Is High pH the Key Factor of Alkali Stress on Plant Growth and Physiology? A Case Study with Wheat (Triticum aestivum L.) Seedlings

Abstract

Salinity and alkalinity stress are two major constraints on plant growth and crop production, limiting sustainable agricultural production. Wheat is a vital cereal crop. It is very important to ensure food security; however, its growth and yield are usually adversely affected by salinity and alkalinity stress. To investigate the differential effects of neutral and alkaline salt stress on the seedling growth of wheat, we set wheat hydroponic culture experiment: CK, neutral salt (NaCl:Na₂SO₄ = 9:1 pH = 6.5), neutral salt with high pH value (NaCl:Na₂SO₄ = 9:1 pH = 8.9), alkaline salt (NaHCO₃:Na₂CO₃ = 9:1 pH = 8.9), all treatments at the same Na⁺ concentration. The results indicated alkaline salt inhibited seedling growth more than neutral salt and neutral salt with high pH value. The results showed that the salt and alkali stresses decreased chlorophyll contents in leaves of wheat seedlings, inhibited photosynthesis and induced osmotic stress, oxidative stress and ion toxicity to wheat seedlings and finally inhibited the growth of wheat seedlings, while the alkaline salt caused a stronger injurious effect on wheat seedlings than the neutral salt, neutral salt with high pH value. Our study results demonstrated that alkaline salt inhibited wheat seedlings more significantly than neutral salt and neutral salt with high pH value. And, the main factor affected seedling growth is not pH alone.

1. Introduction

Salinity and alkalinity stress can adversely affect plant growth and crop production [1]. According to the Food and Agriculture Organization of the United Nations (FAO) and United Nations Educational, Scientific, and Cultural Organization (UNESCO) statistics, there are about 950 million hectares of saline–alkaline soils in the world [2]. In recent years, with the deterioration of the ecological environment and the interference of human factors, the area of salt–alkaline land has further increased, and the problem of salt–alkaline land has become increasingly serious [3]. Soil salinization and alkalization often co-occur. Salinity and alkalinity stress are two different types of stresses that restrict plant growth. Salinity stress is caused by neutral salt, such as NaCl and Na₂SO₄, while alkalinity stress is caused by alkaline salt, such as NaHCO₃ and Na₂CO₃ [4].

Salt-alkali stress can inhibit plant growth; the most conspicuous symptom is the wilting of plants and the yellowing of dehydrated leaves [5]. Photosynthesis is the material and energy basis of plant growth and development. A large number of experimental studies have shown that salt stress could lead to the weakening of photosynthesis, mainly including the reduction in net photosynthetic rate (Pn), stomatal conductance (Gs), and transpiration rate (Tr) and photosynthetic pigments of plants. This is closely associated with a reduction in biomass and yield under salt and alkali stress [6], and alkalinity stress often has more damage to the plant photosynthesis than salinity stress at the same concentration [7]. The excessive salt in the soil could lead to the reduction of the soil water potential and make it difficult for plants to absorb water from the soil [8]. Therefore, salt and alkali stress could cause osmotic stress to plants. Plants can adapt to osmotic stress, by accumulating and synthesizing osmotic substances, and plants usually accumulate more osmotic substances under alkalinity stress than under salinity stress [9,10]. Salt and alkali stress could lead to fierce competition between sodium ions and potassium ions to enter plant cells. Selective absorption of Na⁺ and K⁺ is affected by salt and alkali stress and breaks the ionic balance. Therefore, Na⁺ and K⁺ content and Na⁺/K⁺ are the key index for determining the salt and alkali tolerance of plants. It has been found that lower Na⁺/K⁺ indicates that plants have stronger salt tolerance; plants usually accumulate more Na⁺ and lose more K⁺ under alkalinity stress than under salinity stress [11,12,13]. Plants can produce ROS due to photosynthesis, respiration, and photorespiration but keep the balance between production and elimination of ROS under normal conditions. Both salt and alkali stress can induce excess accumulation of reactive oxygen species (ROS), which mainly include hydrogen peroxide (H₂O₂), superoxide radical, singlet oxygen, and so on, which could attack the plant cell membrane system and cause adverse effects on plant growth [14,15]. To cope with oxidative stress, plants usually through enhancing the activity of superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), ascorbic acid peroxidase (APX), and producing nonenzymatic antioxidants to eliminate excessive ROS, and then protecting the plant from oxidative stress [15], and alkalinity stress can induce more oxidative damage to plants. In order to cope with alkalinity stress, the antioxidant enzyme activity is higher in plants under alkalinity stress than under salinity stress [16]. In soil, the main cations are Na⁺, K⁺, Mg²⁺, and Ca²⁺; the main anions are SO₄²⁻, CO₃²⁻, HCO₃⁻, NO₃⁻, and Cl⁻. Many studies have demonstrated that alkaline salt damage plants more severely than neutral salt. It is generally believed that the main reason for this phenomenon is the high pH factor of alkaline salt, and this viewpoint has been widely accepted [17,18]. However, the high pH may not be the only key factor causing the difference between saline and alkaline stress. but now the understanding of the mechanism of plant response to saline–alkaline stress is not very enough. Especially, the role of CO₃²⁻ and HCO₃⁻ in alkaline stress is rarely reported. Therefore, fully understanding the mechanism of plant response to salt–alkaline stress and determining the key factor in alkaline stress is important for improving the utilization of saline–alkaline soils for agricultural production.

Wheat (Triticum aestivum L.) is an important cereal crop in the world. Approximately one-third of the world’s people live on wheat as their staple food, which provides people with the carbohydrates and calories that they need every day [19,20]. Wheat is planted in saline–alkaline soil and no saline–alkaline soil all over the world; the planting area is about 217 million hectares [21,22]. Salt and alkali stress can pose a threat to wheat production, inhibit wheat growth, and reduce yield; previous studies have shown that alkalinity stress has more injurious effects on wheat than salinity stress at the same concentration [23]. In this study, we take wheat as the test material, investigating the exogenous application of neutral salt, neutral salt with high pH value, and alkaline salt to seedling growth, photosynthesis, physiology, and the distribution of Na⁺ and K⁺ in wheat roots, stems, and leaves. The main objectives of this study were, (1) to evaluate the effects of salinity and alkalinity stress on the growth and physiology of wheat seedlings and (2) to investigate the effects of salt and alkali stress on the distribution of ions in the various parts of wheat seedlings. (3) Is high pH the key factor in determining the effects of alkalinity stress on plant growth and physiology?

2. Materials and Methods

2.1. Plant Material and Experimental Design

The experimental material used in this experiment was wheat (AK58), which is widely planted in China. The experiment was carried out in an artificial light culture room by hydroponics. Selected plump wheat seeds were disinfected with 75% alcohol for 5 min and rinsed with distilled water three times, and the seeds were placed in a customized seedling box which was covered with plastic to prevent water evaporation, and after 5 days of culture then were transplanted to a black plastic bucket containing 1 L of 1/2 concentration Hoagland nutrient solution. Five holes were drilled in each pot. Two seedlings were transplanted into each hole, and a total of 10 seedlings were planted in each pot. LED light 12 h/12 h (L/D) was used for cultivation. The day and night temperatures were 25 and 18 °C, respectively. The stress treatments were carried out after culturing for 25 days. Four experimental treatments were set as CK, A, B, and C: CK (no added NaCl, Na₂SO₄, NaHCO₃, and Na₂CO₃); A: 100 mmol/L neutral salt (NaCl:Na₂SO₄ = 9:1, pH = 6.5); B: 100 mmol/L neutral salt (NaCl:Na₂SO₄ = 9:1, pH = 8.9); C: 100 mmol/L alkaline salt (NaHCO₃:Na₂CO₃ = 9:1, pH = 8.9), the applied stress intensity was 100 mmol L⁻¹ Na⁺ for the A, B, and C treatments, there were four replications for each treatment. The pH was adjusted every day with HCl and NaOH. The nutrient solutions were continuously inflated with air pump, and the nutrient solutions were replaced every 3 days. After 7 days stress treatment, and the samples were collected for growth and physiology index determination.

2.2. Determination of Seedling Growth

Plant height and root length were measured with a graduated ruler, Fresh weight was determined by weighing method, biomass was determined after oven-dried at 105 °C for 10 min and desiccation at 80 °C to a constant weight [24].

2.3. Determination of Photosynthetic Pigment Content

0.2 g fresh leaves were extracted with 80% (v/v) acetone for 48 h, and the contents of chlorophyll a, chlorophyll b, carotenoid, and total chlorophyll were determined at 663, 645, and 470 nm respectively, by a UV–visible spectrophotometer (TU-1810, Puxi, Beijing, China). The chlorophyll contents were calculated using following formula:

$${\mathrm{C}}_{\mathrm{a}}=12.21{\mathrm{A}}_{663}-2.81{\mathrm{A}}_{645}$$

$${\mathrm{C}}_{\mathrm{b}}=20.13{\mathrm{A}}_{645}-5.03{\mathrm{A}}_{663}$$

$${\mathrm{C}}_{\mathrm{x}+\mathrm{c}}=\frac{1000{\mathrm{A}}_{470}-3.27{\mathrm{C}}_{\mathrm{a}}-104{\mathrm{C}}_{\mathrm{b}}}{229}$$

where C_a, C_b, and C_x+c are contents of Chl a, Chl b, and total carotenoids, respectively.

2.4. Gas Exchange Characteristics

Before the harvesting of seedlings, the net photosynthetic rate (P_N) (µmol (CO₂)m⁻² s⁻¹), transpiration rate (Tr) (mmol (H₂O) m⁻² s⁻¹), stomatal conductance (gs) (mol (H₂O) m⁻² s⁻¹), and internal CO₂ concentration (Ci) (µmol (CO₂) mol⁻¹) of wheat seedling leaves were measured on a fully expanded youngest leaf at 9:00 using a 1000 μmol m⁻² s⁻¹ light illumination by a portable open flow gas exchange system, LI-6400. The experiment was repeated for 4 times with 2 blades per pot and 8 leaves per treatment, and the averages were calculated [25].

2.5. Determination of Antioxidant Enzyme Activities

0.5 g fresh leaves was ground in liquid nitrogen, then placed in a 15 mL tube, and homogenized in 10 mL of 50 mM phosphate buffer solution (pH = 7.8). The homogenates were centrifuged at 12,000× g at 4 °C for 20 min, and the supernatants were used for enzymatic assays of peroxidase (POD), catalase ascorbate (CAT), ascorbic acid peroxidase (APX) and superoxide dismutase (SOD) activities. CAT activity was measured as the decline in absorbance at 240 nm due to the decrease in the extinction of H₂O₂. POD activity was determined by assessing the rate of guaiacol oxidation in the presence H₂O₂ at 470 nm, SOD activity was measured by measuring its ability to inhibit the photochemical reduction of nitroblue tetrazolium [26]. APX activity was determined by the decrease in absorbance at 290 nm as ascorbate was oxidized [27].

$$\mathrm{POD}=\frac{(\Delta {\mathrm{A}}_{470}\times {\mathrm{V}}_{\mathrm{T}})}{0.01\times \mathrm{W}\times {\mathrm{V}}_{\mathrm{s}}\times \mathrm{t}}$$

(1)

$$\mathrm{CAT}=\frac{(\Delta {\mathrm{A}}_{240}\times {\mathrm{V}}_{\mathrm{T}})}{0.01\times \mathrm{W}\times {\mathrm{V}}_{\mathrm{s}}\times \mathrm{t}}$$

(2)

$$\mathrm{APX}=\frac{(\Delta {\mathrm{A}}_{290}\times {\mathrm{V}}_{\mathrm{T}})}{0.01\times \mathrm{W}\times {\mathrm{V}}_{\mathrm{s}}\times \mathrm{t}}$$

(3)

$$\mathrm{SOD}=\frac{{(\mathrm{A}}_{\mathrm{ck}}{-\mathrm{A}}_{\mathrm{E}})\times \mathrm{V}}{\frac{1}{2}{\times \mathrm{A}}_{\mathrm{ck}}{\times \mathrm{W}\times \mathrm{V}}_{\mathrm{t}}}$$

(4)

2.6. Determination of Lipid Peroxidation

An amount of 0.5 g of fresh samples were homogenized in 5 mL 5.0% (w/v) trichloroacetic acid (TCA) at 4 °C. The homogenates were centrifuged at 3000 r/min for 10 min. An amount of 1.5 mL of the supernatant was added to 4 mL 20% TCA containing 0.5% (w/v) thiobarbituric acid (TBA). The mixture was incubated at 100 °C for 30 min, and the reaction was stopped by quickly placing the sample in an ice bath. The cooled mixture was centrifuged at 10,000× g for 10 min, and the absorbance of the supernatant was read at 450, 532, and 600 nm [28].

$${\mathrm{C}}_{\mathrm{MDA}}=6.45\left({\mathrm{A}}_{532}-{\mathrm{A}}_{600}\right)-0.56{\mathrm{A}}_{450}$$

2.7. Determination of Membrane Injury (MI)

Briefly, a total of 2 g of fresh samples were submerged in 10 mL of deionized water in a tube and maintained at 25 °C for 1 h and was recorded as (R1), and the same weight of fresh samples was boiled in the water bath for 40 min and cooled to 25 °C, which was recorded as (R2). The degree of injury (ID) of leaves was measured by relative conductivity (RC) with the following calculation formula: RC = R1/R2 × 100% [29].

2.8. Determination of Proline and Soluble Sugar Contents

0.3 g of fresh leaves were placed in a tube and boiled in a solution of 3% (w/v) 5-sulfosalicylic acid for 10 min and cooled to 25 °C and passed through filter paper after homogenization. The homogenized samples were transferred into a 10 mL test tube to which 2 mL glacial acetic acid, 2 mL acid ninhydrin, and 4 mL Toluene was added, and left at 100 °C water bath for 40 min, and the reaction was terminated in ice. The upper phase was taken, and the absorbance was measured the toluene control at a wavelength of 520 nm in the spectrophotometer The Proline content was determined using a calibration curve [30]. 0.3 g of fresh leaves were placed in a tube and boiled in distilled water for 30 min and cooled to 25 °C, and then centrifuged it at 4000× g for 10 min, The reaction included 0.5 mL of supernatant, 0.9 mL of sulfuric acid, and 0.5 mL of anthrone reagent and was incubated in a boiling water bath for 1 min. After cooling, the absorbance of solution was measured at 630 nm. The soluble sugar content was calculated from a standard curve prepared by using glucose [31].

2.9. Statistical Analysis

All data were represented by an average of four replicate measurements with standard error, and statistical analyses were performed using the statistical SPSS 19.0 software (IBM Co., Armonk, NY, USA). Based on the ANOVA results, a DUNCAN-test for mean comparison was performed. Sigma Plot version 10.0 software (Systat Software Inc., San Jose, CA, USA) was used for data presentation in graphics.

3. Results

3.1. Plant Growth of Wheat Seedlings under Salt-Alkaline Stress

Salt and alkali stress can inhibit the growth of wheat seedlings, the harmful effects caused by alkali stress are greater than those of salt stress at the same concentration. As shown in Figure 1, after treatment with salinity and alkalinity stress, the wheat seedlings were significantly suppressed. As shown in Figure 1A,E, salt and alkali stress reduced the aboveground and underground fresh weight of wheat seedlings. The aboveground and underground fresh weights of the wheat seedlings in the C treatment were significantly lower than those of the wheat seedlings in the A and B treatments. In addition, as shown in Figure 1B,F, the aboveground and underground biomasses of wheat seedlings decreased more in the C treatment than in the A and B treatments. The plant height and root length of the wheat seedlings were lower in the C treatment than in the A and B treatments (Figure 1C,G). Figure 1D shows that the leaves of the wheat seedlings were yellow, and the whole plant were wilted in the C treatment. The leaves of wheat seedlings were pale green in the B treatment, and the wheat seedlings grew well in the CK and A treatments.

3.2. Chlorophyll Content of Wheat Seedlings under Salt-Alkaline Stress

Chlorophyll content is a key index to measure the salt–alkali tolerance of plants. Salt and alkali stress can reduce the chlorophyll content of wheat seedlings. As shown in Figure 2, after treatment with salinity and alkalinity stress, the chlorophyll content of wheat seedling leaves decreased, and the contents of wheat seedling leaves were lower under alkali stress than under salt stress. Salt and alkali stress reduced the chlorophyll a content in wheat seedling leaves; the chlorophyll a content was lower in the C treatment than in the A and B treatments (Figure 2A). Salt and alkali stress also reduced the chlorophyll b content in wheat seedling leaves; the chlorophyll b content was lower in the C treatment than in the A and B treatment (Figure 2B). As shown in Figure 2D carotenoids content was highest in A treatment, lowest in C treatment. Salt and alkali stress reduced the total chlorophyll content in wheat seedling leaves; the total chlorophyll content was lower in the C treatment than in the A and B treatments (Figure 2E). As shown in Figure 2C, the chlorophylls extract from the leaves of wheat seedlings were colorless in the C treatment and were yellowish green in the B treatment; the color of the chlorophylls extract from wheat seedlings leaves were green had little difference in the CK and A treatments.

3.3. Gas Exchange of Wheat Seedlings under Salt-Alkaline Stress

Salt and alkali stress had a significantly adverse effect on plant photosynthesis. All values decreased under saline and alkaline conditions except Ci in the A treatment. Figure 3 shows that the photosynthetic parameters in wheat seedling leaves were subjected to salt and alkali stress. The net photosynthetic rate (Pn) of wheat seedling was affected in the A, B, and C treatments, and the Pn was lower in the C treatment than in the A and B treatments (Figure 3A). As seen from Figure 3B, salt and alkali stress reduced the stomatal conductance (Gs), Gs was higher in the A and B treatments than in the C treatment. The Ci value of wheat seedlings was decreased under different treatments except for under the A treatment; the lowest Ci value was in C treatment (Figure 3C). Salt and alkali stress also reduced the transpiration rate (Tr) of wheat seedlings, and Tr was lower in the C treatment than in the A and B treatments. As seen from the above results, compared with neutral salt stress, alkaline salt stress has a stronger inhibition effect on plant photosynthesis at the same concentration.

3.4. Ion Content of Wheat Seedlings under Salt-Alkaline Stress

Salt and alkali stress can cause ion accumulation and break the ion balance of plants. To further investigate the effect of salt and alkali stress on the ion balance of wheat seedlings, the contents of sodium and potassium ions in roots, stems, and leaves of wheat seedlings under salt and alkali stress were measured. As shown in Figure 4A, salt and alkali stress significantly increased Na⁺ in roots, stems, and leaves of wheat seedlings. The accumulation of Na⁺ in the stems and leaves was higher in the C treatment than in the A and B treatments; however, there was no significant difference in roots Na⁺ accumulation in the A, B, and C treatments. Figure 4B shows that K⁺ accumulation was significantly decreased under salt and alkali stress. K⁺ accumulation in the roots of wheat seedlings significantly decreased in the A, B, and C treatments; the lowest K⁺ accumulation in the roots was in the C treatment. As shown in Figure 4B, K⁺ accumulation in the leaves of wheat seedlings also decreased in the A, B, and C treatment; the lowest K⁺ accumulation in the leaves was also in the C treatment. K⁺ accumulation in the stems of wheat seedlings did not show significant difference in the A, B, and C treatments. As shown in Figure 4C, the Na⁺/K⁺ ratio under salt and alkali stress significantly increased. The Na⁺/K⁺ ratio was higher in the roots than in the stems and leaves in the same treatment, and the Na⁺/K⁺ ratio in the roots was higher in the C treatment than in the A and B treatments.

3.5. Osmotic Substance of Wheat Seedlings under Salt-Alkaline Stress

Plants accumulated osmotic substances to copy with osmotic stress, which was induced by salt and alkali stress. As shown in Figure 5A, proline accumulation in wheat seedlings was significantly increased, especially in the C treatment; proline accumulation was the highest. Figure 5B shows that wheat seedlings accumulated more soluble sugars in the A and B treatments than in CK, with the highest accumulation of soluble sugars in the B treatment, the lowest accumulation of soluble sugars was in the C treatment. As shown in Figure 5C, the proline extracted from the leaves of wheat seedlings were crimson in the C treatment and were rosiness and pink in the A and B treatments; the color of the proline extracted from wheat seedlings leaves were almost colorless in CK.

3.6. Oxidation Resistance System of Wheat Seedlings under Salt-Alkaline Stress

The oxidation resistance system of plants can be significantly affected by salt and alkali stress. As shown in Figure 6, salt and alkali stress significantly affected the activities of SOD, POD, CAT, and APX, the MDA accumulation and MI values. The POD activity also showed an upward trend in the A, B, and C treatments, and the highest activity of POD was also shown in the C treatment (Figure 6A). The SOD activity showed an upward trend in the A, B, and C treatments, and the highest activity of SOD was in the C treatment (Figure 6C). As shown in Figure 6B, the CAT activity increased at first and then decreased, the highest activity of CAT was in the A treatment, and the lowest was in the C treatment. Figure 6D shows that the APX activity also increased at first and then decreased, the highest activity of APX was in the A treatment, and the lowest was in the C treatment. The MDA content of wheat seedlings showed an upward trend in in the A, B, and C treatments, and the highest content of MDA were in the C treatment (Figure 6E). The MI value of wheat seedlings showed upward trend in in the A, B, and C treatments, and the highest value of MI was in the C treatment (Figure 6F).

4. Discussion

Soil salinization and alkalization are great environmental problems in the world. Salinity and alkalinity stress pose threat to plant growth and reduce the yield of crops. There are many studies that have reported that alkaline produces greater inhibition in many plants than neutral salt, such as maize [32], rice [16], barley [33], and sunflower [34]. In our study, alkaline salt (NaHCO₃-Na₂CO₃) more strongly inhibited the growth of wheat seedlings than neutral salt (NaCl-Na₂SO₄) at the same concentration. Osmotic stress and ion toxicity are generally considered to be involved in salinity and alkalinity stresses [35,36], and alkalinity stress adds a high pH. Many studies have considered that this is the only key factor that induces the different adverse effects between alkaline salt and neutral salt; however, this conclusion often ignores other factors, such as CO₃²⁻/HCO₃⁻.

Plant growth reflects the life-sustaining activities of the plant, such as fresh weight, biomass, plant height, and root length, which are optimum indexes for evaluating various abiotic stresses on a plant. In the present experiment, both salinity and alkalinity stress induced a significant reduction of fresh weight, biomass, plant height, and root length in wheat seedlings. Although all stresses reduced the fresh weight, biomass, plant height, and root length, the reduction of fresh weight, biomass, plant height, and root length under alkalinity stress was at a higher level than under stress with neutral salt and neutral salt with high pH value. This suggests that alkali salt conditions have a greater injurious effect on plant growth than conditions with neutral salt and neutral salt with high pH value.

Photosynthesis is the basis of plant matter and energy source and is crucial for plant growth and development. The lower accumulation of plant fresh weight and biomass is mainly caused by the inhibition of photosynthesis. Generally, chlorophyll is the main photosynthetic pigment, which plays an important role in the absorption, transmission, and transformation of light energy in plants. The contents of chlorophylls can be used to determine the ability of plants’ tolerance [37]. In the present study, a significant decrease in chlorophyll a, chlorophyll b, carotenoid, and total chlorophyll was observed under salt and alkali stress, especially the level of decrease was greater in alkali stress treatment than in neutral salt and neutral salt with high pH value treatments. The photosynthetic characteristics of plant seedlings under abiotic stress can be optimum indexes to determine the ability of plants coping with salt–alkaline stress [38]. In this experiment, the Pn, gs, Ci, and Tr of wheat seedlings were decreased under salt and alkali stress. Pn, gs, Ci, and Tr were decreased more in alkaline salt stress than stress with neutral salt and neutral salt with high pH value. The results suggested that compared with salt stress, alkali stress has a stronger inhibitory effect on wheat photosynthesis. In this experiment, compared with salt stress, the net photosynthetic rate of wheat under alkali stress was lower, mainly due to the stomatal closure of wheat under alkali stress, thus reducing water the transpiration rate and intercellular carbon dioxide concentration. Therefore, the decrease in photosynthesis of wheat under alkali stress was mainly caused by stomatal factor. Similarly, the decrease in chlorophyll content is also an important factor causing the inhibition of photosynthesis under alkali stress.

Saline and alkaline stress can induce ion toxicity to plants when too many toxic ions enter plant cells, which can harm the plant cytoplasm and organelles; among them, Na⁺ is the main toxicity ion due to the similarity in size of the hydrated ionic radii of Na⁺ and K⁺, which makes them difficult to be discriminated [39]. In this experiment, salt–alkaline stress affected the content of Na⁺, K⁺, and Na⁺/K⁺ in the roots, stems, and leaves of wheat seedlings. In this study, the Na⁺ contents of root, stem, and leaf of wheat seedlings in alkaline salt treatment and both neutral saline-treatments were much higher than in control treatment. The stems and leaves Na⁺ contents of wheat seedlings in alkaline salt treatment were higher than in neutral salt and neutral salt with high pH value treatments. Transportation of Na⁺ from the roots to the stems and from the stems to the leaves is an important physiological response for plant adaption to salt–alkaline stress. This is consistent with previous research result [40]. Additionally, this result suggested that CO₃²⁻ and HCO₃⁻ of alkaline salt can increase more Na⁺ transportation from roots to stems and leaves than pH value in neutral salt treatment. In this study, we found that the roots and leaves K⁺ contents were decreased under saline and alkaline stress. The wheat seedlings showed lower K⁺ contents in roots and leaves in alkaline stress treatment than in neutral salt and neutral salt with high pH value treatments, and the K⁺ contents in stems did not significantly change in different treatments. We also found that Na⁺/K⁺ of roots, stems, and leaves were higher under saline and alkaline stress, and Na⁺/K⁺ in roots was higher than in stems, leaves. Na⁺/K⁺ in roots was higher in alkaline treatment than in neutral salt and neutral salt with high pH treatments. The reason may be that alkali stress weakened the exchange activity of the Na⁺/H⁺ antiporter in the root plasma membrane, which enhanced the accumulation of Na⁺ in the roots of wheat seedlings, and further promoting the transportation of Na⁺ from the roots to the stems and from the stems to the leaves in wheat seedlings [41].

Saline and alkaline ions can reduce the soil water potential, which makes it difficult for plants to absorb water, and then causes osmotic stress. In this study, under saline–alkali stress, more osmotic regulators were accumulated in the leaves of wheat seedlings, and more proline was accumulated in the leaves of wheat seedlings in alkaline salt treatment than in neutral salt and neutral salt with high pH treatments at same concentration, but the content of soluble sugar was decreased. The accumulation of more proline may be that under alkali stress, wheat seedlings must accumulate more proline to cope with osmotic stress, while the decrease in soluble sugar content is mainly due to the fact that under alkali stress the soluble sugar synthesis system of wheat seedlings exceeds the itself tolerance limit, so the soluble sugar content decreased.

In normal growth conditions, plants can keep a dynamic balance between the production and elimination of reactive oxygen species in plants. However, when subjected to salt–alkaline stress, this dynamic balance can be broken out, and plants will accumulate too many reactive oxygen species, which will cause oxidative stress to plants and then produce too much MDA damage to cell membrane [42]. There are many studies that have demonstrated that salt and alkali stress can induce oxidative stress to plants [43]. In order to cope with oxidative stress caused by salt–alkaline stress, plants often increase the activities of various antioxidant enzymes in plants to eliminate excessive reactive oxygen species, and then reduce the damage induced by oxidative stress. In this study, antioxidant enzymes activities of wheat seedlings were higher in alkaline treatment than in treatments of neutral salt, neutral salt with high pH value. The activities of POD and SOD in wheat seedlings increased, and their activities were the highest under alkaline salt stress. The activities of CAT and APX in wheat seedlings increased under stresses with neutral salt and neutral salt with high pH value, but then decreased under alkaline salt stress. The main reason for this phenomenon is that alkaline salt stress exceeds the tolerance of the wheat antioxidant system, which reduces the activities of CAT and APX. In this study, we also found that alkaline stress led to more accumulation of the MDA content of wheat seedlings than stress with neutral salt and neutral with high pH value, and the MI value was highest in the alkaline treatment. The above results suggested that CO₃²⁻ and HCO₃⁻ of alkaline salt can induce more damage to wheat seedlings than the high pH value in the neutral treatment.

In summary, saline–alkaline stress can cause damage to wheat seedlings, and alkali stress can induce more damage than stress with neutral salt and neutral salt with high pH value. Previous studies thought that the main reason for this phenomenon is the high pH value. However, in this study, we found that high pH is not the only key factor for alkaline salt causing more damage to plants than neutral salt, because treatment with neutral salt with high pH induces less damage to plants than alkaline salt treatment. Therefore, we should think that the main key factor may be CO₃²⁻ and HCO₃⁻, and this requires further studies.

5. Conclusions

Summarizing the results of this study, we found that both salt and alkali stress inhibited plant growth in wheat seedlings, However, alkali stress inhibited wheat growth more strongly than stress with neutral salt and neutral salt with high pH value at the same Na⁺ concentration. Salt and alkali stress can suppress the photosynthesis of wheat. The main performance is reduction of the Pn, gs, Ci, Tr, and chlorophyll content of wheat seedlings. Similarly, alkali stress inhibited wheat photosynthesis more strongly than stress with neutral salt and neutral salt with high pH value. Salt and alkali stress can increase the content of Na⁺, decrease the content of K⁺, and increase the content of Na⁺/K⁺ in the roots, stems, and leaves of wheat seedlings compared with control treatment. And this trend is more significant under alkaline stress. Based on the results obtained from this study, we can draw the conclusion that the only key factor inducing the difference between salt and alkali stress may be not high pH, but single salt ion poisoning caused by CO₃²⁻ and HCO₃⁻ anions. Therefore, the toxic effect of CO₃²⁻ and HCO₃⁻ on plants should be the focal point of research in the future.