Physiological and Biochemical Changes Imposed by CeO2

Subscriber access provided by UNIV OF NEBRASKA - LINCOLN

Article

Physiological and Biochemical Changes Imposed by CeO2 Nanoparticles on Wheat: A Life Cycle Field Study Wenchao Du, Jorge L Gardea-Torresdey, Rong Ji, Ying Yin, Jianguo Zhu, Jose R Peralta-Videa, and Hongyan Guo Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b03055 • Publication Date (Web): 14 Sep 2015 Downloaded from http://pubs.acs.org on September 15, 2015

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Environmental Science & Technology is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 40

Environmental Science & Technology

1

Physiological and Biochemical Changes Imposed by CeO2

2

Nanoparticles on Wheat: A Life Cycle Field Study

3

Wenchao Du,† Jorge L. Gardea-Torresdey,‡,£,€ Rong Ji,† Ying Yin,† Jianguo

4

Zhu,§ Jose R. Peralta-Videa‡,£,€ and Hongyan Guo*, †

5 6

†

7

Environment, Nanjing University, Nanjing 210046, China

8

‡

9

79968, United States

State Key Laboratory of Pollution Control and Resource Reuse, School of

Department of Chemistry, The University of Texas at El Paso, El Paso, Texas

10

£

11

at El Paso, El Paso, Texas 79968, United States

12

€

13

(UC CEIN), The University of Texas at El Paso, El Paso, Texas 79968, United

14

States

15

§

16

Science, Chinese Academy of Science, Nanjing 210008, China

17

*Corresponding author. Tel.: +86-25-89680263; Fax: +86-25-89680263.

18

E-mail address: [email protected] (Guo H Y).

Environmental Science and Engineering PhD program, The University of Texas

University of California Center for Environmental Implications of Nanotechnology

State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil

1

ACS Paragon Plus Environment


19

ABSTRACT

20

Interactions of nCeO2 with plants have been mostly evaluated at seedling stage

21

and under controlled conditions. In this study, the effects of nCeO2 at 0 (control),

22

100 (low), and 400 (high) mg/kg were monitored for the entire life cycle (about 7

23

months) of wheat plants grown in a field lysimeter. Results showed that at high

24

concentration nCeO2 decreased the chlorophyll content and increased catalase

25

and superoxide dismutase activities, compared with control. Both concentrations

26

changed root and leaf cell microstructures by agglomerating chromatin in nuclei,

27

delaying flowering by 1 week, and reduced the size of starch grains in endosperm.

28

Exposed to low concentration produced embryos with larger vacuoles, while

29

exposure to high concentration reduced number of vacuoles, compared with

30

control. There were no effects on the final biomass and yield, Ce concentration in

31

shoots, as well as sugar and starch contents in grains, but grain protein increased

32

by 24.8% and 32.6% at 100 and 400 mg/kg, respectively. Results suggest that

33

more field life cycle studies are needed in order to better understand the effects of

34

nCeO2 in crop plants.

35

Keywords: CeO2 nanoparticles, Wheat, Chromatin, Starch, Grain protein

2


Page 2 of 40

Page 3 of 40


36

INTRODUCTION

37

The application of engineered nanoparticles (ENPs) in agriculture and water

38

remediation could lead to their accumulation in ecosystems and entrance into the

39

food chain.1 The safe use of ENPs in agriculture requires a thorough

40

understanding of their interaction with edible plants, including physiological and

41

biochemical responses and accumulation. Increasing numbers of studies

42

concerning the interaction of ENPs with plants have emerged, revealing not only

43

negative but also positive or inconsequential effects.2

44

Cerium oxide nanoparticles (nCeO2) have been widely used in applications

45

such as chemical mechanical planarization, fuel catalysis, UV protection coatings,

46

and paints. Hence, concerns about an unintentional release of these ENPs with

47

possible toxic effects on organisms, particularly plants, have increased.3,4 It is

48

believed that biosolid application to agricultural soils serves as one of the largest

49

environmental pathways for the exposure of terrestrial systems to nCeO2.5

50

Limbach et al. reported a significant amount of nCeO2 (up to 6 % by mass) in the

51

exit stream of a model wastewater treatment plant.6 nCeO2 are stable and

52

undergo a very limited dissolution in environmental media and plant tissues,5, 7-9

53

and may accumulate in vegetative and reproductive organs,10 thereby impacting

54

germination,11,12 growth,13 flowering,14 and production.3 However, most of the

55

existing data has been gathered from short-term hydroponic studies,15 focused

56

primarily on early growth stages.16 In addition, most of these studies have been 3



57

performed in controlled environments that greatly differ from field conditions,

58

which are more complex and constantly changing. There are limited studies

59

investigating the long-term effects of nCeO2 on plants grown in soil. Thus, little is

60

known about the effects of prolonged exposure on plant production, Ce

61

accumulation in edible/reproductive organs, and nutritional value of edible tissues.

62

Rico et al.17 reported that in greenhouse conditions nCeO2 at 500 mg/kg improved

63

wheat grain yield by 36.6%, while at 250 mg/kg reduced accumulation of S and

64

Mn in grains. However, to the authors’ knowledge, field studies about the effects

65

of nCeO2 on wheat grown to full maturity have not been reported yet.

66

Wheat is one of the most important global crops. It is therefore essential to

67

understand the effects of the interaction of this plant with nCeO2 under field

68

conditions. Wheat plants were grown until full maturity in outdoor lysimeters with

69

undisturbed soil monoliths mimicking field conditions.18 The chlorophyll content,

70

malondialdehyde and activities of antioxidant enzymes superoxide dismutase

71

(SOD) and catalase (CAT) were measured in seedlings. Changes in ultrastructure

72

of seedling root and shoot as well as developing process of grain embryo and

73

endosperm were monitored. At harvest, biomass, yield and nutritional quality of

74

wheat grains were analyzed.

75 76 77

EXPERIMENTAL CeO2 nanoparticles nCeO2 have been characterized by Keller et al.19 They are 4


Page 4 of 40

Page 5 of 40


78

rods with a primary size of 8 ± 1 nm, a particle size of 231 ± 16 nm in deionized

79

water, a surface area of 93.8 m2 g−1, and 95.14% purity.19

80

Lysimeters. Lysimeters used were set up in 2000 and are located in Changshu

81

Agroecological Experimental Station, the Chinese Academy of Science

82

(Changshu, Jiangsu, China) (31°32’45”N, 120°41‘57“E), as previously described

83

by Du et al.18 The soil was classified as silty clay with a pH of 7.36. In the top 20

84

cm of the soil, the sand, silt and clay contents were 32.6%, 44.7% and 22.7%,

85

respectively. The organic carbon concentration was 4.6% and the concentration

86

of elemental Ce in soil was 89.9 ± 5.5 mg kg−1. In November 2012, the plowed

87

layers of soil (0–20 cm depth; about 110 kg) were removed from the nine

88

lysimeters, air-dried, crushed, and three of them were mixed thoroughly either

89

with 10 or 45 g of nCeO2, yielding approximately 100 and 400 mg nCeO2 per kg of

90

dry soil. These concentrations are chosen because of previous studies (125-500

91

mg kg−1) carried in greenhouse.3,17 The other three were used as control (no NPs).

92

Each lysimeter was 80 cm in diameter. The prepared soil was added back to the

93

lysimeters. Winter wheat (Triticum aestivum L.) was sown (100 seeds per

94

lysimeter) in November 2012 and harvested in June 2013. Field management of

95

plants followed agricultural practices of the local farmers. Nitrogen fertilizer was

96

provided during tillering stage as urea (150 kg h-1). There was no artificial

97

irrigation during the entire wheat growing season. Analysis of leachates collected

98

from the lysimeters did not show vertical movement of Ce ions or nCeO2. 5



Page 6 of 40

99

Microscopy study of wheat samples. Fresh wheat seedlings were sampled

100

(30 days after germination) and thoroughly washed with deionized water. Root

101

tips and leaves were cut in transverse sections, coated with gold for 60 s(ca. 1 nm

102

gold layer) by using a Sputter Coater (HITACHI E-1010, Japan), and then

103

observed by scanning electron microscopy (SEM; PHILIPS SEM-505, The

104

Netherlands) with Energy Dispersive Spectrometer (EDS) analysis. Root tips and

105

leaves were sliced for transmission electron microscopy using a microtome with

106

diamond knife (TEM; HITACHI H-600, Japan). Grains were sampled at 20, 27,

107

and 34 days after flowering (DAF) and sliced in transverse and longitudinal

108

sections. All samples for TEM were prepared following standard procedures.20

109

Briefly, samples were prefixed in 3% glutaraldehyde, washed in 0.1M phosphate

110

buffer (pH 7.0), postfixed in 1% osmium tetroxide, dehydrated in acetone, and

111

then infiltrated and embedded in epoxy resin.

112

Chlorophyll, malondialdehyde, and enzyme measurement. Fresh wheat

113

seedlings were sampled (30 days after germination) and analyzed for chlorophyll

114

content, malondialdehyde content, and enzyme activities. Chlorophyll was

115

extracted using ethanol and measured at 470, 649 and 665 nm according to the

116

method

117

thiobarbituric acid assay by monitoring contents of thiobarbituric acid reactive

118

substances at 459, 532 and 600 nm according to the method of Ohkawaet al.22

119

Enzymes were extracted following the protocol described by Lin et al.23 The

of

Lichtenthaler.21

Malondialdehyde

was

6


measured

using

the

Page 7 of 40


120

protein content was determined using the Bradford method.24 SOD activity was

121

assayed by the inhibition of nitroblue tetrazolium reduction at 560 nm according to

122

the method of Dhindsa et al.25 One unit of SOD activity was defined as the

123

amount of enzyme that decreased the rate of nitroblue tetrazolium reduction by

124

50%. CAT activity was assayed by determining the degradation of H2O2 at 240 nm

125

according to the method of Cakmak et al.26 One unit of CAT activity was

126

expressed as the amount of enzyme required to degrade 1 µmol of H2O2 per

127

minute.

128

Biomass, Ce and nutritional contents of harvested wheat. Mature plants

129

were harvested, thoroughly washed with tap water followed by rinsing with

130

deionized water, air-dried, weighed, divided into four parts (root, leaf, stem, grain),

131

and then ground to pass through a 0.2-mm sieve. Ce-containing sieved plant

132

materials were digested with HNO3 and HClO4 (1:4, v/v) and quantified using

133

inductively

134

Perkin-Elmer, USA). Nitrogen content was determined using the Kjeldahl method

135

and protein concentration was calculated as total nitrogen content multiplied by

136

5.7.27 Reducing sugar was determined using the 3,5-dinitrosalicylic acid method

137

and calculated using standard calibration curve at 620 nm with glucose as the

138

standard. Glucose oxidase method was used to determine glucose concentration.

139

Starch and total sugar were determined using standard calibration curve at 490

140

nm with glucose as the standard.28

coupled

plasma-mass

spectroscopy

(ICP-MS;

7


NEXION

300,


141

Statistical analyses. The data were expressed as mean ± standard deviation

142

of three or four replicates. Statistical differences among treatments were

143

determined using one-way analysis of variance (ANOVA) followed by Tukey's

144

pair-wise comparisons at a significance level of 0.05. Covariance analysis was

145

also performed.

146 147

RESULTS AND DISCUSSION

148

Effect on structure of seedling organelles. Figure 1 shows TEM micrographs

149

of root tips from control plants and plants exposed to 100 and 400 mg nCeO2 per

150

kg soil. TEM images 1 E and 1 F show that plants exposed to both concentrations

151

had some black particles in nanosize adhered to the cell wall of periderm. In

152

contrast, neither the periderm nor the root cells of control plants show CeO2

153

nanoparticle or aggregates (Fig. 1 D). In addition, the microstructure of root cells

154

of wheat grown with nCeO2 was affected. Winter wheat does not have a period of

155

hibernation, and under normal circumstances, the chromatin is dispersed.29 As

156

seen in Fig. 1A, chromatin in the nucleus of cells in control roots dispersed without

157

agglomeration, which confirmed the status of normality. In contrast, in plants

158

exposed to nCeO2, the chromatin in nuclei was agglomerated into sections (Fig.1

159

B and C). Chromatin condensation is a sign of stress and may cause repression

160

of DNA transcription and mRNA synthesis, thereby inhibiting polyribosome

161

formation and protein synthesis in the cytoplasm, resulting in a reduced growth 8


Page 8 of 40

Page 9 of 40


162

rate that could end in the death of the plant.30 However, in less severe cases,

163

chromatin condensation is temporary and reversible, and it can be an internal

164

mechanism to induce physiological dormancy by repressing transcription.31 It is

165

possible that the nCeO2 effects on wheat were reversible, as the plants completed

166

their life cycle with a delay of 1 week (Fig. S1).

167

nCeO2 also impacted leaf cell organelles (Fig. 2). In the leaves of plants

168

exposed to nCeO2, the chloroplasts were swollen (Fig. 2 B and C) with squeezed

169

nuclei (Fig. 2 I), and thylakoids were bent and loosely arranged (Fig. 2 E and F).

170

Such changes in chloroplasts and thylakoids are an important mechanism used

171

by plants to adapt to adversity by adjusting the light absorption to avoid excessive

172

damage,30 suggesting important disturbances in metabolic functions of these

173

organelles which would result in a low photosynthetic capacity.32

174

Although no nCeO2 aggregates or individual particles were found in TEM

175

images of root cells or leaf mesophyll cells, Ce distribution pattern in cross

176

sections of root tips and leaf were obtained using SEM with EDS analysis (Fig. 3).

177

Ce was internalized in exposed roots, mainly located in epidermis; some was

178

observed in parenchyma region, and not detected in vascular cylinder, indicating

179

that most of nCeO2 remained in root epidermis region, which also conformed

180

those dark particles adhered to root surfaces in TEM images are nCeO2 (Fig. 1 E,

181

F). nCeO2 adsorbed to the wheat root surfaces and located mainly in epidermis

182

region possibly because of the high affinity of the nanoparticles for the epidermis 9



183

and the waxy casparian strips of the roots.33 However, Ce was detected in leaf

184

vein, proving that nCeO2/ionic Ce reached the transport system being acropetally

185

translocated from roots to shoots.34 Previous studies have shown that ENPs in

186

shoots tend to be concentrated or restricted to locations near or within vascular

187

tissues, acropetally translocated via transpiration from roots to shoots, sharing the

188

vascular system with water and nutrients, and consequently, impacting plant

189

development.33,35

190

Effect of nCeO2 on chlorophyll content. Photosynthesis is a sensitive

191

physiological process, vulnerable to environmental stresses.36 Therefore, the

192

photosynthetic performance of a plant under stressful conditions can be indicative

193

of plant adaptability.37 Accordingly, we measured the chlorophyll content in leaves

194

as an indicator of the plant photosynthesis performance. Chlorophyll data from

195

experimental plants is shown in Table 1. As seen in this table, plants exposed to

196

400 mg/kg had significantly less total chlorophyll, and chlorophyll a and b,

197

compared with control and plants exposed to 100 mg per kg. Chlorophyll a is the

198

major photosynthetic pigment in plants, and sensitive to photodegradation. Lower

199

chlorophyll a content indicates a lower photosynthetic rate in wheat plants

200

exposed to nCeO2. These findings are in agreement with previous results that

201

have shown reduction of chlorophyll content in A. thaliana (at 1000 and 2000

202

mg/kg)38 and rice leaves (at 125 and 500 mg/kg)10 due to nCeO2 exposure.

203

Although the mechanism of phytotoxicity of nCeO2 remains unknown, their 10


Page 10 of 40

Page 11 of 40


204

aggregation size was considered to be one of main factors causing the decrease

205

in chlorophyll content.10 A decrease in chlorophyll contents is correlated with

206

disturbance in chloroplast structure and disorganization of thylakoid in leaf

207

mesophyll cells of wheat exposed to nCeO2 (Fig. 2). These changes have the

208

potential to compromise the overall plant growth and vigor.38

209

Effect of nCeO2 on antioxidant defense system. To test the effect of nCeO2

210

on the antioxidant defense system of wheat, we measured CAT and SOD

211

activities and the concentration of thiobarbituric acid reactive substances

212

(TBARS). Both CAT and SOD activities in shoots of plants exposed to 400 mg/kg

213

nCeO2 were significantly higher, compared to the other treatments (Fig. 4 A and

214

B), providing a circumstantial evidence for the production of O2- and H2O2 and the

215

oxidative stress in wheat plants by nCeO2. A similar increase in CAT activity has

216

also been reported for corn (Zea mays) shoots in plants grown with 400 and 800

217

mg nCeO2 per kg soil and in cilantro shoots grown with 62.5 mg nCeO2 per kg

218

soil.39,40 On the other hand, none of the nCeO2 treatments effected the

219

concentration of TBARS (Fig. 4 C), which suggests limited lipid peroxidation and

220

membrane damage. Similar results were found in corn cultivated under

221

greenhouse conditions;39 however, a contrasting result was found in shoots of

222

hydroponically grown rice (Oryza sativa) exposed to 500 mg nCeO2 per liter.10

223

This discrepancy with previous studies could result from differences in plant

224

species, as well as different dissolution levels of nCeO2 in dissimilar experimental 11



225

media. As previously reported, the dissolution of nCeO2 is considerably lower in

226

soil than in liquid.5 It is also possible that wheat plants have different protective

227

mechanisms to combat the stress imposed by nCeO2. On the other hand, nCeO2

228

are known to have a radical scavenging ability and induce a protective cellular

229

response.41,42 The higher CAT activity at high nCeO2 concentrations could

230

probably be explained by the SOD mimetic activity of nCeO2 where the end

231

product is H2O2.10 The high activity of antioxidant enzymes caused a significant

232

reduction in O2- and H2O2 contents and had a preponderant role in controlling the

233

oxidative stress, thereby avoiding the potential membrane damage.10

234

Effect on grain development. Figures 5 and 6 show TEM images of embryo

235

and endosperm of grains at 20 DAF, 27 DAF and 34 DAF. As shown in Figure

236

5(A-C), embryos of plants exposed to 100 mg/kg had larger vacuoles (Fig. 5 B),

237

while at 400 mg/kg had less vacuoles (Fig. 5 C), compared with control grains (Fig.

238

5 A) at 20 DAF. Starch granules (Fig. 5 D–F) were more numerous and larger in

239

plants exposed to 400 mg/kg at 27 DAF. Proteins and starch granules were

240

evenly distributed in control cells at 34 DAF (Fig. 5 G), but larger starch granules

241

were found in embryos of plants exposed to nCeO2 (Fig. 5 H, I). This suggests

242

nCeO2 modify the development and nutritional composition of wheat embryos.

243

Starch granules accumulated in embryos will break down to a very low value in

244

late maturation. No starch granules or only very few and small starch grains were

245

detected in mature wheat embryos.43 Larger starch granules could indicate 12


Page 12 of 40

Page 13 of 40


246

delayed grain maturation induced by nCeO2. These phenomena may be part of

247

the mechanisms that permit the adaptation of wheat embryos to stress.44

248

The number and size of starch granules changed in endosperm cells of nCeO2

249

treated plants. A-type starch granules (corn type, more dense polymorphic

250

structure)45 decreased in number and B/C-type starch granules (B is potato type

251

starch, less dense polymorphic structure; C is legume type)45 increased in

252

number (Fig. 6). Ratio of A-type starch granule number decreased from 14.4 ± 2.1%

253

for control to 8.6 ± 3.6% at 100 mg/kg and 8.7 ± 0.8% at 400 mg/kg. The storage

254

proteins, lipids, and polysaccharides, which are deposited during starchy

255

endosperm development are affected by stress.46,47 Modifications in the size and

256

number of starch granules in endosperm suggest that nCeO2 could have serious

257

implications on the nutritional value of wheat grains. Modifications in size could

258

result in changes in protein occurring both on the surface and embedded within

259

the matrix of starch granules.48 The mechanisms underlying these changes

260

caused by nCeO2 deserve further investigation.

261

Concentration of Ce in plant tissues. Figure 7 shows the concentration of Ce

262

in wheat plant tissues. As expected, Ce concentration in nCeO2 treated roots was

263

significantly higher than in the control (Fig. 7). In contrast, Ce concentrations in

264

nCeO2 treated shoots were very low compared with roots. In addition, there were

265

no differences, compared with control, even when the plants were grown in soil

266

amended with 400 mg/kg (stems: 2.32 ± 0.40 mg/kg; leaves: 5.18 ± 0.83 mg/kg; 13



267

grains: 1.80 ± 0.11 mg/kg). Moreover,no significant increase of Ce in grains was

268

observed, indicating limited acropetal translocation of nCeO2. Similar results were

269

reported by Rico et al.,17 who exposed wheat plants to nCeO2 in a greenhouse.

270

These researchers found that roots accumulate more Ce than shoots, which was

271

mainly retained in cell walls of rice roots, possibly because of nanoparticle

272

aggregation and the physical hindrances of plant tissues.49 The influences of

273

nCeO2 on wheat shoots might be caused by some indirect effects like adsorption

274

to root surface that can affect uptake of nutrients and water.33,35 However, recent

275

studies have demonstrated that a high proportion of Ce absorbed by plants

276

remains in particulate form without biotransformation within tissues,7,9 which

277

suggests these nanoparticles may enter into the food chain when wheat plants

278

are exposed to nCeO2, even at low concentration.

279

Effect of nCeO2 on plant growth and yield. The biomass production, shoot

280

length, and yield data are shown in Figure 8. As seen in this figure, none of the

281

treatments affected these parameters. Earlier studies on the various effects of

282

nCeO2 on crop productivity have shown that it has no impact on soybean pod

283

yield8 but enhances tomato fruit production50 and reduces cucumber yield.51 Rico

284

et al.17 reported that under greenhouse conditions, nCeO2 increased wheat shoot

285

biomass and plant height. In our study, the lower chlorophyll concentration plus

286

impaired microstructure of seedlings are expected to reduce the biomass

287

production, but such changes were not observed under the complex field 14


Page 14 of 40

Page 15 of 40


288

conditions. Results suggest that the yield are not only affected by physiological

289

stress caused by nCeO2 on wheat leaves and grain development, and it is also

290

affected by uptake of nutrients and water and the duration of specific growth

291

stages.14

292

We examined the effect of nCeO2 on the nutritional content of wheat grains in

293

terms of protein, starch and sugar contents (Table 2). As seen in this table, nCeO2

294

did not affect the sugar and starch contents but led to significantly higher protein

295

content (increased by 24.8% and 32.6% at low and high concentration,

296

respectively) compared to the control. In our study, nCeO2 treatments led to a

297

one-week-delay in flowering period (Fig. S1), and consequently shortened the

298

grain filling period. It seems that this phenomenon correlates with the growth

299

conditions. Rico et al.17 reported a 6-day delay in spike formation of wheat plants

300

exposed to nCeO2 in a greenhouse facility and Lin et al.33 reported at least 1

301

month delay in flowering of rice plants incubated with C70-NOM (400 mg/L). It is

302

possible that nanoparticles modify plant gene expression and related biological

303

pathways, interfering with nutrients and water uptake, finally affecting plant

304

development.33 A longer grain filling period is commonly associated with high

305

yield in many species.52 However, plants under stress may have fewer days to

306

reach physiological maturity but can yield higher grain protein content.53 In this

307

study, higher grain protein contents were also found in wheat under exposure to

308

nCeO2 at 100 and 400 mg/kg (Table 2). Other studies have demonstrated that 15



309

ENPs induce similar modifications in the protein levels of plants at the early

310

seedling stage.54-59 Rico et al.17 reported that at low concentration, nCeO2

311

increased the amount of six amino acids in wheat grain. This suggests that nCeO2

312

has the potential to change the nutritional quality of wheat. The mechanism by

313

which nCeO2 alter the protein content in the grain is not clear yet.3 It is possible

314

that nCeO2 affect the gene expression for protein synthesis during grain

315

development,60 or induce stress responsive proteins related to self-protection.61 It

316

could be also due to changes in the number and size of starch granules in

317

endosperm (Fig. 6), mainly the B/C-type starch. These granules have greater

318

surface area associated with more surface protein,50 permitting the adaptation of

319

wheat grains to nCeO2 stress.44

320

In summary, the findings of the present study demonstrate that in complex field

321

conditions, nCeO2 did show toxicity to wheat seedlings, impacting grain

322

development. Although there were no effects on final biomass and yield, Ce

323

concentration in shoots, as well as sugar and starch contents in grains, there was

324

an increase in grain protein content. In addition, as previous studies have shown,

325

most of nCeO2 taken up by plants is stored without modification. Thus, nCeO2

326

stored in wheat grains will enter the food chain with unknown consequences. This

327

long-term field study provided a more realistic and holistic approach for

328

determining the implications of ENPs on growth and yield responses of plants.

329

Further field studies are needed in order to better understand the effects of nCeO2 16


Page 16 of 40

Page 17 of 40

330


on the quality of wheat grains.

331 332

ACKNOWLEDGMENT

333

We gratefully acknowledge the National Natural Science Foundation of China

334

(21307056 and 41372234) and the Program for New Century Excellent Talents in

335

University (NCET-12-0266) for their financial supports. This material is based

336

upon work supported by the National Science Foundation and the Environmental

337

Protection Agency under Cooperative Agreement Number DBI-0830117. Any

338

opinions, findings, and conclusions or recommendations expressed in this

339

material are those of the author(s) and do not necessarily reflect the views of the

340

National Science Foundation or the Environmental Protection Agency. This work

341

has not been subjected to EPA review and no official endorsement should be

342

inferred. This work was also supported by Grant 2G12MD007592 from the

343

National Institutes on Minority Health and Health Disparities (NIMHD), a

344

component of the National Institutes of Health (NIH). Authors also acknowledge

345

the USDA grant number 2011-38422-30835 and the NSF Grants CHE-0840525

346

and DBI-1429708. J. L. Gardea-Torresdey acknowledges the Dudley family for

347

the Endowed Research Professorship, the Academy of Applied Science/US Army

348

Research Office, Research and Engineering Apprenticeship program (REAP) at

349

UTEP, grant # W11NF-10-2-0076, sub-grant 13-7, and STARs programs of the

350

University of Texas System. 17



351 352 353

REFERENCES

354

(1) Gardea-Torresdey, J. L.; Rico, C. M.; White, J. C. Trophic transfer,

355

transformation, and impact of engineered nanomaterials in terriesterial

356

environments. Environ. Sci. Technol. 2014, 48 (5), 2526–2540.

357

(2) Ma, X. M.; Geiser-Lee, J.; Deng, Y.; Kolmakov, A. Interactions between

358

engineered nanoparticles (ENPs) and plants: phytotoxicity, uptake and

359

accumulation. Sci. Total Environ. 2010, 408 (16), 3053–3061.

360

(3) Rico, C. M.; Morales, M. I.; Barrios, A. C.; McCreary, R.; Hong, J.; Lee, W. Y.;

361

Nunez, J.; Peralta-Videa, J. R.; Gardea-Torresdey, J. L. Effect of cerium oxide

362

nanoparticles on the quality of rice (Oryza sativa L.) grains. J. Agri. Food Chem.

363

2013, 61 (47), 11278–11285.

364

(4) Piccino, F.; Gottschalk, F.; Seeger, S.; Nowack, B. Industrial production

365

quantities and uses of ten engineered nanomaterials in Europe and the world. J.

366

Nanopart. Res. 2012, 14, 1109.

367

(5) Cornellis, G.; Ryan, B.; McLaughlin, M. J.; Kirby, J. K.; Beak, D.;

368

Chittleborough, D. Solubility and batch retention of CeO2 nanoparticles in soils.

369

Environ. Sci. Technol. 2011, 45, 2777–2782.

370

(6) Limbach, L. K.; Bereiter, R.; Müller, E.; Krebs, R.; Gälli, R.; Stark, W. J.

371

Removal of oxide nanoparticles in a model wastewater treatment plant: influence 18


Page 18 of 40

Page 19 of 40


372

of agglomeration and surfactants on clearing efficiency. Environ. Sci. Technol.

373

2008, 42 (15), 5828−5833.

374

(7) Zhang, P.; Ma, Y. H.; Zhang, Z. Y.; He, X.; Zhang, J.; Guo, Z.; Tai, R. Z.; Zhao,

375

Y. L.; Chai, Z. Biotransformation of ceria nanoparticles in cucumber plants.

376

Environ. Sci. Technol. 2012, 6, 9943−9950.

377

(8) Priester, J. H.; Ge, Y.; Mielke, R. E.; Horst, A. M.; Cole Moritz, S.; Espinosa, K.;

378

Gelb, J.; Walker, S. L.; Nisbet, R. M.; An, Y.-J.; Schimel, J. P.; Palmer, R. G.;

379

Hernandez-Viezcas, J. A.; Zhao, L. J.; Gardea-Torresdey, J. L.; Holden, P. A.

380

Soybean susceptibility to manufactured nanomaterials: evidence for food quality

381

and soil fertility interruption. Proc. Natl. Acad. Sci. U.S.A. 2012, 109, 2451−2456.

382

(9) Hernandez-Viezcas, J. A.; Castillo-Michel, H.; Andrews, J. C.; Cotte, M.; Rico,

383

C.; Peralta-Videa, J. R.; Ge, Y.; Priester, J. H.; Holden, P. A.; Gardea-Torresdey,

384

J. L. In situ synchrotron X-ray fluorescence mapping and speciation of CeO2 and

385

ZnO nanoparticles in soil cultivated soybean (Glycine max). ACS Nano 2013, 7,

386

1415−1423.

387

(10) Rico, C. M.; Hong, J.; Morales, M. I.; Zhao, L. J.; Barrios, A. C.; Zhang, J. Y.;

388

Peralta-Videa, J. R.; Gardea-Torresdey, J. L. Effect of cerium oxide nanoparticles

389

on rice: a study involving the antioxidant defense system and in vivo fluorescence

390

imaging. Environ. Sci. Technol. 2013, 47(11), 5635–5642.

391

(11) Lopez-Moreno, M. L.; de la Rosa, G.; Hernandez-Viezcas, J. A.;

392

Peralta-Videa, J. R.; Gardea-Torresdey, J. L. X-ray absorption spectroscopy (XAS) 19



393

corroboration of the uptake and storage of CeO2 nanoparticles and assessment of

394

their differential toxicity in fouredible plant species. J. Agri. Food Chem. 2010, 58

395

(6), 3689–3693.

396

(12) Ma, Y. H.; Kuang, L. L.; He, X.; Bai, W.; Ding, Y. Y.; Zhang, Z. Y.; Zhao, Y. L.;

397

Chai, Z. F. Effects of rare earth oxide nanoparticles on root elongation of plants.

398

Chemosphere 2010, 78 (3), 273–279.

399

(13) López-Moreno, M. L.; de la Rosa, G.; Hernández-Viezcas, J. A.;

400

Castillo-Michel, H.;Botez, C. E.; Peralta-Videa, J. R.; Gardea-Torresdey, J. L.

401

Evidence of the differential biotransformation and genotoxicity of ZnO and CeO2

402

nanoparticles on soybean (Glycine max) plants. Environ. Sci. Technol. 2010, 44

403

(19), 7315–7320.

404

(14) Wang, Q.; Ma, X. M; Zhang W.; Pei H. C.; Chen Y. S. The impact of cerium

405

oxide nanoparticles on tomato (Solanum lycopersicum L.) and its implications for

406

food safety. Metallmics 2012, 4, 1105–1112.

407

(15) Mueller,N. C.; Nowack, B. Exposure modeling of engineered nanoparticles in

408

the environment. Environ. Sci. Technol. 2008, 42 (12), 4447–4453.

409

(16) Tiede, K.; Hassellöv, M.; Breitbarth, E.; Chaudhry, Q.; Boxall, A. B. A.

410

Considerations for environmental fate and ecotoxicity testing to support

411

environmental risk assessments for engineered nanoparticles. J. Chromatogr. A

412

2009, 1216 (3), 503–509.

20


Page 20 of 40

Page 21 of 40


413

(17) Rico, C. M.; Lee, S. C.; Rubenecia, R.; Mukherjee, A.; Hong, J.;

414

Peralta-Videa, J. R.; Gardea-Torresdey, J. L. Cerium oxide nanoparticles impact

415

yield and modify nutritional parameters in wheat (Triticum aestivum L.). J. Agri.

416

Food Chem. 2014, 62 (40), 9669–9675.

417

(18) Du, W. C.; Sun, Y. Y.; Ji, R.; Zhu, J. G.; Wu, J. C.; Guo, H. Y. TiO2 and ZnO

418

nanoparticles negatively affect wheat growth and soil enzyme activities in

419

agricultural soil. J. Environ. Monitor. 2011, 13, 822–828.

420

(19) Keller, A. A.; Wang, H.; Zhou, D.; Lenihan, H. S.; Cherr, G.; Cardinale, B. J.;

421

Miller, R.; Ji, Z. Stability and aggregation of metal oxide nanoparticles in natural

422

aqueous matrices. Environ. Sci. Technol. 2010, 44, 1962−1967.

423

(20) Ni, C. Y.; Chen, Y. X.; Lin, Q.; Tian, G. M. Subcellular localization of copper in

424

tolerant and non-tolerant plant. J. Environ. Sci. 2005, 17, 452–456.

425

(21) Lichtenthaler, H. K. Chlorophylls and carotenoids: pigments of photosynthetic

426

biomembranes. In Methods in Enzymology; Packer, R. D. L., Ed.; Academic

427

Press: Waltham, MA, 1987; 148, 350–382.

428

(22) Ohkawa, H.; Ohishi, N.; Yagi, K. Assay for lipid peroxides in animal tissues

429

by thiobarbituric acid reaction. Anal. Biochem. 1979, 95 (2), 351–358.

430

(23) Lin, R. Z.; Wang, X. R.; Luo, Y.; Du, W. C.; Guo, H. Y.; Yin, D. Q. Effects of

431

soil cadmium on growth, oxidative stress and antioxidant system in wheat

432

seedlings (Triticum aestivum). Chemosphere 2007, 69 (1), 89–98.

433

(24) Bradford, M. M. A rapid and sensitive method for the quantitation of 21



434

microgram quantities of protein utilizing the principle of protein-dye binding. Anal.

435

Biochem. 1976, 72, 248−254.

436

(25) Dhindsa, R. S.; Dhinsa, P. P.; Thorpe, T. A. Leaf senescence correlated with

437

increased levels of membrane permeability and lipid peroxidation and decreased

438

levels of superoxide dismutase and catalase. J. Exp. Bot. 1980, 32, 127–132.

439

(26) Cakmak, I.; Strboe, D.; Marschner, H. Activities of hydrogen peroxide

440

scavenging enzymes in germinating wheat seeds. J. Exp. Bot. 1993, 44, 127–

441

132.

442

(27) Sun, W. C.; Cheng, C. H.; Lee, W. C. Protein expression and enzymatic

443

activity of cellulases produced by Trichoderma reesei Rut C-30 on rice straw.

444

Process Biochem. 2008, 43 (10), 1083–1087.

445

(28) Fang, Y.; Wang, L.; Xin, Z. H.; Zhao, L. Y.; An, X. Y.; Hu, Q. H. Effect of foliar

446

application of zinc, selenium, and iron fertilizers on nutrients concentration and

447

yield of rice grain in China. J. Agric. Food Chem. 2008, 56, 2079–2084.

448

(29) Jian, L. C.; Deng, J. M.; Li, J. H.; Li, P. H. Seasonal alteration of the cytosolic

449

and nuclear Ca2+ concentrations in overwintering woody and herbaceous

450

perennials in relation to the development of dormancy and cold hardiness. J. Am.

451

Soc. Hort. Sci. 2003, 128 (1), 29–35.

452

(30) Kratsch, H. A.; Wise, R. R. The ultrastructure of chilling stress. Plant Cell

453

Environ. 2000, 23, 337–350.

454

(31) Paleg, L. G.; Donald A. The Physiology and Biochemistry of Drought 22


Page 22 of 40

Page 23 of 40


455

Resistance in Plants. Academic Press: New York-London. 1981.

456

(32) Sandalio, L. M.; Dalurzo, H. C.; Gomez, M.; Romero‐Puertas, M. C.; Del Rio, L.

457

A. Cadmium‐induced changes in the growth and oxidative metabolism of pea

458

plants. J. Exp. Bot. 2001, 52 (364), 2115–2126.

459

(33) Lin, S. J.; Reppert, J.; Hu, Q.; Hudson, J. S.; Leid, M. L; Ratnikova, T. A.; Rao,

460

A. M.; Luo, H.; Ke, P. C. Uptake, translocation and transmission of carbon

461

nanomaterials in rice plants. Small 2009, 5 (10), 1128–1132.

462

(34) Laurette, J.; Larue, C.; Mariet, C.; Brisset, F.; Khodja, H.; Bourguignon, J.;

463

Carriere, M. Influence of uranium speciation on its accumulation and translocation

464

in three plant species: oilseed rape, sunflower and wheat. Environ. Exp. Bot. 2012,

465

77, 96–107.

466

(35) Ghafariyan, M. H.; Malakouti, M. J.; Dadpour, M. R.; Stroeve. P.; Mahmoudi,

467

M. Effects of magnetite nanoparticles on soybean chlorophyll. Environ. Sci.

468

Technol. 2013, 47 (18), 10645–10652.

469

(36) Hsiao, T. C.; Acevedo, E. Plant responses to water deficits, water-use

470

efficiency, and drought resistance. Agri. Meteorol. 1974, 14 (1), 59–84.

471

(37) Niu, G.; Rodriguez, D. S.; Mackay, W. Growth and physiological response to

472

drought stress in four oleander clones. J. Am. Soc. Hort. Sci. 2008, 133, 188–196.

473

(38) Ma, C. X.; Chhikara, S.; Xing, B. S.; Musante, C.; White, J. C.; Dhankher, O.

474

P. Physiological and molecular response of Arabidopsis thaliana (L.) to

475

nanoparticle cerium and indium oxide exposure. ACS Sustainable Chem. Eng. 23



476

2013, 1 (7), 768–778.

477

(39) Zhao, L. J.; Peng, B.; Hernandez-Viezcas, J. A.; Rico, C.; Sun, Y. P.;

478

Peralta-Videa, J. R.; Tang, X. L.; Niu, G. H.; Jin, L. X.; Varela-Ramirez, A.; Zhang,

479

J. Y.; Gardea-Torresdey, J. L. Stress response and tolerance of Zea mays to

480

CeO2 nanoparticles: Cross talk among H2O2, heat shock protein, and lipid

481

peroxidation. ACS Nano 2012, 6 (11), 9615–9622.

482

(40) Morales, M. I.; Rico, C. M.; Hernandez-Viezcas, J. A.; Nunez, J. E.; Barrios,

483

A. C.; Tafoya, A.; Flores-Marges, J. P.; Peralta-Videa, J. R.; Gardea-Torresdey, J.

484

L. Toxicity assessment of cerium oxide nanoparticles in cilantro (Coriandrum

485

sativum L.) plants grown in organic soil. J. Agric. Food Chem. 2013, 61 (26),

486

6224–6230.

487

(41) Heckert, E. G.; Karakoti, A. S.; Seal, S.; Self, W. T. The role of cerium redox

488

state in the SOD mimetic activity of nanoceria. Biomaterials 2008, 29 (18),

489

2705−2709.

490

(42) Xia, T.; Kovochich, M.; Liong, M.; Madler, L.; Gilbert, B.; Shi, H.; Yeh, J. I.;

491

Zink, J. I.; Nel, A. E. Comparison of the mechanism of toxicity of zinc oxide and

492

cerium oxide nanoparticles based on dissolution and oxidative stress properties.

493

ACS Nano 2008, 2 (10), 2121−2134.

494

(43) Blackig, M.; Corbineau, F.; Grzesikit, M.; Guyi, P.; Come, D. Carbohydrate

495

metabolism in the developing and maturing wheat embryo in relation to its

496

desiccation tolerance. J. Exp. Bot. 1996, 47 (2), 161−169. 24


Page 24 of 40

Page 25 of 40


497

(44) Mocquot, B.; Prat, C.; Mouches, C.; Pradet, A. Effect of anoxia on energy

498

charge and protein synthesis in rice embryo. Plant Physiol. 1981, 68 (3), 636–640.

499

(45) Wang, T. L.; Bogracheva, T. Y.; Hedley, C. L. Starch: as simple as A, B, C? J.

500

Exp. Bot. 1998, 49 (320), 481–502.

501

(46) Olsen, O. A. Endosperm development: cellularization and cell fate

502

specification. Annu. Rev. Plant Biol. 2001, 52 (1), 233–267.

503

(47) Liu, D.; Wang, X.; Lin, Y.; Chen, Z.; Xu, H.; Wang, L. The effects of cerium on

504

the growth and some antioxidant metabolisms in rice seedlings. Environ. Sci.

505

Pollut. Res. 2012, 19 (8), 3282–3291.

506

(48) Tester, R. F.; Karkalas, J.; Qi, X. Starch—composition, fine structure and

507

architecture. J. Cereal Sci. 2004, 39 (2), 151–165.

508

(49) Koller, A.; Washburn, M. P.; Lange, B. M.; Andon, N. L.; Deciu, C.; Haynes, P.

509

A.; Yates, J. R. Proteomic survey of metabolic pathways in rice. Proc. Natl. Acad.

510

Sci. 2002, 99 (18), 11969–11974.

511

(50) Wang, Q.; Ma, X. M.; Zhang, W.; Pei, H. C.; Chen, Y. S. The impact of cerium

512

oxide nanoparticles on tomato (Solanum lycopersicum L.) and its implications for

513

food safety. Metallomics 2012, 4, 1105–1112.

514

(51) Zhao, L. J.; Sun, Y. P.; Hernandez-Viezcas, J. A.; Servin, A. D.; Hong, J.; Niu,

515

G. H.; Peralta-Videa,J. R.; Duarte-Gardea, M.; Gardea-Torresdey, J. L. Influence

516

of CeO2 and ZnO nanoparticles on cucumber physiological markers and

25



517

bioaccumulation of Ce and Zn: a life cycle study. J. Agric. Food Chem. 2013, 61,

518

11945–11951.

519

(52) Fageria, N. K.; Baligar, V. C.; Clark, R. B. Physiology of crop production.

520

Haworth Press Inc. 2006.

521

(53) Li, P.; Chen, J. L.; Wu, P. T. Agronomic characteristics and grain yield of 30

522

spring wheat genotypes under drought stress and nonstress conditions. Agron. J.

523

2011, 103 (6), 1619–1628.

524

(54) Chai, M. W.; Shi, F. C.; Li, R. L.; Liu, L. M.; Liu, Y.; Liu, F. C. Interactive

525

effects of cadmium and carbon nanotubes on the growth and metal accumulation

526

in a halophyte Spartina alterniflora (Poaceae). Plant Growth Reg. 2013, 71, 171–

527

179.

528

(55) Krystofova, O.; Sochor, J.; Zitka, O.; Babula, P.; Kudrle, V.; Adam, V.; Kizek,

529

R. Effect of magnetic nanoparticles on tobacco BY-2cell suspension culture. Int. J.

530

Environ. Res. Pub. Health 2013, 10, 47–71.

531

(56) Krishnaraj, C.; Jagan, E. G.; Ramachandran, R.; Abirami, S. M.; Mohan, N.;

532

Kalaichelvan, P. T. Effect of biologically synthesized silver nanoparticles on

533

Bacopa monnieri (Linn.). Wettst. Plant Metabol. Proc. Biochem. 2012, 47, 651–

534

658.

535

(57) Gao, F. Q.; Liu, C.; Qu, C. X.; Zheng, L.; Yang, F.; Su, M. Y.; Hong, F. S. Was

536

improvement of spinach growth by nano-TiO2 treatment related to the changes of

537

Rubisco activase? Biometals 2008, 21, 211–217. 26


Page 26 of 40

Page 27 of 40


538

(58) Salama, H. M. H. Effects of silver nanoparticles in some crop plants, common

539

bean (Phaseolus vulgaris L.) and corn (Zea mays L.). Int. Res. J. Biotechnol. 2012,

540

3, 190–197.

541

(59) Patra, P.; Choudhury, S. R.; Mandal, S.; Basu, A.; Goswani, A.; Gogoi, R.;

542

Srivastava, C.; Kumar, R.; Gopal, M. Effect of sulfur and ZnO nanoparticles on

543

stress physiology and plant (Vignaradiata) nutrition. Adv. Nanomat. Nanotechnol.

544

2013, 31, 299–307.

545

(60) Mushtaq, R.; Katiyar, S.; Bennett, J. Proteomic analysis of drought

546

stress-responsive proteins in rice endosperm affecting grain quality." J. Crop Sci.

547

Biotechnol. 2008, 11 (4), 227–232.

548

(61) Wang, W. W.; Meng, B.; Ge, X. M.; Song, S. H.; Yang, Y.; Yu, X. M.; Wang, L.

549

G.; Hu, S. N.; Li, S. Q.; Yu, J. Proteomic profiling of rice embryos from a hybrid rice

550

cultivar and its parental lines. Proteomics 2008, 8 (22), 4808–4821.

551 552 553

Figure legends

554

Figure 1. TEM images of wheat root tips from plants grown with 0 (A, D), 100 (B,

555

E), and 400 (C, F) mg nCeO2 per kg soil. A, B, C, nucleus; D, E, F, cell wall.

556

Arrows point particles on cell wall. O, outside; cw, cell wall; cy, cytoplasm; s,

557

starch grain.

558

Figure 2. TEM images of wheat leaves from plants grown with 0 (A, D, G), 100 (B, 27



559

E, H), and 400 (C, F, I) mg nCeO2 per kg soil. A, B, C, chloroplast; D, E, F,

560

thylakoids; G, H, I, nucleus. Ch, chloroplast; n, nucleus; th, thylakoids; s, starch

561

grain; arrow indicates plastoglobuli.

562

Figure 3. SEM image and EDS analysis of cross section from wheat primary root

563

tips and leaf of plants exposed to nCeO2. A, epidermis. B, parenchyma. C,

564

vascular cylinder. D, vein.

565

Figure 4. Activity of the antioxidant enzymes (A) CAT, (B) SOD and (C)

566

concentration of thiobarbituric acid reactive substances (TBARS) in shoots of

567

wheat plants grown with 0, 100, and 400 mg nCeO2 per kg soil. Data are means

568

of three replicates ± standard deviation. Different letters among columns indicate

569

statistically significant differences at p ≤ 0.05.

570

Figure 5. TEM images of embryos of wheat grown with 0 (A, D, G), 100 (B, E, H),

571

and 400 (C, F, I) mg nCeO2 per kg soil. A, B, C, 20 days after flowering (DAF); D,

572

E, F, 27 DAF; G, H, I, 34 DAF. V, vacuoles; s, starch; p, protein.

573

Figure 6. TEM images of endosperm of wheat grown with 0 (A, D, G), 100 (B, E,

574

H), and 400 (C, F, I) mg nCeO2 per kg soil. A, B, C, 20 days after flowering (DAF);

575

D, E, F, 27 DAF; G, H, I, 34 DAF. Sa, A-type starch; sb, B-type starch; sc, C-type

576

starch.

577

Figure 7. Ce concentration in harvested wheat tissues from plants grown with 0,

578

100, and 400 mg CeO2 nCeO2 per kg soil. Data are means of three replicates ±

579

standard deviation. Different letters among columns indicate statistically 28


Page 28 of 40

Page 29 of 40


580

significant differences in Ce content at p ≤ 0.05.

581

Figure 8. (A) Shoot biomass, (B) shoot height, and (C) 1000-seed weight of wheat

582

cultivated with 0, 100, and 400 mg nCeO2 per kg soil. Data are means of three

583

replicates ± standard deviation. Different letters among columns indicate

584

statistically significant differences at p ≤ 0.05.

29



Page 30 of 40

585

Table 1. Chlorophyll content in shoots of wheat plants (30 days after germination) grown in soil amended with 0 (control),

586

100, and 400 mg nCeO2/ kg soil. Different letters in the different columns indicate statistically significant differences at p ≤

587

0.05. nCeO2 Treatments

Chlorophyll a

Chlorophyll b

Total chlorophyll

(mg/kg soil)

(µg/g fresh wt)

(µg/g fresh wt)

(µg/g fresh wt)

Chlorophyll a/b ratio

0

1,104±50a

373±11a

1,478±61a

2.96±0.06ab

100

1,086±70a

360±23a

1,447±99a

3.03±0.06a

400

754±12b

264±23b

1,018± 138b

2.85±0.11b

588 589

30


Page 31 of 40


Table 2. Protein, starch and sugar contents in grains from wheat plants cultivated in soil amended with 0, 100, and 400 mg nCeO2/ kg soil. Data are means of three replicates ± standard deviation. Different letters among columns indicate statistically significant differences at p ≤ 0.05. Starch and sugar

nCeO2 Protein

(mg/g dry wt)

Treatments (mg/g dry wt) (mg/kg)

Total sugar

Reducing sugar

Total starch

0

120.5±5.2a

411.2±51.1a

15.5±2.0a

787.1±50.3a

100

150.4±4.2b

349.8±10.5a

15.7±2.1a

746.7±89.7a

400

159.8±3.5c

412.5±45.8a

13.4±1.6a

705.9±121a

31



Figure 1.

32


Page 32 of 40

Page 33 of 40


Figure 2.

33



Figure 3.

34


Page 34 of 40

Page 35 of 40


Figure 4.

35



Figure 5.

36


Page 36 of 40

Page 37 of 40


Figure 6.

37



Figure 7.

38


Page 38 of 40

Page 39 of 40


Figure 8. 39



TOC/Abstract Art

ASSOCIATED CONTENT Supporting Information. Effect of nCeO2 on wheat flowering of plants grown with 0, 100, and 400 mg nCeO2 per kg soil. This material is available free of charge via the Internet at http://pubs.acs.org.

40


Page 40 of 40

Physiological and Biochemical Changes Imposed by CeO2

Recommend Documents