Food Chemistry 402 (2023) 134290

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Zinc glycerolate (Glyzinc): A novel foliar fertilizer for zinc biofortification

and cadmium reduction in wheat (Triticum aestivum L.)
Jiapan Lian a, Liping Cheng a, Xu Zhai a, Ruofan Wu a, Xiwei Huang a, Dan Chen a, Jianqing Pan b,
M.J.I. Shohag c, Xiaoping Xin d, Xinwei Ren e, Zhenli He d, Xiaoe Yang a, *, 1
a
Ministry of Education (MOE) Key Laboratory of Environmental Remediation and Ecosystem Health, College of Environmental and Resources Sciences, Zhejiang
University, Hangzhou 310058, China
b
Agriculture Bureau of Changxing County, Zhejiang Province, Huzhou 323000, China
c
Department of Agriculture, Faculty of Agricultural Sciences, Bangabandhu Sheikh Mujibur Rahman Science and Technology University, Gopalganj 8100, Bangladesh
d
Department of Soil, Water and Ecosystem Sciences, Indian River Research and Education Center, University of Florida-IFAS, Fort Pierce, FL 34945, United States
e
School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, China

A R T I C L E I N F O A B S T R A C T

Keywords: Sustainable strategies are essential for zinc (Zn) biofortification and cadmium (Cd) reduction in staple food crops.
Biofortification Herein, we evaluated the phytotoxicity of Glyzinc under foliar and root application (FA&RA) in a lab-scale
Wheat experiment, and then investigated its Zn efficiency and Cd reduction through foliar application on wheat (Tri­
Foliar fertilizer
ticum aestivum L.) under field conditions. Compared to RA, FA of Glyzinc exhibited no adverse effect on wheat
Glyzinc
Cadmium
growth and oxidative stresses at all doses. In field conditions, FA of Glyzinc remarkably increased Zn (28.7 %), S
Phytic acid (10.4 %), Cu (17.3 %) and crude protein (9.1 %) content in wheat grain at 100 mg/L without damaging wheat
yield. Furthermore, FA of Glyzinc significantly reduced the grain phytic acid (PA) (23.7 %) and Cd level (19.5 %),
as well as PA to Zn molar ratio (32.3 %). Overall, our results indicate that Glyzinc has great potential as a high-
efficiency foliar fertilizer for Zn biofortification and safe crop production in nano-enabled agriculture.

1. Introduction Agronomic biofortification of staple foods to increase crop yield and

Zinc (Zn) deficiency in humans is a serious global health concern,
especially in developing countries, which is gaining growing attention
worldwide (Hambidge, 2000). It has been reported that more than two
billion people suffering from Zn deficiency across the world (Alloway,
2009; White & Broadley, 2009). As the staple food for over 40 % of the
world’s population, wheat is considered as an important dietary source
of calories, protein and micronutrients such as Zn (Velu et al., 2014).
However, more than half of the world’s wheat is grown on zinc-deficient
soils (Alloway, 2009; Cakmak et al., 2010), resulting in low Zn
bioavailability in wheat grain, which fails to provide the required Zn
needed for human health and nutrition. In addition, under intensive
agriculture model, the adoption of high-yielding wheat varieties would
further reduce the Zn concentration in grain through the dilution effect
(Fan et al., 2008; Liu et al., 2014). Therefore, overcoming the trade-off
between grain yield and Zn content of wheat grain is of great signifi­
cance in addressing Zn deficiency in humans.
nutritional levels of edible parts is an effective and sustainable strategy
to overcome food security (Velu et al., 2014). In this context, foliar
fertilization is widely accepted due to its high efficiency, low cost, time
and effort saving, and environmental friendliness (Niu et al., 2021).
Foliar application of different Zn sources, including Zn sulfate (Yu et al.,
2021; Zou et al., 2012), EDTA-Zn (Doolette et al., 2018; Wang et al.,
2020), and amino acid-cheated Zn (Ghasemi et al., 2013; Souri et al.,
2017) has significantly increased the Zn2+ concentration and bioavail­
ability in wheat grain under Zn-deficiency soil conditions. However,
foliar application of these Zn forms not only resulted limited uptake
efficiency by plants due to the rapid release of Zn2+, but also inadver­
tently led to crop leaf damage, nutrient imbalance, and yield loss (Xu
et al., 2022). Moreover, the degradation by-products of Zn fertilizers
such as EDTA-Zn are environmentally hazardous because of their
persistence, which may induce various environmental hazards in agri­
culture (Garbisu & Alkorta, 2001). Recently, nano-biofortification has
been proposed and proven to be a new approach against nutritional

* Corresponding author at: College of Environmental and Resources Sciences, Zhejiang University, No. 866 Yuhangtang Road, Hangzhou 310058, China.
E-mail address: xeyang@zju.edu.cn (X. Yang).
1
ORCID: 0000-0003-2725-26651.

https://doi.org/10.1016/j.foodchem.2022.134290
Received 21 March 2022; Received in revised form 18 August 2022; Accepted 13 September 2022
Available online 17 September 2022
0308-8146/© 2022 Elsevier Ltd. All rights reserved.
J. Lian et al.
deficiencies (El-Ramady et al., 2021; Raliya et al., 2018), For example,
using ZnO nanoparticles as a foliar nano-fertilizer is more effective and
biocompatible than traditional fertilizers in crops. Therefore, devel­
oping novel nano-enabled fertilizers to control the release and use effi­
ciency of nutrients is imperative and challenging.
In addition, wheat grain-derived products are the important dietary
source of human exposure to cadmium (Cd) (Rizwan et al., 2016).
Previous studies have shown that foliar Zn sprays can modulate the
process of Cd uptake by roots, root-to-shoot translocation, and stem/
leaf-to-grain remobilization in wheat through the antagonistic in­
teractions between Zn and Cd (Li et al., 2020). Recently, Wu et al. (2020)
found that foliar application of Zn (10–40 mg/L) could effectively
mitigate Cd toxicity by activating the antioxidant enzyme systems like
superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), ascor­
bate peroxidase (APX), and reduce Cd concentration in wheat grain by
13 %-50 %. By foliar co-application of different nanomaterials (ZnO,
Fe3O4 and SiO2 NPs), Hussain et al. (2021) achieved an incredible 99 %
reduction in Cd concentration and simultaneously increased Zn by about
55 % in wheat grain under a Cd contaminated field. In this way, foliar Zn
fertilization not only meets the Zn biofortification goals for wheat grain,
but also is an effective way to reduce human exposure to Cd from wheat-
based food.
Zinc glycerolate, a slow-release zinc complex that was first reported
in 1970, is widely applied as light and heat stabilizers for polymer and
drug formulations for health care products (Mir et al., 2012). It is also
often used as a zinc supplement, and an important component of cos­
metics, playing a role in lubrication, UV radiation protection, etc. In
addition, Zn and Cd exhibits similar chemical and biochemical proper­
ties therefore, application of Zn may increase Zn and reduce Cd uptake
by plants. Therefore, we hypothesized that Glyzinc has great potential as
a Zn nano-fertilizer for Zn biofortification and Cd minimization in crops.
As a result, the objectives of this study were to (i) explore the impacts of
Glyzinc on the physiology and biochemistry of wheat seedlings, (ii)
evaluate the effect of foliar Glyzinc application on grain yield, Zn con­
centration and bioavailability in wheat grain under agricultural prac­
tices, (iii) investigate the effect of foliar Glyzinc application on the
uptake, transport and accumulation of Cd in wheat grain. To the best of
our knowledge, this is the first work that comprehensively evaluate the
effectiveness of foliar spraying of Glyzinc on plant growth, gain yield, Zn
fortification and Cd minimization of wheat under field condition. The
results from this study will provide new insights into biofortification and
safe crop production enabled by nanotechnology.
2. Materials and methods
2.1. Materials and Characterization
Zinc glycerolate (C3H6O3Zn, Glyzinc) was provided by Jinheng
Lanhai Technology Co., ltd (Tianjin, China). Glyzinc is a white insoluble
powder, manufactured by the reaction of glycerin and zinc oxide. All
other chemical reagents were purchased from Sinopharm Chemical
Reagent Co., ltd. (Shanghai, China). Wheat seeds (cv. Zhengmai 10), a
relatively low-Cd wheat genotype, were selected through our previous
studies and purchased from local seed store (Huzhou, Zhejiang Province,
China). This cultivar (No. CNA20110802.3) was bred by Zhenjiang
Institute of Agricultural Science (ZIAS) in hilly region of Jiangsu Prov­
ince and is suitable and widely grown in Huainan Jiangsu and northern
Zhejiang regions.
Morphology and particle size of Glyzinc were characterized through
a scanning electron microscopy (SEM) (G300, ZEISS, Germany). Mean­
while, the element distribution mapping and their quantitative contents
were analyzed using energy-dispersive X-ray (EDX). The phase purity
and crystallinity of Glyzinc were determined by X-ray diffraction (XRD)
profile analysis. In addition, the chemical composition of Glyzinc was
determined by Fourier transform infrared spectroscopy (FTIR) spectral
analysis.
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Food Chemistry 402 (2023) 134290
2.2. Hydroponic experiment
2.2.1. Plant cultivation and treatment
Wheat seeds were surface sterilized in a 2 % (v/v) hydrogen peroxide
solution for 30 min, followed by rinsing with deionized water, and then
subjected to germination in the dark for five days. Uniformly growing
wheat seedlings were carefully selected and three of them were
anchored in each hole with plastic foam. Twelve plantlets were subse­
quently transferred to a 750 mL black PVC cup containing 600 mL of
modified 25 % strength Hoagland solution (Text S1). The nutrient so­
lution was maintained in the dark for the duration of the experiment.
Wheat seedlings were allowed to grow in a growth chamber with a day/
night cycle of 14/10 h, a day/night temperature of 26 ◦ C/20 ◦ C, a day/
night humidity of 70 %/85 %, and a light intensity of 400 µmolm-2s− 1.
After a week of pre-cultivation, wheat seedlings were then exposed to
Glyzinc and ZnSO4 solutions for 21 days, respectively. As for root
application, Glyzinc solutions were prepared by adding different
amount of Glyzinc into fresh nutrient solutions (50 % strength) to obtain
a series of concentrations: 50, 100, and 250 mg/L, followed by ultra­
sonication (100 W, 25 kHz) for 30 min. ZnSO4 soultion with an equiv­
alent amount of Zn as the 250 mg/L Glyzinc was also used as control in
this experiment. Four replicates were set for each treatment group.
These culture solutions were replenished with fresh solution every-three
days and thoroughly renewed once a week. In the case of foliar treat­
ment, Glyzinc suspensions (50, 100, and 250 mg/L) were prepared with
deionized water and then dispersed by ultrasound for 30 min (100 W,
25 kHz). Wheat leaves were sprayed every-three days and a total of 50
mL Zn suspensions or solution was applied. Meanwhile, leaves sprayed
with an equivalent amount of deionized water at the same time intervals
were set as the control group.
2.2.2. Morphological and physiological traits measurement
After 21 days, the development status of wheat seedlings was pho­
tographed and plant height was measured from the stem base to the
highest leaf implementation. Afterward, wheat seedlings were gently
removed from the suspensions, rinsed repeatedly with deionized water
and then cleaned with 20 mM EDTA-Na2 solution. Roots were separated
with scissor and scanned with a A4 scanner (ScanMaker i800 Plus,
Microtek, China), and root properties were characterized by RhizoVision
Explorer (Open-source, Noble, USA). The shoots and roots were then
blotted with paper towels and their fresh weights (FW) were recorded
separately. The chlorophyll and carotenoid contents of wheat leaves
were extracted and determined according to the Arnon-Lichtenthaler
method, and the detailed procedures were presented in the Text S2.
Plant tissues were then oven-dried at 65 ◦ C to a constant weight to
measure the dry weights (DW) of shoots and roots. Besides, the root/
shoot biomass ratio (RSR) and water content (WC) were calculated by
the following equation: RSR = DWroot/DWshoot × 100 %; WC=(FW-
DW)/FW × 100 %.
2.2.3. Oxidative stress, proline, antioxidant defense, and nutrients
determination
Measurements of antioxidant enzyme activities, proline and malon­
dialdehyde (MDA) content of wheat leaves in the presence of Glyzinc
were performed following a biological protocol of Chen and Zhang
(2016). Briefly, fresh leaves (0.2 g) were ground in liquid nitrogen and
homogenized with ice-cold phosphate buffer saline (PBS) (100 mM, pH
7.8). The homogenate was then centrifuged at 10,000 g for 20 min at
4 ◦ C. Then, the supernatant was collected as a crude enzyme extract for
further analysis. The proline concentration of the leaves was determined
by a colorimetric method based on the basis of ninhydrin. MDA content
of leaves was determined by the thiobarbituric acid method. Superoxide
dismutase (SOD), catalase (CAT) and peroxidase (POD) activities were
assayed at the absorbance of 560 nm, 240 nm and 470 nm by microplate
reader (Epoch2, BioTek, Thermo Fisher Scientific, USA), respectively. In
addition, electrolyte leakage (EL), a maker of the intact plant cell stress
J. Lian et al.
response, was determined by measuring the electrical conductivity as
described by Lian et al. (2021). These above detailed experimental
protocols were described in the Text S3. Nutrient elements (Ca, P, S, Mg,
Mn, Fe, Zn) in wheat shoots and roots were determined by inductively
coupled plasma optical emission spectroscopy (ICP-OES) where the
detailed acid digestion method and determination procedures are
described in detail in 2.3.3 below.
2.3. Field experiment
2.3.1. Experiment design and sample collection
The field experiment was conducted in a farmland area
(30◦ 53′ 42.56′′ N; 120◦ 4′ 59.93′′ E) located in Huzhou, Zhejiang province,
China. The pilot region is located in the northern subtropical monsoon
climate zone, with an average annual temperature of 12.2–17.3 ◦ C. The
annual precipitation ranges from 761 to 1780 mm and the annual
average relative humidity is above 80 %. The soil type is acidic paddy
soil, and dominated by wheat-rice crop rotation. The basic physical and
chemical properties of soil were presented in Table S1.
The field experiment had five treatments with three replicates in a
randomized complete block design. Agricultural practices were per­
formed according to local farmer habits. After applying chemical basal
fertilizer (500 kg/ha, NPK = 16:16:8), wheat seeds (cv. Zhengmai 10)
were sown in each split plot (2 m × 2.5 m) manually on December 5,
2020. Based on the previous hydroponic study, Glyzinc (50, 100, and
250 mg/L) and equivalent zinc sulfate salt (ZnSO4) were sprayed as a
foliar micronutrient by a hand-held electric sprayer. Foliar formulations
were applied three times at tillering, booting and filling stage, respec­
tively, which are considered to be the best timing for foliar spraying to
improve the quality of wheat. The plots received equal amount of
deionized water spray were used as control group (CK). All solutions
were sprayed to each treatment at a rate of 1000 L/ha in the late af­
ternoon of a sunny day to avoid unnecessary loss. Notably, no adjuvant
was added to the spray solution.
At maturity (May 24, 2021), wheat plant height was first recorded
with a tape measure and then the entire plants were sampled from the
central 1 m2 areas of each plot. Wheat samples were brought back to the
laboratory and separated into grain, glume, leaf, stem and root,
respectively. Subsequently, all samples were thoroughly washed three
times with tap water and soaked in 20 mM EDTA-Na2 for 15 min, fol­
lowed by deionized water to remove the absorbed metal ions. Finally,
plant samples were dried at 65 ◦ C for 48 h, weighted and ground into
fine power, and stored in self-sealing plastic bags for further analysis.
2.3.2. Nutrient element and cadmium analysis
For element analysis, dried plant samples (0.2 g) were digested with
6 mL HNO3/HClO4 (5:1 v/v) at 160 ◦ C for 4 h using a Digiblock digestion
system (EH35A, LabTech, UK). When cooled to room temperature, the
digestive solutions were diluted to 50 mL with ultrapure water and
passed through a 0.22 µm filter membrane. The concentrations of
macro-elements (Mg, P, Ca and S) in wheat grains was determined using
an inductively coupled plasma optical emission spectroscopy (ICP-OES)
(ICP6000, Thermo Fisher Scientific, UK), while other microelements
such as Zn, Fe, Cu, Mo, Mn, B, and Cd in wheat samples were analyzed by
an inductively coupled plasma mass spectrometry (ICP-MS) (Plasma
Quant MS, Analytik Jena, Germany). Digestion blanks, spiked samples
and internal standards of wheat flour (GBW(E)100495, China Standard
Materials Research Center, Beijing, China) were included to check the
accuracy of the digestion procedures. The recovery rates of metals (e.g.
Zn, Fe, Cd, Mn) in certified reference material samples ranged from 95 to
107 %.
2.3.3. Phytic acid and crude protein determination
Phytic acid content of wheat grain was determined according to the
method of Latta and Eskin (1980). Wheat grain (1.0 g) was placed into a
50 mL centrifuge tube with 20 mL of 10 % HCl-Na2SO4. The mixture was
3
Food Chemistry 402 (2023) 134290
first vortexed vigorously for 1 min and then extracted for 2 h (200 rpm,
25 ◦ C) before centrifugation (4000 g, 20 min) at room temperature. The
supernatant was then collected and mixed with an equal amount of 15 %
TCA solution, and placed at 4 ◦ C for 2 h. The reaction solution was
centrifuged (10000 g, 25 ◦ C) for 10 min and the pH was adjusted to
6–6.5 with 0.75 M sodium hydroxide. Supernatant (0.1 mL) were
collected, mixed with 1.4 mL deionized water and then 0.5 mL ferric
chloride-sulfosalicylic acid solution were added on it. Finally, the
mixture was reacted for 20 min and its absorbance was measured at 500
nm using a microplate reader (Epoch2, BioTek, Thermo Fisher Scientific,
USA). The concentration of phytic acid was calculated by using the
standards curve of sodium phytate. Moreover, the molar ratio of phytic
acid to Zn (PA/Zn molar ration) was calculated to assess the Zn
bioavailability in wheat grains.
The crude protein concentration in wheat grains was measured ac­
cording to the Kjeldahl method (Kirk, 1950). Briefly, wheat grain (0.1 g)
was mixed with 5 mL of H2SO4 at temperatures between 350 ◦ C and
380 ◦ C and the concentration of nitrogen in the digestion solution was
determined using a Kjeldahl analyzer. A coefficient of 5.83 was used for
the conversion of nitrogen to protein content in wheat grains.
2.4. Statistical analysis
All data were expressed as mean ± standard deviation (SD) and
statistical analyses were performed using SPSS Statistics software
(Version 26.0, IBM, USA). Statistically significant differences (p < 0.05)
among treatment means were accepted by applying a one-way variance
analysis (ANOVA) followed by a post hoc Duncan test.
3. Results
3.1. Characterization of Glyzinc
The morphology and particle size of Glyzinc were observed by
scanning electron microscopy (SEM), respectively. As shown in Fig. 1a-
b, Glyzincs are unspecified micro-/nanosheets with a thickness of
approximately 175 nm. Energy dispersive spectroscopy (EDS) results
showed that Glyzinc nanosheets consisted of carbon, oxygen and zinc
(Fig. 1c-e), which accounted for 22.58 %, 29.59 % and 43.26 % of the
weight, respectively (Fig. 1f). Consistently, the measured zinc content of
Glyzinc is the same as the information provided by the merchant. The X-
ray diffraction (XRD) pattern in Fig. 1 g displayed typical diffraction
peaks of Glyzinc at 10.9, 17.2, 20.7, 23.8, 27.5, 28.5 and 38.2◦ , corre­
sponding to (1 0 0), (0 1 1), (1 1 1), (1 0 2), ( − 2 1 1), (2 1 1) and (3 1 1)
planes, respectively, which can be indexed to the crystalline phase of
Zinc glycerolate (Glyzinc) (PDF#23–1975). Furthermore, the Fourier
transform infrared spectroscopy (FTIR) spectrum showed that the
dominant absorption bands at 3442, 2881, 2845, 1944, 1465, 1353,
1124, 1064, 991, 876, 652 cm− 1 (Fig. 1h), which were attributed to the
characteristic absorption peaks of C–H, CH2, O–H, C–O, and Zn-O,
respectively.
3.2. Wheat growth under hydroponic conditions
Growth parameters of wheat seedlings induced by Glyzinc were
assessed under two scenarios (foliar spraying and root application).
Generally, foliar spraying of Glyzinc had less disturbance on wheat
seedling growth compared to root application (Fig. 2a). For example,
foliar spraying of both Glyzinc and ZnSO4 had no obvious effects on
plant height and dry weight. In contrast, root application of Glyzinc
significantly (p < 0.01) reduced plant height (by 14.3 %-28.5 %),
pigment content (by 7.1 %-17.7 %), shoot (by 35.9 %-49.2 %) and root
dry weights (by 19.2 %-52.4 %), while the inhibitory effect of ZnSO4 was
more pronounced (38.4 %, 43.6 %, 59.8 %, and 72.9 %, respectively) as
compared to the control (Fig. 2b and c). It was worth noting that foliar
spraying of Glyzinc at low concentrations (50–100 mg/L) could
J. Lian et al. Food Chemistry 402 (2023) 134290

Fig. 1. Characterization of Glyzinc. (a, b) SEM images, (c-e) EDX images of C, O and Zn element, (f) Elemental quantitative analysis, (g) FTIR and (g) XRD spectra
of Glyzinc.

significantly (p < 0.05) increase pigment content compared to the effects of ZnSO4 on root parameters were more intensive than those of
control (Fig. 2d). Additionally, under both exposure modes, Glyzinc had Glyzinc. Therefore, foliar spray of ZnSO4/Glyzinc could be a safer
no significant effect (p > 0.05) on root-to-shoot ration (RSR) and water agronomic practice that does not significantly interfere with root growth
content (WC) of wheat, but root application of ZnSO4 significantly (p < and development of wheat.
0.01) reduced RSR and WC by 10.3 % and 32.5 %, respectively (Fig. S1
and S2). Overall, these above results suggested that foliar spray was less
3.3. Stress responses of wheat seedlings
phytotoxic than root application, and Glyzinc had a greater stimulation
effect than zinc sulfate on wheat growth through foliar spray.
To evaluate the physiological responses of wheat seedlings to Gly­
In our study, root features of wheat were also measured through
zinc, the changes in the antioxidant systems and oxidative damage of
photographs to reveal the impact of zinc application on plant root
wheat leaves were measured (Fig. 3). Obviously, all foliar zinc sprays
development (Fig. S3). Similarly, all foliar zinc sprays exhibited no
produced no significant (p > 0.05) effects on EL rate, MDA content,
significant (p > 0.05) effect on root parameters, but the influence of root
proline content and SOD enzyme activity (Fig. 3 a-d), but Glyzinc
applied Zn was evident (Table S2). In essence, compared to the control,
significantly (p > 0.05) improved POD and CAT enzyme activities at
root application of Glyzinc dramatically (p < 0.01) reduced root tip
100 mg/L when compared to the control (Fig. 3 e, f). However, high
number (27.8 %-71.5 %), total root length (56.1 %-85.6 %), surface area
concentrations of Glyzinc/ZnSO4 root treatment triggered oxidative
(57.8 %-85.3 %) and root volume (46.7 %-63.1 %), while greatly
damage and boosted the activities of non-antioxidant and antioxidant
increasing average root diameter (8.1 %-30.6 %) in a dose-dependent
defense systems. For instance, Glyzinc significantly (p < 0.01) increased
manner. Moreover, it was also important to note that the negative
EL, MDA content, proline content, SOD, POD, and CAT activities by 11.3

4
J. Lian et al. Food Chemistry 402 (2023) 134290

Fig. 2. Effect of Glyzinc on the growth of wheat seedlings under two application modes. (a) phenotypic images of wheat seedlings, (b) plant height, (c) dry weight
and (d) pigment content. * represents the statistically significant at p < 0.05 while **shows the statistically significant at p < 0.01 between the control and Zn
treatments. RA: root application, FA: foliar application. The scale bar represents 10 cm.

Fig. 3. Response of the electrolyte leakage(a), MDA content (b), proline content (c) and antioxidant enzyme system (d-f) of wheat leaves to Glyzinc. * represents the
statistically significant at p < 0.05 while **shows the statistically significant at p < 0.01 between the control and Zn treatments. RA: root application, FA: foliar
application.

%, 35.8 %, 80.9 %, 87.9 %, 96.5 % and 155.9 % at 250 mg/L, respec­
tively, when compared to the control. In particular, ZnSO4 contributed
more to these stress indicators as compared to Glyzinc, indicating a
stronger phytotoxicity to wheat under root exposure.
3.4. Zinc and other nutrient accumulation in hydroponically grown wheat
Compared to the control, foliar spray of Glyzinc not only signifi­
cantly (p < 0.05) increased the shoot Zn content of wheat (by 55.9 %–
323 %), but also raised the concentration of other nutrients such as Ca,

5
J. Lian et al.
Mg, S and Mn (Fig. S3). By contrast, root application of Glyzinc signif­
icantly (p < 0.05) decreased the concentration of Mg, P, S, Mn and Fe in
wheat shoots, whereas dramatically increased root P and Fe concen­
tration. Moreover, there was a clear difference in the accumulation
characteristics of Zn in wheat between Glyzinc and zinc sulfate.
Compared to zinc sulfate, foliar spray of Glyzinc contributed more shoot
Zn but lower root zinc, while fewer both shoot and root zinc were
accumulated when applied at the root (Fig. S3g, h). These showed that
foliar application of Glyzinc could better increase the aboveground Zn
content of wheat without compromising the content of other nutrients.
3.5. Agronomic performance of wheat under field condition
The outcomes of foliar zinc sprays on agronomic traits of wheat
grown in the field are shown in Fig. 4. Despite the fact that foliar spray of
Glyzinc had no significant (p > 0.05) influence on plant height, spike dry
weight, grain number per panicle, grain yield and dry biomass of wheat
compared to the control (Fig. 4 a-c, e-f), they were slightly decreased by
Glyzinc at low to medium concentrations. Notably, foliar spray of Gly­
zinc significantly (p < 0.05) reduced the thousand grain weight by 7.94
% at 250 mg/L, while at 50 and100 mg/L, there was a tendency to in­
crease the thousand grain weight (Fig. 4d). In addition, there were no
significant differences (p > 0.05) in these agronomic indicators between
Glyzinc and ZnSO4 treatments, indicating that the effects of Glyzinc and
ZnSO4 on wheat biomass and productivity were comparable.
3.6. Nutritional quality of wheat grain
Both foliar Glyzinc and ZnSO4 spray had significant positive effects
on Zn uptake by different tissues of wheat plant, except for the glumes
(Fig. 5a). In particular at 100 mg/L, foliar spray of Gyzinc significantly
(p < 0.01) increased the zinc content in wheat grain, stem, leaf and root
by 28.7 %, 47.8 %, 60.8 % and 52.2 %, respectively, as compared to the
control. Likewise, the elevated zinc contents of wheat grain (11.6 %),
leaf (32.1 %) and root (35.6 %) by ZnSO4 were also very pronounced (p
< 0.05), especially in the stem (1.6-fold, p < 0.01). However, interest­
ingly, both low and high concentrations of Glyzinc did not produce a
considerable increase in Zn content of wheat grain and root over the
Fig. 4. Impact of foliar application of Glyzinc on plant height (a), spike dry weight (b), grain number per panicle (c), thousand grain weight (d), grain yield (e), and
dry biomass (f). * represents the statistically significant at p < 0.05 while **shows the statistically significant at p < 0.01 between the control and Zn treatments.
6
Food Chemistry 402 (2023) 134290
control.
The interaction of Glyzinc and nutrient elements in wheat grain is
shown in Fig. 5b, c. Foliar spray of Glyzinc could markedly (p < 0.05)
decrease grain P (9.4 %-17.5 %) and Mo (14.2 %-55.1 %) content, but
significantly (p < 0.01) increased grain S and Cu content by 2.6 %-10.4
% and 13.8 %-17.3 %, respectively, compared to the control. By
contrast, foliar spray of ZnSO4 had no noticeable effect on the content of
almost nutrients of wheat grain such as P, Mg, Ca, Mn, Fe, B, Mo.
Furthermore, regardless of Zn types, foliar application of Glyzinc and Zn
sulfate resulted in higher protein content in wheat grain in comparison
with the control (Fig. 5d). Compared to the control, Glyzinc significantly
(p < 0.01) enhanced grain protein content by 9.1 % at 100 mg/L and 7.9
% at 250 mg/L, respectively. In contrast, Zn sulfate also had the identical
effect of promoting grain protein content (6.0 %).
3.7. Phytic acid and PA/Zn molar ratio
In wheat grain, Zn bind with P in the form of phytate is difficult to
absorbed by humans and the PA to Zn molar ratio is regarded as a valid
indicator of Zn bioavailability (Ghasemi et al., 2013). As shown in Fig. 6,
Glyzinc significantly (p < 0.05) reduced the PA concentration from
15.28 mg/g to 11.65 mg/g in wheat grains under foliar application.
Likewise, foliar spray of ZnSO4 caused a 22.2 % decrease in PA content
of wheat grain. In addition, foliar spray of both Glyzinc and ZnSO4
significantly (p > 0.01) lowered the PA/Zn molar ratio of wheat grain,
and the lowest value of PA/Zn molar ratio was observed at 100 mg/L of
Glyzinc. Furthermore, there were no significant differences (p > 0.05) in
PA content and PA:Zn molar ratio of wheat grain between Glyzinc
(100–250 mg/L) and ZnSO4.
3.8. Cadmium accumulation and transport in wheat
As shown in Fig. S5a, foliar zinc application mainly affected Cd
concentrations in wheat roots, leaves, and grains, but not in the stems
and glumes. Compared to the control, foliar spray of Gyzinc significantly
(p < 0.05) reduced root and grain Cd concentration by 19.5 % and 20.2
% at 250 mg/L, respectively, but considerably (p < 0.05) increased Cd
contents in leaves by 12.3 %-17.9 %. In the same manner, foliar spray of
J. Lian et al.
Fig. 5. Zn concentration (a), nutrient element concentration (b) and protein content (c) of wheat grain under foliar application of Glyzinc in the field experiment. *
represents the statistically significant at p < 0.05 while **shows the statistically significant at p < 0.01 between the control and Zn treatments.
Fig. 6. Phytic acid content and PA:Zn molar ration of wheat grain under foliar
application of Glyzinc in the field experiment. * represents the statistically
significant at p < 0.05 while **shows the statistically significant at p < 0.01
between the control and Zn treatments.
ZnSO4 only altered Cd contents in wheat leaves and grains that were the
same as those of 250 mg/L Glyzinc treatment. In the terms of trans­
location factor (TF) of Cd, foliar spray of Glyzinc only significantly (p <
0.05) declined the value of TFgrain/leaf by 19.1 %–33.9 % in wheat, but
ZnSO4 simultaneously and distinctly reduced the value of TFgrain/leaf and
TFgrain/stem by 31.0 % and 30.6 %, respectively, when compared to the
control (Fig. S5b). Besides, both foliar sprays of Glyzinc and ZnSO4 had
7
Food Chemistry 402 (2023) 134290
no significant effects on TFgrain/glume and TF grain/root.
4. Discussion
To our best knowledge, this is the first study assessing the phyto­
toxicity of Glyzinc on wheat and investigating the effectiveness of foliar
Glyzinc spray on wheat productivity, grain nutritional quality and Cd
minimization in a field trial. The present study demonstrated that foliar
spray of Glyzinc was less disruptive to wheat growth than root appli­
cation, and lower in toxicity than ZnSO4. Moreover, Glyzinc exhibited
better performance than ZnSO4 in improving grain Zn content and
bioavailability, crude protein and reducing anti-nutrient PA content and
Cd in wheat grains under foliar sprays. Thus, these results suggested that
Glyzinc could be a more promising Zn foliar fertilizer than ZnSO4 in
simultaneously enhancing zinc bioavailability and minimizing the
health risks of Cd in wheat grains.
Zinc fertilization appears to be one of the cost-effective and sus­
tainable agronomic interventions for zinc biofortification of wheat, but
the improper application mode and dosage would compromise crop
agronomic performance (Lian et al., 2020; Natasha et al., 2022; Velu
et al., 2014; Xu et al., 2022). For example, Azarin et al. (2022) found that
of ZnO nanoparticles at high concentrations (300 and 2000 mg/L)
substantially reduced plant length and weight of roots and shoots, as
well as significant accumulation of Zn in barley (Hordeum vulgare L.).
Consistently, our results also showed that Glyzinc produced higher
phytotoxicity to wheat seedlings at high concentrations under root
application, as evidenced by the reduction in plant height, dry weight,
pigment content and root morphology parameters (Fig. 2 and Table S2).
In particular, under root application, the different phytotoxic effects of
ZnSO4 and Glyzinc in wheat may be due to the difference in the
J. Lian et al.
accumulation of Zn content (Fig. S4g, h), probably because Glyzinc in
the chelated form released fewer Zn ions than ZnSO4 (Kaewchangwat
et al., 2017). It is well known that metal ions derived from nano­
materials are the important contributor to their primary toxicity (Gao
et al., 2018; Wu et al., 2021). In contrast, foliar application of Glyzinc
had no effect on plant height and dry weight, and even promoted
chlorophyll content, indicating that foliar spray is a safer mode of
fertilization compared to root application (Cao et al., 2022; Raliya et al.,
2018). However, considering the great discrepancies between hydro­
ponic and real environments, the dosage and frequency of using Glyzinc
as a zinc micronutrient requires further study to achieve the targeted
goals of environmental friendliness and optimal efficacy of fertilization
in agriculture.
Zn is essential but excess amount is toxic to the plant, which pro­
duces large amounts of reactive oxygen species (ROS) and causes
oxidative damage (Lian et al., 2020; Lu et al., 2021; Natasha et al.,
2022). In the present study, we revealed that EL and MDA content of
wheat leaves were basically unchanged after all Zn foliar sprays but
were significantly elevated in root treatment of 250 mg/L Glyzinc and
ZnSO4 (Fig. 3a, b). This suggested the low phytotoxicity-induced by
Glyzinc/ZnSO4 on wheat under foliar spray. In line with our findings,
previous study also suggested that wheat leaves had high tolerance to
foliar sprayed ZnSO4 with a concentration threshold of up to 5 g/L (Xu
et al., 2021). This may be explained by the fact that although the high
concentration of the foliar spray solution was high, the amount of spray
applied was very small, collectively resulting in a low intensity of local
exposure to the foliage, and therefore did not provoke any oxidative
damage (Cakmak et al., 2010; Niu et al., 2021). Alternatively, plants
have evolved complex defense systems to against oxidative stresses,
including non-enzymatic antioxidant (e.g. proline) and antioxidant en­
zymes (e.g. SOD, POD, GR and CAT) (Rejeb et al., 2014). In the current
study, enhanced proline and antioxidant enzyme activities (SOD, POD
and CAT) were correspondingly detected at moderate and high con­
centrations of Glyzinc and ZnSO4 under root application. It could be part
of the underlying mechanisms by which plants eliminated excess ROS
and maintain the redox homeostasis at low and moderate concentrations
of Glyzinc, while were not sufficiently effective at high dose as evi­
denced by an increasement in EL and MDA content. However, inter­
estingly, POD and CAT activities of wheat leaves were significantly
stimulated at 100 mg/L of Glyzinc under foliar spray, which may delay
leaf senescence or improve plant disease resistance.
Our results demonstrated that Glyzinc yielded a greater increase in
grain Zn concentration and bioavailability compared to the control and
ZnSO4 treatment, especially at 100 mg/L. This implies that 100 mg/L of
Glyzinc could be the optimal concentration for Zn biofortification in
wheat. There are two plausible factors responsible for the increased Zn
content in wheat: firstly, the glycerol group of Glyzinc assisted the
penetration, uptake and subsequent remobilization of Zn to grain on
hydrophobic wheat leaves (Ghasemi et al., 2013; Kaewchangwat et al.,
2017); secondly, the high concentration of Glyzinc/ ZnSO4 treatment
may have exceeded the Zn absorption sites in the leaves, leading to a
reduction in its absorption efficiency (Xu et al., 2021). Moreover, the
increased Zn bioavailability and decreased phosphorus or PA content in
wheat grain treated with Glyzinc (Fig. 5b and Fig. 6) could be due to the
fact that Zn and phosphorus share a common transport channel,
resulting in an inhibition of phosphorus transport to the grains by the
increased zinc (Hui et al., 2022). In addition, minimizing the negative
impact on crop yield and grain nutritional quality during biofortification
is the fundamental criterion (Velu et al., 2014; Yu et al., 2021). In our
study, Glyzinc did not deteriorate the yield of wheat and also promoted
the micronutrients and protein content in grains, implying that it is a
sustainable agronomic practice.
Micronutrient biofortification coupled with reducing the health risk
of heavy metals in crop grains is a promising strategy and research di­
rection, but not many relevant studies have been available yet. In this
study, for the first time, we found that Glyzinc and ZnSO4 lowered the
8
Food Chemistry 402 (2023) 134290
Cd content in wheat grain via the same mechanism, both by raising the
Cd content in wheat leaves and limiting the re-transport of Cd from
leaves and stems to the grains. In consistent with our findings, Zhen et al.
(2021) also reported that foliar spray of Zn reduced Cd accumulation in
rice grains by enhancing Cd chelation onto cell wall of leaves, thus
inhibiting Cd transport from vegetative organs to grains. It was specu­
lated that this might be involved with sulfur metabolic processes, which
induces the production of sulfur-based substances (glutathione, phy­
tochelatins and non-protein sulfhydryl compounds) (Yang et al., 2020).
In the present study, the elevated sulfur contents in wheat leaves and
grains were also observed after foliar spray of Glyzinc and ZnSO4
(Fig. 5b). Similarly, the higher copper content may be due to elevated
SOD, as copper is an important component of SOD in living organisms.
By contrast, increased Zn would compete with phosphorus for the
common transporter protein binding site, thus reducing the transport of
phosphorus from the leaves or stems to the grains (Natasha et al., 2022).
Furthermore, Cd levels in wheat grains still exceeded China’s national
food safety standards for agricultural products, illustrating that wheat
grown on moderate Cd-contaminated soil cannot achieve safety goals
with foliar application of Glyzinc or ZnSO4 alone. Nevertheless, foliar
spray of Glyzinc cloud be an effective supplement to mitigate Cd accu­
mulation in wheat grains during Zn biofortification.
5. Conclusions
In summary, our results showed that foliar spray is a safer and more
effective mode of fertilization application than root application, and
Glyzinc is less toxic to wheat than ZnSO4 as the negative effects on
growth parameters and oxidative damage were higher in the later one
under the same Zn exposure. In the field trail, Glyzinc was more effective
than ZnSO4 in not only enhancing the Zn concentration and other
nutritional values such as micronutrient and protein content of wheat
grains, but also in increasing Zn bioavailability by lowering PA and PA
to Zn molar ratio after foliar application. Considering that Cd is more
likely to be accumulated in wheat grains, foliar spray of Glyzinc could be
considered as an alternative approach to alleviate the health risk of Cd.
Overall, these results highlight the important benefits of Zn bio­
fortification and Cd minimization of wheat with Glyzinc in nano-
enabled agriculture. However, little knowledge is available on the ab­
sorption and translocation mechanisms in wheat following foliar spray
of Glyzinc. In this way, further and more in-depth work is necessary to
advance on this issue.
CRediT authorship contribution statement
Jiapan Lian: Data curation, Formal analysis, Writing – original
draft, Writing – review & editing. Liping Cheng: Conceptualization,
Methodology, Writing – original draft, Writing – review & editing. Xu
Zhai: Investigation, Methodology, Writing – review & editing. Ruofan
Wu: Investigation, Data curation, Formal analysis. Xiwei Huang:
Investigation, Formal analysis, Data curation. Dan Chen: Investigation,
Methodology. Jianqing Pan: Project administration, Supervision. M.J.
I. Shohag: Formal analysis, Writing – review & editing. Xiaoping Xin:
Formal analysis, Methodology, Validation. Xinwei Ren: Formal anal­
ysis, Writing – review & editing. Zhenli He: Formal analysis, Writing –
review & editing. Xiaoe Yang: Data curation, Funding acquisition,
Project administration, Resources, Supervision, Writing – review &
editing.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
J. Lian et al.
Data availability
Data will be made available on request.
Acknowledgement
This work was financially supported by the National Natural Science
Foundation of China (#41721001 and #31872956), Key project from
Zhejiang Agricultural Bureau (# 2020SNLF009) and Project on safe
utilization of contaminated arable land by Agricultural and Rural Bu­
reau of Changxing County (#KYY-514106-0016). The authors would
like to thank Dr. Peixun Li (Jinheng Lanhai Technology Co., Ltd, Tianjin,
China) for providing Glyzinc and Dr. Hongfei Zhang (Zhejiang Uni­
veristy) for help with the ICP-OES/MS measurements.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.foodchem.2022.134290.
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