JOURNAL OF PLANT NUTRITION
https://doi.org/10.1080/01904167.2022.2067776

Improving wheat (Triticum aestivum L.) antioxidative defense
mechanisms against salinity stress by exogenous applicationof potassium silicate
Faride Feghhenabia, Hashem Hadia, Habib Khodaverdiloob
Genuchtenc,d , and Mohammad Pessaraklie, Martinus Th. vanaDepartment of Plant Production and Genetics, Urmia University, Urmia, Iran; bDepartment of Soil Science,
Urmia University, Urmia, Iran; cDepartment of Earth Sciences, Utrecht University, Utrecht, Netherlands;dCenter for Environmental Studies, CEA, S~ao Paulo State University, UNESP, Rio Claro, Brazil; eSchool of Plant
Sciences, College of Agriculture and Life Sciences, The University of Arizona, Tucson, AZ, USA

ABSTRACTARTICLE HISTORY

The primary objective of this study was to investigate the beneficial effects
of seed priming and foliar spray of potassium silicate on antioxidant activities
under different salinity levels, thereby potentially improving wheat growth.
Seeds were soaked into solutions containing potassium silicate (K2SiO3,
1.5 mM) for 6 h, while foliar spray with K2SiO3 (4 mM) was applied at the early
and the late stages of tillering. Lake Urmia water was used to prepare salinity
levels of 0, 3, 5, 8, 10, 12, and 14 dS m21. For such traits as anthocyanin,
catalase, ascorbate peroxidase, guaiacol peroxidase, and superoxide dismutase activity, an initial increase was observed at lower salinity levels; higher
salinities subsequently decreased these traits or they remained mostly constant. Salinity also increased phenol, malondialdehyde, hydrogen peroxide,and polyphenol oxidase, but decreased flavonoid, nitrate content, and nitrate
reductase activity. Seed priming and foliar spray provided effective
approaches to reduce reactive oxygen species (ROS) manifestation in wheatgrown under saline conditions. The improved antioxidant defense abilities byseed priming and foliar spray alleviated the oxidative damage of proteins
and lipids and improved nitrate content and nitrate reductase activity.

Received 19 July 2021
Accepted 3 November 2021
KEYWORDS

Antioxidants; foliar spray;
radical scavenging; salinity
stress; seed priming

Abbreviations: CAT: catalase; APX: ascorbate peroxidase; FW: Fresh weight;
GPX: guaiacol peroxidase; SOD: superoxide dismutase; PPO: polyphenol oxidase and; PAL: phenylalanine ammonia-lyase; POD: peroxidase; MDA:
malondialdehyde content; NR: nitrate reductase; ROS: reactive oxygen species.

Introduction
High salt concentrations are toxic to plants at all growth stages by causing oxidative damage suchas oxidation of certain macromolecules such as DNA, proteins, and lipids, due to the productionof reactive oxygen species (ROS), ultimately diminishing the growth, economic yield and/or quality of crops (Arora et al. 2008; Bela et al. 2015). To keep ROS levels tightly regulated and to minimize ROS-derived damages, different antioxidant systems have been evolved in aerobic
organisms, including the diverse super-family of peroxidase enzymes (Munns and Gilliham 2015;
Riasat et al. 2018). The primary scavenger in the detoxification of ROS in plants is superoxide
dismutase (SOD), which converts superoxide to hydrogen peroxide (H2O2) and molecular oxygen.
CONTACT H. Hadi

h.hadi@urmia.ac.ir

ß 2022 Taylor & Francis Group, LLC

Department of Plant Production and Genetics, Urmia University, Urmia, Iran.

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F. FEGHHENABI ET AL.

Ascorbate peroxidase (APX), the most important peroxidase in detoxifying H2O2, and catalase (CAT)can reduce H2O2 to water (Bhanuprakash and Yogeesha 2016). Phenylalanine ammonia-lyase (PAL)
is the main enzyme responsible for phenolic synthesis under a variety of stresses. Moreover, polyphenol oxidase (PPO) shares a role in phenolic and lignin synthesis: PPO may remove excess ROS
caused by stress, thus serving a detoxifying role during stress (Ali et al. 2006).
Other antioxidant compounds such as phenolic compounds, flavonoids, and anthocyanin, in concentration and structure-dependent ways, may contribute to antioxidant activity and the scavenging of free
radicals, lipid peroxidation inhibitors, and membrane stabilizers (Saleh and Madany 2015). Previous
reports on the antioxidative response of plants to salt stress indicate an increase in the activities of nonenzymatic and enzymatic antioxidant systems and lipid peroxidation (Tarchoune et al. 2010; Ouhibi
et al. 2014; Panuccio et al. 2014). In contrast, Noreen, Ashraf, and Akram (2010) reported that the imposition of varying levels of salinity could substantially decrease the activities of SOD, peroxidase (POD),
and CAT in turnip cultivars. Some wheat cultivars subjected to long-time exposure to salinity showed anincrease in certain antioxidant enzymes such as POD and SOD (Pessarakli, Haghighi, and Sheibanirad2015). Moreover, Shahzad et al. (2019) reported that salinity stress caused reductions in plant growth due
to increased lipid peroxidation and decreased total phenols in cowpea. Ultimately, the salt tolerance ofplants may be enhanced if ROS could be scavenged by an improved antioxidative defense system. It istherefore important to study antioxidative response during the growth and development of plants undersalinity stress (Turan and Tripathy 2013) and to identify the most effective and easiest techniques that
improve the antioxidative defense system.
Seed priming techniques have been widely used to improve plant growth under different abiotic
stresses. Past studies show that seed priming may increase SOD, POD, and CAT activities in barley (Aliet al. 2013), CAT activity in sunflower (Kibinza et al. 2011), and CAT, POD, and APX activities in wheat(Islam et al. 2015), while also enhancing anthocyanin and PPO; however, seed priming lowered phenol
and the reducing power in rice (Paul et al. 2017). Additionally, seed priming can activate signaling pathways in the early stages of growth, resulting in faster plant defense responses. The biological origin ofenhancing salinity tolerance in plants by seed priming is associated with increased antioxidant enzyme
activities, protection of cell membranes as a result of decreased lipid peroxidation, and improved leaf
photosynthetic rates. However, the exact molecular mechanisms behind priming are not completely
understood (Bhanuprakash and Yogeesha 2016). Moreover, foliar spray, as another beneficial technique,could enhance the activity of antioxidant enzymes (SOD, APX, CAT, and GSH-Px) and reduce the contents of H2O2 and malondialdehyde (MDA) in plants under salinity stress (Subramanyam, Laing, andVan Damme 2019). Bybordi (2012) also reported that foliar spray of K2SiO3 increased APX and nitrate
reductase (NR) activities in canola.
Although the beneficial effects of seed priming and foliar spray against abiotic stresses are evident
from previous studies, not much is known about molecular regulation and the physiological/biochemical mechanisms of seed priming and foliar spray-mediated stress tolerance. Therefore, the hypothesisof our study is that K2SiO3 may enforce the antioxidant system of wheat plants, which ultimately
could scavenge ROS accumulations and improve plant growth, thus inducing a defense against salinity stress. The main objectives of this study hence are to clarify the involvement of exogenous K2SiO3,
as a seed priming and foliar spray agent, in non-enzymatic and enzymatic antioxidant systems andlipid peroxidation processes in wheat subject to varying salinity levels, as well as elucidating the possible mechanisms of K2SiO3-enhanced salt tolerance in wheat.

Materials and methods
Seed materials
Healthy and uniform-sized seeds of wheat (Triticum aestivum L. Var. Chamran) were preparedfor our study by the Cereal Laboratory of the Agricultural Research Center of West Azerbaijan

JOURNAL OF PLANT NUTRITION

3

Province, Urmia, Iran. The mean 100-seed weight and seed moisture content were 3.57 g and 910%, respectively.

Seed priming, foliar spray, and salinity treatments
Prior to the experiments, seeds exhibited a 100% germination rate at 25 C, as assessed using Petridishes. For priming, the seeds were soaked for 6 h in a solution containing K2SiO3 (1.5 mM). This
agent and its specific concentration were selected following Feghhenabi et al. (2020). Onceprimed, the seeds were thoroughly rinsed with tap water and then distilled water, after which
they were dried back to their original moisture content at room conditions (about 25 C and 42%
relative humidity), as determined by the changes in seed weight. All seeds were subsequently surface-sterilized in a 1% sodium hypochlorite solution for 10 min to be used in the main
experiments.
By considering silicate at a concentration of 4 mM as the most effective in modulating theeffects of salinity (Bybordi 2015; Alzahrani et al. 2018), foliar spray at this concentration wasapplied to wheat leaves in two consecutive rounds. Tween 20 at 0.01% (v/v) was used furthermore
as a surfactant to increase foliar spray efficiency by increasing solution penetration. Half of thepots were randomly selected and sprayed separately so that they were not in contact with otherpots. The remaining pots without any treatment were considered as no foliar sprayed pots. Foliar
spray was applied at the early and the late stages of tillering (31 and 45 days after sowing). The
first round was applied before sunset and the second round just before dawn.

The open greenhouse pot experiments
Greenhouse-based pot experiments were undertaken within an open environment. Two-year average maximum and minimum temperatures were 24.86 and 10.50 C, the number of sunny hours
was 9.85 hours, and rainfall throughout the growing season was 18.8 mm. The growth medium
was a field soil collected from a 30-cm deep layer at an alfalfa farm. The field soil was classified
as a loam soil (sand, silt, and clay percentages were 46, 33, and 21%, respectively) that was relatively non saline (the electrical conductivity of the saturation paste was 1.1 dS m21), containing
18.5% equivalent CaCO3, with a pH value of 7.7. The field-collected soil was air-dried, ground,and passed through a 2-mm sieve. In order to maintain the growth of the seedling, a basal doseof urea, di-ammonium phosphate and potassium sulfate was added and mixed well into the soil.
A small amount of urea was also applied as a top dressing. A total of 16.4 kg of the field soil witha bulk density typical of the plow layer of a cultivated field was placed into each 18.5 L plastic
pot. The amount of soil in each pot was measured on a dry weight basis. Pot cultivation was carried out manually at 3 cm depth in an open greenhouse. Watering was performed immediately
after seed sowing using different levels of prepared salt water and continued until harvestingtime. The amount of water required per pot at 30% of field capacity was 2.4 liters. For the experiments we used salinity levels of 0, 3, 5, 8, 10, 12, and 14 dS m21, obtained by diluting saline
water from Lake Urmia, in the northwest of Iran.

Sampling materials
Fresh samples obtained from fully matured flag leaves of wheat at the early flowering stage werefrozen in liquid nitrogen and stored at 80 C. Liquid nitrogen was also used to powder the frozen samples. The whole process of triturating was done in a cold condition.

4

F. FEGHHENABI ET AL.

Measurement of non-enzymatic antioxidants content
Phenol content
Phenol was extracted according to methods outlined by Schaller, Brackhage, and Dudel (2012),
with some modifications. Samples (0.5 g each) were homogenized with 95% (v/v) ethanol. The
extracts were well mixed with an equal volume of Folin-Ciocalteu-reagent for 3 min, after which
10% Na2CO3 was added at half of their volume. After 1 hour’s standing, the absorbance of thereaction solution was read at 760 nm by spectrophotometry. The phenol content was expressed in
mg g21 fresh weight (FW).
Flavonoid content
Flavonoid content was measured employing the colorimetric method described by Zhishen,
Mengcheng, and Jianming (1999). 0.5 ml aliquots of extracts were added to a 10 ml volumetricflask containing 4.5 ml distilled water. 0.3 ml 5% sodium nitrite was added to each aliquot after
5 min, and then also 0.6 ml of 10% aluminum chloride. After 6 min, 2 ml of 1 M sodium hydroxide was added to the mixture followed by the addition of 2.1 ml distilled water. Absorbance wasrecorded at 510 nm, and the flavonoid content was expressed as mg g21 FW.
Anthocyanin content
Determination of anthocyanin contents was carried out using the method of Wagner (1979). To
calculate the content of anthocyanin, an extinction coefficient of 33,000 mol cm was used, withthe resulting anthocyanin content expressed as mg FW.
Measurement of enzymatic antioxidants activities
Fresh samples from fully mature flag leaves of the wheat crop were used for enzyme analysis.
One-gram leaves were homogenized in a 3 ml of 0.05 M sodium phosphate buffer (pH 7.8),
including 1 mM EDTA and 2% (w/v) Polyvinylpyrrolidone (PVP). The homogenates were centrifuged at 13,000  g for 20 min at 4 C. Supernatants were used for enzyme activity and protein
content assays. All steps in the preparation of the enzyme extracts were carried out at 4 C. The
extracts were used for measuring the enzyme activity of CAT, APX, guaiacol peroxidase (GPX),
and SOD.
Catalase, ascorbate peroxidase, guaiacol peroxidase, and superoxide dismutase activities
CAT, APX, and GPX activities were measured according to Cakmak and Marschner (1992),
Nakano and Asada (1981), and Egley et al. (1983), respectively. Activities of the extracts wereexpressed as mM g21 FWmin21. One unit of SOD activity was defined as the amount of enzyme
which caused 50% inhibition of photochemical reduction of nitro blue tetrazolium (NBT) at
560 nm according to the method of Giannopolitis and Ries (1977). SOD activities of the extracts
were expressed as % inhibition g21 FWmin21.
Polyphenol oxidase activity
PPO activity was assayed using the method of Singh et al. (1999). First, samples were homogenized using a sodium phosphate buffer (200 mM, pH 6.5), after which10mM pyrogallol was added
to each test tube. The test tubes subsequently were incubated for 15 min in a water bath at 30 C.
Enzyme extracts were then added to the test tubes. The absorbance of the resulting solutions
were recorded at 334 nm for 2 min with a spectrophotometer. PPO activity of the extract was
expressed as mM g21 FWmin21.

JOURNAL OF PLANT NUTRITION

5

Phenylalanine ammonia-lyase activity
Extractions and assays of PAL (EC 4.3.1.5) were carried out according to methods outlined by
Beaudoin-Eagan and Thorpe (1985). A standard curve of t-cinnamic acid was used; PAL activities
were expressed as nmol t-cinnamic acid mg21 protein min21.
Measurement of signaling molecule content and lipid peroxidation
The H2O2 content, as a signaling molecule, of the samples was determined following Velikova,
Yordanov, and Edreva (2000). Lipid peroxidation (MDA content) was determined using thiobarbituric acid reactions (Bailly et al. 1996), with slight modifications. The MDA content was calculated using: ½ðOD532  OD600 Þ=155  5
MDA content lmol g1 FW ¼
0:1

(1)

where ODx is the absorbance of the extract at a given wavelength x.
Measurement of nitrate and nitrate reductase activity
The nitrate content was obtained using methods described by Cataldo et al. (1975), and the NR
activity using the method of Klepper, Flesher, and Hageman (1971). Nitrate reductase activity was
expressed as lmol h21 FWg21.
Statistical analysis
A randomized complete block design was used with a factorial arrangement of treatments withthree replications. The first factor covered alleviating treatments by K2SiO3 and the Control (noseed priming and foliar spray), and the second was salinity (0, 3, 5, 8, 10, 12, and 14 dS m21).
The combined analysis of variance for two years (a four-way ANOVA, considering the year as arandom effect) was performed using the SAS 9.4 software. Consequently, multiple comparisonswere performed using the protected least significant difference (PLSD) test at the P < 0.05 level ofsignificance. The Excel 2017 software was used to create the figures and regression analyses. Twoyear data averages were used to draw the graphs.

Results and discussion
Results of the two-year data of the analysis of variance showed a significant effect of salinity on
all measured traits (Table 1). Furthermore, treatments (seed priming and foliar spray) had a significant (p < 0.05 or 0.01) effect on all measured traits (Table 1). For some traits (phenol, flavonoid, H2O2, nitrate content, SOD, NR, and PAL), a significant modification was observed with thechange of year. Furthermore, double interactions (treatment  salinity) were significant only for
GPX, NR, and MDA.
Non-enzymatic antioxidants content
Phenol and flavonoid content
The phenol and flavonoid content curves were fitted with a linear regression (Figure 1a and b).
The phenol content demonstrated an upward trend with salinity. Boudet (2007) documented thatthe increased expression and activity of the enzyme PAL, a key enzyme in producing phenylpropanoids, raised phenolic compounds. The phenolic compounds can be oxidized by PPO, leading

df
1
4
1
6
6
1
6
6
108

phen
51.34
4.63
12.67
61.25
0.68 ns
0.08 ns
0.32 ns
0.17 ns
1.29
12.9

flav
0.02
0.05
0.01
0.31
0.003 ns0.007 ns
0.0003 ns
0.0003 ns
0.003
14.0

antho (10 )117ns
3372ns
4795
5478
163ns
13ns
26ns
25ns
2014
17.9
CAT
0.14 ns
1.63
7.08
26.45
0.61 ns0.04 ns
0.02 ns0.01 ns
0.44
14.9

APX
0.003 ns
2.20
3.85
55.78
0.56 ns0.04 ns0.01 ns0.01 ns
0.43
17.5

GPX
1.62 ns
1.87
25.83
99.42
1.55
0.58 ns
1.51 ns0.26 ns
0.75
7.5

SOD
915.54
375.16
3092.10
14215.69
67.02 ns324.53
3.74 ns
3.03 ns106.75
6.2

PPO
2.29 ns11.64
199.47
18.09
3.26 ns
1.95 ns
0.13 ns
0.11 ns
2.95
10.6

Mean Squares
PAL
0.03
0.02
0.22
0.50
0.01 ns0.01 ns
0.0004 ns
0.0002 ns
0.006
16.49

H2O2
20.68
0.90 ns
3.00
60.06
0.43 ns
0.07 ns
0.52 ns0.01 ns
0.46
18.4

MDA
0.28 ns
2.05 ns
30.91
147.61
1.55
0.38 ns
0.31 ns
0.09 ns
0.32
11.5

Nit (10-8)
157
168
212
3032
13ns
1ns
12ns
2ns
11
19.2

NR
39.64
7.10
25.97
116.88
1.64
0.74 ns0.10 ns0.27 ns
0.66
7.11

Note: SOV, sources of variation; Y, year; B, block; T, treatments; S, salinity; phen, phenol; flav, flavonoid; antho, anthocyanin; CAT, catalase; APX, ascorbate peroxidase; GPX, guaiacol peroxidase; SOD, superoxide dismutase; PAL, phenylalanine ammonia-lyase; H2O2, hydrogen peroxide; MDA, malondialdehyde content; Nit, nitrate content. , and ns, denote significant at 5%,1%, and not significant, respectively.

SOV
Y
B
T
S
TS
YT
YS
YTS
Error
CV (%)-8

Table 1. Analysis of variance of the alleviating treatments’ effects on wheat response to salinity stress during two consecutive years (2017-2018).

6
F. FEGHHENABI ET AL.

JOURNAL OF PLANT NUTRITION

7

Figure 1. Effect of seed priming and foliar spray of potassium silicate on phenol (a), flavonoid (b) and anthocyanin (c) content
at different salinity levels. Bars represent two-year mean values with standard deviations.

to the formation of quinones, which are involved in the defense system of a plant. Moreover, bydonating electrons or hydrogen atoms, these compounds can break down the chain reactions thatform free radicals and react with peroxide precursors, thereby preventing the formation of peroxide. Phenolic compounds hence could be regarded as excellent oxygen radical scavengers (Ouhibi
et al. 2014). However, under salinity, the flavonoid content showed a trend opposite to that ofthe phenol content. The slope of the phenol content changes for control was such that the content of phenol became 1.8 times higher at EC ¼ 14 dS m21. Instead, the flavonoid content for thecontrol reduced by half at EC ¼ 14 dS m21. Reduced flavonoid content could be due to channeling to cause anthocyanin overproduction, thus being unavailable for energy required for the

8

F. FEGHHENABI ET AL.

synthesis of flavonoid due to the lower rate of photosynthesis (Chaparzadeh and Hosseinzad-behboud 2015). However, Taïbi et al. (2016) concluded that the high-yielding genotypes may offer
better protection against oxidative damage by increasing the amount of total flavonoids and theactivity of antioxidant enzymes under high salinity. Furthermore, Ouhibi et al. (2014) concluded
that salt stress induced the stimulation of the phenol content, flavonoids, and total antioxidant capacity.
Compared to using seed priming and foliar spray alone, our results showed that their combined use increased the content of flavonoids more effectively. Compared to the control, theapplication of seed priming and foliar spray had a positive effect on the regression slope andintercept of phenol content changes. Our results revealed the significant positive effect of seedpriming and foliar spray on phenol content with 11 and 5% increases (Figure 1a and Table 2).
The application of seed priming along with foliar spray increased phenol content by up to 15%(Table 2). These results are consistent with findings by Dallagnol et al. (2015) who observed significant higher contents of phenolic compounds in K2SiO3-treated plants. Induced accumulationof phenol content can control the production of H2O2, which consequently may play an important role in the oxidative stress tolerance of plants. Moreover, the regression slope of flavonoid
content changes became milder as a result of seed priming and foliar spray. The effects of seedpriming and foliar spray on the flavonoid content caused a decrease of almost 2%. Instead, seedpriming along with foliar spray statistically increased the flavonoid content by up to 5%(Table 2).
Anthocyanin content
The anthocyanin biosynthetic pathway is controlled by both external and internal factors. Its synthesis hence is inducible under stress, as our results showed by the effect of salinity. All data
related to the anthocyanin content were fitted based on quadratic regression (Figure 1c). The
behavior of the anthocyanin for controlling salinity was such that it increased (22%) up to EC ¼ 8
dS m21, and then declined (5%) with a further salinity increase. Moreover, seed priming alongwith foliar spray was most significant for anthocyanin, which increased by 13% (Table 2). On theother hand, seed priming alone had no considerable effect. Anthocyanin has antioxidative properties because of its ability as a hydrogen or electron donor, its ability of the polyphenol-derived
radicals to stabilize and delocalize unpaired electrons, and its ability to chelate transition metal
ions. Increasing anthocyanin hence will be useful in modulating the effects of salinity. However,overproduction of anthocyanin can be associated with decreasing chlorophyll content, biosynthesis of nitrogen-containing compounds, and the content of phenolic compounds such as flavonoids (Chaparzadeh and Hosseinzad-behboud 2015).
Enzymatic antioxidants activities
Catalase activity
According to the regression analysis, a quadratic function describing the CAT activity response toincreasing salinity fitted its behavior best (data not shown). The CAT activity for the control
intensified (2.92 times) up to EC ¼ 12 dS m21, after which it showed a downward trend. Seedpriming had no significant effect on CAT activity. However, CAT activity was enhanced approximately 18% by the combined treatments of seed priming and foliar spray, which statistically was
equal to the application of foliar spray alone (Table 2). In contrast, Bibordy (2016) found that the
CAT and SOD activities were suppressed by supplying silicon (2 or 4 g L1) to canola plants
grown under salinity conditions. The high levels of CAT activity indicated efficient scavenging of
H2O2 (Vaidyanathan et al. 2003). Indeed, foliar spray indirectly reduced H2O2.

8.20 ± 1.87c
9.09 ± 1.98ab
8.60 ± 1.87bc
9.45 ± 1.89a

phen

0.40 ± 0.15ab
0.39 ± 0.11b
0.38 ± 0.12b
0.42 ± 0.12a

flav
0.024 ± 0.004b
0.024 ± 0.004b
0.025 ± 0.004ab
0.027 ± 0.004aantho
4.10 ± 1.26b
4.10 ± 1.26b4.80 ± 1.02a
4.82 ± 1.12aCAT
3.43 ± 1.45b
3.51 ± 1.40b
3.97 ± 1.72a
4.01 ± 1.65a

APX
10.47 ± 1.97c
11.35 ± 2.24b
11.99 ± 1.92a
12.21 ± 2.28a

160.45 ± 27.29b
160.80 ± 24.01b
174.80 ± 24.64a
176.11 ± 23.39a

SOD

Mean of two-year data
GPX

PPO
14.25 ± 1.34b
14.46 ± 1.81b
18.14 ± 1.82a
18.12 ± 2.22a

PAL
0.39 ± 0.16b
0.53 ± 0.16a
0.42 ± 0.16b
0.53 ± 0.14a

Data represent means ± SD of three replicates and two years. Difference in letters indicates significant difference according to LSD’s test (P < 0.05). phen, phenol; flav, flavonoid; antho,
anthocyanin; CAT, catalase; APX, ascorbate peroxidase; GPX, guaiacol peroxidase; SOD, superoxide dismutase; PAL, phenylalanine ammonia-lyase.

control
Seed priming (1)Foliar spray (2)(1)(2)Treatments

Table 2. Influence of alleviating treatments on the antioxidative defense system of wheat.

JOURNAL OF PLANT NUTRITION
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F. FEGHHENABI ET AL.

Ascorbate peroxidase activity
The response of the APX activity to salinity is best demonstrated by quadratic regression(Figure 2a). APX activity increased up to EC ¼ 6 dS m21, and then reduced to its initial value
at EC ¼ 0 dS m21. Indeed, increasing the APX activity produced a more efficient antioxidant
system. Although the application of K2SiO3 in the form of seed priming had no significant
effect on APX activity, an increase in APX activity was observed with the application of
K2SiO3 in the form of foliar spray, even in combination with seed priming, specifically at
EC < 10 dS m21 (Figure 2a and Table 2). The increased APX activity may be the result of
stimulation to upregulate the expression levels of APX genes in salinity conditions (Sheteiwy
et al. 2021).

Guaiacol peroxidase activity
The effect of salinity on the GPX activity was significant. According to the regression analysis,
the relationship between GPX activity and salinity as affected by different treatments could be
best described using quadratic regressions (Figure 2b). GPX activity for the control increased up
to EC ¼ 8 dS m21, followed by a slight decrease to reaching 11.47 mM gFW21 min21 at EC ¼ 14
dS m21. The positive effect of seed priming and foliar spray on GPX activity was significant at all
salinity levels. Foliar spray was more efficient than priming at all levels of salinity. Moreover, thecombination of seed priming and foliar spray had a synergistic effect at EC > 7 dS m21 (Figure
2b and Table 2). Both forms of K2SiO3 had a positive effect on GPX activity at all salinity levels(Figure 2b and Table 2). Increasing GPX activity along with glutathione reductase (GR) will help
maintain a balanced state of reduced glutathione/oxidized glutathione (GSH/GSSG). On the other
hand, improving the ascorbic acid–glutathione cycle and the related activity of enzymes throughK2SiO3 significantly increased the activity of antioxidant enzymes, such as APX and GPX(Pinedo-Guerrero et al. 2020).

Superoxide dismutase activity
The effects of salinity on SOD activity were statistically significant (Figure 2c and Table 2). The
response of the SOD activity to salinity was best demonstrated by quadratic regression. SOD
activity increased linearly (51%) up to EC ¼ 10 dS m21, but then remained mostly constantuntil EC ¼ 14 dS m21. SOD converts superoxide radicles (O2.-) into H2O2. Superoxide radicals
play a crucial role in signaling NaCl-induced up-regulation of antioxidative enzymes. Kovacik
et al. (2009) showed that salinity exposure enhanced superoxide radical accumulation in chamomile plants and that the foliar spray strategy increased SOD activity; superoxide radicles consequently declined. Furthermore, since SOD is involved in the control of H2O2 and hydroxyl
radicals, which are highly reactive causing major damage to protein membranes, part of its
activity may be used to regulate H2O2 and reduce hydroxyl radicals (Bhanuprakash andYogeesha 2016).
Our results show that application of K2SiO3 in the form of foliar spray alone as well as incombination with seed priming, enhanced SOD activity equally. SOD activity increased up to 9%
using these two treatments (Figure 2c and Table 2). Furthermore, seed priming had no significant
effect on SOD activity. In contrast, Soylemezoglu et al. (2009) reported that silicon lowered SOD
and CAT activity, but increased APX in Vitis vinifera L. grown in saline conditions. However, theeffects of silicon on antioxidant enzyme activities have been reported previously. Under salt stress,
silicon significantly increased the activity of antioxidant enzymes such as SOD, GPX, CAT, andAPX, suggesting that silicon may be involved in metabolic or physiological activities under salinity stress (e.g., Manivannan et al. 2016).

JOURNAL OF PLANT NUTRITION

11

Figure 2. Effect of seed priming and foliar spray of potassium silicate on the activity of enzymatic antioxidants at different salinity levels. Bars represent two-year mean values with standard deviations.

12

F. FEGHHENABI ET AL.

Table 3. Influence of alleviating treatments on H2O2, MDA, Nit, and NR of wheat.
Mean of two-year data
Treatments
control
Seed priming
Foliar spray
Seed primingfoliar spray

H2O2

MDA

Nit

NR

3.89 ± 1.81a
3.96 ± 1.79a
3.48 ± 1.44b
3.44 ± 1.51b

5.98 ± 2.86a
4.36 ± 2.20c
5.23 ± 2.53b
4.10 ± 2.01d

0.0014 ± 0.001c
0.0018 ± 0.001b
0.0017 ± 0.001b
0.0020 ± 0.001a

10.37 ± 2.59c
11.39 ± 2.17b
12.04 ± 2.31a12.03 ± 2.16a

Data represent means ± SD of three replicates and two years. Difference in letters indicates significant difference according to
LSD’s test (P < 0.05). H2O2, hydrogen peroxide; MDA, malondialdehyde content; Nit, nitrate content.

Polyphenol oxidase activity
PPO has been shown be involved in phenolics oxidation processes in plants, with a correlation
existing between its activity and the phenolics level, while external factors may also affect its
activity (Dallagnol et al. 2015). While PPO plays a role in defense responses to biotic stresses,
PPO levels are known to vary in response to different forms of abiotic stresses. For instance, ourresults show that salinity has a significant influence on PPO activity; quadratic regression best
described the trend of PPO activity over various salinity levels (Figure 2d). The trends of PPO
activity for the control and seed priming were such that its activity at EC ¼ 14 dS m1 was on
average 14% higher than its initial activity at EC ¼ 0 dS m1, whereas for foliar spray and the
combination of seed priming and foliar spray, this increase was on average 21%. The effect ofseed priming on increasing PPO activity was not comparable to that of foliar spray nor the combination of foliar spray and seed priming (Figure 2d and Table 2). Enhanced PPO activity under
the influence of salinity or K2SiO3 may not be a guarantee for increasing salinity tolerance.
Taranto et al. (2017) claimed that the genotypes in which PPO activity was suppressed by water
stress resulted in more tolerance to stress.
Phenylalanine ammonia-lyase activity
Salinity significantly influenced PAL activity. We found a linear relationship between the increase
in PAL activity and salinity levels for both the control and foliar spray (data not shown). In contrast, the response of PAL activity to salinity under seed priming and the combination of seedpriming and foliar spray was best described using quadratic regression. As salinity increased, PAL
activity for the control increased to a maximum of 0.59 nmol cinamate gFW21 at EC ¼ 14 dS
m21 (data not shown). Despite the significant effects of K2SiO3 in the form of foliar spray onmost traits, it had no effect on PAL activity (Table 2). In contrast, K2SiO3 in the form of seedpriming alone or along with foliar spray had a significant effect on increasing PAL activity.
Overall, these two treatments increased PAL activity by 36% (Table 2). Since phenylalanine is a
substrate for PAL (Kovacik et al. 2009), regarding the increase of PAL, it seems that these twotreatments both increased salinity-reduced phenylalanine. Conversion of phenylalanine into transcinamic acid is a step required to channel the carbon input from primary metabolism to secondary phenylpropanoid metabolism. Although carbon consumption in this way can eventually leadto increased salinity tolerance, it will also reduce yield (Pinedo-Guerrero et al. 2020).
Signaling molecule content (H2O2 content)
H2O2 was influenced by both salinity and foliar spray, but not by seed priming (Table 3).
According to the regression analysis, H2O2 response to different salinity levels under various
treatments could be described better using quadratic regression (Figure 3a). As salinity levels
became more destructive, the H2O2 content increased for all treatments. However, foliar sprayand the combination of seed priming and foliar spray lowered H2O2 on average 11% at all salinity levels, especially at EC > 5 dS m21 (Figure 3a and Table 3). High levels of H2O2 may

JOURNAL OF PLANT NUTRITION

13

Figure 3. Effect of seed priming and foliar spray of potassium silicate on hydrogen peroxide (a) and malondialdehyde content(b) at different salinity levels. Bars represent two-year mean values with standard deviations.

accelerate processes like Haber–Weiss reaction, resulting in the formation of hydroxyl radicals
that could cause lipid peroxidation and increase MDA (Khan et al. 2019). This is reflected by thegreater extent of lipid peroxidation at the higher levels of salinity. These results hence suggestthat at moderate and high levels of salinity stress, wheat suffers mainly from the toxic effects of
H2O2 (due to a lack of efficient H2O2 detoxification mechanisms) as also reported by
Vaidyanathan et al. (2003). The H2O2 increase was alleviated by foliar spray (Table 3). Likewise,
Li et al. (2015) reported increased H2O2 concentrations in salt-stressed tomato seedlings grown ina sand culture; however, silicon application reversed this stress-induced change.
Lipid peroxidation content (MDA content)
Salinity had a significant effect on MDA content, which increased in all treatments over the entire
salinity range (Figure 3b). The MDA content at all salinity levels was the highest for the control,with the differences becoming greater at the higher salinity levels. Compared to the control, thealleviating treatments not only reduced the content of MDA, but also moderated the slope of
MDA content versus salinity. Furthermore, application of seed priming along with foliar spray
showed the greatest effect (31%) on reducing the MDA content. Among the two treatments ofseed priming and foliar spray, seed priming was more effective in decreasing MDA. The MDA
contents obtained with seed priming and foliar spray were 73 and 87% of the control, respectively(Figure 3b and Table 3). Abdel Latef and Tran (2016) also found that seed priming with silicon

14

F. FEGHHENABI ET AL.

Figure 4. Changes in nitrate content (a) and nitrate reductase (b) activities affected by seed priming and foliar spray of potassium silicate at different salinity levels. Bars represent two-year mean values with standard deviations.

improved the growth of plants under stress, which was accompanied by elevated activities of
SOD, CAT, and POD enzymes and lower contents of MDA in stressed plants, relative to plants
subjected to only alkaline stress.
Nitrate content
The response curve of nitrate was fitted by quadratic regression (Figure 4a). The nitrate content
of the control was at its maximum (0.003 mg FW21) at EC ¼ 0 dS m21, and decreased withincreasing salinity to 0.0004 mg FW21. However, seed priming and foliar spray positively influenced the nitrate content. The average increase in nitrate content at all salinity levels for these
two treatments was 25% (Table 3). Furthermore, seed priming along with foliar spray had greatest
effect on enhancing the nitrate content (43%).
Nitrate reductase activity
Increasing the salinity caused the NR activity to drop. The reduction in NR activity of leaves may
be due to an imbalance of salts in cells, enzyme degradation/inactivation, and a reduction in gene
expression and NR protein synthesis (Souza et al. 2016). There is well-documented information
available concerning lower nitrate reductase activity depending on nitrate content. As Botella
et al. (1993) stated, salinity significantly decreased NR activity, with the effects depending on
nitrate content, alteration in nitrate uptake, and the NO3-/Cl- ratio. In addition to NR, SOD and
POD activities could be reduced by nitrogen deficiency (Pessarakli, Haghighi, and Sheibanirad

JOURNAL OF PLANT NUTRITION

152015). Our results showed that the response of NR activity to salinity was best described using
quadratic regression (Figure 4b). The NR activity changes for the control were affected even by
the low salinity and decreased. The changes in NR activity against salinity were affected by seedpriming and foliar spray. The changes of NR activity for the alleviating treatments were milder
up to EC < 5 dS m1, after which they were declined by an average of 40% up to EC ¼ 14 dS
m1. The average NR activity of the Control was 10.4 lmol h21 gFW21, being 91% of that ofseed priming (Figure 4b and Table 3). The highest NR activity was obtained for foliar spray andthe combined seed priming and foliar spray treatment. Overall, the rate of decrease, and the value
and starting point of the reductions, were all affected by the alleviating treatments (Figure 4b andTable 3). Angrish, Kumar, and Datta (2001) reported that the positive effects on salinity stresswere due to the enhanced N status and NR activity by seed priming of wheat seeds. Our results
similarly showed the positive effect of seed priming and foliar spray on NR activity and nitratecontent, which may ultimately help to improve conditions for maintaining wheat yield in saline
environments.
Overall, changes in the activity of antioxidant enzymes and the content of antioxidants
against salinity indicated a significant effect of salinity and increased oxidative stress. One may
conclude that ROS production is enhanced in plants in response to salinity stress. Plants withhigh concentrations of antioxidant enzymes tolerate much better the oxidative damage causedby salinity stress (Pakar et al. 2016). The result of antioxidants changes was reflected in traits
such as nitrate content, NR activity, and MDA content. Increases in scavenging of H2O2
occurred as a result of increased antioxidant enzymes activities (CAT, APX, GPX, and SOD).
Indeed, the free radicals are inhibited by processes between these enzymes (as mentioned in the
introduction). Phenolic compounds such as phenol and anthocyanin were enhanced by increasing the activity of the PAL enzyme and were converted to o-quinones by the PPO enzyme.
These enzymes, along with the content of phenolic compounds, helped to tolerate salinity. Allantioxidants triggered by seed priming and foliar spray were effective in controlling ROS, which
is very important for increasing tolerance to salinity stress. As shown by our findings, silicon
can reduce the oxidative damage by increasing antioxidant enzymes activities, maintaining optimal membrane fluidity, plasma membrane Hþ-ATPase, and reducing the generation of ROS inmany plant species under salinity conditions (Kim et al. 2014; Li et al. 2015; Muneer and Jeong2015). Furthermore, Alzahrani et al. (2018) concluded that enzyme activity (e.g., of SOD, CAT,and POD) improved by applying silicon, which may have been related to elevated antioxidants
and osmoprotectants (e.g., proline, soluble sugars, ascorbic acid, and glutathione) contents,
probably by providing antioxidant defenses against abiotic stress in wheat. Maghsoudi et al.
(2019) also suggested the active role of silicon in plant physiological activities, including of theoxidative defense system and the water status.

Conclusions
Our results show that seed priming and foliar spray with K2SiO3 are effective approaches toreduce reactive oxygen species (ROS) manifestation in wheat grown under saline conditions, particularly at relatively low salinity levels. Indeed, under different salinity levels, seed priming andfoliar application showed varying responses to ROS scavenging by activation of the endogenous
defense systems of plants. The improved antioxidant defense abilities by seed priming and foliar
spray, which were linked to elevated non-enzymatic antioxidants (e.g., phenol and anthocyanin),and enzymatic antioxidants, alleviated the oxidative damage of proteins and lipids and increased
nitrate content and nitrate reductase activities, mostly due to the more pronounced effect of combined application of both alleviators.

16

F. FEGHHENABI ET AL.

Disclosure statement
The authors declare no conflict of interest.

Funding
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit
sectors. It was funded by Urmia University, Urmia, Iran.

ORCID
Habib Khodaverdiloo
http://orcid.org/0000-0003-1436-174X
http://orcid.org/0000-0003-1654-8858
Martinus Th. van Genuchten
Mohammad Pessarakli
http://orcid.org/0000-0002-7662-2258

Author contributions
Faride Feghhenabi, Hashem Hadi, and Habib Khodaverdiloo initiated, designed, and conducted the research.
Faride Feghhenabi also prepared the manuscript. Hashem Hadi, Habib Khodaverdiloo, Martinus van Genuchten,
and Mohammad Pessarakli contributed to manuscript preparation and its improvement.

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