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Agronomy 2019, 9(6), 272; https://doi.org/10.3390/agronomy9060272

Article
Protective Effects of Selenium on Wheat Seedlings under Salt Stress
1
Department of Agronomy, National Taiwan University, Daan, Taipei 101, Taiwan
2
Department of Horticulture and Biotechnology, Chinese Culture University, Shilin, Taipei 114, Taiwan
3
National Research Institute of Chinese Medicine, Ministry of Health and Welfare, Taipei 112, Taiwan
*
Authors to whom correspondence should be addressed.
Received: 21 April 2019 / Accepted: 28 May 2019 / Published: 30 May 2019

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

1. 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 [1], provides the main source of carbohydrates for 35%~40% of the world’s population [2]. However, wheat yields are markedly reduced in saline soils, due to improper fertilization that causes osmotic and drought stresses [3]. Therefore, it is imperative to research the effects of salt stress on wheat’s physiology.
Selenium (Se) is considered a beneficial element to plants [4]. Previous studies indicated that Se can delay senescence [5,6] and promote the vegetative and reproductive growth of plants [7]. Studies reported that Se mitigated disadvantageous phenomena caused by various stressful situations, such as heat [8,9,10], cold [11], heavy metals [12,13], ultraviolet (UV)-B [14,15,16,17], drought [18,19], and salt stress [20,21]. 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 [11]. 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 [11]. Moreover, Se contributes to water status regulation in plants by promoting the water uptake efficiency from roots and reducing water loss from tissues [22]. In addition, Se stimulates plant growth by promoting the integrity of the membrane system which results in root and shoot elongation and biomass accumulation [5,18,23,24,25]. Research by Hawrylak-Nowak [23] reported that Se particularly supported root system development.
Salinization degrades land and is serious environmental stress that limits wheat production [3]. 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 [26]. Few studies have evaluated the mitigation of salt stress in wheat by supplying Se. A study by Yigit et al. [27] reported that organic Se can increase germination percentages and enhance antioxidant activities in wheat exposed to salt stress. Mona et al. [20] 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. [28] 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.

2. Materials and Methods

2.1. 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.

2.2. Experimental Treatments

Se was added in the form of Sodium selenite (Na2SeO3, Sigma-Aldrich Chemie GmbH, Taufkirchen, 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 [29] 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.

2.3. Growth Analysis

Shoot height and root length were measured with a ruler before the measurements of chlorophyll (Chl) fluorescence (ChlF) and sample collection.

2.4. 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−2 s−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 [30,31].

2.5. Measurement of Catalase (CAT) and Ascorbate Peroxidase (APX) Activities

CAT and APX activities were measured according to the methods of Kato and Shimizu [32] and Nakano and Asada [33], 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.

2.6. 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. [34]. 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 [35]. 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).

2.7. Determination of Total Phenols, Total Flavonoids, and Anthocyanin Concentration

Phenolic compounds were determined using the method of Kujala et al. [36]. 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. [37]. 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. [38]. 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.

2.8. Determination of the Photosynthetic Pigment Concentrations

The photosynthetic pigment concentrations were determined using the method of Yang et al. [39]. 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.

2.9. 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).

3. Results

3.1. 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 1a). 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 1b). These results showed that Se effectively promoted the growth of seedlings grown under salt stress.

3.2. 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 2a,b). F0 is a fluorescent signal when the PSII reaction center is fully open [40], and an increase in F0 usually indicates that a plant is under stress [41]. 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 2c,d).
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 [31]. 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. 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).

3.3. 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 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 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 [42]. The method for determining DPPH free radicals is based on the amount of DPPH free radicals removed [43]. 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 1). The reducing power is a method to estimate substances that might own the ability to remove free radicals in a plant [44]. 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 1).

3.4. 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 [45]. Results of total phenols, total flavonoids, and anthocyanin concentrations in seedlings undergoing different treatments in this study are presented in Table 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.

3.5. 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 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.

4. Discussion

Although a beneficial micronutrient, Se exerts a dual effect in plants [5]: 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 [46]. 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 [47]. The recommended Se doses for hydroponic conditions are usually < 1 mg L−1 (29 μM) [4]. 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 [48]. ROS can immediately react with DNA, lipids, and proteins, thereby possibly causing serious cellular damage [49]. Fortunately, the adverse effects of ROS can be diminished by enzymatic and non-enzymatic defense systems in plants [50]. Among the enzymatic defense system, CAT and APX play key roles in quenching H2O2 [49,50]. 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 1). Our results of CAT activity were similar to those of a study by Chu et al. [11] who indicated that Se mitigated cold-induced stress in wheat seedlings. Results of Djanaguiraman et al. [10] and Nawaz et al. [19] also supported our results. However, Iqbal et al. [8] found that Se treatment did not increase CAT activity in every wheat cultivar. Furthermore, Djanaguiraman et al. [5] 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 1). Xue and Hartikainen [17] 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 [9]. Another possibility could be that APX activities in plants might vary in different cultivars and growth stages [5,8]. Our results showed that the Se-induced antioxidative activity in wheat seedlings grown under salt stress did not include mediation of APX activity. The beneficial effects of Se treatment on ChlF, total Chl and Car concentrations, and growth of wheat seedlings grown under salt stress might have been due to the contribution of CAT activity or other enzymes which were not analyzed in this study.
The antioxidant potential was evaluated by the DPPH free radical-scavenging activity and reducing power. Se treatment enhanced the antioxidant potential of wheat seedlings exposed to salt stress (Table 1). Se was also reported to induce enhancement of antioxidant activity in broccoli [51] and turnip [45]. The non-enzymatic antioxidant system diminishes the adverse effects of ROS in plants [8,11,45]. In this study, the concentrations of total phenols, total flavonoids, and anthocyanins in wheat seedlings grown under salt stress were evaluated. Anthocyanins act to reduce photoinhibition and photobleaching of Chl in plants under stress [52]. In addition, flavonoids and anthocyanins also provide functions as osmoprotectants in plants under stress [8]. According to results presented in this study, Se treatment increased the concentrations of total phenols, total flavonoids, and anthocyanins in wheat seedlings grown under salt stress (Table 2). These results were consistent with a study by Yao et al. [15], who evaluated the effect of an exogenous Se supply on wheat seedlings under enhanced UV-B.
The ChlF response can be used to evaluate the physiological condition of photosynthetic tissues in the plant. Fv/Fm and ΦPSII are widely used to estimate the status under stress [40]. Results of ChlF showed that Se treatment enhanced values of Fv/Fm (Figure 2), ΦPSII, and Y(NPQ) (Figure 3) and decreased F0 (Figure 2) and Y(NO) (Figure 3) in salt-stressed wheat seedlings. These results, which illustrated the stimulating effects of Se treatment on the photochemical activity of PSII and photosynthetic activity in wheat seedlings, are in agreement with a previous report by Diao et al. [21]. Se treatment increased the enzymatic and non-enzymatic antioxidative capacities of wheat seedlings grown under salt stress, and reduced ROS accumulation at PSII. Therefore, the salt stress-induced damage in the PSII reaction center complex in wheat seedlings was alleviated [5,12]. On the other hand, the insignificant difference between Fv/Fm of wheat seedlings grown at 100 and 200 mM NaCl suggests that TCS2, the tested cultivar in this study, is a salt-tolerant cultivar. The ChlF results were similar to the results of CAT activity which were enhanced by Se treatment (Table 1). Therefore, we speculated that CAT plays a key role in quenching H2O2 in TCS2, and we propose that application of Se in the soil or coating Se onto seeds might be suitable for TCS2 cultivation in saline areas.
Stress conditions also damage photosynthetic pigments with impaired biosynthesis and/or accelerated degradation of pigments [53,54]. Results of the analysis of photosynthetic pigments in this study (Figure 4) suggested that Se treatment significantly increased the Chl and Car concentrations in wheat seedlings (p < 0.05). The increase in total Chl concentrations might have been due to increases in the Car and anthocyanin concentrations, since these antioxidative metabolites can protect Chl from photo-oxidative destruction and photobleaching under stress conditions [16,52]. Meanwhile, the enzymatic antioxidant system contributed to efficient ROS scavenging [23]. Furthermore, Se treatment positively promoted the integrity of the membrane system of chloroplasts [55]. Se induced enhancements of Chl and Car concentrations in wheat seedlings under stress was also reported in a previous study by Iqbal et al. [8].
Se treatment stimulates root and shoot elongation and biomass accumulation in plants facing salt stress, and the protective effects of Se have been evaluated in sorrel [55], melon [24], and cucumber [23]. In addition, Se treatment promotes the growth of soybeans facing senescence [5]. According to the results of seedling growth in this study (Figure 1), shoot and root elongation were absolutely inhibited under salt stress. However, Se treatment counteracted the stress-induced effects on seedling growth. Se alleviated the disadvantageous effect of salt stress on seedling growth, which might have been associated with enhanced CAT activity and non-enzymatic antioxidants, including total phenols, total flavonoids, and anthocyanins. The Se-induced antioxidant system resulted in activation of antioxidant activity, such as DPPH radical-scavenging activity and reducing power, increased levels of photosynthetic pigments, and effective photosynthesis potency. Moreover, Se supported the integrity of the membrane system in chloroplasts and/or mitochondria which might have also contributed to plant morphogenesis [55].

5. Conclusions

Compared to seedlings without Se treatment, CAT and antioxidative activities of Se-treated seedlings were enhanced, and the accumulation of non-enzymatic antioxidants, including total phenols, total flavonoids, and anthocyanins, increased under salt stress. Meanwhile, the indices of ChlF and degradation of photosynthetic pigments in Se-treated seedlings had stabilized compared to those in untreated seedlings. The elongation of shoots and roots of Se-treated seedlings was preserved under salt stress. The performance of physiological and morphological traits in Se-treated wheat seedlings was better than in untreated seedlings in a saline environment in this study. These results suggest that wheat cultivation with Se treatment could prevent salt stress-induced disadvantageous effects.

Author Contributions

W.-D.H., C.-Y.L., and C.-C.C. conceived and designed the experiments. C.-Y.L. conducted the experiments, collected the data, and performed the analyses. W.-D.H. and K.-H.L. provided the facilities and advised on the preparation of materials. C.-Y.L., and C.-C.C. edited the manuscript. All authors approved the final manuscript.

Funding

This research received no external funding.

Acknowledgments

We thank the Experimental Farm, College of Bioresources and Agriculture, National Taiwan University for donation of ‘Taichung SEL. 2’ seeds.

Conflicts of Interest

The authors declare no competing financial interests.

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Figure 1. Means ± SD (n = 5) of the shoot height (a) and root length (b) of untreated seedlings (No added Se) and Se-treated seedlings (22 μM Se) under different NaCl concentrations. Values followed by different letters statistically significantly differ at p < 0.05. Means with different lowercase letters significantly differ by a t-test between Se treatments (No added Se and 22 μM Se) at the same NaCl concentration. Means with different capital letters significantly differ by an LSD test under different NaCl concentrations in separate untreated and Se-treated groups (p < 0.05).
Figure 1. Means ± SD (n = 5) of the shoot height (a) and root length (b) of untreated seedlings (No added Se) and Se-treated seedlings (22 μM Se) under different NaCl concentrations. Values followed by different letters statistically significantly differ at p < 0.05. Means with different lowercase letters significantly differ by a t-test between Se treatments (No added Se and 22 μM Se) at the same NaCl concentration. Means with different capital letters significantly differ by an LSD test under different NaCl concentrations in separate untreated and Se-treated groups (p < 0.05).
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Figure 2. Images and the mean ± SD (n = 4) of the maximum quantum yield of photosynthetic system II (Fv/Fm) (a,b) and the minimal fluorescence of photosynthetic system II (F0) (c,d) of leaves collected from untreated seedlings (No added Se) and Se-treated seedlings (22 μM Se) under different NaCl concentrations. The false color code depicted on the top of the images ranges from 0.0 (black) to 1.0 (purple). Values followed by different letters statistically significantly differ at p < 0.05. Means with different lowercase letters significantly differ by a t-test between Se treatments (No added Se and 22 μM Se) at the same NaCl concentration. Means with different capital letters significantly differ by an LSD test under different NaCl concentrations in separate untreated and Se-treated groups (p < 0.05).
Figure 2. Images and the mean ± SD (n = 4) of the maximum quantum yield of photosynthetic system II (Fv/Fm) (a,b) and the minimal fluorescence of photosynthetic system II (F0) (c,d) of leaves collected from untreated seedlings (No added Se) and Se-treated seedlings (22 μM Se) under different NaCl concentrations. The false color code depicted on the top of the images ranges from 0.0 (black) to 1.0 (purple). Values followed by different letters statistically significantly differ at p < 0.05. Means with different lowercase letters significantly differ by a t-test between Se treatments (No added Se and 22 μM Se) at the same NaCl concentration. Means with different capital letters significantly differ by an LSD test under different NaCl concentrations in separate untreated and Se-treated groups (p < 0.05).
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Figure 3. Means ± SD (n = 4) of the effective quantum yield of photosystem II under illumination (ΦPSII) (a), quantum yield of regulated energy dissipation of photosystem II (Y(NPQ)) (b), and the quantum yield of non-regulated energy dissipation of photosystem II (Y(NO)) (c) of leaves collected from untreated seedlings (No added Se) and Se-treated seedlings (22 μM Se) under different NaCl concentrations. Values followed by different letters statistically significantly differ at p < 0.05. Means with different lowercase letters significantly differ by a t-test between Se treatments (No added Se and 22 μM Se) at the same NaCl concentration. Means with different capital letters significantly differ by an LSD test under the different NaCl concentrations in separate untreated and Se-treated groups (p < 0.05).
Figure 3. Means ± SD (n = 4) of the effective quantum yield of photosystem II under illumination (ΦPSII) (a), quantum yield of regulated energy dissipation of photosystem II (Y(NPQ)) (b), and the quantum yield of non-regulated energy dissipation of photosystem II (Y(NO)) (c) of leaves collected from untreated seedlings (No added Se) and Se-treated seedlings (22 μM Se) under different NaCl concentrations. Values followed by different letters statistically significantly differ at p < 0.05. Means with different lowercase letters significantly differ by a t-test between Se treatments (No added Se and 22 μM Se) at the same NaCl concentration. Means with different capital letters significantly differ by an LSD test under the different NaCl concentrations in separate untreated and Se-treated groups (p < 0.05).
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Figure 4. Means ± SD (n = 3) of chlorophyll (Chl) a (a), Chl b (b), Chl a+b (c), and carotenoids (d) of leaves collected from untreated seedlings (No added Se) and Se-treated seedlings (22 μM Se) under different NaCl concentrations. Values followed by different letters statistically significantly differ at p < 0.05. Means with different lowercase letters significantly differ by a t-test between Se treatments (No added Se and 22 μM Se) at the same NaCl concentration. Means with different capital letters significantly differ by an LSD test under different NaCl concentrations in the untreated and Se-treated groups (p < 0.05). The concentration of Se treatment was 22 μM.
Figure 4. Means ± SD (n = 3) of chlorophyll (Chl) a (a), Chl b (b), Chl a+b (c), and carotenoids (d) of leaves collected from untreated seedlings (No added Se) and Se-treated seedlings (22 μM Se) under different NaCl concentrations. Values followed by different letters statistically significantly differ at p < 0.05. Means with different lowercase letters significantly differ by a t-test between Se treatments (No added Se and 22 μM Se) at the same NaCl concentration. Means with different capital letters significantly differ by an LSD test under different NaCl concentrations in the untreated and Se-treated groups (p < 0.05). The concentration of Se treatment was 22 μM.
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Table 1. Catalase (CAT) and ascorbate peroxidase (APX) activities, DPPH radical-scavenging activity (%), and reducing the power of wheat seedlings.
Table 1. Catalase (CAT) and ascorbate peroxidase (APX) activities, DPPH radical-scavenging activity (%), and reducing the power of wheat seedlings.
NaCl (mM)Se treatedCAT Activity (μmol H2O2 min−1 mg−1 Protein)APX Activity (μmol AsA min−1 mg−1 Protein)DPPH Radical Scavenging Activity (%)Reducing Power (BHT Equivalent g−1 DW)
0No added1.53 ± 0.05 ns A0.22 ± 0.04 a A33.5 ± 1.1 b B19.2 ± 0.4 b A
22 μM1.56 ± 0.12 ns A0.18 ± 0.01 b A43.2 ± 2.4 a A23.6 ± 0.4 a A
100No added1.39 ± 0.09 b A0.18 ± 0.02 ns AB36.0 ± 1.5 b A20.5 ± 0.9 b A
22 μM1.79 ± 0.14 a A0.16 ± 0.03 ns A40.2 ± 0.2 a B23.1 ± 0.1 a A
200No added1.07 ± 0.19 b B0.17 ± 0.01 a AB28.8 ± 2.2 b C19.7 ± 0.5 b A
22 μM1.62 ± 0.26 a A0.13 ± 0.01 b AB40.2 ± 1.0 a B21.7 ± 0.2 a B
300No added0.92 ± 0.00 ns BC0.13 ± 0.03 ns BC25.4 ± 0.2 b D19.2 ± 0.5 ns A
22 μM0.98 ± 0.19 ns B0.10 ± 0.04 ns B31.9 ± 1.2 a C19.2 ± 0.7 ns C
400No added0.76 ± 0.02 ns C0.10 ± 0.02 ns C28.0 ± 0.2 ns CD15.2 ± 0.3 b B
22 μM0.89 ± 0.14 ns B0.11 ± 0.04 ns B28.9 ± 1.7 ns D18.3 ± 0.4 a D
Values are the mean ± SD (n = 3). Means with different lowercase letters significantly differ by a t-test between untreated (No added) and Se-treated (22 μM) plants at the same salt concentration. Means with different capital letters significantly differ by an LSD test among NaCl concentrations of 0, 100, 200, 300, and 400 mM in untreated (No added) and Se-treated (22 μM) plants (p < 0.05). ns, not significant at the p < 0.05 level. AsA, ascorbate; BHT, butylated hydroxytouene.
Table 2. Total phenols, total flavonoids, and anthocyanin concentrations of wheat seedlings.
Table 2. Total phenols, total flavonoids, and anthocyanin concentrations of wheat seedlings.
NaCl (mM)Se TreatedTotal Phenols Concentration (Gallic Acid Equivalent g−1 DW)Total Flavonoids Concentration (A540 g−1 DW)Anthocyanin Concentration (μmol g−1 DW)
0No added60.9 ± 2.4 b A33.9 ± 3.0 b AB117 ± 2 b A
22 μM65.1 ± 2.8 a A41.5 ± 1.0 a A126 ± 7 a A
100No added56.0 ± 0.9 b B36.0 ± 1.1 b A117 ± 5 b A
22 μM63.4 ± 0.3 a AB42.9 ± 0.6 a A121 ± 1 a A
200No added56.5 ± 0.5 b B31.1 ± 2.4 b B99 ± 0 b B
22 μM65.8 ± 2.5 a A37.9 ± 1.8 a B111 ± 2 a B
300No added58.1 ± 0.1 ns AB22.6 ± 1.3 b C76 ± 2 b C
22 μM59.7 ± 1.7 ns AB27.1 ± 0.9 a C83 ± 0 a C
400No added51.9 ± 2.2 b C17.7 ± 0.7 b C58 ± 1 b D
22 μM59.5 ± 3.2 a B21.8 ± 0.7 a D69 ± 5 a D
Values are the mean ± SD (n = 3). Means with different lowercase letters significantly differ by a t-test between untreated (No added) and Se-treated (22 μM) plants at the same NaCl concentration. Means with different capital letters significantly differ by an LSD test among NaCl concentration of 0, 100, 200, 300, and 400 mM in untreated (No added) and Se-treated (22 μM) plants (p < 0.05). ns, not significant at the p < 0.05 level. DW, dry weight.

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