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Nitricoxideameliorateszincoxidenanoparticles-inducedphytotoxicityinriceseedlings
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Journal of Hazardous Materials 297 (2015) 173–182
Contents lists available at ScienceDirect
Journal of Hazardous Materials
j o ur nal ho me pa ge: www.elsev ier .com/ locate / jhazmat
itric oxide ameliorates zinc oxide nanoparticles-inducedhytotoxicity in rice seedlings
uan 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,artin Simon a, Zhi-Jun Shen a, Qiang Xiao d, Cheng-Cai Chu e, Xin-Xiang Peng f,ai-Lei Zheng a,∗
Key Laboratory for Subtropical Wetland Ecosystem Research of MOE, College of the Environment and Ecology, Xiamen University, Xiamen, Fujian 361005,hinaState Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, Nanjing 210008, ChinaZhejiang Academy of Agricultural Sciences, Hangzhou, Zhejiang Province 310021, ChinaLaboratory of Biological Resources Protection and Utilization of Hubei Province, Hubei Institutes for Nationalities, Enshi, Hubei 445000, ChinaState Key Laboratory of Plant Genomics and National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academyf Sciences, Beijing 100101, ChinaCollege of Life Sciences, South China Agricultural University, Guangzhou 510642, China
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.
r t i c l e i n f o
rticle history:eceived 27 November 2014eceived in revised form 26 April 2015ccepted 27 April 2015vailable online 29 April 2015
eywords:ntioxidantitric oxidehytotoxicity
a b s t r a c t
Nitric oxide (NO) has been found to function in enhancing plant tolerance to various environmentalstresses. However, role of NO in relieving zinc oxide nanoparticles (ZnO NPs)-induced phytotoxicityremains unknown. Here, sodium nitroprusside (SNP, a NO donor) was used to investigate the possibleroles and the regulatory mechanisms of NO in counteracting ZnO NPs toxicity in rice seedlings. Ourresults showed that 10 �M SNP significantly inhibited the appearance of ZnO NP toxicity symptoms. SNPaddition significantly reduced Zn accumulation, reactive oxygen species production and lipid peroxi-dation caused by ZnO NPs. The protective role of SNP in reducing ZnO NPs-induced oxidative damageis closely related to NO-mediated antioxidant system. A decrease in superoxide dismutase activity, aswell as an increase in reduced glutathione content and peroxidase, catalase and ascorbate peroxidase
iceinc oxide nanoparticles
activity was observed under SNP and ZnO NPs combined treatments, compared to ZnO NPs treatmentalone. 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 NOexcess mutant (noe1) and an OsNOA1-silenced plant (noa1) of rice. Together, this study provides the firstevidence indicating that NO functions in ameliorating ZnO NPs-induced phytotoxicity.
© 2015 Elsevier B.V. All rights reserved.
. Introduction
Nanoparticles (NPs) are ultrafine particles that typically have ateast 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.
ttp://dx.doi.org/10.1016/j.jhazmat.2015.04.077304-3894/© 2015 Elsevier B.V. All rights reserved.
trial development at the nanoscale, some metal oxide NPs suchas 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, andantibacterial properties, ZnO NPs are used in various commercial
products including batteries, pigments, catalysts, semiconductors,cosmetics, drug carriers, etc. [2]. The production, use and disposalof a large number of ZnO NPs will inevitably increase their release
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nto the environment and has become a serious threat to biologicalystems including plants [3].
Phytotoxicity of ZnO NPs on seed germination and root devel-pment has recently been studied in lettuce, radish, and cucumber4]. ZnO NPs also seriously inhibited wheat growth under field con-itions [5] and caused genotoxicity to broad bean [6]. A limitedumber of studies on ZnO NPs ecotoxicity suggested several mech-nisms of action. First, the release of Zn2+ from ZnO NPs in exposureedia may be a possible cause for phytotoxicity [3]. Second, ZnOPs may directly disrupt membranes or DNA [2]. Most importantly,nO NPs promote the generation of reactive oxygen species (ROS),hat is superoxide radical (O2
•−) release and hydrogen peroxideH2O2) production, in the absence of photochemical energy [7].xcessive generation of ROS can induce lipid membrane peroxida-ion and cellular damage, which has been suggested as one of therimary reasons contributing to nanotoxicity in general [3]. ZnOPs-induced toxicity via ROS has been extensively demonstratednd clarified in the previous studies [8]. These studies expandednd deepened our knowledge on phytotoxicity of ZnO NPs. How-ver, to date, no information concerning how to ameliorate ZnOPs-induced phytotoxicity is available.
Nitric oxide (NO), an important signaling molecule, mediatesarious plant physiological and developmental processes, includ-ng stomatal closure, flowering, root formation, etc. [9,10]. NO alsolays a critical role in enhancing plant tolerance to environmen-al stresses such as salt, drought, chilling and heavy metals [11].n important mechanism by which NO protects plants againstnvironmental stresses is to eliminate excessive intracellular ROSy increasing the content of antioxidants, as well as regulatingntioxidant enzyme activity [11,12]. For instance, Laspina et al.13] reported that exogenous NO alleviated cadmium-inducedxidative stress in rice and sunflower leaves by increasing theontents of low-molecular-weight antioxidants including ascor-ate and reduced glutathione (GSH). Our recent study also foundhat NO effectively activated antioxidant enzymes and mitigatedxidative damage caused by salt in a mangrove species, Aegicerasorniculatum [14]. Therefore, it is logical to hypothesize that NOould ameliorate phytotoxicity caused by ZnO NPs in plants. If thiss the case, the detoxication mechanism of NO may be related tontioxidant system.
In the light of these questions, we analyzed the effects of sodiumitroprusside (SNP, a widely used NO donor) on growth (e.g., root
ength, shoot height, biomass and total chlorophyll (Chl) content),n accumulation, ROS production, lipid peroxidation, antioxidantnzyme activity and gene transcript abundance in ZnO NPs-treatedice seedlings in the present study. By employing a NO excess
utant (noe1) and an OsNOA1-silenced plant (noa1) of rice, geneticvidences were provided to further confirm the role of NO in medi-ting ZnO NPs-induced phytotoxicity in rice. The objective of thistudy is to investigate whether and how NO functions in amelio-ating phytotoxicity caused by ZnO NPs in plants. This study mightrovide novel and useful information to nanotoxicity studies inlants.
. Materials and methods
.1. Characterization of ZnO NPs
ZnO NPs were purchased from DK Nano Technology Co., Ltd.,eijing, China, with a purity of 99.5%, particle size of 30 nm. The
orphology of ZnO NPs was examined using scanning electronicroscopy (SEM, S-4800, Hitachi, Ltd., Japan) and transmissionlectron microscopy (TEM, H-7650, Hitachi Ltd., Japan). The imagend size distribution of ZnO NPs are shown in Fig. S1, with a size
aterials 297 (2015) 173–182
ranging between 10–70 nm and a mean size of 28 ± 4 nm. The meansize 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% sodiumhypochlorite solution for 15 min, followed by rinsing thoroughlywith sterilized water and germinated on moist filter paper at 35 ◦Cfor 48 h. The germinated seeds were transferred to a hydroponicculture containing Kimura B nutrient solution as described previ-ously [15]. Seedlings were grown in an environmentally-controlledgrowth chamber with a 14 h/27 ◦C day and a 10 h/25 ◦C nightregime, a light intensity of 400 �mol m−2 s−1 photosyntheticallyactive radiation, and relative humidity of about 70%. Three-day-oldseedlings of uniform size were selected and used in the followingexperiments.
Rice (cv. Jiafuzhan) seedlings were divided into the followingthree experimental groups. In the first group, to study the phy-totoxicity of ZnO NPs, the seedlings were transferred to ZnO NPssuspensions prepared in Kimura B nutrient solution as describedpreviously [3]. In brief, the desired ZnO NPs mixtures with con-centration of 0, 50, 100, 250, 500 and 1000 mg L−1 were stirred for5 min and later sonicated by water bath ultrasonic treatment (25 ◦C,100 W, 40 kHz) for 1 h. In the second group, rice seedlings weretransferred to ZnO NPs suspensions containing different concen-trations of SNP (0, 5, 10, 25, 50 and 100 �M) to select an appropriateconcentration of SNP. In the third group, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) was usedas a NO scavenger and K3Fe(CN)6 as additional control for SNPdecomposition. The third group consisted of a control (CK, onlyKimura B nutrient solution), 250 mg L−1 ZnO NPs (N), 10 �MSNP (S), 250 mg L−1 ZnO NPs + 10 �M SNP (N + S), 250 mg L−1 ZnONPs + 10 �M K3Fe(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 �MSNP + 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) ofrice and their corresponding wild-type rice (cv. Nipponbare andZhonghua 11) were exposed to 0 or 250 mg L−1 ZnO NPs. The NOE1mutant obtained from a large T-DNA-tagged population by geneticscreening 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 NOproduction has been observed in noa1 mutant plants [16,17].
All the treatment solutions were freshly prepared before eachexperiment and adjusted to pH 5.8. According to the method of Linand Xing [3], the treatment solutions were stirred three times perday with an 8 h interval, and were renewed every day to maintaina constant ZnO NPs concentration. After various treatments for 3days, fresh roots and shoots of rice seedlings were collected forfurther measurements.
2.3. Plant growth measurement
Root length and shoot height were photographed and mea-sured using Image J 1.4 software (Wayne Rasband National Instituteof Health, Bethesda, MD, USA). Biomass was measured after dry-ing at 70 ◦C for 48 h and was recorded by weighing individualseedling. Total chlorophyll (Chl) content in rice shoot was deter-mined 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 mmpieces and prefixed in 2.5% glutaraldehyde for 4 h. After washingwith phosphate buffer solution (PBS, 0.1 M, pH 7.0), the samples
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J. Chen et al. / Journal of Hazar
ere post fixed in 1% OsO4 for 4 h. Then the samples were dehy-rated in a series of acetone, embedded in Spurr’s resin, and finally,ectioned with an ultramicrotome (Leica EM UC6 and Reichertltracut S., Leica Microsystems GmbH, Wetzlar, Germany). A TEM
H-7650 Hitachi, Tokyo, Japan) was used for observing the ultra-tructures of rice roots and shoots.
.5. Zn concentration determination
According to the method described previously [7], the roots andhoots were washed with HNO3 (10 mM) for 10 min, followed byinsing with deionized water to remove ZnO NPs stuck on the sur-ace. Subsequently, the washed roots and shoots were dried at 70 ◦Cor 48 h. Next, the dried samples (200 mg) were ashed for 12 ht 550 ◦C in a muffle furnace and then dissolved in 1 ml concen-rated HNO3. After digestion, the acid solution was diluted to anppropriate volume with deionized water. Zn concentration in theolution was determined by atomic absorption spectrophotometryAAS, Thermo Element MKII-M6, Thermo Electron, USA). Finally, Znoncentration in roots or shoots was expressed by mg per g of dryeight.
.6. NO content measurement
NO content in the roots and shoots of rice seedlings wasisualized using the highly specific NO fluorescent probe 4-amino--aminomethyl-2′,7′-difluorofluorescein diacetate (DAF-FM DA,
nvitrogen, Carlsbad, CA, USA) as described previously [12]. Theoot or shoot slices were loaded with 10 �M DAF-FM DA in0 mM HEPES-NaOH buffer (pH 7.5) for 30 min. Thereafter, theamples were washed four times in fresh buffer to wash offxcess fluorophore. DAF-FM DA fluorescence was imaged using aaser confocal scanning microscope (LSM 510, Zeiss, Oberkochen,ermany) with 495 nm excitation and 515 nm emission. The
mages acquired from the microscope were analyzed using ImageJ.4 software as described above. At least six root or shoot slices wereeasured in each treatment and the NO content was expressed as
he means of relative intensity of fluorescence over the control.
.7. Measurement of lipid peroxidation, GSH and ROS production
Level of lipid peroxidation was measured by estimating mal-ndialdehyde (MDA) content using thiobarbituric acid reaction asescribed by Tewari et al. [18]. GSH content was determined usingn assay kit purchased from Nanjing Jiancheng Bioengineeringnstitute, China. O2
•− and H2O2 content was measured followinghe methods of Nair and Chung [19].
.8. Assay of antioxidant enzyme activity
The enzymes were extracted according to Chen et al. [14].uperoxide dismutase (SOD) activity was determined according toeyer and Fridovich [20]. The definition of one unit of SOD washe enzyme amount causing 50% decrease in the rate of nitriteormation from the oxidation of hydroxylamine by superoxide.eroxidase (POD) was assayed as described by Dimkpa et al. [8].atalase (CAT) activity was determined spectrophotometrically byeasuring the rate of H2O2 decrease at 240 nm [21]. Ascorbate per-
xidase (APX) activity was assayed as described by Nakano andsada [22]. The activity of SOD, POD, CAT or APX was expressed asnits per mg of protein. Protein concentration in the enzyme extractas determined using the method described previously [23].
aterials 297 (2015) 173–182 175
2.9. Total RNA extraction and quantitative real-time PCR analysis
Total RNA was extracted from root or shoot samples usingRNA purification reagent (Invitrogen Inc. CA, USA) according tothe manufacturer’s procedure. The extracted RNA was quanti-fied 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 first-strand cDNAs using PrimescriptTM First-Strand cDNA Synthesis Kit(TaKaRa Bio Inc., Dalian, China). The cDNA was amplified using spe-cific primers (Table S1). The quantitative real-time PCR (qRT-PCR)reaction was performed using an ABI Prism 7300 Sequence Detec-tion System (Applied Biosystems, CA, USA). The qRT-PCR reactionincluded 1 �l of cDNA (equivalent to 10 ng of mRNA), 0.2 �M offorward and reverse primers, and 5 �l of Faststart Universal SYBRGreen Master (ROX Molecular Biochemicals, Mannheim, Germany)in a final reaction volume of 10 �l. Amplifications were performedaccording 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. Theactin gene of rice (acquired from NCBI, Genbank accession num-ber AB047313.1) was selected as the internal standard. Relativetranscript abundance of each target gene was expressed as valuerelative to corresponding control sample after normalization toactin using the ��Ct method [24].
2.10. Statistical analysis
At least 15 seedlings were randomly selected and used for rootlength, shoot height and biomass measurement. For other measure-ments, four replicates were conducted in each treatment. Values infigures 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 subjectedto the Bonferroni correction using SPSS 19.0 (Chicago, IL, USA). Aprobability 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 increasedwith increasing concentration of ZnO NPs (Fig. 1A). The seedlinggrowth was markedly inhibited by ZnO NPs, especially under higherthan 250 mg L−1 ZnO NPs. After 3-day of 250 mg L−1 ZnO NPstreatment, root length, shoot height and biomass were 16.8, 35.6and 12.3% lower, respectively, than those in ZnO NPs-free control(Fig. 1B–D). Similarly, ZnO NPs also decreased dramatically the totalChl content (Fig. 1E). Due to the obvious inhibition on seedlinggrowth, 250 mg L−1 was used as the treatment concentration of ZnONPs in the subsequent experiments.
TEM images of rice seedlings show the presence of dark dots inthe roots and shoots under ZnO NPs treatment (Fig. S2). NPs wereobserved to concentrate on the cell wall surface. Such dark dotswere not observed in the seedlings without ZnO NPs treatment (Fig.S2A and C). Thus, it can be concluded that ZnO NPs have enteredinto the roots and shoots of rice seedlings.
3.2. NO ameliorates the growth inhibition caused by ZnO NPs inrice seedlings
The inhibition on growth caused by ZnO NPs was markedlymitigated by low concentrations of SNP (1, 5 and 10 �M), espe-
cially 10 �M SNP (Figs. S4 and S5). However, high concentrationsof SNP (25, 50 and 100 �M) inhibited seedling growth (Fig. S4).Therefore, 10 �M SNP was used to investigate the role of NO inamelioration of ZnO NPs toxicity in the subsequent experiments.
176 J. Chen et al. / Journal of Hazardous Materials 297 (2015) 173–182
F nt (E)(
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ig. 1. Symptoms (A), root length (B), shoot height (C), biomass (D) and Chl conte0–1000 mg L−1).
s shown in Fig. 2B–D, root length, shoot height and biomass of riceeedlings grown under ZnO NPs and SNP treatment were higher by2.8, 23.5 and 10.2%, respectively, than those under ZnO NPs treat-ent alone. SNP also significantly increased Chl content in ZnOPs-stressed seedlings (Fig. 2E). However, no significant improve-ent was found after the addition of K3Fe(CN)6, which was as
control to SNP. The seedling growth was comparable betweenhe control and cPTIO treatment alone, whereas, the applicationf cPTIO strongly aggravated ZnO NPs-induced growth inhibition.hen cPTIO added together with SNP, the role of SNP in the allevia-
ion of ZnO NPs-induced growth inhibition was blocked (Fig. 2). NOevels in roots and shoots with different treatments were detectedy fluorescence imaging as shown in Fig. 3. Compared with theontrol (CK, only Kimura B nutrient solution), a slight increase ofelative intensity of NO fluorescence was induced by ZnO NPs. Asxpected, compared with other treatments, more intense greenuorescence was observed in both roots and shoots of seedlingsupplied with SNP (Fig. 3A). However, no significant increase ofO fluorescence was observed after K3Fe(CN)6 addition, and cPTIOlmost completely inhibited the appearance of fluorescence (Fig. 3).hese results clearly suggest that NO specifically contributed to theffects of SNP on ZnO NPs tolerance response in rice.
.3. Effects of NO on O2•−, H2O2, MDA and GSH content and Zn
ccumulation
As shown in Fig. 4, ZnO NPs treatment stimulated the produc-ion of O2
•− and H2O2 in rice seedlings. For instance, H2O2 content
of rice seedlings after 3-day exposure to ZnO NPs at the different concentrations
in ZnO NPs-treated rice roots and shoots increased by 21.7 and51.9%, respectively, compared with the control. However, ZnO NPs-induced increase of O2
•− and H2O2 content was reversed by SNPaddition. 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 leavesincreased by 25.3%, compared with the control, while this effect wasreversed by SNP addition (Fig. 5B). As shown in Fig. 5C and D, ZnONPs increased significantly the GSH content in rice seedlings. Inter-estingly, SNP addition substantially strengthened the increasingtendency of GSH in roots and shoots, being 10.0 and 15.1% higher,respectively, than those with ZnO NPs treatment alone (Fig. 5C andD). ZnO NPs dramatically increased Zn accumulation in roots andshoots, and this increase was effectively inhibited by SNP addition(Fig. 5E and F).
3.4. NO regulates activity and gene transcript abundance ofantioxidant enzymes
Under ZnO NPs treatment, SOD activity increased by 18.4 and39.1% in roots and shoots, respectively, compared with the control(Table 1). However, the increases in SOD activity were reversed bySNP addition. In contrast to SOD, ZnO NPs resulted in decreases inCAT, APX and POD activity in roots and shoots of rice seedlings. It
is noteworthy that SNP significantly increased CAT, APX and PODactivity, compared with ZnO NPs treatment alone (Table 1).To better understand the molecular mechanism of NO-mediatedantioxidant response in ZnO NPs-stressed rice seedlings, we inves-
J. Chen et al. / Journal of Hazardous Materials 297 (2015) 173–182 177
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 K3Fe(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 K3Fe(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 �MSNP + 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).
178 J. Chen et al. / Journal of Hazardous Materials 297 (2015) 173–182
Fig. 4. O2•− content in root (A) and shoot (B) and H2O2 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 1Effects 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 exposedto 250 mg L−1 ZnO nanoparticles (ZnO NPs) for 3 days.
Treatment 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
Control 3.8 ± 0.1b 9.2 ± 0.1c 48.2 ± 3.7a 213 ± 6.2b 20.4 ± 2.7a 322 ± 13.6a 89.7 ± 6.2a 1006 ± 51bSNP 3.9 ± 0.2b 9.1 ± 0.1c 42.0 ± 3.6b 258 ± 14.2a 21.2 ± 0.8a 330 ± 20.8a 85.6 ± 5.6a 1371 ± 29aZnO NPs 4.5 ± 0.1a 12.8 ± 1.2a 26.5 ± 1.1c 124 ± 8.8c 10.3 ± 1.0b 283 ± 7.0b 44.8 ± 3.6c 945 ± 28cZnO NPs + SNP 4.1 ± 0.1b 11.1 ± 0.2b 40.1 ± 2.3b 193 ± 7.6b 23.7 ± 0.8a 341 ± 7.6a 65.9 ± 6.9b 1179 ± 62b
Values followed by the same letter in a column are not significantly different (p = 0.05) as described by one-way ANOVA.
J. Chen et al. / Journal of Hazardous Materials 297 (2015) 173–182 179
F (D), APS
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tornptcdsZApwwoo
ig. 6. Relative transcript abundance of Cu/Zn-SOD (A), Mn-SOD (B), CATa (C), CATb
NP, 250 mg L−1 of ZnO NPs and 250 mg L−1 of ZnO NPs + 10 �M SNP for 3 days.
igated the effect of SNP on the relative transcript abundance oforresponding antioxidant genes including Cu/Zn-SOD, Mn-SOD,ATa, CATb, APX and POD using qRT-PCR. As shown in Fig. 6,he relative transcript abundance of these genes was compara-le between the control and SNP treatment alone. In addition,nO NPs treatment alone increased the relative transcript abun-ance of Cu/Zn-SOD and Mn-SOD gene, but decreased the genexpression of CATa, CATb, APX and POD in both roots and shootsf rice seedlings compared with the control. These increases inu/Zn-SOD and Mn-SOD gene or decreases in CATa, CATb, APX andOD gene were effectively inhibited or reversed by SNP additionFig. 6).
To confirm the role of NO in mediating ZnO NPs-induced phy-otoxicity in rice at the genetic level, two NO producing mutantsf rice, noe1 and noa1, were used and treated with ZnO NPs. Ouresults showed that the noe1 mutant was more tolerant but theoa1 mutant was more vulnerable to ZnO NPs treatment com-ared with their corresponding wild-type plants, as measured byhe responses of root length, shoot height, biomass and total Chlontent (Fig. S6). We next examined the relative transcript abun-ance of genes encoding several antioxidant enzymes in roots andhoots of the mutants and wild-type plants. As shown in Fig. 7, uponnO NPs treatment, the relative transcript abundance of CATa, CATb,PX and POD gene was, with few exceptions, decreased in wild-typelants compared with their untreated controls, and this decreaseas further strengthened in the noa1 mutant. In contrast, comparedith the wild-type of noe1, significant increases in the expression
f these antioxidant genes were observed in both roots and shootsf the noe1 mutant after exposure to ZnO NPs.
X (E) and POD (F) in root and shoot of rice seedlings exposed to the control, 10 �M
4. Discussion
The accumulation, persistence and impact of NPs on plantgrowth depend on the size and applied concentration of NPs, as wellas 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 concentrationsoften lead to significant toxicity on plants. For example, Lin andXing [4] reported that the suspensions of ZnO NPs at the concen-tration of 2000 mg L−1 practically terminated root elongation of sixcrops including radish, rape, ryegrass, lettuce, corn and cucumber.Significant retardation of root growth was also observed when soy-bean seedlings were exposed to 500 mg L−1 ZnO NPs [26]. DissolvedZn2+ was reported to be a possible cause for the toxicity of ZnO NPs[27], because high level of Zn2+ significantly inhibit plant growthand development [28]. Many previous studies showed phytotoxicdose of Zn2+ ranging from 43 to 996 mg Zn L−1 to various plantspecies [29]. In this study, toxic symptoms were observed in riceseedlings exposed to Zn2+ with concentrations higher than 80 mgZn L−1 by dissolving ZnSO4·7H2O into the nutrient solution (Fig.S3). However, the soluble Zn2+ in all ZnO NPs treatment solutionwas less than 3 mg Zn L−1 (Table S2), which was far lower than thetoxic threshold of Zn2+ to rice in this study. Thus, the phytotoxicityobserved in this study is likely attributed to ZnO NPs rather thanZn2+. In agreement with previous finding by Lin and Xing [3], TEMimages showed that NPs were absorbed and agglomerated on thecell wall surface of rice root and shoot cells after ZnO NPs treatment
(Fig. S2), implying that the phytotoxicity of ZnO NPs was likely dueto the NPs per e.
180 J. Chen et al. / Journal of Hazardous Materials 297 (2015) 173–182
F ot andt nt; W
mmfGssrcssiwie(s
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iscZeaT
ig. 7. Relative transcript abundance of CATa (A), CATb (B), APX (C) and POD (D) in roo 0 and 250 mg L−1 ZnO NPs for 3 days. WT-noe1, the wild-type plant of noe1 muta
NO has emerged as a crucial signaling molecule involved inultiple resistant responses to environmental stresses [11]. Theethodology using exogenous NO donor (SNP) provides a use-
ul tool to investigate the biological role of NO in plants [13,14].reen fluorescence caused by NO was observed in rice roots and
hoots (Fig. 3), suggesting that the increased endogenous NO waspecially induced by SNP. To pinpoint whether NO functions inesisting ZnO NPs-induced phytotoxicity in rice, the effects of SNPoncentrations (0–100 �M) on the growth of ZnO NPs-stressed riceeedlings were considered in this study. Our results clearly demon-trated that 10 �M SNP reversed ZnO NPs-induced plant growthnhibition (Fig. 2, Figs. S4 and S5). This finding was in agreement
ith NO-induced plant tolerance to various environmental stressesncluding salinity, heat, heavy metal, drought, etc. [14,17]. How-ver, high concentrations of SNP (>50 �M) had the opposite effectFig. S3), indicating that the protective role of NO during ZnO NPstress was in a dosage-dependent manner [30].
It has been clearly and extensively demonstrated that NPs cannduce toxicity via ROS-mediated cellular damage [7]. ZnO NPsave the ability to generate ROS, due to their photocatalytic activ-
ty [2]. Thwala et al. [31] reported that ZnO NPs induced theverproduction of ROS in Spirodela punctuta, and similar resultas also observed in ZnO NPs-treated rice seedlings in this study
Fig. 4). Excessive generation of ROS can induce cell membraneipid peroxidation and/or facilitate accumulation and internaliza-ion of the NPs into cells, and eventually lead to cell death [2,25].hus, ZnO NPs-induced ROS accumulation appears to be an impor-ant cause of plant growth inhibition. MDA, a cytotoxic product ofipid peroxidation, has been considered as an indicator of oxida-ive damage induced by ROS [19]. Enhanced lipid peroxidation inlants exposed to various environmental stresses has been largelyeported [14,18]. In the present study, an increase in the MDA con-ent in ZnO NPs-treated rice shoots implied the oxidative damagef ZnO NPs (Fig. 5B).
NO generation by SNP supplement effectively scavenged ROSn both roots and shoots, as well as decreased MDA level in ricehoots under ZnO NPs treatment (Figs. 4 and 5). These results indi-ated that exogenous NO reduced oxidative damage that caused by
nO NPs and exhibited a protective effect on rice seedlings. How-ver, MDA content was decreased in ZnO NPs-treated rice roots,nd SNP supplement did not significantly affect MDA level (Fig. 5A).he decline of MDA content in roots may be a result of increasingshoot of the noe1, noa1 mutants, and their corresponding wild-type plants exposedT-noa1, the wild-type plant of noa1 mutant.
antioxidant defense capacity and/or the possible biomodification ofNPs in the root cells [32,33]. The activation of antioxidant systemis an important adaptive strategy to reduce ROS production andrelieve oxidative damage in plant stress response [18,34]. Somelow-molecular-weight antioxidants including GSH play importantroles in the control of the cellular redox homeostasis and H2O2elimination [35]. In this study, we noticed that ZnO NPs treatmentincreased the GSH content (Fig. 5C and D), a similar trend has alsobeen reported in the leaves of rice subjected to CuO NPs stress[1]. Interestingly, SNP induced even higher GSH level in ZnO NPs-stressed rice roots and shoots (Fig. 5C and D), indicating that NOcould reduce H2O2 accumulation and alleviate oxidative damageby 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 andmaintaining cellular redox steady state in plants under environ-mental stresses [8,31]. Effects of NPs on antioxidant enzymes arevery complex and related to the properties of NPs, treatment timeand concentration, plant species and genotypes [27]. Thwala et al.[31] reported that 100 mg L−1 ZnO NPs significantly increased SODactivity in Spirodela punctuta. However, CeO2 NPs at low concentra-tions (≤125 mg L−1) resulted in an obvious decrease in CAT activityin rice roots, but no significant change was observed in SOD, PODand APX activity [36]. In this study, after ZnO NPs treatment, riceroots and shoots had a significantly higher SOD activity comparedwith the control (Table 1), which might be attributed to the highlevel of O2
•− generated by ZnO NPs stress (Fig. 4A and B). SNP addi-tion resulted in the reduced induction of SOD activity (Table 1),which coincided with the lower H2O2 and O2
•− content in riceseedlings exposed to SNP and ZnO NPs combined treatment (Fig. 4).POD, CAT and APX are the principal H2O2 scavenging enzymesin plants [34]. Unlike SOD, the activity of POD, CAT and APX wasdecreased by ZnO NPs, but this decrease was remarkably reversedby SNP supplement (Table 1), suggesting that NO was involved inreducing H2O2 accumulation by activating POD, CAT and APX.
Although the varying activity of antioxidant enzyme has beenconsidered as a quick activation of defense response to combatNPs-induced oxidative damage [1], knowledge on the genetic and
molecular mechanisms responsible for the tolerance of plants tonanotoxicity is still very limited. Several recent studies providednew insight into the transcriptomic responses of plants upon expo-sure of nanomaterials. Khodakovskaya et al. [37] investigated the
J. Chen et al. / Journal of Hazardous Materials 297 (2015) 173–182 181
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ig. 8. Proposed model for the role of NO in alleviating ZnO NPs-induced oxidativeolor, decreased component is marked by a downward arrow and blue color. CK, thef the references to color in this figure legend, the reader is referred to the web ver
ffects of carbon nanotubes on gene expression in tomato roots byicroarray analysis, and discovered a number of genes involved in
tress-related processes. The transcript expression of genes encod-ng antioxidant enzymes including APX and SOD were up-regulatedn Arabidopsis thaliana exposed to ZnO NPs [38]. On the contrary,ilver NPs decreased the gene expression level of APX in rice roots19]. In this study, significant transcript up-regulation of Cu/Zn-SODnd Mn-SOD gene, as well as down-regulation of CATa, CATb, APXnd POD gene were observed in rice seedlings upon ZnO NPs expo-ure (Fig. 6). The different expression patterns of genes encodingntioxidant enzymes reflect a complex gene regulation pathwayn plants in response to various types of NPs [19,38]. It is knownhat NO plays an important role in counteracting stress-inducedytotoxic processes in plants through mediating transcript changesf genes involved in ROS production and degradation [9,10]. Xiet al. [12] found that the SNP pretreatment could block the down-egulation of antioxidant-related gene expressions in A. thalianander salt stress. Similarly, in this study, SNP addition signifi-antly up-regulated the expression of CATa, CATb, APX and PODene (Fig. 6), which perfectly matched the changes in correspond-ng antioxidant enzyme activity (Table 1). These results, along withhe lower ROS level and the alleviation on growth inhibition ofice seedlings, clearly indicate that NO generated by SNP can helpo reestablish the redox balance and to protect against oxidativeamage in rice seedling upon ZnO NPs stress.
To further confirm the alleviating role of NO in ZnO NPs-inducedhytotoxicity from the genetic viewpoint, two NO producingutants of rice, noe1 and noa1, were used in this study. The noe1utant accumulated more NO than its wild-type plant [16]. On the
ontrary, the noa1 mutant with defect in NO synthesis-associatedrotein1 (NOA1) reduced NO biosynthesis [15]. Genetic mutants
ith altered endogenous NO levels provide a powerful tool forissecting the physiological function of NO in plants [9,17]. Forxample, He et al. [9] reported NO repressed the floral transition
ge in rice seedlings. Increased component is marked by an upward arrow and redol; N, ZnO NPs treatment alone; N + S, ZnO NPs + SNP treatment. (For interpretationf this article.)
in A. thaliana by using a mutant overproducing NO (nox1). In rice,by using noe1 mutant, Lin et al. [16] demonstrated that NO wasan important mediator in H2O2-induced leaf cell death. In thisstudy, compared with the wild-type plant of rice, the inhibitionson growth and gene expression of CATa, CATb, APX and POD causedby ZnO NPs were more severe in the noa1 mutant (Figs. S6 and 7). Onthe contrary, due to more NO accumulation, the noe1 mutant wasmore tolerant to ZnO NPs stress and exhibited the increased expres-sion of antioxidant genes than its wild-type plant (Figs. S6 and 7).These results support the conclusion that NO is involved in enhanc-ing ZnO NPs tolerance in rice through modulating antioxidant geneexpression.
5. Conclusion
This study provides the first evidence, to our knowledge, thatNO functions in alleviating ZnO NPs-induced growth inhibitionand oxidative damage in rice. We proposed a schematic model toexplain the regulatory mechanism of NO against ZnO NPs stress(Fig. 8). ZnO NPs stimulated ROS production and destroyed H2O2scavenging system in rice seedlings through inhibiting the activityand gene expression of antioxidant enzymes including APX, CATand POD. It is clear that NO supplied by SNP could decrease ROSaccumulation, increase GSH level and reverse the effects of ZnONPs on antioxidant enzymes, finally enhancing antioxidant defenseto ZnO NPs stress in rice seedlings. Genetic analysis by using noe1and noa1 mutants further confirmed the alleviating role of NO inZnO NPs-induced toxicity in rice. All together, we can concludethat NO has an anti-stress property during the establishment ofZnO NPs tolerance in plants. This novel finding indicates that the
usage of NO donor may be an effective approach for enhancing ZnONPs tolerance in plants. However, there are many unsolved butvery important questions regarding the effects of NO on ZnO NPsuptake and transport. Further genetic and molecular study will be
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cknowledgments
We are grateful to Dr. Ming-Zhi Guo for critically editing theanuscript. This study was financially supported by the Natu-
al Science Foundation of China (NSFC) (31300505, 31260057,0930076), Research Fund of State Key Laboratory of Soil andustainable Agriculture, Nanjing Institute of Soil Science, Chinesecademy of Science (Y412201449), China Postdoctoral Scienceoundation (2012M521278).
ppendix A. Supplementary data
Supplementary data associated with this article can be found, inhe online version, at http://dx.doi.org/10.1016/j.jhazmat.2015.04.77.
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