Journal of Plant Nutrition

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Influence of seed priming with ZnO nanoparticles

on the salt-induced damages in wheat (Triticum
aestivum L.) plants

Hanan Mahmoud Abou-Zeid , Ghada Saber Mohamed Ismail & Salwa Ahmed
Abdel-Latif

To cite this article: Hanan Mahmoud Abou-Zeid , Ghada Saber Mohamed Ismail & Salwa
Ahmed Abdel-Latif (2021) Influence of seed priming with ZnO nanoparticles on the salt-induced
damages in wheat (Triticum�aestivum L.) plants, Journal of Plant Nutrition, 44:5, 629-643, DOI:
10.1080/01904167.2020.1849288

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Published online: 19 Nov 2020.

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JOURNAL OF PLANT NUTRITION
2021, VOL. 44, NO. 5, 629–643
https://doi.org/10.1080/01904167.2020.1849288

Influence of seed priming with ZnO nanoparticles on the salt-

induced damages in wheat (Triticum aestivum L.) plants
Hanan Mahmoud Abou-Zeida, Ghada Saber Mohamed Ismaila, and Salwa Ahmed
Abdel-Latifb
a
Botany and Microbiology Department, Faculty of Science, Alexandria University, Alexandria, Egypt; bBiology
and Geology Department, Faculty of Education, Alexandria University, Alexandria, Egypt

ABSTRACT ARTICLE HISTORY

Salinity is a worldwide issue of intimidating land productivity and the pro- Received 16 February 2020
duction of food. Seed priming can be an effective approach to enhance Accepted 17 April 2020
the performance of seedlings growing in salinity stressed conditions. The
KEYWORDS
present investigation examined whether the ZnO-nano priming (ZnONPs;
leaf ultrastructure;
50, 100 and 500 mg L
1) has a role in the alleviation of the salt-induced photosynthesis; priming;
toxicity of Triticum aestivum L. plants grown under 150 mM NaCl. protein profile; salinity; ZnO
Treatment with all concentrations of ZnONPs significantly improved wheat nanoparticles
growth biomarkers. Furthermore, salt-induced alterations of growth, photo-
synthetic pigments, photosynthetic efficiency, and leaf ultrastructure were
lesser upon ZnO-nano priming. Besides, ZnO-nano priming induced various
changes in electrophoretic profiles of shoot proteins.

Introduction
Over the last 20 years, cultivated areas in various parts of the world have been severely affected
by different abiotic stresses like salinity and drought which inhibit plant growth and crop prod-
uctivity (Acosta-Motos et al. 2017). Salinity stress altered the anatomy and ultrastructure of some
plant organs which are sometimes interlinked with changes in a variety of biochemical, physio-
logical and molecular responses within the plant cells. Salinity consequences in growth retard-
ation, reduction in photosynthetic efficiency and photosynthetic pigments and achieves
physiological disturbance of the whole plant like oxidative, osmotic and ionic stress (Ahmad et al.
2018; Kholghi et al. 2018). Furthermore, salinity contributes to a major increase within the level
of reactive oxygen species (ROS), causing serious oxidative damage on membrane lipids, proteins,
and nucleic acids (Kaya et al. 2018; Rani et al. 2019).
Currently, the interest in sustainable agriculture has drawn attention to some strategies that
are developed to induce abiotic stress tolerance in crops supported by genetic control and physio-
logical approaches (Akram et al. 2018). Among these various strategies, seed priming is consid-
ered as an innovative attractive approach to overcome the growth retardation of young plants
within the salt-affected soils. Pre-sowing seeds treatment is a simple, low risk and cost-effective
technique to mitigate the salinity hazards of agricultural lands. Various seed priming techniques
including hydropriming, osmopriming, chemical priming, nutrient priming, hormonal-priming,
and redox priming have been reported to reinforce rapid and uniform emergence, increase ger-
mination-enhancing metabolites, vigor seedling growth, change in gene expression throughout
chromosomal variations, and increase within the synthesis and mobilization of proteins in many

CONTACT Hanan Mahmoud Abou-Zeid Hananmahmoud93@yahoo.com Botany and Microbiology Department, Faculty
of Science, Alexandria University, Alexandria, Egypt
ß 2020 Taylor & Francis Group, LLC
630 H. M. ABOU-ZEID ET AL.

crops, particularly under adverse environmental conditions (Hussain et al. 2016; Sheteiwy et al.
2017, 2019). Nevertheless, the efficiency of a variety of priming agents diverges under different
stresses and in numerous crop species. Recently, seed priming with various growth regulators as
well as bio-nanoparticles has been reported to enhance the performance of assorted crops (Abou-
Zeid and Moustafa 2014; Abou-Zeid and Ismail 2018).
Nowadays, nano fertilizers are the foremost technically advanced approach for supplying
mineral nutrients to plants compared to common fertilizers (Jayarambabu, Sivakumari, and
Prabhu 2014). It causes various morphological and physiological changes when interacting with
plants dependent on the chemical structure, size, reactivity and most importantly the dose at
which they’re useful (Fireman et al. 2017). Zinc is an essential micronutrient for plant metabol-
ism, nerveless, its deficiency causes physiological disturbance resulting from several metabolic
disruptions interconnected to Zn (Baybordi 2006). Recently, the interest is diverting toward the
utilization of metal-based nanoparticles in the agriculture sector as a fertilizer which can be
effective because of decreased nutrient losses in fertilization. Zinc oxide nanoparticles
(ZnONPs) are the most widely used NPs worldwide and their potential detrimental as well as
positive effects on the physio-biochemical attributes of plants have been reported (Munir et al.
2018). Furthermore, Abdel Latef, Abu Alhmad, and Abdelfattah (2017) reported that the appli-
cation of ZnNPs has been found quite effective in improving the resistance of lupin plants
toward salinity.
Wheat (Triticum aestivum L.) grains and its above-ground biomass are among the extensively
used crops all over the globe owing to its greater nutritional values and adaptability under various
environmental conditions. With world utilization predictable to extend beyond production, wheat
inventories are expected to decline (FAO 2018). Water and salt stress are the most important lim-
iting factors in wheat production in arid and semi-arid regions all over the world (Almaghrabi
2012). In Egypt, there’s an urgent national target for increasing wheat production to cope with
the population increment. This needs a continuous scientifically based implementation of effective
agricultural practices. Thus, this study was conducted to amend the role of seed priming with dif-
ferent concentrations of ZnONPs in mitigating the adverse impacts of salt stress on wheat plants.
The changes in some growth parameters, photosynthetic pigments, photosynthetic efficiency, cell
ultrastructure, and protein profile were evaluated. This work could appreciably contribute to fur-
ther perceptive the mechanism induced by priming wheat grains with ZnONPs under salin-
ity stress.

Materials and methods

ZnONPs were purchased with a purity of 99.5%, the particle size of <100 nm from Sigma
Aldrich Chemical Co. (St. Louis, MO, USA).

Preparation of ZnONPs suspensions

The nano-particles were suspended directly in double-distilled water and dispersed by using
mechanical stirrer and ultrasonic vibration (100 W, 40 kHz) for 30 min before use to avoid aggre-
gation of the particles.

Characterization of ZnONPs
The chemical composition and the functional groups present in the ZnONPs were studied by
using Fourier transform infrared (FT-IR) spectroscopy (Perkin-Elmer Spectrum 1000). ZnONPs
were characterized by UV-visible spectrophotometer (T80 UV–vis spectrophotometer–double
beam) at a scanning speed of 200-800 nm. Transmission electron microscope (TEM) of the
 JOURNAL OF PLANT NUTRITION 631

suspension containing ZnONP was sampled by using (JEM-1400), the shape and size of ZnONP
were determined from TEM micrographs. The dried nano-pellet was used for XRD analysis and
confirmed the presence of ZnONPs. Energy Dispersive X-ray (EDS) spectrometer using Link ISIS
analyzer programmed attached with Scanning Electron Microscope (SEM) was employed for the
data collection.

Plant material, growth conditions, and treatments

Wheat grains (Triticum aestivum L., cv. Giza 168) were purchased from Agricultural Research
Center, Giza, Egypt. Prior to germination, the grains were soaked in 0.1% sodium hypochlorite
solution for 3 min then washed thoroughly several times with distilled water. Sterilized grains were
primed with different concentrations of ZnONP (0, 50, 100 and 500 mg L
1) for 24 h at room tem-
perature. Primed grains were germinated in Petri plates using filter paper moistened with 5 ml of
de-ionized water for 24 h, and incubated at 25 ± 1  C. To determine the effect of seed priming with
ZnONP on wheat growth under salinity stress, a factorial laboratory pot experiment of a completely
randomized design with 4 replicates was carried out. Uniform 24 h germinating seeds were selected
and transplanted to pots containing sandy clay loam soil. The pots were incubated in natural envir-
onmental conditions (photoperiod of 16 h/8 h light/dark, 28/23 ± 2  C light/dark temperature; light
intensity PPFD, 23 lmol m-2s
1) and irrigated with distilled water two days intervals till field cap-
acity for 7 days. Thereafter, pots were divided into two sets, the first set was irrigated with distilled
water and the second one was irrigated two times with 150 mM NaCl during the next 7 days. On
the 18th day of the ZnONPs treatment seedlings were harvested. They were rinsed carefully in water
and pressed gently between blotting paper to remove excess water, dissected to shoots and roots
and quickly saved for estimation of the various growth parameters and chemical analyses.

Determination of growth biomarkers

The growth biomarkers [shoot and root length, shoot and root fresh (FM) and dry mass (DM)]
were determined.

Estimation of photosynthetic pigments

The photosynthetic pigments were determined according to methods described by Moran (1982)
using N,N-dimethyl formamide (DMF), total carotenoids content was calculated according to
Wellburn (1994) and related to leaf weight.

Measurement of photosynthesis parameters

Fluorescence parameters were calculated by the method of Genty, Briantais, and Baker (1989)
and Van Kooten and Snel (1990). Before measurement, the leaf samples were kept in darkness
for 20 minutes. Actinic light of 400 mmol m
2 s
1 and saturating pulse light of 8000 mmol m
2
s
1 were used. Fluorescence was measured with OS-30P pulse modulated chlorophyll fluorimeter
(Opti-sciences, Hudson, and USA) and determined the coefficient of the effective quantum yield
of photochemical energy conversion of PSII (UPSII), photochemical quenching (qP) and photo-
chemical efficiency of PSII (Fv/Fm).

Leaf ultrastructure
The leaves fragment from both control and treated (50 mg L
1 ZnONPs, 500 mg L
1 ZnONP,
150 mM NaCl, 50 mg L
1 ZnONPsþ 150 mM and 500 mg L
1 ZnONPs þ 150 mM NaCl) samples
632 H. M. ABOU-ZEID ET AL.

Figure 1. Characterization of ZnONPs by (A) FT-IR spectrum, (B) UV-visible absorption spectra, (C & D) TEM Images, (E) XRD pat-
tern and (F) EDS spectra.

were fixed according to the method described by Spurr (1969). Ultra-thin sections of leaves were
cut on an ultramicrotome (Leica EM UC6, Germany) with a diamond knife and were mounted
on copper grids with 300 square mesh).The cell ultrastructure visualization and photographing
were carried out using the transmission electron microscope at the Electron Microscopic Unit,
Faculty of Science, Alexandria University.
 JOURNAL OF PLANT NUTRITION 633

Figure 2. Effects of salinity stress and seed-priming with ZnONPs on growth biomarkers in 18-day-old wheat shoots. Different
letters indicate significant difference by Duncan’s multiple range tests at p  0.05. Values are means ± SE (n ¼ 4). 1:Control, 2:
50 mg L-1 ZnONPs, 3: 100 mg L-1 ZnONPs, 4: 500 mg L-1 ZnONPs,5: 150 mM NaCl, 6: 50 mg L-1 þ150 mM NaCl,7: 100 mg L-1þ150
and 8: 500 mg L-1 þ150 mM NaCl.

Protein extraction and gel electrophoresis

For sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), shoot tissues of each
treatment were ground to powder under liquid nitrogen and melted in ice-cold extraction buffer
(50 mM NaH2PO4, pH 7; 10 mM EDTA, pH 8; 10 mM b-mercaptoethanol; 0.2% Triton X-100)

per g of tissue, followed by centrifugation at 14,000 rpm, 4 C for 15 min. The supernatant was
used for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The protein
quantification was done according to the method given by Bradford (1976). Electrophoresis was
performed on 12.5% SDS-gel after Laemmli (1970). Gels were stained in 0.5% Coomassie Brilliant
Blue R 250 in ethanol and 10% 3-chloroacetic acid. Gel documentation system (Geldoc-it, UVP,
England), was applied to analyze banding pattern, molecular mass and band percentage using
Totallab analysis software (ww.totallab.com, Ver.1.0.1).
634 H. M. ABOU-ZEID ET AL.

Figure 3. Effects of salinity stress and seed-priming with ZnONPs on photosynthetic pigments and chlorophyll fluorescence
parameters in 18-day-old wheat shoots. Different letters indicate significant difference by Duncan’s multiple range tests at
p  0.05. Values are means ± SE (n ¼ 4). 1:Control, 2: 50 mg L-1 ZnONPs, 3: 100 mg L-1 ZnONPs, 4: 500 mg L-1 ZnONPs,5: 150 mM
NaCl, 6: 50 mg L-1 þ150 mM NaCl,7: 100 mg L-1þ150 and 8: 500 mg L-1 þ150 mM NaCl.

Statistical analysis
The results statistical analysis was carried out according to Duncan’s multiple range tests
using SPSS-20. Data were subjected to one-way ANOVA following the method of Sokal and
Rohlf (1995). Differences between treatment-means were considered statistically significant
at p  0.05.

Results
Characterization of ZnONPs
The FT-IR spectrum of ZnONPs (Figure 1A) showed peaks at 3440.06, 1633.58, 872.95 and
442.4458 wave number cm
1, the UV–vis absorption spectra of the ZnONPs peak that was
recorded in a range of 360–380 nm (Figure 1B), TEM image for a ZnONPs sample (Figure 1C
&D) indicated that the ZnONPs had a uniform spherical shape with varying sizes ranging from
12.0 to 21.0 nm. The XRD analysis (Figure 1E) produced strong intense peaks in the spectrum of
2q values ranging from 25 to 55 and indicted the crystalline nature of zinc ions. The presence of
Zn element signal was confirmed by EDS as shown in Figure 1F. An optical absorption
bands were observed at 0.887 to 1.128, 8.448 to 8.828 and 9.387 to 9.767 Kev reveals the presence
of pure metallic ZnONPs. The spectrum showed mainly Zn, 15.3%, 74.5%, and 10.2%,
respectively.
 JOURNAL OF PLANT NUTRITION 635

Figure 4. Ultrastructure of leaf mesophyll cells of 18-day-old wheat plants grown under salinity stress and priming with ZnONPs.
A: Control, B:500 mg L-1 ZnONPs,C:150 mMNaCl and D: 500 mg L-1 þ150mM NaCl. Bar :2 mm and MAG: 3000
(A1,A2,B1,B2,C1,C2,D1,D2). Bar: 500 nm and MAG: 10 K (A3, B3, C3, D3) and MAG: 25 K (A4,B4, C4, D4).

Plant growth, biomass, photosynthetic pigments, and chlorophyll fluorescence parameters

The shoots heights, as well as shoots and roots biomass (FM and DM) of wheat plants, were
assessed to evaluate the impact of priming with different concentrations of ZnONPs on plant
growth under salinity stress. The results of the present study depicted that salinity stress provoked
a significant suppression in shoot height, FM and DM (Figure 2). For example, the DM of shoots
and roots was 27.18% and 37.96% respectively, compared to the control. Seed-priming with
ZnONPs mostly induced an elevation in the formerly mentioned tested growth traits compared
with the control plant; the plants were healthy, greenish and grew well over the control plants. In
636 H. M. ABOU-ZEID ET AL.

Figure 5. (A) SDS-PAGE protein electrophoretic patterns and (B) phyllogenetic tree based on protein finger printing patterns for
wheat shoots under salinity stress and seed-priming with ZnONPs. 1:Control, 2: 50 mg L-1 ZnONPs, 3: 100 mg L-1 ZnONPs, 4:
500 mg L-1 ZnONPs,5: 150 mM NaCl, 6: 50 mg L-1 þ150 mM NaCl,7: 100 mg L-1þ150 and 8: 500 mg L-1 þ150 mM NaCl.

saline conditions with the highest level of ZnONPs (500 mg L
1), wheat growth was improved
and the values of shoot height and FM were 23.3 cm and 0.209 g respectively (Figure 2). These
observations revealed that the application of ZnONPs might improve -to some extent- the growth
biomarkers of wheat plants under salt stress.
Prolonged exposure to salinity significantly decreased the total pigment content of wheat
plants. At 150 mM NaCl treatment, the decrease in chl.a, chl.b, total chlorophylls and carot. was
40.5%, 39.9%, 40.18%, and 25.6% respectively compared to the control. Priming with various con-
centrations of ZnONPs markedly increased total pigments content in salt-stressed plants versus
those of non-primed ones (Figure 3). It is interesting to demonstrate that in the absence of salt
stress, ZnONPs priming additionally increased the content of the photosynthetic pigments com-
pared to non-primed control.
Exposing plants to 150 mM NaCl resulted in changes to the photosynthetic parameters, UPSII,
qP and Fv/Fm in leaves as shown in Figure 3. These parameters were significantly declined as a
result of salt stress and the reduction values were 26.55%, 31%, and 21.4%, respectively. ZnONPs-
primed wheat plants showed higher values of UPSII and qP reaching 1.40- and 2.36-fold,
 JOURNAL OF PLANT NUTRITION 637

Table 1. Occurrence of protein bands and their molecular masses (MM) as revealed by SDS-PAGE of total protein in the shoots
of wheat plant treated with different concentrations of ZnONPs (0, 50, 100 and 500 mg L
1) in absence or presence of
150 mM NaCl.
50 mg L
1 100 mg L
1 500 mg L
1
50 mg 100 mg 500 mg ZnONPs ZnONPs ZnONPs þ
L
1 L
1 L
1 þ150 mM þ150 mM 150 mM
Band No Control ZnONPs ZnONPs ZnONPs 150 mM NaCl NaCl NaCl NaCl
MW
1 116.9 135.6 133.5 131.3 152.9 150.8 111.4 157.3
2 109.7 108.1 120.9 115.0 92.5 142.2 93.1 144.3
3 93.3 93.9 98.9 93.6 89.2 116.9 88.1 133.5
4 73.4 64.6 91.7 91.3 84.9 95.9 69.9 116.9
5 58.7 57.6 84.9 56.5 96.9 93.9 64.6 108.1
6 51.7 45.4 62.9 53.0 57.6 91.3 52.2 93.9
7 50.4 43.8 44.4 51.3 52.6 57.6 51.1 92.8
8 41.0 35.4 42.7 49.5 50.9 55.6 47.9 91.0
9 32.7 23.8 37.7 41.6 48.8 51.7 45.4 83.8
10 31.7 20.9 36.1 33.0 44.4 50.2 42.1 46.9
11 30.9 19.5 35.4 31.7 41.0 49.1 325 38.1
12 29.3 18.8 31.9 29.7 33.9 44.9 29.8 34.2
13 23.0 13.6 30.5 25.7 29.5 34.8 27.2 33.3
14 21.8 12.1 24.6 24.4 25.7 31.5 23.8 31.9
15 20.2 20.7 21.3 23.8 30.2 21.8 27.9
16 15.3 18.6 19.3 20.2 27.5 19.3 26.7
17 12.7 14.5 16.5 19.3 25.4 16.2 21.8
18 10.2 12.5 13.8 16.2 21.5 13.8 16.9
19 10.2 12.32 15.6 16.0 9.8
20 10 12.32 13.6
21 7.6

respectively compared with the control values. However, ZnONPs had no significant effects on
maximum quantum yield of PSII (Fv/Fm). Maximal amelioration of salinity mediated decline was
observed in the photosynthetic parameters in plants primed with ZnONPs over the NaCl
stressed plants.

Leaf ultrastructure
Ultrastructural micrograph of Triticum aestivum leaf was observed with TEM (Figure 4). Leaf
mesophyll cells with a delimited cell wall and continuous cell membranes were observed.
Chloroplasts exhibited a typical structure with an ellipsoidal shape and well-arranged granum,
well-arranged thylakoid membranes as well as many starch grains and few plastoglobules (Figure
4A). The mitochondrion structure observed was typical with good membranes and a clear nucle-
olus and well-developed nuclear envelope were noticed in the nucleus. Plants treated with ZnOPs
alone showed a typical chloroplast ultrastructure with no significant changes (Figure 4B). On the
contrary, salt-affected plants showed some noticeable ultrastructural alteration of the organelles
and cellular injuries like nucleus condensation and smaller organelles. Under saline conditions,
chloroplasts often had abnormal spherical shapes with various degrees of damage such as
unorganized grana, swollen thylakoid in some parts. Additionally, many severely damaged chloro-
plasts in which they were collapsed and their envelopes were disrupted. In addition, these severely
damaged chloroplasts did not contain starch granules and few plastoglobules were noticed. The
mitochondria were very small; the nucleus was elongated with a distorted nuclear envelope
(Figure 4C). Observing NaCl-stressed plants primed with ZnONPs revealed that the cell ultra-
structure seems to be similar to that of control as the shape of the chloroplast was ellipsoidal
form, close to the cell wall, the distribution of grana was along the long axis of chloroplast and
well aligned internal lamellar system was noticed (Figure 4D). These results suggested that
638 H. M. ABOU-ZEID ET AL.

Figure 6. (A): Total fractions, polymorphic and monomorphic and (B): % polymorphism of electrophoretic protein patterns of
wheat shoots under salinity stress and seed-priming with ZnONPs. 1:Control, 2: 50 mg L
1 ZnONPs, 3: 100 mg L
1 ZnONPs, 4:
500 mg L
1 ZnONPs,5: 150 mM NaCl, 6: 50 mg L
1 þ150 mM NaCl,7: 100 mg L
1þ150 and 8: 500 mg L
1 þ150 mM NaCl.

priming with ZnONPs helped to maintain the integrity of chloroplast ultrastructure, thus per-
formed a normal physiological functions -to some extent- of the plant exposed to salt stress.

Changes of protein profiles

The electrograms of the SDS-PAGE protein profiles of Triticum aestivum shoots exhibited dis-
tinctive quantitative and qualitative alterations compared to untreated ones. As shown in Figure
5(A) and Table 1, these protein alterations were detected based on changes in polypeptides
molecular weights (MWs), bands intensities, fractionation of some bands, and appearance of new
polypeptides and disappearance of some others. The total number of bands detected was 18 bands
for control leaves with molecular weight ranged between 116.9 to 10.2 kDa. Application of NaCl
showed disappearance of 3 polypeptides with MW 116.9, 73.4 and 12.7 KDa and de novo induc-
tion of 5 new polypeptides with MW 152.9, 48.8, 44.4, 13.6 and 7.6 KDa. Also, high MW poly-
peptides of 135.6, 133.5 and 131.3 KDa were detected only in leaves primed with different
concentrations of ZnONPs. At 500 mg L
1 þ150 mM NaCl two unique bands with 157.3 and
144.3 KDa were detected. In general priming with different concentrations of ZnONPs in the
absence or presence of NaCl increased of protein pattern polymorphism comparing with the con-
trol (Figure 6A and B). Based on electrophoretic pattern polymorphism, using 50 mg L
1
ZnONPs þ150 mM NaCl reflected the highest polymorphism (79%) while salt stress showed the
lowest (48%). Dendrogram cluster analysis of wheat proteins profile resulted from SDS-PAGE
(Figure 5B) elucidated the relationships among different treatments, three groups were discrimi-
nated. The first group comprised both samples treated with 50 mg L
1 ZnONPs þ 150 mM NaCl
 JOURNAL OF PLANT NUTRITION 639

and the control. In the other two groups, the sample of the control was separated from the rest
of the samples. Salt treated leaves primed with 500 mg L
1 ZnONPs located in a relation with the
leaves primed with 100 mg L
1 ZnONPs without the salt stress.

Discussion
Nano-priming of seeds is a new technique developed to improve seed germination and seedling
growth especially under stressful conditions including abiotic stresses (Mahakham et al. 2017). In
the present investigation, seed-priming with ZnONPs considerably enhanced growth biomarkers
of wheat plants (shoot height, FM and DM). These observations were consistent with other
reported for several plant species including Vigna radiata and Cicer arietinum (Mahajan, Dhoke,
and Khanna 2011) and Triticum aestivum (Munir et al. 2018). Zinc is a necessary micro-nutrient
that has important roles in many physiological processes in plants, serving as a cofactor for many
enzymes and as the key structural motif in transcriptional regulatory proteins (Ishimaru, Bashir,
and Nishizawa 2011). In this connection, the enhancement of wheat growth is likely to be due to
the increased rate of micronutrient uptake, notably zinc, when treated with ZnONPs which was
related to the inherently small size the associated large surface area and high mobility of ZnONPs
(Prasad et al. 2012). Furthermore, several studies showed that ZnONPs have many beneficial
effects on germination leads to hormone biosynthesis, especially auxins and gibberellins that con-
sequently activating cell division and enlargement, promotes the degradation of seed reserves and
increased seed vigor (El-Kereti et al. 2014; Al-Harbi, Abdelhaliem, and Araf 2019).
Under the prevailing experimental conditions, salinity stress caused a significant reduction in
wheat growth biomarkers, this might be attributed to physiological and biochemical imbalance as
water deficit which leads to abnormal changes in plant morphology, osmotic stress, nutritional
disorders and alterations in photosynthetic activity (Soliman, El-Fiky, and Dariesh 2015; Ahmad
et al. 2018). Moreover, it is well documented that imposition of salinity causes damage to plant
tissues as a result of excessive ROS like H2O2 which leads to lipid oxidation and has a detrimental
effect on the membrane integrity (Munns, James, and L€auchli 2006)
In the present study, salt stress triggered a significant reduction in the content of the photo-
synthetic pigments as well as the photosynthetic parameters (UPSII, qP, and Fv/Fm) of wheat
plants. These observations are in agreement with earlier findings of Weisany et al. (2011) and
Iqbal, Umar, and Khan (2015) working on different plant species. The suppression in photosyn-
thetic pigments content has been reported to be related to the inhibitory effect of salinity on spe-
cific enzymes responsible for their synthesis, decrease in the absorption of minerals needed for
chlorophyll biosynthesis and induction of some degradative enzymes such as chlorophyllase as
well as the destruction of the photosynthetic machinery and pigment-protein complex instability
(El-Tayeb 2005). Moreover, Muranaka, Shimizu, and Kato (2002) suggested that the salt-induced
osmotic effect may induce a steady decline in photosynthesis owing to stomata closure.
Moreover, uptake of excessive amounts of Naþ may affect the electron transport causing a reduc-
tion in photosynthetic ability and a reduction in the chlorophyll content. A poor PSII activity
during salt stress had been also suggested to be attributed to the disturbed ionic composition of
stroma resulting from the entry of Naþ and Cl
which could cause unstacking and distortion of
grana and swelling of thylakoids as in rice and wheat (Rahman et al. 2000; Salama et al. 1994)
and the damage of chloroplast can lead to restricting photosynthetic rate (Acosta-Motos et al.
2017). In agreement with this view, chloroplasts of the salt-stressed wheat plant in the present
study often had abnormal spherical shapes with varying degrees of damage. Previously Shu et al.
(2013) observed that in salt-stressed Cucumis sativus L. chloroplast lamella disordered, slackened,
and even vague, with reduction in grana stacking on account of inhibition of protein synthesis.
The results of the present investigation demonstrated that seed-priming with ZnONPs posi-
tively affected the growth attributes, cell ultrastructure and photosynthetic parameters in salt-
640 H. M. ABOU-ZEID ET AL.

stressed wheat plants as Zn is an essential element and plays vital roles in growth and develop-
ment of plants (Pathak, Gupta, and Pandey 2012). It has been reported that ZnONPs can improve
the synthesis of protein and the translocation of nutrients from the aged cells to newborn cells
(Ebrahimian and Bybordi 2011), decrease the uptake of the excess of Naþ and Cl- (Ibrahim and
Faryal 2014) and has the ability to scavenge free oxygen radicals (Jiang et al. 2014). Increased
concentrations of chloroplastic pigments in the wheat plants primed with ZnONPs were in
accordance with earlier observations on Vigna radiate (Samreen et al. 2017), Rosmarinus officina-
lis (Mehrabani, Hassanpouraghdam, and Shamsi-Khotab 2018) and Triticum aestivum (Rani et al.
2019). Hao, Wei, and Dang (2003) observed that plants with zinc application have enhanced
photosynthetic rate owing to the synthesis of the chloroplast, increased synthesis of chlorophyll,
oxidation of water at PSII and predominated activities of Calvin cycle enzymes in addition to
increased absorption of CO2 as a result of stomatal opening and increased nutrient uptake. In
this connection, the current study showed that the application of ZnONPs under salt stress
enhanced photosynthesis by improving the chlorophyll synthesis and chlorophyll fluorescence in
wheat plants. Additionally, a marked amelioration or modulation in the damages of the ultra-
structure of chloroplasts and nuclei in salt-stressed wheat leaves primed with ZnONPs was
noticed. Zinc regulates the functioning and development of chloroplasts and it regulates the
repair of PSII by transferring the photodamaged protein D1 (Hansch and Mendel 2009). On con-
trary, Sheteiwy et al. (2015) findings were that in the rice cultivars primed with 750 mg L
1
ZnONPs the leaf mesophyll cells were significantly damaged.
SDS-PAGE is frequently used to visualize preliminary changes of polypeptide patterns by dif-
ferent types of abiotic stresses. Figure 5 showed that salt stress brought about some changes in
the polypeptide patterns of wheat plants either primed or non-primed with ZnONPs. Salt stress
with or without ZnONPs priming caused induction of some polypeptides while other polypepti-
des disappeared. Similarly, salinity has been reported to cause a complete loss of present proteins
and/or the synthesis of new proteins in wheat and flax (Yıldız and Terzi 2008, Talei et al.
2015).In the present study, the induction of the 48 kDa and 7.6 kDa protein was observed in
shoots of wheat plants in response to salinity. Recently, Razavizadeh (2015) reported the induc-
tion of a new polypeptide of 48.61 kDa in canola plant exposed to salt stress. Earlier Mikolajczyk
et al. (2000) suggested that the 48 kDa protein induction may be considered to symbolize a pro-
tein kinase under osmotic stress in plants. El-Bassiouny and Sadak (2015) reported the induction
of a small molecular weight (9 KDa) protein in flax plants under salinity stress and suggested to
be Ubquitin which protects the protein from degradation by protease. It is worth noting the
induction of 4 unique polypeptides of 157.3, 144.3, 26.7 and 16 KDa in response to 500 mg L
1
ZnONPs þ150 mM NaCl (the 16 kDa polypeptide was induced at all ZnONPs treated wheat
plants in presence of NaCl). Similar changes were reported for the induction of a 26 kDa protein
by Razavizadeh (2015) and the induction of a 16 kDa protein by Przymusinski, Rucinska, and
Gwozdz (2004) in canola and lupin plants, respectively. Moreover, El-Bassiouny and Sadak (2015)
reported the induction of new polypeptide of 144 kDa in flax plants grown under salt stress.
Thus, it can be generally speculated that the observed alterations in the protein synthesis pattern
by salinity and/or ZnONPs might predict the presence of several osmoresponsive genes for the
production of osmoprotective compounds, which are known as compatible solutes (Hussain et al.
2008). Furthermore, high MW polypeptides of 135.6, 133.5 and 131.3 KDa were detected only in
shoots primed with different concentrations of ZnONPs. It has been reported that zinc modulates
protein synthesis and defines the function and three-dimensional structure of many proteins e.g.,
Zn finger proteins (Feinauer et al. 2013). Moreover, it helps to bridge amino acid residues
(methionine and cysteine) as it is strongly bound to the –SH group and limits disulfide bond per-
oxidation (Marschner 2012). Liu et al. (2017) reported that zinc fertilization increases the protein
content and quality of wheat grains by increasing the concentrations of albumin, glutenin, glia-
dins, and globulin. In summary, in the present investigation, the occurrence of new protein bands
 JOURNAL OF PLANT NUTRITION 641

could be stress proteins produced to overcome the toxic effect of salinity. On the other hand, the
disappearance of some protein bands could be due to lowered protein synthesis and/or the deple-
tion of the reserve.

Conclusion
The results of the present investigation suggest that seed priming with ZnONPs can improve the
growth of wheat plants subjected to salt stress. The better growth performance of wheat seedlings
after seed priming was associated with a better ultrastructure, more photosynthetic attributes and
changes of polypeptide patterns under saline conditions. However, further research is necessary
to test the efficiency of Zn priming under field conditions with varying salt stress conditions.
Furthermore, the exact cascade of changes at the molecular level and the particular genes that are
induced to bring such an effect needs to be further elucidated.

Conflict of interest
No potential conflict of interest was reported by the authors.

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