antioxidant enzymes including APX, CAT and POD. It is clear that NO supplied by SNP could decrease ROS accumulation, increase GSH level and reverse the effects of ZnO NPs on antioxidant enzymes, finally enhancing antioxidant defense to ZnO NPs stress in rice seedlings. Genetic analysis by using noe1 and noa1 mutants further confirmed the alleviating role of NO in ZnO NPs-induced toxicity in rice. All together, we can conclude that NO has an anti-stress property during the establishment of ZnO NPs tolerance in plants. This novel finding indicates that the usage of NO donor may be an effective approach for enhancing ZnO NPs tolerance in plants. However, there are many unsolved but very important questions regarding the effects of NO on ZnO NPs uptake and transport. Further genetic and molecular study will be

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required to better understand the detailed molecular mechanisms of NO-induced ZnO NPs tolerance in plants.

[16]

Acknowledgments [17]

We are grateful to Dr. Ming-Zhi Guo for critically editing the manuscript. This study was financially supported by the Natural Science Foundation of China (NSFC) (31300505, 31260057, 30930076), Research Fund of State Key Laboratory of Soil and Sustainable Agriculture, Nanjing Institute of Soil Science, Chinese Academy of Science (Y412201449), China Postdoctoral Science Foundation (2012M521278). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jhazmat.2015.04. 077.

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