Accepted Manuscript

Zinc oxide nanoparticles alter the wheat physiological response and reduce the
cadmium uptake by plants

Afzal Hussain, Shafaqat Ali, Muhammad Rizwan, Muhammad Zia ur Rehman,

Muhammad Rizwan Javed, Muhammad Imran, Shahzad Ali Shahid Chatha, Rashid
Nazir
PII: S0269-7491(18)33113-0
DOI: 10.1016/j.envpol.2018.08.036
Reference: ENPO 11474

To appear in: Environmental Pollution

Received Date: 5 July 2018

Revised Date: 4 August 2018
Accepted Date: 13 August 2018

Please cite this article as: Hussain, A., Ali, S., Rizwan, M., Zia ur Rehman, M., Javed, M.R., Imran, M.,
Chatha, S.A.S., Nazir, R., Zinc oxide nanoparticles alter the wheat physiological response and reduce
the cadmium uptake by plants, Environmental Pollution (2018), doi: 10.1016/j.envpol.2018.08.036.

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Graphical Abstract

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1.4

Grain Cd concentration
a A
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1.0 b B

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(mg kg-1)
0.8 b C
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50 a A
Zinc oxide B
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Grain Zn concentration

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nanoparticle c C
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30 D
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20 d E
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0
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Cd-contaminated soil 0 25 50 75 100

ZnO NPs concentrations
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1 Zinc oxide nanoparticles alter the wheat physiological response and reduce the cadmium

2 uptake by plants

3 Afzal Hussaina, Shafaqat Alia, Muhammad Rizwana,*, Muhammad Zia ur Rehmanb,

4 Muhammad Rizwan Javedc, Muhammad Imrand, Shahzad Ali Shahid Chathae, Rashid

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5 Nazirf

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a
6 Department of Environmental Sciences and Engineering, Government College University,

7 Allama Iqbal Road, 38000 Faisalabad, Pakistan

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8 Institute of Soil & Environmental Sciences, University of Agriculture Faisalabad, Pakistan
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9 Department of Bioinformatics & Biotechnology, Government College University, Allama Iqbal

10 Road, 38000 Faisalabad, Pakistan

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11 Department of Environmental Sciences, COMSATS University Islamabad, Vehari Campus,

Pakistan
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13 Department of Chemistry, Government College University, Allama Iqbal Road, 38000
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14 Faisalabad, Pakistan
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15 Department of Environmental Sciences, COMSATS University Islamabad, Abbottabad Campus,

16 Tobe Camp University Road Abbottabad 22060 Pakistan

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17 *Corresponding Author: [email protected]

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18 Abstract
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19 An experiment was performed to explore the interactive impacts of zinc oxide nanoparticles

20 (ZnO NPs) and cadmium (Cd) on growth, yield, antioxidant enzymes, Cd and zinc (Zn)

21 concentrations in wheat (Triticum aestivum). The ZnO NPs were applied both in Cd-

22 contaminated soil and foliar spray (in separate studies) on wheat at different intervals and plants

23 were harvested after physiological maturity. Results depicted that ZnO NPs enhanced the

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24 growth, photosynthesis, and grain yield, whereas Cd and Zn concentrations decreased and

25 increased respectively in wheat shoots, roots and grains. The Cd concentrations in the grains

26 were decreased by 30-77%, and 16-78% with foliar and soil application of NPs as compared to

27 the control, respectively. The ZnO NPs reduced the electrolyte leakage while increased SOD and

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28 POD activities in leaves of wheat. It can be concluded that ZnO NPs (levels used in the study)

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29 could effectively reduce the toxicity and concentration of Cd in wheat whereas increase the Zn

30 concentration in wheat. Thus, ZnO NPs might be helpful in decreasing Cd and increasing Zn

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31 biofortification in cereals which might be effective to reduce the hidden hunger in humans owing

32 the deficiency of Zn in cereals.

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33 Keywords: Zinc biofortification, cadmium, antioxidants, wheat, grain yield

34 Capsule: ZnO NPs increased growth and yield while decreased Cd in wheat grains
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45 1. Introduction

46 The soils contaminated with toxic metals such as cadmium (Cd) have become a widespread

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47 environmental problem due to their toxicity in plants and humans (Nagajyoti et al., 2010). The

48 Cd contamination in agricultural soils is increasing due to the anthropogenic sources of Cd such

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49 as mining, electroplating and agricultural activities (Gallego et al., 2012; Rizwan et al., 2012).

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50 Cadmium is nonessential metal and is easily taken up by plants and is toxic to living things

51 (Rizwan et al., 2016a). In Plants, Cd caused oxidative stress and reduced the plant growth and

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52 quality (Rehman et al., 2017a). Numerous studies have reported the toxic effects of Cd in crop
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53 plants at various levels (Abbas et al., 2017; Rehman et al., 2018a; Rizwan et al., 2018).

54 Therefore, there is an urgent need to reduce the Cd accumulation in crops.

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55 Previously, several approaches have been used to decrease Cd and other heavy metal
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56 concentrations in agricultural crops (Etesami, 2018; Keller et al., 2015; Rizwan et al., 2016b;

57 Rehman et al., 2017b). Researchers have shown that zinc (Zn), being an essential trace element
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58 for plants and humans, could be used to decrease Cd in plants (Rizwan et al., 2017a).
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59 Nevertheless, Zn deficiency is prevailing in crops, mainly cereals and people consuming the food

60 obtained from these crops may suffer Zn deficiency (Gomez-Coronado et al., 2016; Joy et al.,
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61 2017). Thus, biofortification of crops using Zn fertilizers not only increase grain Zn
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62 concentrations, but also decrease Cd concentrations in grains (Rizwan et al., 2017a; Murtaza et

63 al., 2017).

64 In recent years, nanotechnology has been receiving the attention of the researchers because of

65 their wide use in many sectors such as agriculture (Rizwan et al., 2017b). Nanoparticles (NPs)

66 can also be used as a source of nutrient, especially micronutrients, for plants called

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67 nanofertilizers which might be a promising approach as plants use only a trace amount of the

68 chemical fertilizers applied in the soil and remaining fertilizers in the soil may pose serious risks

69 to the surrounding environment (Liu and Lal, 2015; Raliya et al., 2017). Thus, the use of

70 nanofertilizers for the controlled release of concerned nutrients for the crops might be a potential

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71 strategy to solve such issues of soil contamination and low agronomic productivity (Liu et al.,

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72 2016; Raliya et al., 2017). Similarly, NPs have also been used to reduce abiotic stresses in plants

73 including Cd and other metal stress (Awad et al., 2017; Mohamed et al., 2017). Among NPs, zinc

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74 oxide nanoparticles (ZnO NPs) are considered among the most highly used NPs in several

75 industrial products (Mukherjee et al., 2015). The ZnO NPs have been suggested as a Zn fertilizer

76
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at lower levels to supply Zn to plants (Milani et al., 2012; Rizwan et al., 2017b). It was observed
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77 that ZnO NPs (1 ∼ 20 ppm) increased the growth of mungbean and chickpea (Mahajan et al.,
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78 2011). The ZnO NPs increased the cotton growth and reduced the oxidative stress in plants

79 (Venkatachalam et al., 2017a). On the other hand, phycomolecules loaded ZnO NPs increased
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80 the growth, photosynthesis, Cd, and Pb uptake whereas reduced the oxidative stress in Leucaena
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81 leucocephala seedlings (Venkatachalam et al., 2017b). The interaction between ZnO NPs and

82 metal may affect the bioavailability of toxic metals such as Cd and Pb (Venkatachalam et al.,
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83 2017b). However, the studies regarding the use of NPs for metal remediation are at initial stages

84 and the mechanisms of metal remediation by using NPs are less known.
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85 Wheat (Triticum aestivum L.) is the predominant cereal crop and is considered as a staple food
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86 over 50% of population at world level (FAO, 2014) and could accumulate higher Cd contents in

87 aerial parts (Naeem et al., 2016). On the other hand, wheat suffers Zn deficiency mainly in

88 developing countries which is the main problem of lower Zn contents in humans whose staple

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89 food is wheat (Joy et al., 2017; Qaswar et al., 2017). Therefore, wheat was selected as a test plant

90 in the current study.

91 In the present study, it was hypothesized that ZnO NPs may alleviate Cd toxicity in wheat by

92 enhancing wheat growth and Zn concentrations while reducing Cd concentrations in plants.

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93 Overall, the effects of ZnO NPs, applied in the soil and foliar spray, were measured on wheat dry

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94 weights, grain yield, chlorophyll contents, electrolyte leakage (EL), superoxide dismutase (SOD)

95 and peroxidase (POD) activities, Cd and Zn contents in wheat. This study will provide the new

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96 insights into the application of ZnO NPs for Zn biofortification and Cd uptake in plants as well

97 as potential applications of nanofertilizers under metal stress.

98 2. Materials and methods

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99 2.1. Materials
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100 A field used for growing agricultural crops was selected for this study which was under raw

101 effluent irrigation for more than 30 years and, thus, polluted with toxic heavy metals mainly Cd.
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102 This field is located in Multan, Pakistan (Rehman et al., 2018). The random soil sampling was
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103 done at a depth of 0-20 cm by using a stainless steel spade. The samples were pooled and

104 homogenized and then air-dried without direct sunlight and sieved (2 mm). Soil was
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105 characterized for different parameters such as particle size (Bouyoucos, 1962), pH, EC, sodium
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106 adsorption ratio (US Salinity Lab. Staff, 1954; Page et al., 1982) and ammonium bicarbonate
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107 diethylene triamine pentaacetic acid (AB-DTPA) extractable metals (Soltanpour, 1985). The

108 properties of the soil used in the present study are presented in Table 1. Zinc oxide NPs were

109 purchased from Alfa Aesar with a purity of 99%, 20-30 nm APS powder, and a density of 5.606.

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111 2.2. Experimental design

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112 Surface sterilization of wheat seeds was done with sodium hypochlorite for 5 min and then

113 washed thoroughly with distilled water. The experiment was conducted in the botanical garden

114 of the Government College University, Faisalabad, Pakistan. Initially, eight healthy and uniform

115 size wheat seeds (cv. Lasani-2008) were transferred to each plastic pot with 5.0 kg of Cd-

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116 contaminated soil. The design of the experiment was completely randomized design whereas the

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117 experiment was conducted under natural environmental conditions with 24-29 °C and about 68%

118 humidity during the sowing time and about 29-43 °C and about 54% humidity at the time of

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119 harvesting. Day and night duration at the time of sowing was 10:30/13:30 and during harvesting

120 was about 13:00/11:00 respectively. The pots were protected from rain during the entire growth

121
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period. For ZnO NPs exposure, half of the pots were treated with different treatments (0, 25, 50,
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122 75, and 100 mg kg-1) of NPs by thoroughly mixing and wheat seeds were sown after 15 days of

NPs application in the soil to avoid the immediate effects of NPs on seed germination.
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124 Remaining half pots were foliar sprayed with NPs (0, 25, 50, 75, and 100 mg L-1) at different
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125 time intervals by using Tween-20 as sticking agent. Different treatments of NPs were prepared in
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126 distilled water and were ultra-sonicated for about 30 min to separate and homogenize the

127 particles. The plants were thinned to 5 seedlings in each pot after 15 days of sowing and first
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128 foliar spray was done just after thinning the plants between 10 to 11 am whereas 2nd, 3rd, and 4th

129 foliar sprays were done at 4th, 6th, and 8th weeks of sowing respectively. During foliar spray, soil
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130 of each pot and neighbor plants were covered to avoid NPs direct entry to soil and on
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131 surrounding plants due to foliar spray whereas controlled plants were sprayed with distilled

132 water at the same time. A total volume of 2.0 L per treatment was used. Four replicates of each

133 treatment were maintained in the experiment. Each pot was fertilized with NPK (120-50-25 kg

134 ha-1 respectively) in the form of urea, diammonium phosphate and potassium sulfate. The soil

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135 moisture contents of 70% of soil water holding capacity were maintained during the entire

136 growth of plants.

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138 2.3. Plant harvesting and sampling

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139 Wheat plants were harvested after 125 days of seed sowing when the plants were reached at

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140 maturity. A stainless steel meter scale was used to measure the plant height and spike length.

141 Plants were separated into grains, husk, shoots and roots. The washing of the roots was done

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142 with 0.1 M HCl to remove the metals on the root surface and then all the roots were carefully

143 washed with distilled water. Oven drying of the samples was done at 70 °C for 72 h. Samples

144
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were crushed into small pieces after recording the dry weight and stored for metal determination.
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145

2.4. Determination of chlorophyll and gas exchange parameters

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147 Chlorophyll contents were extracted from leaf samples after 80 days of sowing with acetone
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148 (85% v/v) by placing the samples in the dark at 4 °C. A spectrophotometer was used to record
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149 the reading at different wavelengths and chlorophyll contents were calculated by using equation

150 (Lichtenthaler, 1987). Infra-Red Gas Analyzer (IRGA) was used to record the photosynthetic
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151 rate, stomatal conductance, and transpiration rate from the leaves after 80 days of sowing during

152 sunny days assuming that the plants were fully functional at that time.
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153
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154 2.4. Determination of electrolyte leakage and SOD and POD activities

155 Electrolyte leakage, SOD, POD activities in leaves of wheat was measured after 80 days of

156 growth. For EL determination, the leaves were cut into small pieces and were placed in tubes

157 containing deionized water. Initial EC1 of the samples was recorded after incubating the samples

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158 at specific temperature and time period (32 °C for 2 h) and then EC2 was recorded after again

159 incubating these samples at specific temperature and time period (121 °C for 20 min) (Dionisio-

160 Sese and Tobita, 1998).

161 EL = (EC1/EC2) × 100 (1)

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162 Superoxide dismutase and POD activities in leaves were estimated after crushing the samples in

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163 liquid nitrogen and then standardizing in 0.5 M phosphate buffer (pH 7.8) (Zhang, 1992).

164

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165 2.5. Determination of Cd and Zn contents in plants

166 The plant sample (1.0 g each) digestion was done with HNO3-HClO4 (3:1, v:v) by using hot plate

167
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as reported by Rehman et al. (2015). An atomic absorption spectrophotometer was used to
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168 determine the Cd and Zn concentrations in the digested samples.
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170 2.6. Post-harvest soil analyses

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171 Soil sampling was done just after harvesting the wheat plants and oven drying was done at 40 °C
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172 for about 72 h for obtaining the constant dry weight of the soil. Then, soil was analyzed for pH

173 and bioavailable Cd (AB-DTPA extractable) with standard protocols (section 2).
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175 2.7. Statistical analyses

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176 One-way ANOVA at the 5% probability level was used to analyse the data with IBM SPSS
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177 Statistics for Windows, Version 21.0. The multiple comparisons of means were performed with

178 Tukey’s HSD post hoc test.

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180 3. Results

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181 3.1 Wheat growth and photosynthesis

182 To investigate the impacts of ZnO NPs on wheat growth under Cd stress, the plant growth

183 parameters of wheat were recorded. The results pertaining to plant height, spike length, and dry

184 weights of shoot, roots, spikes and grains are depicted in figure 1 and 2. The plant height and

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185 spike length were significantly increased with the ZnO NPs application either in soil or foliar

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186 spray. The increasing trend was higher with the rates of NPs applied. Shoot length increased by

187 21, 35, 50, and 55% in foliar treatments and increased by 10, 23, 33, and 43% in soil application

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188 of 25, 50, 75, and 100 mg kg-1 ZnO NPs treatments over control respectively. At the highest NPs

189 treatment, spike length increased by 59 and 54% in foliar and soil treatments as compared to the

190
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respective control treatments. At the highest foliar application of ZnO NPs (100 mg L-1), dry
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191 weights of shoots, roots, spikes and grains were increased by 72, 59, 90, and 97% over control

respectively. At 75 and 100 mg kg-1 ZnO NPs treatments, the shoot dry weights were increased
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193 by 42 and 52%, root dry weights were increased by 43, and 50%, spike dry weights were
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194 increased by 59, and 69% and grain dry weights were increased by 60, and 74% over control in
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195 soil application of NPs respectively.

196 Chlorophyll and carotenoid concentrations were significantly increased either foliar or soil
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197 application of ZnO NPs as compared to the respective control treatments (Figure 3). The highest

198 chlorophyll and carotenoid concentrations were found in the highest levels of NPs used whereas
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199 the lowest values of these parameters were observed in the control treatments. Chlorophyll a
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200 concentration increased by 60, and 53% over the control in the highest treatment of NPs in both

201 foliar and soil applications, respectively. Chlorophyll b and carotenoid concentrations were

202 increased by 139, 119% in the highest foliar treatment and increased by 138, and 111% in the

203 highest soil application of NPs over control respectively. Photosynthetic rate, transpiration rate,

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204 and stomatal conductance were linearly increased with increasing NPs levels in both foliar and

205 soil applications over control (Figure 3). The photosynthetic rate increased by 25, 61, 90, and

206 102% in 25, 50, 75, and 100 mg L-1 foliar NPs application over control whereas this increase was

207 about 18, 46, 77, and 89% in soil application of NPs over control respectively. At the highest

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208 treatment of NPs, transpiration rate increased by 116, and 107% over the control in foliar and

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209 soil application respectively.

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211 3.2. Electrolyte leakage and antioxidant enzymes

212 The EL in wheat leaves was determined in order to evaluate the impacts of ZnO NPs on

213
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oxidative damage in Cd-stressed wheat leaves. The highest level of EL was observed in control
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214 leaves and ZnO NPs application reduced the EL in the leaves and the decreasing trend was

pronounced with the increase in NPs levels in the growth medium (Figure 4a). The changes in
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216 SOD and POD activities in Cd-stressed wheat leaves with ZnO NPs are reported in figure 4.
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217 Relative to the control, SOD and POD activities were increased in wheat leaves exposed to
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218 different treatments of NPs. The increase in antioxidant enzyme activities was more remarkable

219 at higher levels of NPs applied over control. The SOD activity increased by 40, and 46% in the
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220 highest treatment (100 ppm) over control in foliar and soil application respectively, whereas

221 POD activity was increased by 46, 54% over control in the treatment in foliar and soil
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222 application respectively.

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224 3.3. Cadmium and Zn concentration wheat

225 To investigate the effects of ZnO NPs on metal concentrations, the Cd and Zn accumulation in

226 wheat grains, shoots and roots were determined (Figure 5). The highest Cd concentrations were

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227 observed in the control plants and the lowest Cd concentrations were observed in the highest NPs

228 treatment in both application methods whereas the opposite trend was found for Zn

229 concentrations in wheat. Foliar application of ZnO NPs treatments decreased the Cd

230 concentrations in grains by 30-77%, in shoots by 10-55%, and in roots by 25-64% over the

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231 control whereas soil application of ZnO NPs decreased the Cd concentrations in grains by 16-

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232 78%, in shoots by 17-68%, and in roots by 23-60% over the control.

233 For a better assessment of ZnO NPs effects on Cd uptake by wheat, Zn concentrations in shoot,

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234 roots and grains were determined. Zinc is an essential micronutrient and thus some Zn

235 concentrations were observed in wheat tissues. ZnO NPs increased the Zn content in wheat

236
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tissues and this increase was more pronounced as the ZnO NPs concentrations increased either in
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237 soil or foliar spray. This enhancement was more pronounced with foliar spray than soil

application of NPs. Foliar ZnO NPs treatments increased the Zn content in grains by 40-185%, in
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239 shoots by 10-52%, and in roots by 7-60% over the control. Soil application of 100 mg kg-1 NPs
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240 treatment increased the Zn concentrations in shoots, roots and grains by 58, 68, and 190% over
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241 control respectively.

242
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243 3.4. Soil pH and available Cd

244 Soil pH was not significantly affected by NPs treatments either in soil application or foliar spray
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245 (Table 2). The AB-DTPA extractable Cd concentrations from soil were slightly decreased with
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246 increasing NPs treatments (Table 2). However, the results are non-significant when compared

247 with the control treatment in both soil and foliar NPs treatments.

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249 4. Discussion

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250 The main aim of our study was to test the hypothesis if ZnO NPs can be used to reduce the

251 toxicity and uptake of Cd in wheat as the effect of NPs on the growth of wheat and metal uptake

252 is not largely known. It was assumed that metal based NPs could decrease toxic metal uptake and

253 their toxicity in plants by enhancing the plant growth and photosynthesis. The results of the

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254 present study indicated that ZnO NPs increased the wheat growth and dry weights under Cd

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255 stress (Figure 1 and 2). Different types of NPs increased the growth of Cd-stressed plants. For

256 example, according to Venkatachalam et al. (2017a), phycomolecules coated ZnO NPs enhanced

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257 the growth of Leucaena leucocephala seedlings under Cd and Pb stress over the control.

258 Nanoscale zerovalent iron increased the leaf, stem and root dry weight of Boehmeria nivea under

259
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Cd stress (Gong et al., 2017). Nano-TiO2 particles increased the growth and biomass of Cd-
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260 stressed soybean plants (Singh and Lee, 2016). Our results clearly indicated that ZnO NPs are

involved in increasing wheat growth and yield and enhancing wheat tolerance to Cd stress. Our
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262 results further depicted that the effect of the ZnO NPs foliar application was higher than soil
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263 applications (Figure 1 and 2). No studies are available in the literature regarding the comparison
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264 of foliar vs soil application of NPs on wheat growth under metal stress. The higher production of

265 the dry weight of wheat with foliar spray might be due to the increased concentrations of Zn in
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266 plants (Figure 5). This higher Zn concentration in plants may reduce to Cd toxicity in due to

267 different mechanisms such as co-precipitation of Cd and Zn in metabolically inactive parts and
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268 reducing the shortage of Zn leading to better plant growth.

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269 Heavy metal-induced toxicity in plants can be assessed by measuring the photosynthetic pigment

270 concentrations in the plants (Rizwan et al., 2016a; Hussain et al., 2018). The current study

271 indicated that chlorophyll concentration and gas exchange attributes were increased in wheat

272 leaves by the application of ZnO NPs than control (Figure 3). Phycomolecules loaded ZnO NPs

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273 increased the photosynthetic pigments in L. leucocephala than control (Venkatachalam et al.,

274 2017a). Similarly, other NPs also increased the photosynthetic pigments in several plant species

275 under metal stress (Rizwan et al., 2017b). The higher photosynthetic pigments in wheat leaves

276 may be due to decreased Cd contents in wheat (Figure 5).

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277 It is well demonstrated that heavy metals caused oxidative stress in plants by increasing the level

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278 of ROS, which ultimately impairs macromolecules at the cellular level (Mittler, 2002). On the

279 other hand, higher levels of antioxidase activity may reduce the oxidative stress caused by Cd by

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280 scavenging the excess ROS depending upon plant species and type of metal stress and duration

281 of exposure (Habiba et al., 2015; Rizwan et al., 2017c). Our results depicted that ZnO NPs

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reduced the EL and modified the SOD and POD activities in leaves of wheat over control (Figure
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283 4). This indicated that ZnO NPs could alleviate Cd-induced oxidative damage in wheat leaves.

This reduced EL and enhanced antioxidants resulted from ZnO NPs may also help to reduce
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285 oxidative stress in wheat. ZnO NPs reduced the MDA level and enhanced the CAT and SOD
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286 activities in metal-stressed L. leucocephala than respective metal treatments alone

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287 (Venkatachalam et al., 2017b). ZnO NPs increased antioxidant enzyme activity in Prosopis

288 juliflora-velutina plants (Hernandez-Viezcasa et al., 2011). The response of ZnO NPs on MDA
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289 contents and antioxidant enzyme activities in green pea and sugar beet varies with the plant

290 species, soil types, doses of NPs, and duration of exposure. However, the response of ZnO NPs
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291 did not vary significantly as compared to the bulk ZnO and ZnSO4 applications (Garcia-Gomez et
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292 al., 2018). The EL concentration was lower whereas antioxidant enzyme activities were higher in

293 leaves with foliar applied ZnO NPs than soil application (Figure 4). This variation in EL and

294 antioxidant levels may be due to the decreased Cd contents in foliar applied plants than

295 respective treatments with soil applications (Figure 5).

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296 The higher Cd concentration was observed in control plants and ZnO NPs decreased the Cd

297 contents in different parts of wheat than control (Figure 5). The higher Cd concentration in

298 control plants showed that Cd could be absorbed by wheat roots and is translocated to aerial

299 parts, as reported in previous studies (Rizwan et al., 2017c, 2018). The Cd concentrations were

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300 higher in roots than other plant parts (Figure 5) which may be one of the protective mechanisms

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301 in plants (Rizwan et al., 2018) and the direct contact of roots with Cd in the soil. The effect of

302 NPs on metal uptake in plants is diverse (Singh and Lee, 2016; Gong et al., 2017; Konate et al.,

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303 2017). Phycomolecules loaded ZnO NPs increased the Cd and Pb concentrations in L.

304 leucocephala plants than the plants treated with only Cd or Pb (Venkatachalam et al., 2017a).

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Similarly, nano zerovalent Fe increased the Cd concentrations in B. nivea than control and also
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306 changed the subcellular distribution of Cd (Gong et al., 2017). TiO2 NP (100 to 300 mg kg-1)

increased the Cd uptake in soybean (Singh et al., 2016). Contrarily, Fe2O3 NP reduced the heavy
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308 metal (Cd, Pb, Zn, and Cu) concentrations in wheat shoot and roots than control (Konate et al.,
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309 2017). This showed that metal uptake by plants with NPs application depends upon on the type
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310 of NPs, plant species, growth conditions and so on. Nanoparticles could immobilize metals

311 through surface complexation and absorption processes. The Cd content linearly decreased with
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312 the doses of ZnO NPs which indicate that these NPs may serve as a Cd barrier to reduce Cd

313 uptake by plants. However, this phenomenon may not be applicable when the NPs were foliar
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314 sprayed.
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315 Cadmium toxicity may cause the shortage of essential plant nutrients which might be due to the

316 competition of the Cd and other divalent ions during uptake by plants (Rizwan et al., 2016a). Our

317 results depicted that ZnO NPs increased the Zn concentrations and decreased the Cd

318 concentrations in shoot, root, and grains of wheat than control (Figure 5). It has been

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319 demonstrated that ZnO NPs enhanced the soil Zn bioavailability and its concentration in leaves

320 of green pea and beet over control (Garcia-Gomez et al., 2018). Several previous studies

321 revealed that higher Zn levels in plants suppressed the Cd uptake by plants (Rizwan et al., 2017a;

322 Munir et al., 2018). Zinc has an antagonistic effect on Cd uptake by plants and the application of

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323 Zn reduced the Cd concentrations in plants and also alleviated the toxic effects of Cd in crop

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324 plants (Saifullah et al., 2014; Qaswar et al., 2017; Rizwan et al., 2017a). The higher Zn may

325 partially alleviate the Cd toxicity in wheat due to different mechanisms such as co-precipitation

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326 of Cd and Zn in metabolically less active parts such as cell wall (Rizwan et al., 2016a). Soil

327 application of ZnO NPs may immobilize Cd in the soil and reduced its bioavailability to plants

328
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(Figure 5). Our results showed that the effect of Zn on Cd uptake is largely concentration-
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329 dependent. Not like soil application, foliar application of ZnO NPs is unlikely to modify the soil

properties and thus immobilization of Cd in the soil may not be involved in a foliar application
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331 of ZnO NPs. It is expected that ZnO NPs can penetrate from the root or leaf surface and could
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332 transport to different parts of the plant. However, studies are required to measure the Zn
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333 speciation and biotransformation of ZnO NPs in plants. The higher Zn concentrations may

334 increase the wheat growth which may cause the reduction of Cd per unit biomass. Recently, it
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335 was reported that Zn NPs and Zn salts positively affected the sorghum growth and nutritional

336 quality with NPK inputs and increased the nitrogen contents in plants (Dimkpa et al., 2017). Our
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337 results showed no significant increase in Zn concentrations in plants with soil application of 25
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338 mg kg-1 ZnO NPs than control but there was a significant decrease in Cd content in plants with

339 this treatment than control (Figure 5). This showed that increased uptake of Zn is only partially

340 related to the decrease in Cd content in plants and more studies are required to explore the

341 mechanisms by which NPs affect Cd uptake and its translocation in plants.

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342 The higher Zn concentrations in cereal grains are required to avoid the Zn hidden hunger in

343 humans (Cakmak et al., 2017; Qaswar et al., 2017). This higher concentration of Zn in cereal

344 grains can be achieved through several ways, such as the application of Zn fertilizers and

345 development of high Zn accumulating varieties. A level of > 40 mg Zn per kg of grains is

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346 required for Zn biofortification (Cakmak et al., 2017). This target level of Zn was nearly

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347 achieved when ZnO NPs were applied at a level of 75 mg kg-1 or higher (Figure 5F). On the

348 other hand, Cd concentration blow 0.2 mg Cd per kg of grains is required for reducing Cd risk in

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349 humans (FAO, 2012). This required level of Cd in grains was achieved with the soil or foliar

350 application of 75 mg kg-1 or higher level of ZnO NPs in the current study (Figure 5C). Thus, the

351
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highest level of ZnO NPs produced the grains by achieving the both targets of Zn and Cd levels
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352 in grains under current experimental conditions, wheat cultivar, and metal levels in the soil used

for the experiment. However, further studies at field levels are required, especially under various
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353

354 environmental conditions and pollution levels before practical application of ZnO NPs on metal
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355 contaminated soils.

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356

357 5. Conclusion
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358 The results revealed that ZnO NPs decreased and increased the Cd and Zn concentrations in

359 plants respectively and triggered the metal tolerance mechanisms in plants. ZnO NPs enhanced
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360 the grain yield and photosynthesis whereas reduced the oxidative stress in plants. These results
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361 suggested that ZnO NPs at suitable levels may reduce the Cd toxicity and its uptake by wheat

362 and fortified the wheat grains with Zn. However, so far, very less is known about the ZnO NPs

363 application on plant growth and metal uptake and further comprehensive field studies are needed

364 for the practical application of ZnO NPs in metal contaminated soils.

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365

366 Acknowledgement

367 The authors are thankful for the financial support from the Government College University,

368 Faisalabad and Higher Education Commission (HEC) of Pakistan under HEC Project No. 20-

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369 3653/NRPU/R&D/HEC/14/437 and NRPU Project No. 5634/Punjab/NRPU/R&D/HEC/2016.

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370 References

371 Abbas, T., Rizwan, M., Ali, S., Rehman, M.Z., Qayyum, M.F., Abbas, F., Hannan, F., Rinklebe,

SC
372 J., Ok, Y.S., 2017. Effect of biochar on cadmium bioavailability and uptake in wheat
373 (Triticum aestivum L.) grown in a soil with aged contamination. Ecotoxicol. Environ. Saf.
374 140, 37-47.
375 Awad, Y.M., Vithanage, M., Niazi, N.K., Rizwan, M., Rinklebe, J., Yang, J.E., OK, Y.S., Lee,

U
376 S.S., 2017. Potential toxicity of trace elements and nanomaterials to Chinese cabbage in
377 arsenic- and lead-contaminated soil amended with biochars. Environ. Geochem. Health
AN
378 doi:10.1007/s10653-017-9989-3
379 Bouyoucos, G.J., 1962. Hydrometer method improved for making particle- size analyses of soils.
380 Agron. J. 54, 464-465.
Cakmak, I., McLaughlin, M.J., White, P., 2017. Zinc for better crop production and human
M

381
382 health. Plant Soil. 411, 1-4.
383 Dimkpa, C.O., White, J.C., Elmer, W.H., Gardea-Torresdey, J., 2017. Nanoparticle and Ionic Zn
384 Promote Nutrient Loading of Sorghum Grain under Low NPK Fertilization. J. Agri. Food
D

385 Chem. 65, 8552-8559.

386 Dionisio-Sese, M.L., Tobita, S., 1998. Antioxidant responses of rice seedlings to salinity stress.
TE

387 Plant Sci. 135, 1–9.

388 Etesami, H., 2018. Bacterial mediated alleviation of heavy metal stress and decreased
389 accumulation of metals in plant tissues: mechanisms and future prospects. Ecotoxicol.
EP

390 Environ. Saf. 147, 175-191.

391 FAO, 2012. ProdStat. Core Production Data Base, Electronic resource under
392 〈http://faostat.fao.org/〉. (Accessed 30 June 2015).
393 FAO, 2014. ProdStat. Core Production Data Base, Electronic resource under
C

394 http://faostat.fao.org/ (assed 30.05.2017).

395 Gallego, S.M., Pena, L.B., Barcia, R.A., Azpilicueta, C.E., Iannone, M.F., Rosales, E.P.,
AC

396 Benavides, M.P., 2012. Unravelling cadmium toxicity and tolerance in plants: insight into
397 regulatory mechanisms. Environ. Exp. Bot. 83, 33-46.
398 Garcia-Gomez, C., García, S., Obrador, A.F., González, D., Babín, M., Fernández, M.D., 2018.
399 Effects of aged ZnO NPs and soil type on Zn availability, accumulation and toxicity to
400 pea and beet in a greenhouse experiment. Ecotoxicol. Environ. Saf. 160, 222-230.
401 Gomez-Coronado, F., Poblaciones, M.J., Almeida, A.S., Cakmak, I., 2016. Zinc (Zn)
402 concentration of bread wheat grown under Mediterranean conditions as affected by
403 genotype and soil/foliar Zn application. Plant soil. 401, 331-346.

17
 ACCEPTED MANUSCRIPT

404 Gong, X., Huang, D., Liu, Y., Zeng, G., Wang, R., Wan, J., Zhang, C., Cheng, M., Qin, X., Xue,
405 W., 2017. Stabilized Nanoscale Zerovalent Iron Mediated Cadmium Accumulation and
406 Oxidative Damage of Boehmeria nivea (L.) Gaudich Cultivated in Cadmium
407 Contaminated Sediments. Environ. Sci. Technol. 51, 11308-11316.
408 Habiba, U., Ali, S., Farid, M., Shakoor, M.B., Rizwan, M., Ibrahim, M., Abbasi, G.H., Hayat, T.,
409 Ali, B., 2015. EDTA enhanced plant growth, antioxidant defense system, and
410 phytoextraction of copper by Brassica napus L. Environ. Sci. Pollut. Res. 22, 1534–1544.

PT
411 Hernandez-Viezcasa, J.A., Castillo-Michelb, H., Servina, A.D., Peralta-Videaa, J.R., Gardea-
412 Torresdey, J.L., 2011. Spectroscopic verification of zinc absorption and distribution in
413 the desert plant Prosopis juliflora-velutina (Velvet mesquite) treated with ZnO

RI
414 nanoparticles. Chem. Eng. J. 170, 346-352.
415 Hussain, A., Ali, S., Rizwan, M., Rehman, M.Z., Hameed, A., Hafeez, F., Alamri, S.A.,
416 Alyemeni, M.N., Wijaya, L., 2018. Role of zinc–lysine on growth and chromium uptake

SC
417 in rice plants under Cr stress. J. Plant Growth Regul. doi:10.1007/s00344-018-9831-x
418 Joy, E.J., Ahmad, W., Zia, M.H., Kumssa, D.B., Young, S.D., Ander, E.L., Watts, M.J., Stein,
419 A.J., Broadley, M.R., 2017. Valuing increased zinc (Zn) fertiliser-use in Pakistan. Plant
420 Soil. 411, 139-150.

U
421 Keller, C., Rizwan, M., Davidian, J.C., Pokrovsky, O.S., Bovet, N., Chaurand, P., Meunier, J.D.,
422 2015. Effect of silicon on wheat seedlings (Triticum turgidum L.) grown in hydroponics
AN
423 and exposed to 0 to 30 mM Cu. Planta 241, 847-860.
424 Konate, A., He, X., Zhang, Z., Ma, Y., Zhang, P., Alugongo, G.M., Rui, Y., 2017. Magnetic
425 (Fe3O4) Nanoparticles Reduce Heavy Metals Uptake and Mitigate Their Toxicity in
Wheat Seedling. Sustainability. 9, 1-16.
M

426
427 Lichtenthaler, H.K., 1987. Chlorophylls and carotenoids—pigments of photosynthetic
428 biomembranes. In: Colowick SP, Kaplan NO (eds) Methods in enzymology, vol 148.
429 Academic Press, San Diego, pp 350–382.
D

430 Liu, R., Lal, R., 2015. Potentials of engineered nanoparticles as fertilizers for increasing
431 agronomic productions. Sci. Total Environ. 514, 131–139.
TE

432 Liu, R., Zhang, H., Lal, R., 2016. Effects of stabilized nanoparticles of copper, zinc, manganese,
433 and iron oxides in low concentrations on lettuce (Lactuca sativa) seed germination:
434 nanotoxicants or nanonutrients?. Water, Air, & Soil Pollut. 227, 1-14.
EP

435 Mahajan, P., Dhoke, S.K., Khanna, A.S., 2011. Effect of nano-ZnO particle suspension on
436 growth of mung (Vigna radiata) and gram (Cicer arietinum) seedlings using plant agar
437 method. J. Nanotechnol. 696535, 1–7. doi:10.1155/2011/696535.
438 Milani, N., McLaughlin, M.J., Stacey, S.P., Kirby, J.K., Hettiarachchi, G.M., Beak, D.G.,
C

439 Cornelis, G., 2012. Dissolution kinetics of macronutrient fertilizers coated with
440 manufactured zinc oxide nanoparticles. J. Agric. Food Chem. 60, 3991–3998.
AC

441 Mittler, R., 2002. Oxidative stress, antioxidants and stress tolerance. Trends Plant Sci. 7, 405–
442 410.
443 Mohamed, A.K.S., Qayyum, M.F., Abdel-Hadi, A.M., Rehman, R.A., Ali, S., Rizwan, M., 2017.
444 Interactive effect of salinity and silver nanoparticles on photosynthetic and biochemical
445 parameters of wheat. Arch. Agron. Soil Sci. 63, 1736-1747.
446 Mukherjee, A., Sun, Y., Morelius, E., Tamez, C., Bandyopadhyay, S., Niu, G., White, J.C.,
447 Peralta-Videa, J.R., Gardea-Torresdey, J.L., 2015. Differential toxicity of bare and hybrid
448 ZnO nanoparticles in green pea (Pisum sativum L.): a life cycle study. Front. Plant Sci. 6,
449 1242.

18
 ACCEPTED MANUSCRIPT

450 Munir, T., Rizwan, M., Kashif, M., Shahzad, A., Ali, S., Amin, N., Zahid, R., Alam, M.F.E.,
451 Imran, M., 2018. Effect of zinc oxide nanoparticles on the growth and Zn uptake in wheat
452 (Triticum aestivum L.) by seed priming method. Digest J. Nanomater. Biostruc. 13, 315-
453 323.
454 Murtaza, G., Javed, W., Hussain, A., Qadir, M., Aslam, M., 2017. Soil-applied zinc and copper
455 suppress cadmium uptake and improve the performance of cereals and legumes. Int. J.
456 Phytorem. 19, 199-206.

PT
457 Naeem, A., Saifullah, Rehman, M.Z.U., Akhtar, T., Ok, Y.S., Rengel, Z., 2016. Genetic variation
458 in cadmium accumulation and tolerance among wheat cultivars at the seedling
459 stage. Commun. Soil Sci. Plant Anal. 47, 554-562.

RI
460 Nagajyoti, P.C., Lee, K.D., Sreekanth, T.V.M., 2010. Heavy metals, occurrence and toxicity for
461 plants: a review. Environ. Chem. Lett. 8, 199-216.
462 Page, A.L., Miller, R.H., Keeny, D.R., 1982. Methods of soil analysis (Part 2). Chem. Microbiol.

SC
463 Proper. Agron 9. SSSA, Madison.
464 Qaswar, M., Hussain, S., Rengel, Z., 2017. Zinc fertilisation increases grain zinc and reduces
465 grain lead and cadmium concentrations more in zinc-biofortified than standard wheat
466 cultivar. Sci. Total Environ. 605, 454-460.

U
467 Raliya, R., Saharan, V., Dimkpa, C., Biswas, P., 2017. Nanofertilizer for precision and
468 sustainable agriculture: current state and future perspectives. J. Agri. Food Chem. doi:
AN
469 10.1021/acs.jafc.7b02178
470 Rehman, M.Z., Khalid, H., Akmal, F., Ali, S., Rizwan, M., Qayyum, M.F., Iqbal, M., Khalid,
471 M.U., Azhar M., 2017b. Effect of limestone, lignite and biochar applied alone and
combined on cadmium uptake in wheat and rice under rotation in an effluent irrigated
M

472
473 field. Environ. Pollut. 227, 560-568.
474 Rehman, M.Z., Rizwan, M., Ali, S., Ok, Y.S., Ishaque, W., Saifullah, Nawaz, M.F., Akmal, F.,
475 Waqar M. 2017a. Remediation of heavy metal contaminated soils by using Solanum
D

476 nigrum: A review. Ecotoxicol. Environ. Saf. 143, 236-248.

477 Rehman, M.Z., Rizwan, M., Ghafoor, A., Naeem, A., Ali, S., Sabir, M., Qayyum, M.F., 2015.
TE

478 Effect of inorganic amendments for in situ stabilization of cadmium in contaminated soils
479 and its phyto-availability to wheat and rice under rotation. Environ. Sci. Pollut. Res. 22,
480 16897-16906.
EP

481 Rehman, M.Z., Rizwan, M., Hussain, A., Saqib, M., Ali, S., Sohail, M.I., Shafiq, M., Hafeez, F.,
482 2018a. Alleviation of Cd toxicity and minimizing its uptake in wheat (Triticum aestivum)
483 by using organic carbon sources in Cd-spiked soil. Environ. Pollut. 241, 557-565.
484 Rehman, M.Z., Rizwan, M., Khalid, H., Ali, S., Naeem, A., Yousaf, B., Liu, G., Sabir, M.,
C

485 Farooq, M., 2018b. Farmyard manure alone and combined with immobilizing
486 amendments reduced cadmium accumulation in wheat and rice grains grown in field
AC

487 irrigated with raw effluents. Chemosphere, 199, 468-476.

488 Rizwan, M., Ali, S., Abbas, T., Rehman, M.Z., Hannan, F., Keller, C., Al-Wabel, M.I., Ok, Y.S.,
489 2016a. Cadmium minimization in wheat: A critical review. Ecotoxicol. Environ. Saf. 130,
490 43–53.
491 Rizwan, M., Ali, S., Adrees, M., Ibrahim, M., Tsang, D.C.W., Rehman, M.Z., Zahir, Z.A.,
492 Rinklebe, J., Tack, F.M.G., Ok, Y.S., 2017c. A critical review on effects, tolerance
493 mechanisms and management of cadmium in vegetables. Chemosphere 182, 90-105.

19
 ACCEPTED MANUSCRIPT

494 Rizwan, M., Ali, S., Hussain, A., Ali, Q., Shakoor, M.B., Rehman, M.Z., Farid, M., Asma, M.,
495 2017a. Effect of zinc-lysine on growth, yield and cadmium uptake in wheat (Triticum
496 aestivum L.) and health risk assessment. Chemosphere 187, 35-42.
497 Rizwan, M., Ali, S., Qayyum, M.F., Ibrahim, M., Rehman, M.Z., Abbas, T., OK, Y.S., 2016b.
498 Mechanisms of biochar-mediated alleviation of toxicity of trace elements in plants: a
499 critical review. Environ. Sci. Pollut. Res. 23, 2230-2248.
500 Rizwan, M., Ali, S., Qayyum, M.F., Ok, Y.S., Adrees, M., Ibrahim, M., Rehman, M.Z., Farid,

PT
501 M., Abbas, F., 2017b. Effect of metal and metal oxide nanoparticles on growth and
502 physiology of globally important food crops: A critical review. J. Hazard. Mater. 322, 2-
503 16.

RI
504 Rizwan, M., Ali, S., Rehman, M.Z., Rinklebe, J., Tsang, D.C.W., Bashir, A., Maqbool, A., Tack,
505 F.M.G., Ok, Y.S., 2018. Cadmium phytoremediation potential of Brassica crop species:
506 A review. Sci. Total Environ. 631-632, 1175-1191.

SC
507 Saifullah, Sarwar, N., Bibi, S., Ahmad, M., Ok, Y.S., 2014. Effectiveness of zinc application to
508 minimize cadmium toxicity and accumulation in wheat (Triticum aestivum L.). Environ.
509 Earth Sci. 71, 1663–1672.
510 Singh, J., Lee, B.K., 2016. Influence of nano-TiO2 particles on the bioaccumulation of Cd in

U
511 soybean plants (Glycine max): A possible mechanism for the removal of Cd from the
512 contaminated soil. J. Environ. Manage. 170, 88−96.
AN
513 Soltanpour, P.N., 1985. Use of AB-DTPA soil test to evaluate elemental availability and toxicity.
514 Commun. Soil Sci. Plant Anal. 16, 323-338.
515 US Salinity Laboratory Staff, 1954. Diagnosis and Improvement of Saline and Alkali Soils.
Agriculture Handbook 60. United States Salinity Laboratory, USDA, Washington DC, p.
M

516
517 160.
518 Venkatachalam, P., Jayaraj, M., Manikandan, R., Geetha, N., Rene, E.R., Sharma, N.C., Sahi,
519 S.V., 2017b. Zinc oxide nanoparticles (ZnONPs) alleviate heavy metal-induced toxicity
D

520 in Leucaena leucocephala seedlings: A physiochemical analysis. Plant Physiol.

521 Biochem. 110, 59-69.
TE

522 Venkatachalam, P., Priyanka, N., Manikandan, K., Ganeshbabu, I., Indiraarulselvi, P., Geetha,
523 N., Muralikrishna, K., Bhattacharya, R.C., Tiwari, M., Sharma, N., Sahi, S.V., 2017a.
524 Enhanced plant growth promoting role of phycomolecules coated zinc oxide
EP

525 nanoparticles with P supplementation in cotton (Gossypium hirsutum L.). Plant Physiol.
526 Biochem. 110, 118-127.
527 Zhang, X.Z., 1992. The measurement and mechanism of lipid peroxidation and SOD, POD and
528 CAT activities in biological system. In: Zhang, X.Z. (Ed.), Research Methodology of
C

529 Crop Physiology. Agriculture Press, Beijing, pp. 208–211.

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Table 1. Selected initial physic-chemical properties of the soil used in the pot study
Parameters Unit Values
Textural class Sandy Loam
Sand % 78
Silt % 12
Clay % 10

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pHs - 7.85
ECe dS m-1 2.11
Soluble ions

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CO32- mmolc L-1 -
HCO3- mmolc L-1 1.21
Cl- mmolc L-1 6.75
*SO42- mmolc L-1

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12.4
Ca2+ + Mg2+ mmolc L-1 7.51
Na+ mmolc L-1 9.26
K+ mmolc L-1 1.24
(mmolc L-1)1/2

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SAR 4.91
CEC cmolc kg-1 3.86
CaCO3 % 1.71
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OM % 0.92
(a)
Metal concentrations Total Available
-1
Cd mg kg 7.38 0.93
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Pb mg kg-1 45.76 7.24

Zn mg kg-1 41.15 5.69
Cu mg kg-1 14.7 3.98
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Mn mg kg-1 52.14 6.57

Ni mg kg-1 4.45 0.46
mg kg-1
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Fe 198.4 52.04
*By difference = TSS - (CO32- + HCO3- + Cl-)
(a)
AB-DTPA extractable
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NPs Treatments 0 25 50 75 100

pH
Foliar 7.92 ± 0.03a 7.85 ± 0.04 a 7.89 ± 0.05 a 7.95 ± 0.02 a 7.84 ± 0.05 a
a a a a
Soil 7.97 ± 0.04 7.87 ± 0.07 7.89 ± 0.08 8.01 ± 0.05 7.98 ± 0.02 a
-1
AB-DTPA extractable Cd (mg kg )
Foliar 0.87 ± 0.05 a 0.82 ± 0.02 a 0.81 ± 0.01 a 0.79 ± 0.06 a 0.75 ± 0.05 a

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a a a a
Soil 0.86 ± 0.03 0.83 ± 0.03 0.81 ± 0.04 0.78 ± 0.03 0.77 ± 0.04 a
Table 2. Post-harvest soil pH and bioavailable Cd. Values are means of four replicates.

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Figure 1. Phenotypic images of wheat plants after 80 days of growth in Cd-contaminated soil
with soil (a), and foliar (b) application of different concentrations of ZnO NPs.
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80 A 12 ab
Foliar Soil ab
ab B a A
Shoot length (cm plant-1)

Spike length (cm plant-1)

bc 10 b A
A B
60 c B c
d D C 8 d C
D
40 6

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30 C 8 ab
ab D ab A
a bc A
Shoot dry weight (g pot-1)

Root dry weight (g pot-1)

25 A
A B
b AB 6 c B

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B C
d C
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10

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Spike husk dry weight (g pot-1)

ab
Grain dry weight (g pot-1)

b A a
8 A 20 A
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c B B
BC c C
6 d C 15 D
d
E
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4 10
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ZnO NPs concentrations ZnO NPs concentrations

Figure 2. Effect of ZnO NPs on shoot length, spike length, and dry weights of shoot, root, spikes
and grains of wheat plants grown in aged Cd-contaminated soil. Values presented are means of
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differences among various treatments at at P < 0.05.
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5 A 2.0 B
Foliar Soil
ab ab
ab A A
4 bc A a A
1.5

Chlorophyl b
(mg g-1 FW)
c B b B
Chlorophyl a
(mg g-1 FW)

3 d C
D c C
1.0
2 d D

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1.5 C ab 20
ab A D
b A ab
a

Photosynthetic rate
(µmol CO2 m-2 S-1)
B 15 A A
b
(mg g-1 FW)

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Carotenoids

1.0 c
C B
c
d D 10 d D C

0.5

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4.0 3.0 F ab
E ab a A
3.5 a A
A 2.5 b
Stomatal conductance

A
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B
(mmol H2O m-2 s-1)

3.0 b
Transpiration rate

B c
(mol m-2 s-1)

2.0 C
2.5 c
C d C
2.0 d 1.5
D
D

1.5 1.0
1.0
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0.5
0.5
0.0 0.0
0 25 50 75 100 0 25 50 75 100
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ZnO NPs concentration ZnO NPs concentration

Figure 3. Effect of ZnO NPs on chlorophyll a (a), chlorophyll b (b), carotenoids concentrations
(c), photosynthetic rate (d), transpiration rate (e), and stomatal conductance (f) of wheat plants
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A Foliar
100 a A

Electrolyte leakage (%)

ab b Soil
B C c
75 D d
E

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500 B
SOD (Units g-1 FW)

400 ab A
ab BC a B

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c D

200

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1800 C ab A
a A
b B
POD (Units g-1 FW)

1500 c C
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d
1200 D
900
600
D

300
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ZnO NPs concentrations
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Figure 4. Effect of ZnO NPs on electrolyte leakage (a), superoxide dismutase (b), and
peroxidase (c) of wheat leaves grown in aged Cd-contaminated soil. Values presented are means
of four replicates with standard deviation. Different letters on the bars represent significant
differences among various treatments at at P < 0.05.
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6 A A 16
Foliar B
a 14 a A
Shoot Cd concentration

Root Cd concentration
5 b B Soil
12
b B

(mg kg-1 DW)

(mg kg-1 DW)

4 c C 10
d
c C
3 D 8 d D
e E
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Shoot Zn concentration
Grain Cd concentration

a A 80 B
1.2 B b
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(mg kg-1 DW)

(mg kg-1 DW)

1.0 b B 60 cd c

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0.8 b C
0.6 40
D
0.4 c c D 20

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175 50 a A
E A F B
150 a b
Grain Zn concentration

40
Root Zn concentration

b B c C
125 C
(mg kg-1 DW)

c
(mg kg-1 DW)

d D D
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100 d D 30
d
75 20 d E
50
D

10
25
0 0
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0 25 50 75 100 0 25 50 75 100
ZnO NPs concentrations ZnO NPs concentrations

Figure 5. Effect of ZnO NPs on Cd concentration in shoot (a), roots (b), grains (c) and Zn
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concentrations in shoot (d), roots (e), and grains (f) of wheat plants grown in aged Cd-
contaminated soil. Values presented are means of four replicates with standard deviation.
Different letters on the bars represent significant differences among various treatments at at P <
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Research Highlights
• Zinc oxide nanoparticles (ZnO NPs) increased the wheat yield under cadmium (Cd) stress
• ZnO NPs increased chlorophyll contents and gas exchange attributes under Cd stress
• ZnO NPs enhanced antioxidant enzyme activities in wheat leaves under Cd stress
• ZnO NPs reduced the Cd concentrations and increased zinc concentrations in wheat

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