Acta agriculturae Slovenica, 118/3, 1–15, Ljubljana 2022
doi:10.14720/aas.2022.118.3.2529 Original research article / izvirni znanstveni članek
Seed priming with ZNPs reduced expression of salinity tolerance genes 
in Glycine max L. and improved yield traits
Reda Mohamed GAAFAR 1, 2, Mohamed Lotfi HALAWA 1, Adel Ramadan EL-SHANSHORY 1, Abdelhamid 
Abdelrahim EL-SHAER 3, Rana Hosny DIAB 1, Marwa Mahmoud HAMOUDA 1
Received January 18, 2022; accepted July 17, 2022.
Delo je prispelo 18. januarja 2022, sprejeto 17. julija 2022
1 Botany Department, Faculty of Science, Tanta University, Egypt
2 Corresponding author, e-mail: redagaafar@science.tanta.edu.eg
3 Physics Department, Faculty of Science, Kafrelsheikh University, Kafrelsheikh, Egypt
Seed priming with ZNPs reduced expression of salinity toler-
ance genes in Glycine max L. and improved yield traits
Abstract: Little has been done to evaluate the molecular 
role of ZnO nanoparticles (ZNPs) in regulating biochemical 
processes and plant yield in response to salt-induced stress. 
In this study, the molecular response of salt-stressed soybean 
(‘Giza111’) was assessed under different concentrations of 
ZNPs (25, 50, 100, and 200 mg l-1) by measuring some osmo-
lytes, yield parameters, and Na+ and K+ content. The impact of 
salinity on the mRNA expression levels of three key salt-toler-
ance related genes (GmCHX1, GmPAP3, and GmSALT3) using 
qRT-PCR was also determined. The high level of salinity (250 
mM NaCl) led to a significant increase in Na+ content, total sol-
uble proteins, and total soluble carbohydrates and significantly 
upregulated gene expression of GmCHX1, GmPAP3, and Gm-
SALT3, while reducing K+ content, K+/Na+ ratio and all yield 
parameters compared to control plants. Soaking soybean seeds 
in various ZNP concentrations, on the other hand, increased 
K+ content and K+/Na+ ratio while decreasing Na+ content, to-
tal soluble proteins, and total soluble carbohydrates in stressed 
plants, particularly at 50 mg l-1 ZNPs. Furthermore, GmCHX1, 
GmPAP3, and GmSALT3 expressions were all downregulated 
at 50 mg l-1 ZNPs, which ultimately improved soybean yield 
parameters. Accordingly, these results recommend the applica-
tion of 50 mg l-1 ZNPs for improving the productivity of soy-
bean cultivated in saline soils.
Key words: ZnO; nanoparticles; salinity; soybean; gene 
expression; qRT-PCR ; productivity
Predtretiranje semen s cinkovimi nano delci je zmanjšalo iz-
ražanje genov tolerance na slanost pri soji (Glycine max L.) in 
izboljšalo lastnosti pridelka
Izvleček: Malo je bilo narejenega za ovrednotenje mole-
kularne vloge nano delcev ZnO (ZNPs) pri uravnavanju bio-
kemičnih procesov in pridelka rastlin kot odziva na slanostni 
stres. V tej raziskavi je bil ocenjen molekularni odziv na sol-
ni stres pri soji (‘Giza111’) pri uporabi različnih koncentracij 
ZNPs (25, 50, 100, in 200 mg l-1) z meritvami nekaterih osmoti-
kov, parametrov pridelka in vsebnosti Na+ in K+. Vpliv slanosti 
na količino mRNK treh ključnih s toleranco na slanost poveza-
nih genov (GmCHX1, GmPAP3, in GmSALT3) je bil določen z 
uporabo qRT-PCR metode. Velika slanost (250 mM NaCl) je 
vodila k znatnemu povečanju vsebnosti Na+, celokupnih topnih 
beljakovin, celokupnih topnih ogljikovih hidratov in značilno 
povečala izražanje genov GmCHX1, GmPAP3, in GmSALT3, 
med tem ko, je zmanjšala vsebnost K+, razmerja K+/Na+ in vse 
parameter pridelka v primerjavi s kontrolo. Namakanje semen 
soje v različnih koncentracijah ZNP je povečalo vsebnost K+ in 
razmerje K+/Na+ v rastlinah pod stresom in hkrati zmanjšalo 
vsebnost Na+, celokupnih topnih beljakovin in celokupnih to-
pnih ogljikovih hidratov, še posebej pri uporabi 50 mg l-1 ZNPs. 
Dodatno je bilo pri tem obravnavanju zmanjšano izražanje ge-
nov GmCHX1, GmPAP3,in GmSALT3, kar je na koncu izboljša-
lo parametre pridelka soje. Skladno s temi rezultati priporoča-
mo uporabo 50 mg l-1 ZNPs za izboljšanje pridelka soje, gojene 
na slanih tleh.
Ključne besede: ZnO; nano delci; slanost; soja; izražanje 
genov; qRT-PCR; produktivnost
Acta agriculturae Slovenica, 118/3 – 20222
R. M. GAAFAR et al.
1 INTRODUCTION
Soybean (Glycine max L.) is one of the important 
food and industrial crops worldwide because of its con-
tent of cholesterol-free oil (30  %) and proteins (40  %), 
which are similar in their nourishing value to animal 
proteins (Van Zanten et al., 2016). The fractions and de-
rivatives of soybean seeds have major economic impor-
tance in a wide range of industrial, food, pharmaceutical, 
and agricultural products (Chen et al., 2012).
Salinity of the soil is a serious problem all over the 
world. It has been estimated that around 954 million hec-
tares are already salinized (Qadir et al., 2014). It usually 
causes a reduction of water potential, ion imbalances or 
disturbances in ion homeostasis, resulting in a reduction 
of plant growth and crop productivity (Han et al., 2019). 
Mittler (2002) observed that the oxidative demolition of 
the cell (oxidative stress) occurs by injuring membranes 
(lipid peroxidation), proteins, RNA, and DNA molecules 
as a result of elevated ROS levels in the cells. DNA dam-
age is caused by OH· and O2- radicals, and this damage 
results in heritable changes (Fatima et al., 2017). Moreo-
ver, these signals play an important role in the adapta-
tion process of plants to abiotic stress (Choudhury et al., 
2017).
Plant tolerance to salinity stress includes physiologi-
cal and molecular changes such as accumulation of or-
ganic solutes, antioxidant enzymes, and inorganic ions as 
well as gene expression responses (Ahanger et al., 2017). 
These alterations include either the induction of some 
polypeptides, the disappearance of others, or the overex-
pression of other sets of proteins (El-Mashad et al., 2012). 
Therefore, linking the expression of a gene to a higher 
degree of tolerance within a genotype offers an impera-
tive argument for a role in plant adaptation (Abreu et 
al., 2013). Numerous reports suggest that the harm-
ful effect of salinity stress was manifested by relatively 
higher expression of salt-related genes in soybean, such 
as GmP5CS, GmDREB1a, GmGOLS, GmBADH and Gm-
NCED1 (Liu et al., 2017), GmERF3 (Zhang et al., 2009), 
GmMYB genes, GmMYB76, GmMYB92 and GmMYB177 
(Liao et al., 2008), GmPAP3 (Liao et al., 2003), GmCHX1 
(Patil et al., 2016) and GmSALT 3 (Guan et al., 2014). 
Previous studies in soybean determined that a QTL 
on chromosome 3 is the major genomic region that dic-
tates salinity tolerance in soybean (Patil et al., 2016; Chen 
et al., 2018). This gene locus carries the dominant func-
tional sodium/hydrogen exchanger family gene in wild 
(GmCHX1) and cultivated soybean (GmNcl/GmSALT3), 
which explains more than 64 % of the phenotypic varia-
tion (Qi et al., 2014). Normally, the GmCHX1 gene is ex-
pressed under high salt conditions in root stellar cells and 
limits salt transport to shoot tissues (Guan et al., 2014). It 
has been described that the full-length GmSALT3 protein 
is closely correlated to the Arabidopsis thaliana AtCHX20 
(a Cation/Proton Exchanger), which is a function-
ally characterized member of the CPA2 (Cation/Proton 
Antiporter2) family of transporters (Padmanaban et al., 
2007; Qu et al., 2020). Functional studies of AtCHXs have 
shown that they might play a role in modulating cation 
and pH homeostasis within the endomembrane system 
(Chanroj et al., 2011). The ER-localized AtCHX20 was 
suggested to be an endomembrane K+ transporter in-
volved in the osmoregulation of guard cells (Padmana-
ban et al., 2007). Purple acid phosphatases (PAPs) rep-
resent a diverse group of acid phosphatases in animals, 
microorganisms, and plants (Vogel et al., 2001; Olczak 
et al., 2003). The primary biochemical reaction of PAPs 
is to catalyze the hydrolysis of phosphate esters and an-
hydrides. The physiological role of GmPAP3 might be re-
lated to the adaptation of soybean to NaCl stress, possibly 
through its involvement in reactive oxygen species (ROS) 
forming and/or scavenging or stress-responding signal 
transduction pathways (Liao et al., 2003; Soleimani et al., 
2017).
Zinc (Zn) is a metallic cofactor for more than 300 
enzymes. The Zn-finger proteins that attach to deoxyrib-
onucleic acid (DNA) are clear evidence of the usefulness 
of Zn in biological systems (Hezaveh et al., 2019). Zinc is 
a structural component of ribosomes and is essential for 
their structural integrity. On the other hand, it has other 
indirect effects on the control of stomatal opening and 
closing and ROS detoxification (Haliloglu et al., 2020). 
Currently, nanotechnology has broad perspectives in all 
fields of science (Dewdar et al., 2018). The application 
of nanoparticles to plants can be beneficial for growth 
and development due to their greater absorbance and 
high reactivity (Fraceto et al., 2016). ZnO nanoparticles 
(ZNPs) are one of the most frequently used nanoprod-
ucts (Samei et al., 2019). Interestingly, priming of seeds 
with ZNPs positively affected the yield traits in salt-
stressed plants, whereas ZNPs stimulated natural auxin 
(IAA), thus activating cell division and enlargement and 
also increasing K+ ion content, which increases storage 
of food in seeds (Ali and Mahmoud, 2013), maintain-
ing the structural integrity of biomembranes (He et al., 
2015), improving protein synthesis and DNA replication 
(Landa et al., 2015), scavenging free oxygen radicals and 
decreasing the uptake of excess Na+ and Cl− (Farhangi-
Abriz and Torabian, 2018), as well as augmentation of 
photosynthesis, total soluble proteins, total soluble car-
bohydrates, and total phenols in stressed plants (Abdel 
Latef et al., 2017).
Transcription factors are the primary regulators of 
gene expression in a variety of genes that are involved in 
reducing and/or protecting against cellular stress damage 
Acta agriculturae Slovenica, 118/3 – 2022 3
Seed priming with ZNPs reduced expression of salinity tolerance genes in Glycine max L. and improved yield traits
(Linh et al., 2020). The catalytic activity of RNA polymer-
ases, which is essential for gene expression, is well known 
to require Zn2+ ions. Zn stabilizes several structural mo-
tifs in transcriptional regulatory proteins, such as Zn fin-
ger domains (Albert et al., 1998). Zn has been shown to 
upregulate gene expression, particularly in Zn-controlled 
genes, in numerous studies. Plants treated with ZnO, for 
example, had the highest OsZIP1 expression in their 
roots after 7 days when compared to no-zinc controls 
(Selvaraj and Dananjeyan, 2016). Recently, ZNPs boost-
ed the expression of the wheat drought-tolerance genes 
DREB2 and Wdhn13, catalase activity (CAT1), proline 
biosynthesis (P5CS), and proline biosynthesis (P5CS) 
genes (Raeisi Sadati et al., 2022).
It was concluded that priming with ZNPs, particu-
larly at 60 mg l-1, improved photosynthetic pigments, al-
tered osmoregulation, and decreased MDA and Na con-
centrations in lupine plants (Abdel Latef et al., 2017). So, 
the current study was conducted to investigate the effect 
of seed-priming using different concentrations of ZNPs 
on the expression of three salinity-tolerance genes. In 
addition, their impacts on alleviating salinity stress and 
improving productivity in soybean plants were assessed.
2 MATERIALS AND METHODS
2.1 PLANT MATERIALS 
Seeds of a soybean cultivar (‘Giza 111’) were pro-
vided by the Food and Legumes Research Department, 
Field Crops Research Institute, Agricultural Research 
Center, Giza, Egypt.
2.2 SYNTHESIS AND CHARACTERIZATION OF 
ZNO NANOPARTICLES
In this study, ZnO nanoparticles were synthesized 
using the chemical bath deposition (CBD) method as 
described by El-Shaer et al. (2018). The crystalline struc-
ture and optical properties of the prepared ZnO nano-
structures were examined with X-ray Diffraction (XRD, 
Shimadzu 6000), while the samples’ morphology was in-
vestigated using a scanning electron microscope (SEM, 
JSM-651OLV). As shown in Fig. 1A, ZnO nanostruc-
tures are formed as nano-rods with a hexagonal quartzite 
crystal structure. These nano-rods accumulate to form 
the surface morphology of grains, similar to flowers. The 
XRD pattern of ZnO nano-rods is shown in Fig. 1B. The 
diffraction peaks at 32°, 34.5°, 36.4°, 47.5°, 57°, 62.7°, 
67.9°, and 69.3° correspond to the (100), (002), (101), 
(102), (110), (103), (112), and (201) lattice planes, re-
spectively (Fig. 1B).
2.3 PLANT GROWTH CONDITIONS
Priming and growing of the soybean seeds (‘Giza 
111’) were performed as described by Gaafar et al. (2020). 
The seeds were sterilized with 70  % ethanol for 5 min 
and sodium hypochlorite (10 %) for 10 min, followed by 
washing several times with distilled water. Four concen-
trations of ZnO nanoparticles (ZNPs) of 25 (ZNPs25), 
50 (ZNPs50), 100 (ZNPs100), and 200 (ZNPs200) mg l-1 
were used to prime the seeds for two hours at room tem-
perature, and distilled water was used as a control (0). 
Previous studies have found that low concentrations of 
Fig. 1: (A) SEM image and (B) XRD chart of ZnO nanostructures prepared by CBD method
Acta agriculturae Slovenica, 118/3 – 20224
R. M. GAAFAR et al.
ZnO NPs are beneficial to plant growth, whereas concen-
trations equal to or greater than 200 mg l-1 are detrimen-
tal. Therefore, the used ZNP concentrations were chosen 
(Liu et al., 2015; Abdel Latef et al., 2017). After priming, 
the seeds were sown (20 seeds/pot) in plastic pots (45 cm 
x 40 cm) filled with 24 kg of 2:1 (clay: sandy) soil. Based 
on the preliminary experiment results, 250 mM NaCl (S) 
was chosen as a sub-lethal salinity level and used in this 
study.
The pots were irrigated with tap water until seed 
germination (emergence), then with tap water and with 
250 mM NaCl solution to 80 % field capacity for 21 days 
(seedling stage) and 90 days (yield stage). Three pots were 
used as replicates for each treatment. The germinated 
soybean seeds were let to grow in the green house under 
the following environmental conditions: 29 ± 2 oC/25 ± 2 
oC day/night and 16h/8h light/dark regimes. The 21-day-
old seedlings of all treatments were collected, washed, 
and used for further analyses, and the productivity of 
yielded seeds was determined on 90-day-old plants.
2.4 DETERMINATION OF SODIUM AND POTAS-
SIUM CONTENT 
According to Allen et al. (1974), the mixed acid 
digestion method was used for element determination. 
The concentration of Na+ and K+ (mg g-1 d.m.) was deter-
mined by using Inductively Coupled Plasma (ICP, STI) at 
the central laboratory of Tanta University.
2.5 QUANTITATIVE ESTIMATION OF TOTAL 
SOLUBLE PROTEINS AND TOTAL SOLUBLE 
CARBOHYDRATES
The total soluble proteins were extracted according 
to the method described by Naguib et al. (1968). Then 
the protein content was determined as described by 
Bradford (1976), and the phenol-sulfuric acid method 
has been used for estimation of total soluble carbohy-
drates according to Dubois et al. (1956).
2.6 QUANTITATIVE REAL TIME PCR (QRT-PCR) 
RNA EXTRACTION AND PURIFICATION
For the extraction of total RNA, approximately 100 
mg of ground plant fresh leaves were used, and RNA was 
extracted using the RNeasy Plant Mini Kit (Qiagen, Ger-
many) according to the manufacturer’s protocol. The total 
RNA was then quantified and assessed for quality using 
a Nanodrop (ScanDrop, Analytik, Jena, Germany). Total 
RNA samples were kept at -80 °C until further analysis.
2.7 CDNA SYNTHESIS
The cDNA synthesis was performed using the Sen-
siFAST cDNA synthesis kit (Ameridian Life Science, 
USA) using the protocol of the manufacturer. The cDNA 
synthesis reaction contained the following components: 
1 µg total RNA, 1 µl reverse transcriptase enzyme, 4 µl 
5 × Trans Amp buffer, which was completed to a total 
volume of 20 µl. The conditions for cDNA synthesis were 
as follows: primer annealing for 10 min at 25 °C, reverse 
transcription for 15 min at 42  °C and finally 5 min at 
85 °C for enzyme inactivation. After being diluted in 10 
mM Tris-HCl (pH = 8) and 0.1 mM EDTA, the cDNA 
reaction products were stored at -20 °C.
2.8 GENE EXPRESSION ANALYSIS (QRT- PCR)
In order to measure the gene expression of the three 
targeted genes, the reaction mix was prepared by mixing 
10 µl of TOP real qPCR2x premix (SYBR Green with low 
ROX), 1 µl of each of the cDNA template, forward and 
Primer name Sequence (5’→3’) Length (bp) Annealing temp. (ºC) Reference
GmPAP3F GTGGCCGGCAGTTGACATCC 20 55.5 Liao et al. (2003)
GmPAP3R GCTGTGCCCTGGCTCTTCTGTG 22 55.5
GmCHX1F GATTTGTTTTCGGGCTAACG 20 49.5 Gutierrez Gonzalez et al. (2010)
GmCHX1R ATCCACCACGCTTCGTAACT 20 49.5
GmSALT3F CGGTTGATGAAGGGAAAAC 19 48.5 Hu et al. (2009)
GmSALT3R TCCTTGACGCTTGGAGTGTT 20 48.5
GmTublinF GAGAAGAGTATCCGGATAGG 20 50 Gutierrez Gonzalez et al. (2010)
GmTublinR GTTTCCGAACACTCAAGCTC 20 50
Table 1: List of sequences of the primers used for gene expression study by qRT-PCR
Acta agriculturae Slovenica, 118/3 – 2022 5
Seed priming with ZNPs reduced expression of salinity tolerance genes in Glycine max L. and improved yield traits
reverse primer (10 pmol μl-1) and was completed up to 
20 µl. The Rotor-Gene Q5 plex (Qiagen, Germany) was 
used, and the PCR conditions were as follows: an initial 
denaturation step at 95 °C for 10 min; a denaturation step 
at 95 °C for 10 s; an annealing step at 60 °C for 15 s; and 
an elongation step at 72 °C for 15 s. The thermal cycler 
steps were repeated 35 times. The sequences of the prim-
ers used for qRT-PCR analysis are shown in Table 1. The 
relative gene expression was calculated using the 2-∆∆CT 
method according to Livak and Schmittgen (2001).
2.9 YIELD TRAITS
The yield parameters, including length of pods/
plant, mass of pods/plant, the mass of 1000 seeds, the 
number of pods/plant, the number of seeds/pod, the 
mass of seeds/pod, and the mass of seeds/plant, were 
determined at the end of the growing season. (approxi-
mately 3 months from cultivation). The maturity (num-
ber of viable - nonviable seeds in pods * 100) and the 
productivity of soybean (weight of the yielded seeds/pot 
in grams) were also calculated.
2.10 STATISTICAL ANALYSIS
The statistical analyses were carried out according 
to a completely randomized design (CRD) using analysis 
of variance. The significance was determined using LSD 
values at p = 0.05 and 0.01 according to Bishop (1983). 
The results were analyzed using a one-way ANOVA test 
to determine the degree of significance. The statistical 
analyses were performed using CoStat Software version 
6.311 (CoHort Software, CA, USA). The heatmap of the 
gene expression data and Pearson correlation were con-
structed using R software (ver. 4.1.1).
3 RESULTS
3.1 SODIUM AND POTASSIUM CONTENT
The results in Table 2 show the effect of salinity 
stress on mineral ion content (Na+, K+, and K+/Na+) in 
21-day old soybean seedlings after soaking of soybean 
seeds in different concentrations of ZNPs (0, 25, 50, 100, 
and 200 mg l-1). The salinity stress (250 mM NaCl) se-
verely decreased the content of potassium by 68 % com-
pared to control. Similarly, it reduced the K+/Na+ ratio by 
90 % compared to control. In contrast, the content of Na+ 
was highly increased by 2.16-fold compared to control. 
On the other hand, the combination of ZNPs50+S (50 
mg l-1 + 250 mM NaCl) significantly increased the con-
tent of potassium by 1.67-fold compared to salt-stressed 
seedlings and ameliorated the harmful effect of salinity 
Treatments
K+ 
(mg g-1 d. m.)
Na+ 
(mg g-1 d. m.) K+/Na+ ratio
Salinity level 
(0 mM NaCl)
Control 4.287 ± 0.04 a 2.346 ± 0.0008 f 1.827
ZNPs25 4.043 ± 0.04 b 2.160 ± 0.007 f 1.871
ZNPs50 4.242 ± 0.01 a 1.761 ± 0.022 g 2.409
ZNPs100 3.942 ± 0.04 b 2.247 ± 0.009 f 1.754
ZNPs200 3.756 ± 0.04 c 2.286 ± 0.048 b 1.642
Salinity level 
(S = 250 mM NaCl)
Salinity (S) 1.336 ± 0.01 g 7.419 ± 0.08 a 0.180
ZNPs25+S 2.539 ± 0.14 e 5.425 ± 0.13 d 0.468
ZNPs50+S 3.569 ± 0.11 d 4.061 ± 0.03 e 0.878
ZNPs100+S 2.011 ± 0.008 f 6.629 ± 0.28 c 0.303
ZNPs200+S 1.118 ± 0.004 h 7.120 ± 0.06 b 0.157
F-value 689.8 894.8 -
LSD (0.05) 0.138 0.226 -
Significance * -
Table 2: Effect of salinity (S = 250 mM NaCl) on the content of Na+, K+ and K+/Na+ ratio of 21-day old soybean (‘Giza 111’) 
seedlings grown in clay-sandy soil (2:1 w/w) after soaking of soybean seeds in four different concentrations of ZnO nanoparticles 
(ZNPs) (ZNPs25 = 25, ZNPs50 = 50, ZNPs100 = 100, and ZNPs200 = 200 mg/L)
Values are the mean of three replicates ± SD. Values within the same column for each factor designated by different letters are significant at p ≤ 0.05, 
while values with identical letters are non-significant. *: significant at p ≤ 0.05
Acta agriculturae Slovenica, 118/3 – 20226
R. M. GAAFAR et al.
stress. Also, the combination of ZNPs25 + S (25 mg l-1 + 
250 mM NaCl) increased the content of potassium ions 
by only 1.07-fold.
Moreover, results indicated that the combination of 
ZNPs200 + S (200 mg l-1 + 250 mM NaCl) exhibited a se-
vere harmful effect compared to other treatments; thus, it 
reduced the potassium and K+/Na+ ratio content by 16 % 
and 12  %, respectively, compared to salt stressed seed-
lings (Table 2).
3.2 TOTAL SOLUBLE PROTEINS AND TOTAL 
SOLUBLE CARBOHYDRATES 
The results in Figure 2 (A and B) indicated that high 
salinity stress (S = 250 mM NaCl) caused a highly signifi-
cant increase in total soluble carbohydrates and protein 
content by 75.1 % and 76.1 %, respectively, compared to 
control. However, the results showed a general decrease 
in total soluble carbohydrates and protein content for all 
Fig. 2: Effect of NaCl (S = 250 mM) on the total soluble carbohydrates (A) and total soluble proteins (B) of 21-day old soybean 
(‘Giza 111’) seedlings grown in clay-sandy soil  (2:1 w/w) after soaking of soybean seeds in four different concentrations of ZnO 
nanoparticles (ZNPs) (ZNPs25 = 25, ZNPs50 = 50, ZNPs100 = 100, and ZNPs200 = 200 mg l-1)
Acta agriculturae Slovenica, 118/3 – 2022 7
Seed priming with ZNPs reduced expression of salinity tolerance genes in Glycine max L. and improved yield traits
ZNPs (25, 50, 100, and 200 mg l-1) combined with salin-
ity, except for the combination of ZNPs25+S (25 mg l-1 
+ 250 mM NaCl), which exhibited the least reduction in 
total soluble carbohydrates and protein content with a 
percentage of 10 % and 23 %, respectively, compared to 
control plants, which were irrigated with water. The high-
est reduction in total soluble carbohydrates and protein 
content was recorded in the case of ZNPs50+S (50 mg 
l-1 + 250 mM NaCl) with 29  % and 43  %, respectively, 
compared to control plants irrigated with salt only (Fig. 
2A and B).
3.3 GENE EXPRESSION ANALYSIS (QRT- PCR)
3.3.1 GmCHX1
The results of qRT-PCR analysis showed that Gm-
CHX1 expression was increased by ZNPs alone and the 
highest increase was with ZNPs100+S (100 mg l-1 + 250 
mM NaCl) compared to control (no salinity) by about 
1.2-fold (Fig. 3). Also, 250 mM NaCl (S) alone showed the 
highest increase in GmCHX1 gene expression by 1.9-fold 
compared to control (no salinity and no ZNPs). How-
ever, ZNPs25+S (25 mg l-1 + 250 mM NaCl) decreased 
gene expression by 0.4-fold, and then it was increased 
with ZNPs50+S (50 mg l-1 + 250 mM NaCl). Also, Gm-
CHX1 gene expression was decreased by 0.08-fold with 
ZNPs100+S (100 mg l-1 + 250 mM NaCl). In contrast, 
ZNPs200+S (200 mg l-1 + 250 mM NaCl) showed an in-
crease of 1.8-fold, which was similar to that of salinity (S 
= 250 mM NaCl) (Fig. 3).
3.3.2 GmPAP3
GmPAP3 expression was slightly increased by ZNPs 
treatments, and the two concentrations (ZNPs100 and 
ZNPs200 mg l-1) showed the highest increases of about 
0.73 and 0.70-fold, respectively, compared to control (no 
salinity and no ZNPs) (Fig. 3). In the case of salt treat-
ment (S = 250 mM NaCl), GmPAP3 expression increased 
by 3-fold. ZNPs25+S (25 mg l-1 + 250 mM NaCl), ZNPs50 
+ S (50 mg l-1 + 250 mM NaCl), ZNPs100 + S (100 mg l-1 
+ 250 mM NaCl), and ZNPs200 + S (200 mg l-1 + 250 mM 
NaCl) all reduced GmPAP3 expression. The highest de-
crease was by 0.46-fold and was recorded with ZNPs25+S 
(25 mg l-1 + 250 mM NaCl) treatment (Fig. 3).
3.3.3 GmSALT3 
In the case of GmSALT3, gene expression was in-
creased by ZNP treatment using a concentration of 100 
mg l-1 compared to control (no salinity and no ZNPs) by 
about 2-fold, as shown in Fig. 3. However, salinity stress 
(250 mM NaCl) showed the highest increase in Gm-
SALT3 gene expression by 7.7-fold. The concentration of 
25 mg l-1 of ZNPs with salinity decreased gene expression 
by 1.56-fold, and then it was increased by the concentra-
tion of 50 mg l-1 of ZNPs with salinity by 4.5-fold. Then, in 
comparison to the salinity stress alone (250 mM NaCl), 
gene expression decreased by 3.9-fold with ZNPs100 + S 
(100 mg l-1 + 250 mM NaCl) treatment and by 3.5-fold 
with ZNPs200 + S (200 mg l-1 + 250 mM NaCl). In con-
trast to the salinity stress alone (250 mM NaCl), which 
increased gene expression by 8-fold (Fig. 3).
Fig. 3: Heatmap of relative expression of three soybean salinity-linked genes (GmPAP3, GmCHX1, and GmSALT3) in 21-day 
old soybean (‘Giza 111’) seedlings after soaking of soybean seeds in four different concentrations of ZnO nanoparticles (ZNPs) 
(ZNPs25 = 25, ZNPs50 = 50, ZNPs100 = 100, and ZNPs200 = 200 mg l-1)
Acta agriculturae Slovenica, 118/3 – 20228
R. M. GAAFAR et al.
3.4 YIELD PARAMETERS
The results given in Table 3 show the effect of 250 
mM NaCl and ZNPs (25, 50, 100, and 200 mg l-1) treat-
ments on yield parameters. These results revealed an ob-
servable increase in all measured yield parameters, spe-
cifically in the case of ZNPs25 (25 mg l-1) and ZNPs50 
(50 mg l-1) treatments without salinity, where these treat-
ments increased the pod length, pod mass, number of 
pods/plants, and mass of pods/plant, number of seeds/
pods, mass of seeds/pods, and mass of seeds/plant. The 
most significant increase was with ZNPs50 (50 mg l-1) by 
29 %, 27.8 %, 62.8 %, 39.9 %, 15.3 %, 47.8 %, and 78.8 %, 
respectively, compared to control. In contrast, results 
showed that the application of 200 mg l-1 ZNPs (ZNPs200) 
caused a highly significant decrease in all measured yield 
parameters: pod length, pod mass, number of pods/
plant, mass of pods/plant, number of seeds/pod, mass of 
seeds/pods, and mass of seeds/plant by 37.8 %, 16.1 %, 
13.89 %, 20.9 %, 26.9 %, 11.5 %, and 18.1 %, respectively 
compared to control.
Similarly, a remarkable increase in all measured 
yield parameters in the case of treatments ZNPs25 + S, 
ZNPs50 + S, and ZNPs100 + S was observed (Table 3). 
The most significant increases in pod length, pod mass, 
number of pods/plants, and mass of pods/plant, number 
of seeds/pods, mass of seeds/pods, and mass of seeds/
plant were with ZNPs50+S (50 mg l-1 + 250 mM NaCl) 
by 50.11 %, 85.4 %, 42.6 %, 47.7 %, 341.6 %, 100 %, and 
119  %, respectively, compared to control. Whereas, a 
decrease in these yield parameters was observed with 
ZNPs200+S (200 mg l-1 + 250 mM NaCl) by 53.2  %, 
15.2 %, 23.2 %, 14.5 %, 100 %, 15.7 %, and 4.7 %, respec-
tively, compared to control.
Similarly, the most significant increases in mass of 
the seeds (g/plant), mass of 1000 seeds, maturity per-
centage, and productivity index (g/pot) were with treat-
ment ZNPs50 + S (50 mg l-1 + 250 mM NaCl) by 14.8 %, 
31.5 %, 32.6 %, and 118.9 %, respectively, compared to 
control. In contrast, treatment with ZNPs200 + S (200 
mg l-1 + 250 mM NaCl) decreased these parameters by 
3 %, 14.2 %, 30.3 %, and 17.8 %, respectively, compared 
to control. These results proved the efficiency of 50 mg l-1 
(ZNPs50) for increasing the productivity of the soybean 
plant under high salinity levels (S = 250 mM NaCl).
3.5 PEARSON CORRELATION ANALYSIS
As shown in Figure 4, the Na+ was only positively 
corrected with Zn (r = 1.0*), total proteins (r = 0.89*), and 
total carbohydrates (r = 92*), while it was negatively cor-
rected with the rest of the studied characters, with dif-
ferent correlation coefficients. The pod weight was posi-
tively correlated with the weight of seeds/plant (r = 0.9*), 
K+ (r = 0.91*), K+/Na+ ratio (r = 0.88*), and the number of 
seeds/pod (r = 0.79 ns).
4 DISCUSSION
As sessile organisms, plants have adapted a variety 
of signal perception mechanisms as well as pathways to 
control molecular responses in order to respond effec-
tively to abiotic stress situations (Dudziak et al. 2019). 
Exposure of soybean seedlings to high salinity stress (250 
mM NaCl) imposed a significant depletion in K+ content 
and in K+/Na+ ratio by 68 % and 90 %, respectively, com-
pared to control. Similar findings were also found by Taf-
fouo et al. (2009) and Khan et al. (2017) in cowpea and 
soybean, respectively. Contrarily, the amount of Na+ was 
2.16-fold more than it was in the control plants. It is pos-
sible that high salinity promoted the uptake of Na+ due to 
its adverse effects on membrane integrity. In this regard, 
a similar conclusion was also made by Abdel Latef et al. 
(2017) on lupine plants.
According to reports, K+ is required for maintaining 
osmotic balance and is an essential co-factor for many 
enzymes. Therefore, K+ reduction negatively affects the 
growth and productivity of plants (Hauser and Horie, 
2010). The results of this study indicated that K+ content 
in soybean seedlings showed a highly significant reduc-
tion under acute salinity stress. However, treatment with 
ZNPs increased K+ by 1.67-fold and decreased Na+ by 
45 % compared to salt-stressed seedlings, which is com-
parable to the findings of Abdel Latef et al. (2017) on lu-
pine plants (Lupine termis L.). This is due to the fact that 
Zn+2 helps in maintaining the structural and functional 
integrity of root cell membranes and therefore controls 
the influx and efflux of Na+ across the plasma membranes 
(Rezaie and Abbasi, 2014).
The application of ZnO is associated with a remark-
able increase in K+ uptake from soil to roots (Weisany 
et al., 2012; Soliman et al., 2015). As a consequence of 
enhanced K+ uptake, plants treated with ZnO had great-
er K+/Na+ ratios than those under salinity stress alone. 
A high K+/Na+ ratio is often reported as a good indica-
tor of a high tolerance to salt stress conditions (Khan et 
al., 2017). Thus, applications of ZNPs could be a useful 
strategy for achieving increased macronutrient uptake by 
plants (Dimkpa and Bindraban, 2016), which is similar 
to what was observed in this study, where ZNPs50+S (50 
mg l-1+ 250 mM NaCl) treatment significantly increased 
the content of potassium by 1.67-fold compared to salt-
stressed seedlings and ameliorated the harmful effect of 
salinity stress.
Acta agriculturae Slovenica, 118/3 – 2022 9
Seed priming with ZNPs reduced expression of salinity tolerance genes in Glycine max L. and improved yield traits
Tr
ea
tm
en
ts
Po
d 
le
ng
th
 
(c
m
)
Po
d 
m
as
s 
(g
)
N
o.
 o
f p
od
s/
pl
an
t
M
. o
f p
od
s/
pl
an
t (
g)
N
o.
 o
f s
ee
ds
/ 
po
d
M
. o
f s
ee
ds
/
po
d 
(g
)
M
. o
f s
ee
ds
/ 
pl
an
t (
g)
M
. o
f 1
00
0 
se
ed
 (g
)
Pe
rc
en
ta
ge
 o
f 
m
at
ur
ity
 (%
)
Pr
od
uc
tiv
ity
 
in
de
x 
(g
/p
ot
)
Sa
lin
ity
 le
ve
l 
(0
 m
M
 N
aC
l)
C
on
tr
ol
6.
71
 ±
 0
.1
2 
e
2.
41
 ±
 0
.0
5 
d
5.
11
 ±
 0
.4
4 
d
8.
42
 ±
 0
.1
7 
d
5.
2 
± 
0.
18
 c
1.
38
 ±
 0
.1
2 
e
13
4.
72
 ±
 0
.2
2 
c
61
.1
2 
± 
4.
5 
h
36
 ±
 4
.1
 f
5.
42
 ±
 0
.1
7 
d
ZN
Ps
25
7.
34
 ±
 0
.2
6 
c
2.
92
 ±
 0
.0
5 
b
6.
16
 ±
 0
.4
4 
c
10
.6
6 
± 
0.
25
  b
5.
8 
± 
0.
16
 ab
1.
58
 ±
 0
.0
3 
d
15
4.
22
 ±
 0
.1
3 
b
92
.3
3 
± 
4.
4 
b
55
.8
 ±
 1
.6
 b
8.
37
 ±
 0
.1
1 
b
ZN
Ps
50
8.
68
 ±
 0
.1
7 
a
3.
08
 ±
 0
.1
3 
a
8.
32
 ±
 0
.6
8 
a
11
.7
8 
± 
0.
16
 a
6.
0 
± 
0.
27
 a
2.
04
 ±
 0
.1
2 
a
17
5.
8 
± 
0.
15
 a
94
.6
5 
± 
4.
8 
a
69
.9
 ±
 3
.2
 a
10
.4
8 
± 
0.
14
 a
ZN
Ps
10
0
7.
72
 ±
 0
.2
0 
b
2.
12
 ±
 0
.1
5 
e
4.
6 
± 
0.
93
 e
6.
30
 ±
 0
.3
2 
f
4.
6 
± 
0.
43
 d
1.
68
 ±
 0
.0
6 
c
12
4.
4 
± 
0.
20
 d
74
.2
4 
± 
4.
8 
d
29
.4
 ±
 3
.0
 g
4.
35
 ±
 0
.1
1 
g
ZN
Ps
20
0
4.
17
 ±
 0
.1
0 
i
2.
02
 ±
 0
.0
6 
f
4.
4 
± 
0.
44
 f
6.
66
 ±
 0
.4
1 
e
3.
8 
± 
0.
18
 e
1.
22
 ±
 0
.0
6 
f
11
7.
0 
± 
0.
12
 f
66
.3
4 
± 
4.
4 
f
27
.1
 ±
 3
.4
 h
4.
64
 ±
 0
.1
7 
f
Sa
lin
ity
 le
ve
l 
(S
=5
0 
m
M
 N
aC
l)
Sa
lin
ity
 (S
)
4.
51
 ±
 0
.1
1 
h
1.
31
 ±
 0
.1
 h
4.
43
 ±
 0
.4
4 
f
6.
62
 ±
 0
.1
1 
e
1.
2 
± 
0.
12
 g
0.
95
 ±
 0
.1
1 
h
10
6.
2 
± 
0.
42
 h
70
.1
2 
± 
0.
63
 e
41
.9
 ±
 2
.6
 c
3.
65
 ±
 0
.3
6 
h
ZN
Ps
25
+S
5.
22
 ±
 0
.1
2 
g
1.
87
 ±
 0
.1
g
3.
46
 ±
 0
.4
4 
h
8.
46
 ±
 0
.1
3 
d
4.
3 
± 
0.
15
 de
1.
01
 ±
 0
.0
2 
gh
11
3.
4 
± 
0.
20
 g
74
.2
3 
± 
0.
48
 d
39
.3
 ±
 3
.0
 d
5.
35
 ±
 0
.4
1 
e
ZN
Ps
50
+S
6.
77
 ±
 0
.1
5 
d
2.
43
 ±
 0
.1
1 
c
6.
32
 ±
 0
.6
8 
b
9.
78
 ±
 0
.1
6 
c
5.
3 
± 
0.
11
 bc
1.
9 
± 
0.
15
 b
12
2.
0 
± 
0.
72
 e
92
.2
2 
± 
0.
40
 c
55
.6
 ±
 3
.3
 b
7.
99
 ±
 0
.4
6 
c
ZN
Ps
10
0+
S
5.
55
 ±
 0
.1
1 
f
1.
15
 ±
 0
.1
2 
i
3.
6 
± 
0.
93
 g
4.
30
 ±
 0
.3
3 
h
4.
6 
± 
0.
13
 d
1.
08
 ±
 0
.0
3 
g
10
7.
0 
± 
0.
30
 h
66
.1
 ±
 1
.0
 g
37
.6
 ±
 3
.4
 e
4.
64
 ±
 0
.4
7 
f
ZN
Ps
20
0+
S
2.
11
 ±
 0
.1
3 
j
1.
11
 ±
 0
.1
2 
j
3.
4 
± 
0.
44
 h
5.
66
 ±
 0
.2
3 
g
2.
4 
± 
0.
18
 f
0.
80
 ±
 0
.0
4 
i
10
3.
0 
± 
0.
31
 i
60
.1
 ±
 0
.3
1 
i
29
.2
 ±
 0
.3
1 
g
3.
0 
± 
0.
31
 i
F-
va
lu
e
11
46
55
.2
14
56
8.
5
17
94
.3
80
91
.5
62
.9
25
7.
5
40
36
.3
25
26
64
.1
53
84
.5
15
56
7.
3
LS
D
 (0
.0
5)
0.
01
7
0.
01
7
0.
10
8
0.
07
7
0.
56
2
0.
07
7
1.
08
0.
07
7
0.
56
2
0.
05
6
Si
gn
ifi
ca
nc
e
*
Ta
bl
e 
3:
 E
ffe
ct
 o
f s
al
in
ity
 (S
 =
 2
50
 m
M
 N
aC
l) 
on
 th
e 
yi
el
d 
pa
ra
m
et
er
s o
f 9
0-
da
y 
ol
d 
so
yb
ea
n 
(c
v. 
G
iz
a 
11
1)
 p
la
nt
s g
ro
w
n 
in
 cl
ay
-s
an
dy
 so
il 
(2
:1
 w
/w
) a
fte
r s
oa
ki
ng
 o
f s
oy
be
an
 
se
ed
s i
n 
fo
ur
 d
iff
er
en
t c
on
ce
nt
ra
tio
ns
 o
f Z
nO
 n
an
op
ar
tic
le
s (
ZN
Ps
) (
ZN
Ps
25
 =
 2
5,
 Z
N
Ps
50
 =
 5
0,
 Z
N
Ps
10
0 
= 
10
0,
 a
nd
 Z
N
Ps
20
0 
= 
20
0 
m
g 
l-1
Va
lu
es
 ar
e t
he
 m
ea
n 
of
 th
re
e r
ep
lic
at
es
 ±
 S
D
. V
al
ue
s w
ith
in
 th
e s
am
e c
ol
um
n 
fo
r e
ac
h 
fa
ct
or
 d
es
ig
na
te
d 
by
 d
iff
er
en
t l
et
te
rs
 ar
e s
ig
ni
fic
an
t a
t p
 ≤
 0
.0
5,
 w
hi
le
 v
al
ue
s w
ith
 id
en
tic
al
 le
tte
rs
 ar
e n
on
-s
ig
ni
fic
an
t
Acta agriculturae Slovenica, 118/3 – 202210
R. M. GAAFAR et al.
Fig. 4: Correlogram based Pearson correlation analysis of 
Na+, K+, K+/Na+, Zn and yield parameters of 90-day old 
soybean (‘Giza 111’) plants grown in clay-sandy soil (2:1 w/w) 
after soaking of soybean seeds in different concentrations of 
ZNPs (25, 50, 100, and 200 mg l-1). On the right hand side of 
the correlogram, the legend color shows the correlation coef-
ficients and the corresponding colors. The positive correla-
tions are displayed in blue, while the negative correlations are 
shown in red. The color intensity and the size of the circle are 
proportional to the correlation coefficients
In this study, high salinity stress (250 mM NaCl) 
caused a highly significant increase in total soluble pro-
teins and carbohydrates content by 76 % and 75 %, re-
spectively, in salt-stressed soybean seedlings. Similar 
results were reported by Sadeghipour (2017) in Vigna 
unguiculata (L.), Karimi et al. (2019) in Vitis vinifera (L.), 
and Cardoso et al. (2019) in two varieties of cowpea. It 
is well known that osmotic stress induced by salt stress 
leads to the synthesis of proteins, which play an impor-
tant role in plant salt tolerance through cytosolic calcium 
signal. This signal activates the calcium sensor protein 
for activation of the protein kinase to regulate Na+/H+ 
antiporter in plasma membranes and tonoplasts, thus 
the osmo-sensory histidine kinase regulates osmotic ho-
meostasis and ROS scavenging (Chinnusamy et al., 2005; 
Abdel Latef et al., 2017). In addition, total soluble carbo-
hydrates are key osmolytes in the osmotic adjustment of 
all plants, ROS scavenging, and maintaining ion homeo-
stasis under salinity stress (Chen and Jiang, 2010) and 
have a direct relationship with physiological processes in 
plants (Tombesi et al., 2019).
In this study, treatment with ZNPs reduced total 
soluble proteins and total soluble carbohydrates content 
under salinity stress, and the highest reduction was re-
corded in the case of ZNPs50+S (50 mg l-1 + 250 mM Na 
Cl) with 43  % and 36  %, respectively. This result indi-
cates that ZNPs alleviated the harmful impacts of salin-
ity stress. The ZNPs treatment might cause an inhibition 
of oxidative stress, decreasing the content of Na+ in the 
shoot tissues (Haidera et al., 2019). Indeed, ZNPs have 
been shown to increase CO2 fixation, photosynthetic pig-
ments, photosynthetic efficiency, and plant growth res-
toration in response to salt stress (Soliman et al., 2015; 
Kasim et al., 2017; Mathur et al., 2019).
In addition, in this study, the expression levels of 
three key salt-tolerance related genes (GmCHX1, Gm-
PAP3, and GmSALT3) were determined under 250 mM 
of NaCl salt alone. The gene expression was increased 
for all three genes (GmCHX1, GmPAP3, and GmSALT3) 
by 1.9-, 3-, and 7.7-fold, respectively. Generally, stress 
results in changes in the cellular program that involve 
significant transcriptional alterations aimed at increas-
ing the chances of survival (Diédhiou et al., 2008). A 
study by Dang et al. (2014) proved that overexpression of 
GmPAP3 improved rice salt tolerance by increasing the 
ROS-scavenging ability and decreasing oxidative dam-
age. Similarly, a possible tolerance role of GmPAP3 under 
oxidative stress was demonstrated in soybean, indicating 
that the GmPAP3 gene expression is regulated by salinity, 
osmotic, and oxidative stresses (Liao et al., 2003; Li et al., 
2008b; Soleimani et al., 2017). It can be concluded that 
salinity induces the formation of ROS, which in turn ac-
tivates GmPAP3, leading to an increase in ROS degrada-
tion till reaching the proper level in the mitochondria, at 
which point the activity of the GmPAP3 gene is decreased 
(Francisca, 2005; Li et al., 2008a).
As mentioned above, the results of this study re-
vealed that GmCHX1 expression was increased under 
salinity stress by 1.9-fold, which is parallel to the results 
of Patil et al. (2016), who reported that salinity stress 
(200 mM NaCl) significantly induced the expression of 
the GmCHX1 gene in soybean, maintaining ion homeo-
stasis by lowering the Na+/K+ ratio. This result is also con-
sistent with data from this study, which showed a high 
reduction in K+/Na+ ratio by 90 % compared to control. 
Furthermore, the GmCHX1 gene was highly expressed in 
the leaves and roots of soybean seedlings in response to 
salinity stress (Do et al., 2016). It was reported that low 
Na+ accumulation in shoot tissues of soybean plants may 
be due to the powerful function of the GmCHX1 gene, 
which was highly expressed in salt-stressed soybean 
roots, forming Na+ exclusion proteins in root tissues and 
preventing Na+ entrance from soil to roots (Guan et al., 
2014; Qu et al., 2020). This function of the GmCHX1 
gene has been documented in other plant species such as 
cotton (Wu et al., 2004), rice (Ren et al., 2005), Arabidop-
sis (Møller et al., 2009) and wheat (Munns et al., 2012).
Acta agriculturae Slovenica, 118/3 – 2022 11
Seed priming with ZNPs reduced expression of salinity tolerance genes in Glycine max L. and improved yield traits
Moreover, the results of this study indicated that 
GmSALT3 gene expression was significantly increased 
in response to salinity stress by 7.7-fold in salt-stressed 
soybean seedlings. It was reported that the GmSALT3 
gene is the major salt tolerance gene in soybean belong-
ing to the cation/H+ exchanger (CHX) family (Patil et al., 
2016), which is mainly expressed in root cells associated 
with the phloem and xylem, leading to limiting the ac-
cumulation of sodium ions in leaves (Pardo et al., 2006), 
which improved the physiological and morphological 
parameters and ultimately increased soybean yield under 
saline conditions (Do et al., 2016). As GmSALT3 is local-
ized in the endoplasmic reticulum (ER), it plays a direct 
role in the retrieval of salt from the xylem (Padmanaban 
et al., 2007; Cao et al., 2019). It has been reported that 
GmSALT3 exerts a positive effect on soybean salt toler-
ance by exclusion of Na+ in plant shoots and therefore 
prevents the toxic accumulation of Na+ in photosynthetic 
tissues (Maathuis et al., 2014). Furthermore, Do et al. 
(2016) suggest that CHX1/GmSALT3 controls Na+, K+, 
and Cl- accumulation and may function as a cation-chlo-
ride co-transporter.
Application of nanoparticles alters the levels of ex-
pression of certain transcription factors, making it pos-
sible to modify plant tolerance to salinity stress (Yamagu-
chi et al., 2013). In particular, application of ZNPs could 
upregulate or downregulate the stress-tolerant genes de-
pending on their function by cascade reactions, thereby 
enhancing salt tolerance (Jonak et al., 2002). 
The results of this study showed that application of 
ZNPs in combination with salt-stress downregulated the 
expression of the three studied salinity-tolerant genes in 
soybean seedlings compared to salt-stressed ones. The 
expression of GmCHX1, GmPAP3, and GmSALT3 was 
decreased by 0.4, 0.46, and 1.56-fold, respectively, par-
ticularly with 25 mg l-1 ZNPs in combination with high 
salinity stress (250 mM NaCl). Interestingly, this finding 
confirms the ameliorative role of ZNPs in improving soy-
bean plant tolerance in response to salinity, which was 
reflected in enhancement effects on mineral uptake, total 
soluble proteins, total soluble carbohydrates, and yield 
characteristics. This finding is in accordance with that 
of Almutairi (2019) and Alharby et al. (2016) in tomato 
plants, where ZNPs imposed a positive response on plant 
metabolism under salt stress. It was reported that the dif-
ferential response of GmPAP3 expression in soybean to 
different ZNPs treatments under salinity stress could be 
as a result of reverted effects caused by NPs (Zhang et 
al., 2020) by excluding sodium ions from the roots, thus 
preventing the accumulation of toxic concentrations in 
the stem and leaves (Munns and Tester, 2008; Zhang et 
al., 2020).
5 CONCLUSIONS
The results of the present study indicated the im-
portance of Zn+2 in increasing soybean tolerance to salt 
stress. Soaking seeds of soybean cultivar Giza 111 in 
ZNPs at 50 mg l-1 reduced oxidative damage caused by 
salinity stress, downregulated salt-tolerant gene expres-
sion, and increased soybean plant yield under high salin-
ity stress (250 mM NaCl). Additionally, gene expression 
analysis of GmCHX1, GmPAP3, and GmSALT3 con-
firmed their roles in salt tolerance in the soybean cultivar 
Giza 111. Moreover, the significant downregulation of 
these genes under combined treatments (250 mM NaCl 
and ZNPs) suggests that soybean plants favor ZNPs as 
an antioxidant. Therefore, application of low concentra-
tions of ZNPs, particularly 50 mg l-1, is recommended 
before planting as a nano-fertilizer stimulator and could 
be a strategy to energize the growth and economic yield 
in plants growing in salinized soils, where it increases the 
adaptation capability of soybeans under such conditions.
6 REFERENCES
Abdel Latef, A. A., Alhmad, M. F. A. and Abdelfattah, K. E. 
(2017). The possible roles of priming with ZnO nanopar-
ticles in mitigation of salinity stress in lupine (Lupinus ter-
mis) plants. Journal of Plant Growth Regulation, 36, 60-70. 
https://doi.org/10.1007/s00344-016-9618-x
Abreu, I. A., Farinha, A. P., Negrão, S., Gonçalves, N., Fon-
seca, C., Rodrigues, M. and Oliveira, M. M. (2013). Cop-
ing with abiotic stress: proteome changes for crop im-
provement. Journal of Proteomics, 93, 145-168. https://doi.
org/10.1016/j.jprot.2013.07.014
Ahanger, M. A., Tomar, N. S., Tittal, M., Argal, S. and Agarwal, 
R. M. (2017). Plant growth under water/salt stress: ROS 
production; antioxidants and significance of added po-
tassium under such conditions. Physiology and Molecular 
Biology of Plants, 23(4), 731-744. https://doi.org/10.1007/
s12298-017-0462-7
Albert, I.L.; Nadassy, K.; Wodak, S.J. (1998). Analysis of zinc 
binding sites in protein crystal structures. Protein Science, 
7, 1700-1716. https://doi.org/10.1002/pro.5560070805
Alharby, H. F., Metwali, E. M., Fuller, M. P. and Aldhebiani, A. 
Y. (2016). The alteration of mRNA expression of SOD and 
GPX genes, and proteins in tomato (Lycopersicon esculen-
tum Mill.,) under stress of NaCl and/or ZnO nanoparticles. 
Saudi Journal of Biological Sciences, 23(6), 773-781. https://
doi.org/10.1016/j.sjbs.2016.04.012
Ali, E. A. and Mahmoud, A. M. (2013). Effect of foliar spray 
by different salicylic acid and zinc concentration on seed 
yield components of mungbean in sandy soil. Asian Jour-
nal of Crop Science, 5(1), 33-40. https://doi.org/10.3923/
ajcs.2013.33.40
Allen, S.G., Girmshaw, H.M., Parkinson, J.A. and Quarmby, C. 
Acta agriculturae Slovenica, 118/3 – 202212
R. M. GAAFAR et al.
(1974). Chemical analysis of ecological materials. Blackwell 
Scientific Publications, Oxford, London, 565.
Almutairi, Z. M. T. (2019). Plant molecular defense mecha-
nisms promoted by nanoparticles against environmental 
stress. International Journal of Agriculture and Biology, 21, 
259-270.
Bishop, O. N. (1983). Statistics in biology. Longman Penguin 
London, 56-63.
Bradford, M. M. (1976). A rapid and sensitive method for the 
quantitation of microgram quantities of protein utilizing 
the principle of protein-dye binding. Analytical Biochem-
istry, 72(1-2), 248-254. https://doi.org/10.1016/0003-
2697(76)90527-3
Cao, Y., Liang, X., Yin, P., Zhang, M. and Jiang, C. (2019). A 
domestication‐associated reduction in K+‐preferring HKT 
transporter activity underlies maize shoot K+ accumula-
tion and salt tolerance. New Phytologist, 222(1), 301-317. 
https://doi.org/10.1111/nph.15605
Cardoso, K. P. S., Conceição, S. S. and de Araújo Brito, A. E. 
(2019). Biochemical metabolism of two cultivars of cow-
pea treated with 24-Epibrassinolide and subjected to saline 
stress. Australian Journal of Crop Science, 13(3), 444. htt-
ps://doi.org/10.21475/ajcs.19.13.03.p1390
Chakraborty, K., Basak, N., Bhaduri, D., Ray, S., Vijayan, J., 
Chattopadhyay, K. and Sarkar, R. K. (2018). “Ionic basis of 
salt tolerance in plants: Nutritional homeostasis and oxida-
tive stress tolerance,” in Plant Nutrients and Abiotic Stress 
Tolerance, eds M. Hasanuzzaman, M. Fujita, H. Oku, K. Na-
har, and B. Hawrylak-Nowak (Berlin: Springer), 325-362. 
https://doi.org/10.1007/978-981-10-9044-8_14
Chanroj, S., Lu, Y., Padmanaban, S., Nanatani, K., Uozumi, N., 
Rao, R., et al.(2011) Plant-specific cation/H+ exchanger 17 
and its homologs are endomembrane K+ transporters with 
roles in protein sorting. Journal of Biological Chemistry, 286, 
33931-33941. https://doi.org/10.1074/jbc.M111.252650
Chen, C., Yu, Y., Ding, X., Liu, B., Duanmu, H., Zhu, D. and 
Zhu, Y. (2018). Genome-wide analysis and expression pro-
filing of PP2C clade D under saline and alkali stresses in 
wild soybean and Arabidopsis. Protoplasma, 255(2), 643-
654. https://doi.org/10.1007/s00709-017-1172-2
Chen, H. and Jiang, J. G. (2010). Osmotic adjustment and plant 
adaptation to environmental changes related to drought 
and salinity. Environmental Reviews, 18, 309-319. https://
doi.org/10.1139/A10-014
Chen, K. I., Erh, M. H., Su, N. W., Liu, W. H., Chou, C. C. and 
Cheng, K. C. (2012). Soy foods and soybean products: from 
traditional use to modern applications. Applied microbiol-
ogy and biotechnology, 96(1), 9-22. https://doi.org/10.1007/
s00253-012-4330-7
Chinnusamy, V., Jagendorf, A. and Zhu, J. K. (2005). Under-
standing and improving salt tolerance in plants. Crop 
Science, 45(2), 437-448. https://doi.org/10.2135/crop-
sci2005.0437
Choudhury, F. K., Rivero, R. M., Blumwald, E. and Mittler, R. 
(2017). Reactive oxygen species, abiotic stress and stress 
combination. The Plant Journal, 90(5), 856-867. https://doi.
org/10.1111/tpj.13299
Dang, Z. H., Qi, Q., Zhang, H. R., Li, H. Y., Wu, S. B. and 
Wang, Y. C. (2014). Identification of salt-stress-induced 
genes from the RNA-Sequencing data of Reaumuria 
trigyna using differential-display reverse transcription 
PCR. International Journal of Genomics, 1-7. https://doi.
org/10.1155/2014/381501
Dewdar, M., Abbas, M., Hassanin, A. and Aleem, H. (2018). Ef-
fect of nano micronutrients and nitrogen foliar applications 
on sugar beet (Beta vulgaris L.) of quantity and quality traits 
in marginal soils in Egypt. International Journal of Current 
Microbiology and Applied Sciences, 7(8), 4490-4498. https://
doi.org/10.20546/ijcmas.2018.708.475
Diédhiou, C. J., Popova, O. V., Dietz, K. J. and Golldack, D. 
(2008). The SNF1-type serine-threonine protein kinase 
SAPK4 regulates stress-responsive gene expression in 
rice. Biomed Central Plant Biology, 8(1), 1-13. https://doi.
org/10.1186/1471-2229-8-49
Dimkpa, C. O. and Bindraban, P. S. (2016). Fortification of mi-
cronutrients for efficient agronomic production: a review. 
Agronomy for Sustainable Development, 36(1), 7. https://
doi.org/10.1007/s13593-015-0346-6
Do, T. D., Chen, H., Hien, V. T. T., Hamwieh, A., Yamada, T., 
Sato, T. and Xu, D. (2016). Ncl synchronously regulates Na+, 
K+, and Cl− in soybean and greatly increases the grain yield 
in saline field conditions. Scientific Reports, 6, 1-10. https://
doi.org/10.1038/srep19147
Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. T. and 
Smith, F. (1956). Colorimetric method for determination of 
sugars and related substances. Analytical Chemistry, 28(3), 
350-356. https://doi.org/10.1021/ac60111a017
El-Mashad, A. A.  and Mohamed, H. I. (2012). Brassinolide al-
leviates salt stress and increases antioxidant activity of cow-
pea plants (Vigna sinensis). Protoplasma, 249(3), 625-635. 
https://doi.org/10.1007/s00709-011-0300-7
El-Shaer, A., Abdelfatah, M., Basuni, A. and Mosaad, M. (2018). 
Effect of KOH molarity and annealing temperature on ZnO 
nanostructure properties. Chinese Journal of Physics, 56(3), 
1001-1009. https://doi.org/10.1016/j.cjph.2018.03.015
Farhangi-Abriz, S. and Torabian, S. (2018). Nano-silicon alters 
antioxidant activities of soybean seedlings under salt toxic-
ity. Protoplasma, 255(3), 953-962. https://doi.org/10.1007/
s00709-017-1202-0
Fatima, U., Khan, M. F., Fatima, J., Shahab, U., Ahmad, S. and 
Yusuf, M. A. (2017). DNA damage, response, and repair 
in plants under genotoxic stress: stress signaling in plants. 
Genomics and Proteomics Perspective, 2, 151-171. https://
doi.org/10.1007/978-3-319-42183-4_7
Fraceto, L. F., Grillo, R., de Medeiros, G. A., Scognamiglio, V., 
Rea, G. and Bartolucci, C. (2016). Nanotechnology in ag-
riculture. Frontiers in Environmental Science, 4, 20. https://
doi.org/10.3389/fenvs.2016.00020
Francisca, L. W. Y. (2005). Characterization and Functional 
Studies of GmPAP3, a Novel Purple Acid Phosphatase-like 
Gene in Soybean Induced by NaCl Stress but not Phosphorus 
Deficiency (Doctoral dissertation, The Chinese University 
of Hong Kong).
Gaafar R.M., Diab R., Halawa M., Elshanshory A., El-Shaer 
A., Hamouda A. (2020). Role of zinc oxide nanoparticles 
in ameliorating salt tolerance in soybean. Egyptian Jour-
nal of Botany, 60(3), 733-747. https://doi.org/10.21608/
ejbo.2020.26415.1475
Acta agriculturae Slovenica, 118/3 – 2022 13
Seed priming with ZNPs reduced expression of salinity tolerance genes in Glycine max L. and improved yield traits
Guan, R., Chen, J., Jiang, J., Liu, G., Liu, Y., Tian, L. and Qiu, L. 
J. (2014). Mapping and validation of a dominant salt tol-
erance gene in the cultivated soybean (Glycine max) vari-
ety Tiefeng 8. The Crop Journal, 2(6), 358-365. https://doi.
org/10.1016/j.cj.2014.09.001
Haider, M. S., Jogaiah, S., Pervaiz, T., Yanxue, Z., Khan, N. and 
Fang, J. (2019). Physiological and transcriptional variations 
inducing complex adaptive mechanisms in grapevine by 
salt stress. Environmental and Experimental Botany, 162, 
455-467. https://doi.org/10.1016/j.envexpbot.2019.03.022
Haliloglu, K., Hosseinpour, A., Cinisli, K. T., Ozturk, H. I., 
Ozkan, G., Pour-Aboughadareh, A. and Poczai, P. (2020). 
Investigation of the protective roles of zinc oxide nanopar-
ticles and plant growth promoting bacteria on DNA dam-
age and methylation in tomato (Solanum lycopersicum L.) 
under salinity stress. Horticulture Environment and Biotech-
nology. 7(2), 23-41.
Han, G., Yuan, F., Guo, J., Zhang, Y., Sui, N. and Wang, B. (2019). 
AtSIZ1 improves salt tolerance by maintaining ionic home-
ostasis and osmotic balance in Arabidopsis. Plant Science, 
285, 55-67. https://doi.org/10.1016/j.plantsci.2019.05.002
Hauser, F. and Horie, T. (2010). A conserved primary salt toler-
ance mechanism mediated by HKT transporters: a mecha-
nism for sodium exclusion and maintenance of high K+/Na+ 
ratio in leaves during salinity stress. Plant, Cell and Envi-
ronment, 33(4), 552-565. https://doi.org/10.1111/j.1365-
3040.2009.02056.x
He, X. C., Lin, M., Li, F., Sha, B. Y., Xu, F., Qu, Z. G. and Wang, 
L. (2015). Advances in studies of nanoparticle-biomem-
brane interactions. Nanomedicine, 10(1), 121-141. https://
doi.org/10.2217/nnm.14.167
Hezaveh, T. A., Pourakbar, L., Rahmani, F. and Alipour, H. 
(2019). Interactive effects of salinity and ZnO nanoparti-
cles on physiological and molecular parameters of rapeseed 
(Brassica napus L.). Communications in Soil Science and 
Plant Analysis, 50(6), 698-715. https://doi.org/10.1080/001
03624.2019.1589481
Jonak, C., Ökrész, L., Bögre, L. and Hirt, H. (2002). Complexity, 
cross talk and integration of plant MAP kinase signaling. 
Current Opinion in Plant Biology, 5(5), 415-424. https://doi.
org/10.1016/S1369-5266(02)00285-6
Karimi, R., Mohammadparast, B. and Minazadeh, R. (2019). 
Phytochemical responses and antioxidant activity of potas-
sium-treated grapevines (Vitis vinifera L.) in salinity stress 
condition. Journal of Plant Process and Function, 245-260. 
Kasim, W. A. E. A., AboKassem, E. M. and Ragab, G. A. A. 
(2017). Ameliorative effect of yeast extract, IAA and green-
synthesized nano zinc oxide on the growth of Cu-stressed 
Vicia faba seedlings. Egyptian Journal of Botany, 1-16. htt-
ps://doi.org/10.21608/ejbo.2017.885.1065
Khan, M. S. A., Karim, M. A., Haque, M. M., Karim, A. J. M. 
S. and Mian, M. A. K. (2017). Effect of salt and water stress 
on gas exchange, dry matter production and K+/Na+ ions 
selectivity in soybean. Bangladesh Journal of Agricultural 
Research, 42(3), 487-501. https://doi.org/10.3329/bjar.
v42i3.34507
Landa, P., Prerostova, S., Petrova, S., Knirsch, V., Vankova, 
R. and Vanek, T. (2015). The transcriptomic response of 
Arabidopsis thaliana to zinc oxide: A comparison of the im-
pact of nanoparticle, bulk, and ionic zinc. Environmental 
Science and Technology, 49(24), 14537-14545. https://doi.
org/10.1021/acs.est.5b03330
Li, C., Fang, G., Li, J., Ai, L., Dong, B. and Zhao, X. (2008a). Ef-
fect of seed layer on structural properties of ZnO nanorod 
arrays grown by vapor-phase transport. The Journal of Phys-
ical Chemistry C, 112(4), 990-995. https://doi.org/10.1021/
jp077133s
Li, W. Y. F., Shao, G. and Lam, H. M. (2008b). Ectopic expres-
sion of GmPAP3 alleviates oxidative damage caused by sa-
linity and osmotic stresses. New Phytologist, 178(1), 80-91. 
https://doi.org/10.1111/j.1469-8137.2007.02356.x
Liao, H., Wong, F. L., Phang, T. H., Cheung, M. Y., Li, W. Y. F., 
Shao, G. and Lam, H. M. (2003). GmPAP3, a novel purple 
acid phosphatase-like gene in soybean induced by NaCl 
stress but not phosphorus deficiency. Gene, 318, 103-111. 
https://doi.org/10.1016/S0378-1119(03)00764-9
Liao, Y., Zou, H. F., Wang, H. W., Zhang, W. K., Ma, B., Zhang, J. 
S. and Chen, S. Y. (2008). Soybean GmMYB76, GmMYB92, 
and GmMYB177 genes confer stress tolerance in trans-
genic Arabidopsis plants. Cell Research, 18(10), 1047-1060. 
https://doi.org/10.1038/cr.2008.280
Linh TM, Mai NC, Hoe PT, Lien LQ, Ban NK, Hien LTT, Chau 
NH, Van NT. (2020). Metal-based nanoparticles enhance 
drought tolerance in soybean. Journal of Nanomaterials, 
1-13. https://doi.org/10.1155/2020/4056563
Liu, H. R., Sun, G. W., Dong, L. J., Yang, L. Q., Yu, S. N., Zhang, 
S. L. and Liu, J. F. (2017). Physiological and molecular re-
sponses to drought and salinity in soybean. The Plant Bi-
ology, 61(3), 557-564. https://doi.org/10.1007/s10535-017-
0703-1
Liu, X., Wang, F., Shi, Z., Tong R., Shi X. (2015).Bioavailability 
of Zn in ZnO nanoparticle-spiked soil and the implications 
to maize plants. Journal of Nanoparticle Research, 17, 175. 
https://doi.org/10.1007/s11051-015-2989-2
Livak, K. J. and Schmittgen, T. D. (2001). Analysis of relative 
gene expression data using real-time quantitative PCR 
and 2− ΔΔCT method. Methods, 25(4), 402-408. https://doi.
org/10.1006/meth.2001.1262
Maathuis, F. J., Ahmad, I. and Patishtan, J. (2014). Regulation of 
Na+ fluxes in plants. Frontiers in Plant Science,5, 1-9. https://
doi.org/10.3389/fpls.2014.00467
Mathur, S., Raikalal, P. and Jajoo, A. (2019). Physiological re-
sponses of wheat to environmental stresses. Wheat Pro-
duction in Changing Environments, 31-61. https://doi.
org/10.1007/978-981-13-6883-7_2
Mittler, R. (2002). Oxidative stress, antioxidants and stress tol-
erance. Trends in Plant Science, 7(9), 405-410. https://doi.
org/10.1016/S1360-1385(02)02312-9
Møller, I. S., Gilliham, M., Jha, D., Mayo, G. M., Roy, S. J., 
Coates, J. C. and Tester, M. (2009). Shoot Na+ exclusion and 
increased salinity tolerance engineered by cell type-specific 
alteration of Na+ transport in Arabidopsis. The Plant Cell, 
21(7), 2163-2178. https://doi.org/10.1105/tpc.108.064568
Munns, R. and Tester, M. (2008). Mechanisms of salinity toler-
ance. Annual Review of Plant Biology, 59, 651-8. https://doi.
org/10.1146/annurev.arplant.59.032607.092911
Munns, R., James, R. A., Xu, B., Athman, A., Conn, S. J., Jor-
dans, C. and Plett, D. (2012). Wheat grain yield on saline 
Acta agriculturae Slovenica, 118/3 – 202214
R. M. GAAFAR et al.
soils is improved by an ancestral Na+ transporter gene. Na-
ture Biotechnology, 30(4), 360-364. https://doi.org/10.1038/
nbt.2120
Naguib, M. I., Youssef, E. and Kinawy, M. M. (1968). Effect of 
kinetin on the nitrogen metabolism of radish plants. Bul-
letin of The Faculty of Science, Cairo University, 42, 13-22.
Olczak, M., Morawiecka, B. and Watorek, W. (2003). Plant pur-
ple acid phosphatases genes, structures and biological func-
tion. Acta Biochimica Polonica, 50, 1245-1256. https://doi.
org/10.18388/abp.2003_3648
Padmanaban, S., Chanroj, S., Kwak, J. M., Li, X., Ward, J. M. 
and Sze, H. (2007). Participation of endomembrane cation/
H+ exchanger AtCHX20 in osmoregulation of guard cells. 
Plant Physiology, 144(1), 82-93. https://doi.org/10.1104/
pp.106.092155
Pardo, J. M., Cubero, B., Leidi, E. O. and Quintero, F. J. (2006). 
Alkali cation exchangers: roles in cellular homeostasis and 
stress tolerance. Journal of Experimental Botany, 57(5), 
1181-1199. https://doi.org/10.1093/jxb/erj114
Patil, G., Do, T., Vuong, T. D., Valliyodan, B., Lee, J. D., Chaud-
hary, J. and Nguyen, H. T. (2016). Genomic-assisted haplo-
type analysis and the development of high-throughput SNP 
markers for salinity tolerance in soybean. Scientific Reports, 
6(1), 1-13. https://doi.org/10.1038/srep19199
Qadir, M., Quillérou, E., Nangia, V., Murtaza, G., Singh, M., 
Thomas, R. J. and Noble, A. D. (2014). Economics of salt‐
induced land degradation and restoration. Natural Resourc-
es Forum, 38(4), 282-295. https://doi.org/10.1111/1477-
8947.12054
Qi, X., Li, M. W., Xie, M., Liu, X., Ni, M., Shao, G., Song, C., 
and Yim, K.Y. (2014). Identification of a novel salt toler-
ance gene in wild soybean by whole-genome sequencing. 
Nature Communications, 5, 4340. https://doi.org/10.1038/
ncomms5340
Qu, Y., Guan, R., Bose, J., Henderson, S. W., Wege, S., Qiu, L. J. 
and Gilliham, M. (2020). GmSALT3 confers shoot Na+ and 
Cl− exclusion in soybean via two distinct processes. Fron-
tiers in Plant Science, 1-7.
Raeisi Sadati, S.Y., Godehkahriz S. J., Ebadi A. and Sedghi M. 
(2022). Zinc Oxide Nanoparticles Enhance Drought Toler-
ance in Wheat via Physio-Biochemical Changes and Stress 
Genes Expression. Iranian Journal of Biotechnology, 20(1), 
e3027. 
Ren, Z. H., Gao, J. P., Li, L. G., Cai, X. L., Huang, W., Chao, D. Y. 
and Lin, H. X. (2005). A rice quantitative trait locus for salt 
tolerance encodes a sodium transporter. Nature Genetics, 
37(10), 1141-1146. https://doi.org/10.1038/ng1643
Rezaei, M. and Abbasi, H. (2014). Foliar application of nano-
chelate and non-nanochelate of zinc on plant resistance 
physiological processes in cotton (Gossipium hirsutum L.). 
Iranian Journal of Plant Physiology. 4, 1137-1144.
Sadeghipour, O. (2017). Nitric oxide increases Pb tolerance by 
lowering Pb uptake and translocation as well as phytohor-
monal changes in cowpea (Vigna unguiculata (L.) Walp.). 
Sains Malaysiana, 46(2), 189-195. https://doi.org/10.17576/
jsm-2017-4602-02
Samei, M., Sarrafzadeh, M. H. and Faramarzi, M. A. (2019). The 
impact of morphology and size of zinc oxide nanoparticles 
on its toxicity to the freshwater microalga, Raphidocelis 
subcapitata. Environmental Science and Pollution Research, 
26(3), 2409-2420. https://doi.org/10.1007/s11356-018-
3787-z
Selvaraj, K., and Dananjeyan, B. (2016). Expression of Zinc 
Transporter Genes in Rice as Influenced by Zinc-Solubi-
lizing Enterobacter cloacae Strain ZSB14. Frontiers in Plant 
Science, 7, 446. https://doi.org/10.3389/fpls.2016.00446
Soleimani, V., Ahmadi, J., Sadeghzadeh, B. and Golkari, S. 
(2017). The expression of GmP5CS, GmPAP3, and Gm-
BZIP50 genes under saline condition in soybean us-
ing real-time PCR. Genetics, 49(2), 483-494. https://doi.
org/10.2298/GENSR1702483S
Soliman, A. S., El-feky, S. A. and Darwish, E. (2015). Alleviation 
of salt stress on Moringa peregrina using foliar application 
of nanofertilizers. Journal of Horticulture and Forestry, 7, 
36-47. https://doi.org/10.5897/JHF2014.0379
Taffouo, V. D., Kouamou, J. K., Ngalangue, L. M. T., Ndjeudji, 
B. A. N. and Akoa, A. (2009). Effects of salinity stress on 
growth, ions partitioning and yield of some cowpea (Vigna 
unguiculata L. Walp.) cultivars. International Journal of Bot-
any, 5(2), 135-143. https://doi.org/10.3923/ijb.2009.135.143
Tombesi, S., Cincera, I., Frioni, T., Ughini, V., Gatti, M., Pal-
liotti, A. and Poni, S. (2019). Relationship among night 
temperature, carbohydrate translocation and inhibition of 
grapevine leaf photosynthesis. Environmental and Experi-
mental Botany, 157, 293-298. https://doi.org/10.1016/j.en-
vexpbot.2018.10.023
Van Zanten, H. H., Mollenhorst, H., Klootwijk, C. W., Van Mid-
delaar, C. E. and De Boer, I. J. (2016). Global food supply: 
land use efficiency of livestock systems. The International 
Journal of Life Cycle Assessment, 21(5), 747-758. https://doi.
org/10.1007/s11367-015-0944-1
Vogel, A., Spener, F. and Krebs, B. (2001). Purple acid phos-
phatase. Handbook of metalloproteins. Chichester, 752-766.
Weisany, W., Sohrabi, Y., Heidari, G., Siosemardeh, A. and 
Ghassemi-Golezani, K. (2012). Changes in antioxidant en-
zymes activity and plant performance by salinity stress and 
zinc application in soybean (Glycine max L.). Plant Omics, 
5(2), 60.
Wu, C. A., Yang, G. D., Meng, Q. W. and Zheng, C. C. (2004). 
The cotton GhNHX1 gene encoding a novel putative ton-
oplast Na+/H+ antiporter plays an important role in salt 
stress. Plant and Cell Physiology, 45(5), 600-607. https://doi.
org/10.1093/pcp/pch071
Yamaguchi, T., Hamamoto, S. and Uozumi, N. (2013). Sodium 
transport system in plant cells. Frontiers in Plant Science, 4, 
410. https://doi.org/10.3389/fpls.2013.00410
Zhang, G., Chen, M., Li, L., Xu, Z., Chen, X., Guo, J. and Ma, 
Y. (2009). Overexpression of the soybean GmERF3 gene, 
an AP2/ERF type transcription factor for increased toler-
ances to salt, drought, and diseases in transgenic tobacco. 
Journal of Experimental Botany, 60(13), 3781-3796. https://
doi.org/10.1093/jxb/erp214
Zhang, X., Zörb, C. and Geilfus, C. M. (2020). The root as a 
sink for chloride under NaCl-salinity. Plant Physiology 
and Biochemistry, 155,161-168. https://doi.org/10.1016/j.
plaphy.2020.06.036