Using Synchrotron-Based Approaches To Examine the Foliar

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Using synchrotron-based approaches to examine the foliar application of ZnSO4 and ZnO nanoparticles for field-grown winter wheat Teng Zhang, Hongda Sun, Zhiyuan Lv, Lili Cui, Hui Mao, and Peter M Kopittke J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b04153 • Publication Date (Web): 01 Nov 2017 Downloaded from http://pubs.acs.org on November 7, 2017

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Journal of Agricultural and Food Chemistry

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Using synchrotron-based approaches to examine the foliar application of ZnSO4 and

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ZnO nanoparticles for field-grown winter wheat

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Teng Zhang1, 2, Hongda Sun1, Zhiyuan Lv1, Lili Cui1, Hui Mao1, 2, *, Peter M. Kopittke3

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5 6 7 8 9

College of Natural Resources and Environment, Northwest A&F University, Yangling,

712100, Shaanxi, China 2

Key Laboratory of Plant Nutrition and the Agri-environment in Northwest China, Ministry

of Agriculture, Yangling, 712100, Shaanxi, China 3

School of Agriculture and Food Sciences, The University of Queensland, St. Lucia,

Queensland 4072, Australia

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*

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Address: College of Natural Resources and Environment, Northwest A&F University,

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Corresponding author: Hui Mao

Yangling, 712100, Shaanxi, China

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Tel.: +86 29 8708 0055; Fax: +86 29 8708 0055

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E-mail: [email protected]

1

ACS Paragon Plus Environment


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ABSTRACT

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The effects of foliar-applied ZnO nanoparticles (NPs) and ZnSO4 on winter wheat

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(Triticum aestivum L.) grain yield and grain quality were studied under field conditions, with

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the distribution and speciation of Zn within the grain examined using synchrotron-based

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X-ray fluorescence microscopy and X-ray absorption spectroscopy. Although neither of the

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two Zn compounds improved grain yield or quality, both increased the grain Zn concentration

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(average increments were 5 and 10 mg/kg for ZnSO4 and ZnO NPs treatments, respectively).

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Across all treatments, this Zn was mainly located within the aleurone layer and crease of the

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grain, although the application of ZnO NPs also slightly increased Zn within the endosperm.

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This Zn within the grain was found to be present as Zn phosphate, regardless of the form in

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which the Zn was applied. These results indicate that the foliar-application of ZnO NPs

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appears to be a promising approach for Zn bio-fortification as required to improve human

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health.

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KEYWORDS: ZnO nanoparticles, foliar, wheat, Zn biofortification, field experiment, X-ray

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absorption spectroscopy, X-ray fluorescence microscopy

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2


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INTRODUCTUION

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Zinc (Zn) is an essential micronutrient for the growth of plants, animals, and humans.

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However, Zn deficiency is widespread due to its inherently low concentration and poor

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bio-availability in a range of soils, leading to an inadequate dietary intake.1 Indeed,

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approximately 50 % of the world’s important agricultural soils are deficient in Zn,2 with Zn

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deficiency being one of the most widespread public health problems.3 Worldwide, Zn

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deficiency ranks eleventh most important of the factors causing illness and disease, being fifth

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most important in developing countries.4 Worldwide, approximately one third of the human

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population has an inadequate dietary intake of Zn, causing a loss of 28 million life-years

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annually.5-6

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To address this problem, the agronomic and genetic biofortification of Zn are of

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increasing interest. Although the genetic biofortification of crops is a sustainable and

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cost-effective approach, it is suitable only for the longer-term. In contrast, the application of

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Zn-containing fertilizers is a suitable approach for the short-term. Application of Zn fertilizers,

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either to soils or directly to the leaves, can improve the Zn nutrition of crops and further

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protect human health. Traditionally, bulk Zn sulfate (ZnSO4) has been used to overcome Zn

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deficiency, but fertilization using ZnSO4 does not always result in the desired increase in

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tissue Zn concentrations.7 Thus, it is useful to consider if novel Zn-containing compounds are

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potentially more efficient in improving the Zn status of the plant. One such compound is

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Zn-containing nanoparticles, which are of particular interest in the present study. Indeed, with

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the development of nanotechnology and the increasing use of nanoparticles, the use of

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nano-fertilizers is gaining increased attention.8-9 It is estimated that the annual value of 3



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nanotechnology related products is expected to reach US$3 trillion by 2020.10 Among these

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nanomaterials are ZnO nanoparticles (ZnO NPs) which are already used widely in sunscreen

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products, cosmetics, semiconductors, catalysts, solar cells and pharmaceuticals. Due to their

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smaller size, NPs have many unique properties, including a large surface area and higher

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reactivity compared to particles of larger size.11

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Nanoparticles potentially provide an option for not only improving crop nutrition but

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also for reducing adverse environmental outcomes.12 Zhao et al.13-14 reported that the

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application of ZnO NPs to soil increased the Zn content of tissues of cucumber (Cucumis

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sativus) and maize (Zea mays). In addition, in contrast to bulk ZnO, foliar application of ZnO

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NPs significantly increased Zn concentration in tissues of sunflower (Helianthus annuus L.).15

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The foliar application of fertilizers is of particular interest for alkaline soils, where Zn applied

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directly to the soil is rapidly converted into forms that are unavailable for plant roots.16

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Furthermore, the rates of Zn applied to soils are generally substantially greater than those

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applied to the foliage.17-19

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Recently, synchrotron-based techniques, including X-ray fluorescence microscopy

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(µ-XRF) and X-ray absorption spectroscopy (XAS), have been used increasingly for the in

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situ study of biological samples, both to investigate elemental distribution and speciation.20-21

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For example, Peng et al.22 examined the distribution and speciation of Cu and Zn in grains of

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rice (Oryza sativa L.) when the plants were supplied with CuO NPs in a glasshouse pot

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experiment. However, we are aware of only a few studies that have used synchrotron-based

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approaches for the in situ analysis of the distribution or speciation of Zn in plant tissues

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following the foliar-application of Zn compounds. For example, Ajiboye et al.23 used µ-XRF 4


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to examine the distribution of Zn in grains of wheat following the foliar-application of ZnSO4,

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finding that the Zn was mainly present in the aleurone layer, crease tissue, and to a lesser

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extent in endosperm. In addition, Du et al.24 used µ-XRF to examine changes in Zn

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distribution in leaves following foliar-application of ZnSO4 and Zn hydroxide nitrate to

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tomato (Solanum lycopersicum) and citrus (Citrus reticulatus). However, it remains unknown

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whether the speciation of Zn in the grains of wheat differs between plants depending upon the

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form of Zn supplied. In this regard, the use of synchrotron-based XAS can assist in

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understanding the role of Zn fertilizer in Zn bio-fortification, as required to improve human

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health.

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The aim of the present study was to use a field experiment to examine how the

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foliar-application of ZnO NPs influenced plant behavior, including the Zn concentration of

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wheat grain Zn. Synchrotron-based XAS was used to determine the speciation of Zn within

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the grain following this application of ZnO NPs and compared to plants supplied with ZnSO4.

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In a similar manner, synchrotron-based µ-XRF was used to compare the distribution of Zn

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within the wheat grain. Finally, the concentrations of proteins, starch and carbohydrates were

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also examined.

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MATERIALS AND METHODS

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Preparation of ZnO NPs and ZnSO4. The ZnO NPs (purity 99.6 %) was obtained from

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Hongsheng Material Sci & Tech Co. (China), and the ZnSO4·7H2O from Klamar (China). The

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surface area of the ZnO NPs was calculated by the multipoint Brunauer-Emmett-Teller (BET)

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method.25 The morphology of the ZnO NPs was examined in deionized (DI) water using 5



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transmission electron microscopy (TEM, JEOL 100CX, Japan), with the hydrodynamic size

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examined using dynamic light scattering (DLS, Wyatt Technology Dynapro Titan TC, US).

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Site Description and Experiment Design. The experimental field site is located in

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Yujiagong Village, Yongshou County, Shaanxi Province, China (latitude 34°49′ N, longitude

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108°11′ E, elevation 1127 m above sea level), being a typical Zn deficient (DTPA-Zn < 0.50

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mg/kg) area within the Loess Plateau (Table 1). The soil at the trial site is classified as

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Earth-cumuli-Orthic Anthrosols (Udic Haplustalf in the US Soil Taxonomy). The annual

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precipitation is 610 mm and the average annual temperature is 10 °C. Approximately 65 % of

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the annual precipitation falls between July and September.

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The experimental design was a randomized complete block with four replicates. The

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three treatments consisted of: no Zn fertilizer (CK), foliar-applied ZnO NPs (0.2 % w/v, i.e. 2

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g/L) at a rate of 1.2 kg/ha (FZnO), and foliar-applied ZnSO4·7H2O (0.7 % w/v, i.e. 7 g/L) at a

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rate of 4.2 kg/ha (FZn). Therefore, 12 plots were arranged, with each having an area of 4 m2

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(2 m × 2 m). The nanoparticles that were applied did not have a surface coating. The foliar

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application rates of ZnO NPs and ZnSO4·7H2O were calculated to correspond to the same

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level of Zn, being 0.96 kg Zn ha-1, with the rate at which ZnSO4·7H2O applied corresponding

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to the recommended rate for this region.7 In stem elongation and early milk stages, the two Zn

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compounds were sprayed with a rate of half, respectively. In addition to this foliar-application

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of Zn, basal nutrients were applied to the soil prior to sowing, with 120 kg N ha-1 (as urea)

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and 90 kg P2O5 ha-1 (as superphosphate) applied to each plot. These field experiments were

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conducted during winter from September 20 to June 15 in 2015-2016 (first growing season,

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Year 1) and from September 22 to June 18 in 2016-2017 (second growing season, Year 2) 6


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with wheat cultivar luohan 6. These same treatments were applied in both cropping seasons.

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Grain Yield and Zn Concentration. The wheat grain was harvested in mid-June of

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each year, with yield determined. Some of the samples were thoroughly washed with DI water

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and oven-dried at 65 °C for 48 h. Then the dried grains were ground using a ball miller

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(RETSCH MM400, Germany) and digested with 5 mL HNO3 and 1 mL H2O2 by a microwave

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digestion system (PreeKem WX-800, Shanghai, China). A standard wheat flour GBW10046

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(GSB-24) was also used for quality control, with the average recovery being 93 %. The bulk

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Zn concentration of the grain was determined by using graphite furnace atomic absorption

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spectrophotometry (HITACHI Z2000, Japan). To examine the dissolution of the ZnO NPs, the

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concentration of soluble Zn concentration was examined in supernatants of 2 g/L ZnO NPs

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suspensions as described by Lin and Xing26. The suspensions were first prepared using DI

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water before being sonicated for 30 min (SHUMEI KQ-500DE, Zhejiang, China).

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Determination of Total Soluble Sugars and Starch. The soluble sugars and starch were

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extracted as described by Verma and Dubey.27 For soluble sugars, 0.1 g of the ground grain

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was homogenized in 10 mL of 80 % ethanol and placed in a water bath heated to 80 °C for 30

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min. Thereafter, the contents were centrifuged at 22,000 g for 20 min. This process was

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repeated three times, with the three supernatants combined. Thereafter, the residue was

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removed for the measurement of starch. Briefly, the residue was oven-dried at 80 °C for 24 h,

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then 2 mL of DI water was added to boil the dried residue with a water bath for 15 min. The

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contents were mixed with 2 mL of concentrated H2SO4, and stirred with a glass rod for 15 min.

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The extracts were centrifuged at 3000 g for 20 min, and the extraction performed with 50 %

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H2SO4. The supernatants were combined, and the content of soluble sugars and starch were 7



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determined as described by Dubois and Gilles.28

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Determination of Crude Protein and Components. The protein components were

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extracted as described by Chen and Bushuk.29 Briefly, 0.5 g of the ground grain material was

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extracted using: DI water, 0.5 M NaCl, 70 % ethanol, and 0.05 M acetic acid. The albumin,

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globulin, prolamin, and glutelin were obtained sequentially, with their content determined as

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described by Bradford.30 The crude protein content was measured by DigiPREP TKN Systems

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(FOSS KjeltecTM 8400, Sweden) with a coefficient of 5.7.

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µ-XRF Analysis of Elemental Distribution. The wheat grain was washed with DI water

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three times before being placed in liquid nitrogen for 30 min. Samples were transversely

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sectioned (150 µm thick) with a Lecia CM1950 cryomicrotome at -20 °C with Tissue Tek

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(Sakura Finetek USA, Torrance, CA) embedding medium. Then a series of thin sections were

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obtained and placed on Kapton tape. Samples were freeze-dried for 1 h (Telstar

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LYOQUEST-85 plus, Spain) at -53 °C and 0.140 mBar pressure.

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The µ-XRF mapping was conducted at beamline 4W1B of the Beijing Synchrotron

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Radiation Facility (BSRF). The beam was focused to 50 µm × 50 µm (vertical × horizontal)

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using Kirkpatrick-Baez mirrors and a polycapillary lens. The two dimensional mapping was

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conducted at an incident energy of 15 keV in step-mode, with a step size of 500 µm. The XRF

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signal was collected using a Si (Li) solid state detector with a live time of 60 s. The XRF data

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were analyzed using the PyMCA software.31

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Bulk XAS Analyses of Zn Speciation. The grain samples were placed in liquid nitrogen

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for 30 min after harvest and freeze-dried for 72 h. Afterwards, the grain samples were

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homogenized using an agate mortar and pestle, formed into pellets, placed on Kapton film, 8


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and placed on sample holders to allow for analysis. The X-ray absorption near edge structure

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(XANES) spectra were collected at the Zn K-edge (9.659 keV) at the XAS beamline of the

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Australian Synchrotron in fluorescence mode with a 100-element solid-state Ge detector. To

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minimize beam-induced artifacts and thermal disorder, all samples were analyzed in a cryostat

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(ca. 12 K, liquid helium). The spectra were calibrated using a Zn foil measured

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simultaneously in transmission.

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In addition to the grain samples, XANES spectra were also collected for eight standard

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compounds, being ZnO NPs, Zn phosphate (Zn3(PO4)2), Zn phytate, Zn cysteine, Zn histidine,

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Zn citrate, Zn oxalate, and aqueous (Zn2+), as described previously.32 The XANES spectra

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were processed using ATHENA version 0.8.56, with linear combination fitting (LCF) used for

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sitting the sample spectra.

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Data Analysis. Statistical analyses were performed using SPSS (Version 19.0, SPSS

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Inc.). Means were compared using analyses of variance (ANOVA), with results reported as

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mean values ± standard deviation (SD). A probability level of p < 0.05 was considered as

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significant in all measured traits.

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RESULTS AND DISCUSSION

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Characterization of ZnO NPs. The BET surface area of the ZnO NPs was measured to

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be 52 m2/g. Using TEM, the ZnO NPs were found to be nearly spherical to oblong in shape,

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with some aggregation observed due to drying on the TEM grid (Figure 1A). From the TEM

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micrographs, the average diameter was measured for > 150 single particles, with the average

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size being 20 ± 5 nm. These measurements from TEM are in agreement with the data 9



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provided by the manufacturer. The DLS analysis was used to examine hydrodynamic size,

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with a mean hydrodynamic diameter of 406 nm (Figure 1B).

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Effects of the Foliar-application of Zn on Grain Yield and Zn Concentration.

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Overall, average yield was higher in Year 1 (7,817 kg/ha) than in Year 2 (5,210 kg/ha) (Table

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2). However, the foliar-application of Zn compounds did not increase yield, either as ZnSO4

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or as ZnO NPs (Table 2). In contrast, the concentration of Zn within the grain was increased

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significantly by the foliar-application of both of these compounds. Specifically, the grain Zn

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concentration increased from 18 mg/kg in Year 1 and 24 mg/kg in Year 2 in the control, to 21

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mg/kg in Year 1 and 30 mg/kg in Year 2 in the ZnSO4 treatment, and to 27 mg/kg in Year 1

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and 35 mg/kg in Year 2 in the ZnO NPs treatment (Table 2).

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The observation in the present study that the foliar application of Zn did not increase

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yield is in general agreement with previous studies, although some studies have also reported

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that yield does actually increase upon foliar fertilization.33-34 For example, studying seven

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countries, Ram et al.34 reported that foliar-application of Zn generally increased yield in the

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trial site in Pakistan, but not in India, Brazil, China, Turkey, or Zambia – this pattern of grain

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yield response was not related to concentrations of DTPA-Zn in the soil. In a similar manner,

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Zhang et al.17 reported that foliar application of ZnSO4·7H2O did not increase wheat grain

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yield in China. In contrast to the grain yield, the foliar-application of Zn compounds

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significantly increased the grain Zn concentration, with these findings being similar to those

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reported previously in wheat.33-35 We noted, however, that the application of ZnO NPs

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increased the grain Zn concentration to a greater extent than did the application of ZnSO4,

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with average values being 21 mg/kg for the control, 26 mg/kg for ZnSO4, and 31 mg/kg for 10


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ZnO NPs (Table 2). Cakmak et al.36 and Pfeiffer et al.37 have stated that the increase in the Zn

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concentration of the grain should be ≥ 10 mg/kg to have a measurable biological influence on

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human health. Thus, in the present study, the increase in the Zn concentration in the grain for

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the ZnO NPs treatment is similar to that reported to be beneficial by Cakmak et al.36 and

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Pfeiffer et al.37 . Comparing against control, the foliar-application of ZnSO4 increased grain

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Zn concentration by 3 mg/kg in Year 1 and 6 mg/kg in Year 2, while foliar-applied ZnO NPs

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increased concentrations by 9 mg/kg in Year 1 and 11 mg/kg in Year 2, thereby indicating the

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greater efficacy of ZnO NPs. It is possible that the higher efficiency of the ZnO NPs relative

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to the ZnSO4 is due to the smaller size effect of the nanoparticles which increases adhesion on

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the leaf surface,38 with the slow dissolution of the ZnO NPs (the concentration of soluble Zn

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in a ZnO NP suspension was measured to be 4.2 mg/L), with this then serving as a sustained

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Zn pool to provide Zn nutrition for plant growth. However, further experiments would be

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required to examine the adhesion of the NPs to the leaf surface.

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In the present study, the ZnSO4 and ZnO NPs were applied in stem elongation and milk

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stages, with these having been identified previously as the being the best for the

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bio-fortification of Zn.23, 36 However, whilst we found that the ZnO NPs were more effective

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for increasing the grain Zn concentration, the grain Zn concentrations for both the ZnO NPs

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(average of 31 mg/kg) and ZnSO4 (average of 26 mg/kg) were < 40 mg/kg (with this being

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the value recommended by Food and Agriculture Organization of the United Nations), and so

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further work is required in this regard.

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Effects of ZnO NPs and ZnSO4 on Grain Quality Factors. It was hypothesized that

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the foliar fertilization of both ZnSO4 and ZnO NPs would improve grain yield and Zn 11



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concentration, as well as improving grain quality. Starch and sugars have important effects on

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grain quality, and are the major carbohydrate components in wheat grains, being mostly stored

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in the endosperm.14, 39 The grain protein content is also an important factor in determining

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quality.40 Normally, prolamin and glutelin components account for 66-87 % of the total

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protein in wheat grain. Although the proportions of albumin and globulin are comparatively

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small, they are rich in lysine and have higher nutritional value. In the present study, we

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examined the effects of the foliar-application of ZnO NPs and ZnSO4 on these grain quality

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factors (Tables 3 and 4). Neither the application of ZnO NPs or ZnSO4 markedly influenced

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any of the measured grain quality parameters, indicating that although the foliar application of

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Zn increased grain Zn concentrations, it did not alter concentrations of carbohydrates

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(protein).

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The finding that the foliar-application of Zn did not impact upon grain quality is in

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general agreement with previous studies.41-42 For example, although Li et al.43 also found that

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the foliar-application of Zn increased the Zn concentration of the grain, it had no influence on

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the grain protein concentration. However, where the foliar-application of Zn increases yield, it

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is possible that this will also impact upon grain quality. For example, in a field experiment,

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Amiri et al.44 found an inverse relationship between the grain Zn concentration, protein

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concentration, and grain yield. Specifically, the addition of Zn decreased the protein

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concentration due to a dilution effect resulting from increased yield, but the addition of Zn did

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not increase yield in the present study (Table 2).

250 251

Synchrotron

µ-XRF

Analysis

of

Zn

Localization

in

Wheat

Grains.

Synchrotron-based µ-XRF was used for the in situ examination of the distribution of Zn in 12


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sections of wheat grain. In all three treatments, the Zn was mainly found within the aleurone

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layer and the crease region (Figures 2 and 3). These results are in agreement with the studies

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of both De Brier et al.20 and Ajiboye et al.23 who also examined wheat grain, with it being

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reported that the Zn within the grain was mainly present within the aleurone and crease.

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However, as expected based upon bulk measurements (Table 2), we found that the

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concentration of Zn within these tissues differed between treatments, being highest for the

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ZnO NPs treatment and lowest in the control. Of particular importance, it was also found that

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the Zn concentration in the endosperm increased slightly for the ZnO NPs treatment (Figures

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2 and 3). The endosperm fraction of the grain is widely consumed worldwide and an increase

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in the endosperm Zn concentration would be important for human nutrition. In a similar

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manner, Cakmak et al.36 examined Zn distribution within wheat grain using laser ablation

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inductively coupled plasma mass spectrometry (LA-ICP-MS), also finding that foliar-applied

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Zn could potentially increase the Zn concentration within the endosperm. Recently, using

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µ-XRF analyses, Ajiboye et al.23 also found similar result, with the Zn concentration of the

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endosperm increasing slightly. Indeed, It is known that Zn is primarily transported from the

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crease vascular parenchyma into the nucellar projection and modified aleurone, then

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distributes to the aleurone layer, the endosperm and the embryo.20, 45 However, due to inherent

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barriers of aleurone layer (with transported Zn, Fe and other elements occurring as phosphates

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in this layer), this limits Zn from entering the endosperm.46-47

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Although the production of wheat flour for human consumption results in the removal of

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the seed coat, embryo, and the aleurone layer,48 the increased Zn observed in the crease region

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and the endosperm in the present study (along the whole vertical section of wheat grains, 13



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Figures 2 and 3) would presumably result in a substantial improvement in Zn delivery for

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human health, especially following the foliar-application of ZnO NPs.

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Speciation of Zn within Wheat Grains. First, we compared the Zn K-edge spectra of

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the various standard compounds (Figure 4A). It was noted that these spectra had various

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distinctive features, including the (i) energy corresponding to the white-line peak, (ii) height

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of the white-line peak, and (iii) presence of other spectral features. First, the energy

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corresponding to the white-line peak were examined. For all forms of Zn complexed by

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carboxyl groups (such as Zn citrate and Zn oxalate) and uncomplexed Zn2+ (i.e. the Zn(NO3)2

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solution), the white-line peak was found to correspond to an energy of 9,669 eV. However,

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differences were found for the remaining standard compounds examined, being at ca. 9,668.4

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eV for Zn histidine, 9,667 eV for Zn phosphate and Zn phytate, and at 9,665 eV for Zn

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cysteine, whilst ZnO NPs had a shoulder at 9,665 eV and a peak at ca. 9,671 eV. Next, the

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height of the white-line peak was considered. Again, it was found that the spectra were similar

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in this regard for all forms of Zn complexed by carboxyl groups and for uncomplexed Zn2+,

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with these spectra having a comparatively high peak. Indeed, in approximate descending

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order of peak height: Zn-carboxyl / free Zn2+ > ZnO NPs ~ Zn phytate ~ Zn histidine > Zn

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phosphate > Zn cysteine. Thus, it was clear that the spectra for the various forms of Zn

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complexed by carboxyl groups and the spectrum for uncomplexed Zn2+ were visually similar,

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and it was not possible to distinguish between these various forms of Zn.

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Next, we examined the grain samples. It was firstly noted that the spectra from all wheat

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grain samples were similar in appearance (Figure 4B), with the foliar application of Zn not

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influencing the speciation of Zn in the grains regardless of the form in which the Zn was 14


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added. Next, it was noted that the height of the white-line peak and its energy (9,667 eV) for

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the samples corresponded most closely with that of Zn associated with P (i.e. either Zn

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phosphate or Zn phytate) (Figure 4C). However, the spectrum was not identical to that of Zn

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phosphate, indicating that although likely dominated by Zn phosphate, some Zn was also

300

present in other forms. Indeed, using linear combination fitting (LCF), it was estimated that

301

70-80 % of the Zn was present as Zn phosphate but that 20-30 % was present as other forms.

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However, the form of the remaining 20-30 % of the Zn remains unclear, with several different

303

compounds yielding similar R-factors. Certainly, it was apparent that the Zn in the wheat

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grains was not present as ZnO NPs despite the foliar application of this compound, with the

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spectrum for the grain differing markedly from the spectrum for ZnO NPs (Figures 4A and

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4B).

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The data suggest that the uptake of Zn in leaves treated with ZnO NPs occurred as

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soluble Zn rather than as NPs per se. This result is consistent with the soil application of ZnO

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NPs in cowpea (Vigna unguiculata) and wheat,9, 11 as well as with the addition of ZnO NPs to

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the nutrient solution for growth of maize.49 Although several studies have examined the

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potential uptake of NPs by roots,11,

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absorption through leaves.8 Thus, we hypothesize that the dissolution of the ZnO NPs on the

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leaf surface (the concentration of Zn in a saturated suspension of ZnO NPs being 4.2 mg/L)

314

allowed the movement of soluble Zn across the leaf surface, before being translocated to the

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grain in a similar manner to that occurring when the Zn was supplied as ZnSO4. In this regard,

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it is known that Zn in the leaf and stem tissues of wheat can be remobilized and transported

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into wheat grains during the period of seed formation.36, 51. However, further studies are still

49-50

comparatively few studies have examined their

15



318

required in this regard in order to understand the interactions between leaf tissues and NPs.

319

Furthermore, additional studies would also be required to consider any potential adverse

320

effect of ZnO NPs on human health during their application (spraying) within the field by

321

farmers.

322

Another issue that deserves consideration is the observation that Zn was primarily

323

associated with P in all treatments, seemingly largely as Zn phosphate (Figure 4C). The

324

speciation of Zn appears to differ markedly between plant species and between tissues. For

325

example, in seeds of cowpea, Wang et al.9 reported that Zn histidine and Zn cysteine were the

326

dominant compounds. In a soil-based study examining soybean (Glycine max), Zn was found

327

to largely be associated with citrate in the pods.52 In another study, Dimkpa et al.11 examined

328

wheat seedlings grown in sand culture and found that the addition of bulk ZnO, ZnO NPs and

329

Zn2+ resulted in the formation of Zn phosphate (hopeite, Zn3(PO4)2·4H2O) in the shoots of

330

wheat.11 Finally, Hatzack et al.53 and Persson et al.54 both found the accumulation of phytic

331

acid had no effect on Zn, and the Zn was not complexed by phytate in barley (Hordeum

332

vulgare L.) grains.

333

In summary, across the two cropping seasons of the present study, although the foliar

334

application of ZnSO4 and ZnO NPs did not increase grain yield, starch, soluble sugars, or

335

proteins, their application did significantly increased the concentration of Zn in the grain. The

336

magnitude of this increase was greater for ZnO NPs than for ZnSO4, with the increase in Zn

337

concentration for the ZnO NPs treatment (up to 10 mg/kg) being sufficient to have a

338

measurable influence on human health. Using µ-XRF mapping to examine the distribution of

339

Zn within the wheat grains, it was found that Zn was mainly present within the aleurone layer 16


Page 16 of 41

Page 17 of 41


340

and within crease region, regardless of treatment. However, for ZnO NPs treatment, the Zn

341

concentration in the endosperm also increased somewhat, with this being particularly

342

important for human Zn nutrition. Although the seed coat, embryo, and aleurone layer are

343

removed for human consumption, the increased Zn within the crease region is also likely to

344

still improve Zn delivery for human health. Furthermore, XANES analysis suggested that

345

although the use of ZnO NPs increased Zn concentrations in the grain, this Zn that

346

accumulated within the grain was not present as ZnO NPs, but rather, it was in the same form

347

as in the two other treatments. To the best of our knowledge, this is the first report of the

348

speciation of Zn in wheat grain following the foliar-application of ZnSO4 and ZnO NPs

349

following growth in field conditions. The foliar-application of ZnO NPs appears to be a

350

promising approach for Zn bio-fortification as required to improve human health, particularly

351

in developing countries.

352 353

ABBREVIATIONS

354

LCF: Linear Combination Fitting

355

µ-XRF: Synchrotron-based X-ray Fluorescence Microscopy

356

XAS: X-ray Absorption Spectroscopy

357

DI water: deionized water

358

ZnO NPs: ZnO nanoparticles

359

TEM: transmission electron microscopy

360

DLS: dynamic light scattering

361

LA-ICP-MS: laser ablation inductively coupled plasma mass spectrometry 17



362 363

ACKNOWLEDGEMENTS

364

The staff members at 4W1B beamline of Beijing Synchrotron Radiation Facility,

365

Institute of High Energy Physics, Chinese Academy of Sciences are acknowledged for their

366

support in measurements and data reduction. Components of this research were undertaken on

367

the XAS beamline at the Australian Synchrotron, part of the Australian Nuclear Science and

368

Technology Organisation (ANSTO).

369 370 371

FUNDING The authors gratefully acknowledge the National Natural Science Foundation of China

372

(41571282), the Fundamental Research Funds for the Central Universities (2452015047), the

373

Natural Key Technologies R&D Program (2015BAD23B04), and the Special Fund for

374

Ago-scientific Research in the Public Interest (201503124).

375 376 377

NOTES The authors declare that they have no competing interests.

378

18


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379

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Smolders, E. Distribution of minerals in wheat grains (Triticum aestivum L.) and in roller

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Peralta-Videa, J. R.; Ge, Y.; Priester, J. H.; Holden, P. A.; Gardea-Torresdey, J. L. In Situ 25



531

synchrotron X-ray fluorescence mapping and speciation of CeO2 and ZnO nanoparticles in

532

soil cultivated soybean (Glycine max). ACS Nano 2013, 7, 1415-1423.

533

(53) Hatzack, F.; Johansen, K. S.; Rasmussen, S. K. Nutritionally relevant parameters in

534

low-phytate barley (Hordeum vulgare L.) grain mutants. J. Agric. Food Chem. 2000, 48,

535

6074-6080.

536

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537

Simultaneous iron, zinc, sulfur and phosphorus speciation analysis of barley grain tissues

538

using SEC-ICP-MS and IP-ICP-MS. Metallomics 2009, 1, 418-26.

539 540

FIGURE CAPTIONS

541

Figure 1. (A) A transmission electron microscopy (TEM) image of the ZnO NPs. (B)

542

Hydrodynamic size distribution of the ZnO NPs in deionized water using dynamic light

543

scattering (DLS) analysis.

544

Figure 2. A light micrograph of a cross-section of a wheat grain (as shown in Figure 3).

545

Figure 3. Distribution of Zn within transverse sections of wheat grain in Year 2 from the

546

control (CK), foliar ZnO NPs (FZnO), and Foliar ZnSO4 (FZn).

547

Figure 4. (A) Normalized Zn K-edge XANES spectra of standard compounds. (B)

548

Normalized Zn K-edge XANES spectra of wheat grains from the three treatments, being CK,

549

FZn, FZnO. (C) The comparisons of Zn K-edge XANES spectra for the grains of FZnO with

550

the Zn-phosphate (solid, Zn3(PO4)2). The vertical dotted lines in (A) and (B) correspond to

551

9.667, 9.669, and 9.671 keV, being the white-line peaks for Zn associated with P (9.667 keV),

552

Zn associated with carboxyl groups or free Zn2+ (9.669 keV), and ZnO (9.671 keV). 26


Page 26 of 41

Page 27 of 41


553 554

TABLES

555

Table 1. Basic Physical and Chemical Properties of the soil from the field sitea

556 557

Properties Value pH (Soil:water 1:2.5) 8.31 ± 0.38 Calcium carbonate (%) 9.75 ± 0.15 Organic C (%) 1.55 ± 0.08 Sand (%) 27 ± 0.7 Silt (%) 39 ± 0.9 Clay (%) 34 ± 0.0 18.6 ± 0.10 Cation exchange capacity (cmolc/kg) DTPA-Zn (mg/kg) 0.49 ± 0.14 Total Zn (mg/kg) 63.6 ± 8.12 Total N (mg/kg) 1040 ± 61.23 Available P (mg/kg) 18.8 ± 7.16 Available K (mg/kg) 154.5 ± 14.17 a Data are means of four replicates ± standard deviation. Available P was extracted by 0.5 M NaHCO3, and available K was extracted using 1 M NH4AC.

27



558 559

Table 2. Effects of ZnO NPs and ZnSO4 on Grain Yield and Zn Concentrationa Year 1

Year 2

treatment

560 561

grain yield Zn grain yield Zn (kg/ha) (mg/kg) (kg/ha) (mg/kg) CK 7881 ± 216 a 18.4 ± 0.13 c 5412 ± 279 a 23.6 ± 0.83 c FZnO 7750 ± 437 a 26.5 ± 0.60 a 5062 ± 288 a 34.6 ± 1.40 a FZn 7820 ± 692 a 21.1 ± 0.05 b 5157 ± 485 a 29.5 ± 0.30 b a Data are means of four replicates ± standard deviation. Different letters within same column indicate significant difference at 0.05 level.

28


Page 28 of 41

Page 29 of 41


562 563

564 565 566

Table 3. Effects of ZnO NPs and ZnSO4 on Soluble Sugars and Starcha Year 1 Year 2 treatment soluble sugars starch soluble sugars starch (%) (%) (%) (%) CK 27.45 ± 2.13 a 51.45 ± 0.43 a 24.00 ± 3.12 a 50.27 ± 0.69 a FZnO 28.17 ± 3.15 a 52.33 ± 0.13 a 24.46 ± 1.44 a 48.66 ± 0.75 a FZn 26.98 ± 2.46 a 52.02 ± 0.99 a 24.08 ± 5.47 a 48.64 ± 2.40 a a Data are means of four replicates ± standard deviation. Different letters within same column indicate significant difference at 0.05 level.

29



567

Table 4. Effects of ZnO NPs and ZnSO4 on Crude Protein and Its Fraction (%)a Year 1 Year 2 CK FZnO FZn CK FZnO FZn crude protein 11.33 ± 0.23 a 11.49 ± 0.07 a 11.51 ± 0.48 a 11.96 ± 0.30 a 12.03 ± 0.25 a 12.22 ± 0.46 a albumin 2.27 ± 0.09 a 2.32 ± 0.10 a 2.33 ± 0.05 a 2.39 ± 0.06 a 2.41 ± 0.05 a 2.44 ± 0.09 a globulin 0.91 ± 0.06 a 0.93 ± 0.07 a 0.96 ± 0.03 a 0.96 ± 0.02 a 0.96 ± 0.02 a 0.98 ± 0.04 a prolamin 3.39 ± 0.03 a 3.45 ± 0.12 a 3.43 ± 0.04 a 3.59 ± 0.09 a 3.61 ± 0.08 a 3.67 ± 0.14 a glutelin 2.83 ± 0.06 a 2.88 ± 0.05 a 2.84 ± 0.02 a 2.99 ± 0.07 a 3.01 ± 0.66 a 3.05 ± 0.12 a a Data are means of four replicates ± standard deviation. Different letters within same row indicate significant difference at 0.05 level. Trait

568 569

30


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Page 31 of 41


570

FIGURE GRAPHICS

571 572

Figure 1. (A) A transmission electron microscopy (TEM) image of the ZnO NPs. (B)

573

Hydrodynamic size distribution of the ZnO NPs in deionized water using dynamic light

574

scattering (DLS) analysis.

575

31



576 577

Figure 2. A light micrograph of a cross-section of a wheat grain (as shown in Figure 3).

32


Page 32 of 41

Page 33 of 41


578 579

Figure 3. Distribution of Zn within transverse sections of wheat grain in Year 2 from the

580

control (CK), foliar ZnO NPs (FZnO), and Foliar ZnSO4 (FZn).

33



581 582

Figure 4. (A) Normalized Zn K-edge XANES spectra of standard compounds. (B)

583

Normalized Zn K-edge XANES spectra of wheat grains from the three treatments, being CK,

584

FZn, FZnO. (C) The comparisons of Zn K-edge XANES spectra for the grains of FZnO with

585

the Zn-phosphate (solid, Zn3(PO4)2). The vertical dotted lines in (A) and (B) correspond to

586

9.667, 9.669, and 9.671 keV, being the white-line peaks for Zn associated with P (9.667 keV),

587

Zn associated with carboxyl groups or free Zn2+ (9.669 keV), and ZnO (9.671 keV).

588

34


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589

TOC

590 591

For Table of Contents Only

35



Figure 1. (A) A transmission electron microscopy (TEM) image of the ZnO NPs. 59x51mm (300 x 300 DPI)


Page 36 of 41

Page 37 of 41


Figure 1. (B) Hydrodynamic size distribution of the ZnO NPs in deionized water using dynamic light scattering (DLS) analysis. 90x60mm (300 x 300 DPI)



Figure 2. A light micrograph of a cross-section of a wheat grain (as shown in Figure 3). 80x60mm (300 x 300 DPI)


Page 38 of 41

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Figure 3. Representative Zn µ-XRF mapping of the grain transverse sections in Year 2 from the control (CK), foliar ZnO NPs (FZnO), and Foliar ZnSO4 (FZn). 80x80mm (600 x 600 DPI)



Figure 4. (A) Normalized Zn K-edge XANES spectra of standard compounds. (B) Normalized Zn K-edge XANES spectra of wheat grains from the three treatments, being CK, FZn, FZnO. (C) The comparisons of Zn K-edge XANES spectra for the grains of FZnO with the Zn-phosphate (solid, Zn3(PO4)2). The vertical dotted lines in (A) and (B) correspond to 9.667, 9.669, and 9.671 keV, being the white-line peaks for Zn associated with P (9.667 keV), Zn associated with carboxyl groups or free Zn2+ (9.669 keV), and ZnO (9.671 keV). 90x98mm (600 x 600 DPI)


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For Table of Contents Only 47x26mm (600 x 600 DPI)


Using Synchrotron-Based Approaches To Examine the Foliar

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