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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

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Environmental Science & Technology

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Physiological and Biochemical Changes Imposed by CeO2

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Nanoparticles on Wheat: A Life Cycle Field Study

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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

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

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

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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.

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Exposed to low concentration produced embryos with larger vacuoles, while

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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

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

34

nCeO2 in crop plants.

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

2


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INTRODUCTION

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

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

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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

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negative but also positive or inconsequential effects.2

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

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

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

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

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

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

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

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

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

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

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

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

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



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performed in controlled environments that greatly differ from field conditions,

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

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

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

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

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

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

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

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

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

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

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

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

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malondialdehyde and activities of antioxidant enzymes superoxide dismutase

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

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

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

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wheat grains were analyzed.

75 76 77

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


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

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

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

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Agroecological Experimental Station, the Chinese Academy of Science

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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



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Microscopy study of wheat samples. Fresh wheat seedlings were sampled

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

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

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

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

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

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leaves were sliced for transmission electron microscopy using a microtome with

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

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

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

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

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

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then infiltrated and embedded in epoxy resin.

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Chlorophyll, malondialdehyde, and enzyme measurement. Fresh wheat

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

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content, malondialdehyde content, and enzyme activities. Chlorophyll was

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

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method

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thiobarbituric acid assay by monitoring contents of thiobarbituric acid reactive

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

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

of

Lichtenthaler.21

Malondialdehyde

was

6


measured

using

the

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

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

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

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

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

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

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

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minute.

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

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

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

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

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

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inductively

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

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and protein concentration was calculated as total nitrogen content multiplied by

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

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

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

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

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nm with glucose as the standard.28

coupled

plasma-mass

spectroscopy

(ICP-MS;

7


NEXION

300,


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

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of three or four replicates. Statistical differences among treatments were

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

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

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also performed.

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RESULTS AND DISCUSSION

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

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

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

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

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

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

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

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

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

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

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

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

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of DNA transcription and mRNA synthesis, thereby inhibiting polyribosome

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


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

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

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

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

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

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

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

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

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Such changes in chloroplasts and thylakoids are an important mechanism used

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

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damage,30 suggesting important disturbances in metabolic functions of these

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organelles which would result in a low photosynthetic capacity.32

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Although no nCeO2 aggregates or individual particles were found in TEM

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

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

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

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

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

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

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

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



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

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

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

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

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

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

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development.33,35

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Effect of nCeO2 on chlorophyll content. Photosynthesis is a sensitive

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

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photosynthetic performance of a plant under stressful conditions can be indicative

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

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

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

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

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

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

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chlorophyll a content indicates a lower photosynthetic rate in wheat plants

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

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

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

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


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

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

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disturbance in chloroplast structure and disorganization of thylakoid in leaf

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

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potential to compromise the overall plant growth and vigor.38

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

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

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activities and the concentration of thiobarbituric acid reactive substances

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

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

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

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

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

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

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

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

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membrane damage. Similar results were found in corn cultivated under

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

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

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This discrepancy with previous studies could result from differences in plant

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



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

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

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

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

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

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

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

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

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oxidative stress, thereby avoiding the potential membrane damage.10

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

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

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

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

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

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

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

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

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nCeO2 modify the development and nutritional composition of wheat embryos.

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

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

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


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

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

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

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

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

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

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

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

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

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

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

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

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

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

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caused by nCeO2 deserve further investigation.

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

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

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

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

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

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



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

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

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

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

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

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

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

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

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

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

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

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are exposed to nCeO2, even at low concentration.

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

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

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

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

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

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

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

286

impaired microstructure of seedlings are expected to reduce the biomass

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


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

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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

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

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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

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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

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

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

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possible that nanoparticles modify plant gene expression and related biological

303

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

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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

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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

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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

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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

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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


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on the quality of wheat grains.

331 332

ACKNOWLEDGMENT

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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

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

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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


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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


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Figure 2.

33



Figure 3.

34


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Figure 4.

35



Figure 5.

36


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Figure 6.

37



Figure 7.

38


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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.

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Physiological and Biochemical Changes Imposed ... - ACS Publications

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