Lan CY, Lin KH, Huang WD, Chen CC (2019). Protective Effects of Selenium on Wheat Seedlings under Salt Stress. Agronomy, 9(6): 272.
Abstract
Wheat is a staple food worldwide, but its productivity is reduced by salt stress. In this
study, the mitigative effects of 22 μM selenium (Se) on seedlings of the wheat (Triticum
aestivum L.) cultivar Taichung SEL. 2 were investigated under different salt stress levels
(0, 100, 200, 300, and 400 mM NaCl). Results of the antioxidative capacity showed that
catalase (CAT) activity, non-enzymatic antioxidants (total phenols, total flavonoids, and
anthocyanins), 1,1-Diphenyl-2-Picryl-Hydrazyl (DPPH) radical-scavenging activity, and
the reducing power of Se-treated seedlings were enhanced under saline conditions. The
more-stabilized chlorophyll fluorescence parameters (maximal quantum yield of
photosystem II (Fv/Fm), minimal chlorophyll fluorescence (F0), effective quantum yield
of photosystem II (ΦPSII), quantum yield of regulated energy dissipation of photosystem
II (Y(NPQ)), and quantum yield of non-regulated energy dissipation of photosystem II
(Y(NO)) and the less-extensive degradation of photosynthetic pigments (total chlorophyll
and carotenoids) in Se-treated seedlings were also observed under salt stress. The
elongation of shoots and roots of Se-treated seedling was also preserved under salt stress.
Protection of these physiological traits in Se-treated seedlings might have contributed to
stable growth observed under salt stress. The present study showed the protective effect
of Se on the growth and physiological traits of wheat seedlings under salt stress.
Keywords: wheat; selenium; salt stress; enzymatic and non-enzymatic activities;
antioxidant activity
Introduction
Wheat (Triticum stivum L.), the third most important primary cereal with more than
600 million tons of global production, preceded only by corn and rice (Asseng et al.,
2011), provides the main source of carbohydrates for 35%~40% of the world’s population
(Chen et al., 2017). However, wheat yields are markedly reduced in saline soils, due to
improper fertilization that causes osmotic and drought stresses (Egamberdieva, 2009).
Therefore, it is imperative to research the effects of salt stress on wheat’s physiology.
Selenium (Se) is considered a beneficial element to plants (Pilon-Smits et al., 2009).
Previous studies indicated that Se can delay senescence (Djanaguiraman et al., 2005; Xue
et. al., 2001) and promote the vegetative and reproductive growth of plants (Hajiboland
et al., 2012). Studies reported that Se mitigated disadvantageous phenomena caused by
various stressful situations, such as heat (Iqbal et al., 2015; Shang et al., 2005;
Djanaguiraman et al., 2010), cold (Chu et al., 2010), heavy metals (Khan et al., 2015;
Mroczek-Zdyrska and Wójcik, 2012), ultraviolet (UV)-B (Breznik et al., 2005; Yao et al.,
2010; Yao et al., 2011; Xue and Hartikainen, 2000), drought (Xiaoqin et al., 2009; Nawaz
et al., 2015), and salt stress (Mona et al., 2017; Diao et al., 2014). According to previous
research, possible protective mechanisms of Se in plants against stresses include its
enhancement of antioxidant enzyme activities (peroxidase (POD), catalase (CAT), etc.)
and increasing antioxidant compounds (anthocyanins, flavonoids, phenolic compounds,
etc.), and these antioxidant systems thus reduce stress-induced oxidative situations (Chu
et al., 2010). In addition, Se can improve plant photosynthesis by increasing the efficiency
of photosystem II (PSII), enhancing chlorophyll fluorescence, and reducing the
degradation of chlorophyll concentration (Chu et al., 2010). Moreover, Se contributes to
water status regulation in plants by promoting the water uptake efficiency from roots and
reducing water loss from tissues (Kuznetsov et al., 2003). In addition, Se stimulates plant
growth by promoting the integrity of the membrane system which results in root and shoot
elongation and biomass accumulation (Djanaguiraman et al., 2005; Xiaoqin et al., 2009;
Nowak, 2009; Hu et al., 2013 ; Sun et al., 2010). Research by
Hawrylak-Nowak (2009) reported that Se particularly supported root system development.
Salinization degrades land and is serious environmental stress that limits wheat
production (Egamberdieva, 2009). There are two phases when plants encounter salt stress.
Initially within a few minutes, plants are subjected to osmotic changes which reduce the
roots’ ability to absorb water. Gradually, the toxicity of NaCl inhibits ion transport and
results in leaf senescence and reduced photosynthesis (Läuchli and Grattan, 2007). Few
studies have evaluated the mitigation of salt stress in wheat by supplying Se. A study by
Yigit et al. (2012) reported that organic Se can increase germination percentages and
enhance antioxidant activities in wheat exposed to salt stress. Mona et al. (2017)
evaluated the effect of Se on wheat under salt stress, and their results suggested that
supplying Se led to increased germination percentages and growth, and also an
enhancement of total soluble sugars. A study by Sattar et al. (2017) indicated that foliar
application of Se improved the growth and physiological status of wheat seedlings under
stressed conditions.
All of the above results provide evidence that supplying Se can alleviate the
disadvantageous effects of salt stress on wheat, but the physiological mechanisms which
Se trigger need to be further evaluated. In this study, Se played a role as a bioregulator to
remediate the physiological status in wheat seedlings cultivated under salt stress. The
hypothesis was a hydroponic solution with Se application might improve the growth and
physiological performance of wheat subjected to salt stress.
Materials and Methods
Plant and Growth Conditions
Wheat (Triticum aestivum L.) cultivar Taichung SEL. 2 (TCS2), one of the most
widely cultivated wheat cultivars in Taiwan, was used in this study. Seeds used in the
present study were obtained from the Department of Agronomy, National Taiwan
University (Taipei, Taiwan). The seeds were sterilized with 1% hydrogen peroxide for 5
min, washed with distilled water, and germinated in Petri dishes on wetted filter paper at
25 °C in the dark. After 24 h of incubation, uniformly germinated seeds were selected and
cultivated in 150 mL beakers containing complete Hoagland’s nutrient solution
(PhytoTech, Lenexa, KS, USA), which was replaced every 3 days. Hydroponically
cultivated wheat seedlings were raised in growth chambers with fluorescent lamp lighting
at 25 and 20 °C during the day and night, respectively, under a 12-h photoperiod. The
photosynthetic photon flux density (PPFD) was uniformly set to 300 μmol m−2 s−1.
Experimental Treatments
Se was added in the form of Sodium selenite (Na2SeO3, Sigma-Aldrich Chemie
GmbH, Taufk irchen, BY, Germany) to the nutrient solution (pH = 4.6) with the treated
concentration of 22 μM once the germinated seeds were cultivated in 150 mL beakers.
No treatment consisted of the nutrient solution without Se supplementation.
Hydroponically grown seedlings that had reached stage Z1.0 (Zadoks et al., 1974) on day
6 were treated with sodium chloride (NaCl) at concentrations of 0, 100, 200, 300, and 400
mM for 7 days. The experiment was independently performed three times for a
randomized design of growth conditions.
Growth Analysis
Shoot height and root length were measured with a ruler before the measurements of
chlorophyll (Chl) fluorescence (ChlF) and sample collection.
Measurements of ChlF
Fluorescence parameters in seedling leaves were determined after Se and salt
treatments. ChlF was measured in the middle portion of the first leaf of each seedling
taken at ambient temperature with Chl fluorometer imaging-PAM (Walz, Effeltrich,
Germany). Actinic light and saturating light intensities were set to 185 and 7200 μmol
m−2s−1 of photosynthetically active radiation (PAR), respectively. The minimal (F0) and
maximal (Fm) ChlF, the maximum quantum yield of PSII (Fv/Fm), the effective quantum
yield of PSII (ΦPSII), the quantum yield of regulated energy dissipation of PSII (Y(NPQ))
and the quantum yield of non-regulated energy dissipation of PSII (Y(NO)) were
measured and calculated according to previously described methods (Van and Snel, 1990;
Kramer et al., 2004).
Measurement of Catalase (CAT) and Ascorbate Peroxidase (APX) Activities
CAT and APX activities were measured according to the methods of Kato and
Shimizu (1987) and Nakano and Asada (1981), respectively. Briefly, 0.06 g of the latest
newly expanded leaf was placed in 2 mL of sodium phosphate buffer (50 mM, pH 6.8) in
an ice bath for extraction and centrifuged at 4 °C and 12,000 rpm for 20 min. For CAT
activity, the supernatant (0.2 mL) was collected, followed by the addition of 2.7 mL of
sodium phosphate buffer (100 mM, pH 7.0), 0.05 mL of H2O, and 0.05 mL of H2O2 (1
M), and then mixed well. The absorbance of the sample solution at 240 nm (A240) was
determined every 15 s for 1 min. A blank containing the same mixture with no enzyme
extract was also measured. For APX activity, the supernatant (0.1 mL) was collected,
followed by the sequential addition of 2.7 mL of potassium phosphate buffer (150 mM,
pH 7.0), 0.4 mL of ethylenediaminetetraacetic acid (EDTA, 0.75 mM), 0.5 mL of H2O2
(6 mM), 0.5 mL of H2O, and 0.5 mL of ascorbate (1.5 mM) and then mixed well. The
absorbance at 290 nm of the sample solution was determined every 15 s for 1 min using
a spectrophotometer (Hitachi U3010, Tokyo, Japan). The blank containing the same
mixture with no enzyme extract was also measured.
Measurement of 1,1-Diphenyl-2-Picryl-Hydrazyl (DPPH)-Scavenging Capacity and the Reducing Power
The methanol extract for analyzing the DPPH scavenging capacity and the reducing
power was prepared by adding 12 mL of 100% methanol to 0.02 g of lyophilized sample
powder. The mixture was oscillated in an ultrasonic oscillator for 1 h and extracted
overnight at 4 °C. The mixture was then centrifuged at 3000 rpm for 20 min, and the
supernatant was collected for the following measurement.
The DPPH-scavenging capacity was determined using the method of Shimada et al.
(1992). Briefly, 160 μL of a methanol extract of the sample combined with methanol or a
standard solution of butylated hydroxytoluene (BHT) was added to 40 μL of a freshly
prepared DPPH solution (1 mM) to initiate the antioxidant-radical reaction at room
temperature. The control was 160 μL of sample extract, methanol, or BHT solution diluted
to 200 μL. The absorbance of the reaction mixture was determined at 517 nm during the
30 min reaction time. The DPPH-scavenging capacity was calculated by the percentage
of the free radical-scavenging activity.
The reducing power was determined using the method of Oyaizu (1986). Briefly, 0.3
mL of a methanol extract from a leaf was placed in 0.3 mL of sodium phosphate buffer
(0.2 M, pH 6.6) and 0.3 mL of 1% K3Fe(CN)6 in a water bath at 50 °C for 20 min,
immediately placed in 0.3 mL of 10% trichloroacetic acid (TCA) in an ice bath, and then
centrifuged at 9000 rpm for 10 min. The supernatant (0.5 mL) was well mixed with 0.5
mL distilled water and 0.1 mL FeCl3·H2O (0.1%). The absorbance of the reaction mixture
was determined at 700 nm during the 10 min reaction. The reducing power was calculated
using a curve of BHT standards. Results are expressed as mg BHT equivalents g−1 dry
weight (DW).
Determination of Total Phenols, Total Flavonoids, and Anthocyanin Concentration
Phenolic compounds were determined using the method of Kujala et al. (2000).
Briefly, 0.01 g of lyophilized sample powder was extracted with 1 mL of a 0.3% HCl in
a 60% methanol solution, and then centrifuged at 4000 rpm for 10 min. The supernatant
(200 µL) was added to 2 mL of 1 N Folin-Ciocalteau reagent (Sigma, St. Louis, MO,
USA), mixed well, and allowed to sit for 10 min. Na2CO3 (sodium carbonate; 10%) was
added to the solution and allowed to sit for 2.5 h. The absorbance was determined at 750
nm. The total phenolic concentration was calculated using a curve of gallic acid standards.
Results are expressed as mg gallic acid equivalents (GAE) g−1 DW.
The flavonoid concentration was determined according to Chen et al. (2015). Sample
powder of 0.01 g was extracted with 1 mL of a 1% HCl solution in ethanol and centrifuged
at 3000 rpm for 10 min at 4 °C, and the absorbance at a wavelength of 540 nm was
measured with a spectrophotometer.
The anthocyanin concentration was determined using the method of Mancinelli et al.
(1975). Lyophilized sample power (0.01 g) was extracted with 6 mL of 1% (v/v) HCl of
a methanol solution and then centrifuged at 2000 rpm for 15 min. The supernatant of the
sample extract was tested to determine the absorbance of 530 and 657 nm, respectively.
Determination of the Photosynthetic Pigment Concentrations
The photosynthetic pigment concentrations were determined using the method of
Yang et al. (1998). Briefly, 0.01 g of lyophilized sample powder was extracted with 12
mL of an 80% acetone solution, and then centrifuged at 4500 rpm for 5 min. The
supernatant of the sample extract was tested to determine the absorbance extents of Chl
a, Chl b, and carotenoids in acetone at 663.6, 646.6, and 440.5 nm, respectively.
Statistical Analyses
All measurements were evaluated for significance using an analysis of variance
(ANOVA) followed by a least significant difference (LSD) test and t-test at p < 0.05. All
statistical analyses were conducted using R i386 3.5.1 software
(https://cran.r-project.org/bin/windows).
Results
Growth Analysis
Shoot heights of seedlings without Se treatment gradually declined when the NaCl
concentration exceeded 100 mM. Seedlings with Se treatment also showed a similar trend,
but shoot heights of Se-treated seedlings were significantly (p < 0.05) higher than those
without Se when the NaCl concentration exceeded 100 mM (Figure 3-1). Root lengths of
Se-treated seedlings also declined with an increase in the NaCl concentration. A similar
declining trend was observed in Se-treated seedlings. However, root lengths of Se-treated
seedlings were significantly longer (p < 0.05) than those of seedlings without Se treatment
(Figure 3-1). These results showed that Se effectively promoted the growth of seedlings
grown under salt stress.
ChlF
The response of ChlF can be applied as an index to evaluate the physiological
condition of the photosynthetic tissues of plants. Fv/Fm in leaves was determined after
dark adaption. Fv/Fm in leaves of seedlings without Se treatment suddenly declined as the
NaCl concentration exceeded 200 mM, but Fv/Fm in leaves of Se-treated seedlings was
significantly enhanced (p < 0.05) under salt stress (Figure 3-2). F0 is a fluorescent signal
when the PSII reaction center is fully open (Sun et al., 2006), and an increase in F0 usually
indicates that a plant is under stress (Song et al., 2013). F0 in leaves of seedlings without
Se treatment gradually increased with an increase in the NaCl concentration, but F0 in
Se-treated seedlings was stable (Figure 3-2).
ΦPSII reflects the effective quantum yield of PSII under illumination. Y(NPQ) and
Y(NO) are important fluorescence parameters of photo-protection and photodamage,
respectively (Kramer et al., 2004). In this study, ΦPSII, Y(NPQ), and Y(NO) were
determined at an illumination of 185 μmol m−2 s−1, and results are presented in Figure
3-3.The value of ΦPSII in leaves of seedlings without Se treatment dramatically decreased
with an increase in the NaCl concentration. A similar trend was also observed in results
of ΦPSII in leaves of Se-treated seedlings, but ΦPSII values of Se-treated seedlings at 300
and 400 mM NaCl significantly improved (p < 0.05). Y(NPQ) in leaves of seedlings
without Se treatment was significantly (p < 0.05) enhanced by NaCl of < 200 mM, but
dramatically declined under more-severe salt stress (> 300 mM NaCl). A similar Y(NPQ)
dynamic was determined in Se-treated seedlings, but the value at 400 mM NaCl was
significantly higher than that without Se (p < 0.05). Y(NO) in leaves of seedlings without
Se was maintained at a level of around 0.26-0.30 under NaCl of < 200 mM and was
significantly enhanced at 300 and 400 mM (p < 0.05). The Y(NO) result for Se-treated
seedlings also presented a similar trend, but Y(NO) values at 300 and 400 mM NaCl were
significantly lower than that without Se (p < 0.05).
Activities of CAT and APX, DPPH-Scavenging Capacity, and Reducing Power
Results of the antioxidant enzyme activity and capacity in wheat seedlings of this
study are presented in Table 3-1. CAT and APX play important roles in quenching H2O2.
In this study, results for CAT activity in seedlings without Se treatment showed a
descending trend with an increasing NaCl concentration, while CAT activities in
Se-treated seedlings significantly (p < 0.05) remained at a level of around 1.79~1.56 μmol
H2O2 min−1 mg−1 protein until NaCl exceeded 300 mM (Table 3-1). On the other hand,
results of APX activities in both Se-treated seedlings and untreated seedlings showed
descending trends with an increasing NaCl concentration.
The removal of DPPH radicals and reduction in the reducing power are methods for
measuring antioxidant activities (Erel, 2004). The method for determining DPPH free
radicals is based on the amount of DPPH free radicals removed. In this study, the ability
to clear DPPH in untreated seedlings was reduced from 33.5% to 28.0% with an
increasing NaCl concentration, while this ability was significantly induced (p < 0.05) at
around 31.9% ~ 43.2% in Se-treated seedlings until NaCl exceeded 300 mM (Table 3-1).
The reducing power is a method to estimate substances that might own the ability to
remove free radicals in a plant (Jayanthi and Lalitha, 2011). In this study, the reducing
power in untreated seedlings was maintained at a level of 20.5 ~ 19.2 BHT equivalent g−1
DW until NaCl exceeded 300 mM, but the reducing power in Se-treated seedlings was
significantly enhanced (p < 0.05) except at 300 mM NaCl (Table 3-1).
Total Phenols, Total Flavonoids, and Anthocyanin Concentrations
Plants contain a variety of non-enzymatic antioxidants which can scavenge free
radicals, including phenols, flavonoids, and anthocyanins (Thiruvengadam and Chung,
2015). Results of total phenols, total flavonoids, and anthocyanin concentrations in
seedlings undergoing different treatments in this study are presented in Table 3-2. Total
phenols contained in untreated wheat seedlings declined from 60.85 to 51.89 mg GAE
g−1 DW with an increase in the NaCl concentration, while in Se-treated seedlings, they
were effectively enhanced. Similar trends were also observed in the dynamics of total
flavonoid and anthocyanin concentrations in seedlings in this study.
Photosynthetic Pigments
Chls and Cars are both involved in the light reaction of photosynthesis. Chl a, Chl b,
and their sum, and carotenoid concentrations in leaves of seedlings from all treatments in
this study are presented in Figure 3-4. In this study, Chl a and Chl b concentrations and
their sum in leaves of Se-treated seedlings and untreated seedlings were slightly enhanced
at 100 mM NaCl, but these Chl concentrations significantly (p < 0.05) and sharply
declined when seedlings were grown under NaCl of more than 200 mM. A similar trend
was also observed in Car concentrations in leaves of Se-treated seedlings and untreated
seedlings. However, all values of photosynthetic pigments of Se-treated seedlings were
higher than those of untreated seedlings. These results revealed that Se treatment served
as a protectant for photosynthetic pigments to prevent their salt-stress induced
degradation in wheat seedlings.
Discussion
Although a beneficial micronutrient, Se exerts a dual effect in plants (Djanaguiraman
et al., 2005): It can stimulate plant growth and provide beneficial effects at low
concentration, but it is harmful to plants at higher concentrations. Positive effects of Se
depend on its form, dose and the chosen plant genotype (Sieprawska et al., 2015).
According to the pH and redox potential of soil, two kinds of inorganic Se forms can be
found: One is selenite, and the other one is selenate. Each of them exhibits different
availabilities and effects to plant. In our study, sodium selenite (Na2SeO3) was treated in
the acidic nutrient solution, which existed primarily as HSeO3- (Guerrero et al., 2014).
The recommended Se doses for hydroponic conditions are usually < 1 mg L−1 (29 μM)
(Pilon-Smits et al., 2009). According to results of our preliminary study for wheat (T.
aestivum L.) cultivar Taichung SEL. 2, the growth rate of wheat seedlings was strongly
retarded at 10 mg Se L−1 (294 μM), but was not promoted at 0.5 mg Se L−1 (14.7 μM). Se
at 1 mg L−1 (29 μM) might exceed the plant’s threshold, resulting in a slightly
disadvantageous effect on the plant. Therefore, 0.75 mg Se L−1 (22 μM) was an
appropriate concentration for Se treatment in this study.
When plants experience salt stress, electron leakage from chloroplasts and
mitochondria might react with O2 during normal aerobic metabolism to produce reactive
oxygen species (ROS), such as singlet oxygen (1O2), hydrogen peroxide (H2O2),
superoxide (∙O2-), and hydroxyl (∙OH) radicals (Dionisio-Sese and Tobita, 1998). ROS
can immediately react with DNA, lipids, and proteins, thereby possibly causing serious
cellular damage (Sato et al., 2001). Fortunately, the adverse effects of ROS can be
diminished by enzymatic and non-enzymatic defense systems in plants (Gondim et al.,
2010). Among the enzymatic defense system, CAT and APX play key roles in quenching
H2O2 (Sato et al., 2001 ; Gondim et al., 2010). In our study, we found that applying Se
improved CAT activities in wheat seedlings grown under all salt treatments, especially at
100 and 200 mM NaCl (Table 3-1). Our results of CAT activity were similar to those of
a study by Chu et al. (2010) who indicated that Se mitigated cold-induced stress in wheat
seedlings. Results of Djanaguiraman et al. (2010) and Nawaz et al. (2015) also supported
our results. However, Iqbal et al. (2015) found that Se treatment did not increase CAT
activity in every wheat cultivar. Furthermore, Djanaguiraman et al. (2005) observed that
CAT did not participate in active H2O2 reduction irrespective of the sampling date (on the
80th and 90th days after sowing). These previous studies suggested that CAT activity was
cultivar-specific and variable according to the growth period. In this study, CAT
absolutely played an important role in the enzymatic defense system when wheat was
suffering from salt stress.
Nonetheless, we found that Se treatment did not promote APX activity in wheat
seedlings that were suffering from salt stress at 200 mM NaCl (p < 0.05) (Table 3-1). Xue
and Hartikainen (2000) also reported that APX activities in ryegrass and lettuce were not
enhanced by Se treatment. This phenomenon could be explained by APX and CAT
sharing the same substrate, and while Se might increase CAT activity, it would effectively
reduce H2O2, which might also weaken the substrate to induce APX activity (Shang et.
reduce H2O2, which might also weaken the substrate to induce APX activity (Shang et.