Antioxidant defense responses of hulled wheat varieties to the addition of sodium and potassium salts and exogenous glycine-betaine, and evaluation of the usability of these hulled wheats in the remediation of saline soils

doi:10.21203/rs.3.rs-4368507/v1

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Antioxidant defense responses of hulled wheat varieties to the addition of sodium and potassium salts and exogenous glycine-betaine, and evaluation of the usability of these hulled wheats in the remediation of saline soils

https://doi.org/10.21203/rs.3.rs-4368507/v1

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Soil salinization is an important factor that reduces crop yield by causing soil degradation, severely restricting plant growth and development. We examined the usability of four types of ancient ancestral hulled wheat plants in saline soils based on the enzymatic and non-enzymatic responses of the wheat plants to salt stress and evaluated the ability of these varieties to be used in saline soils. We investigated the extent to which four different ancient hulled wheat species from Turkey can be cultivated in saline soils based on their antioxidant response to different salt stresses and the contribution of exogenously applied GB to this response. The fresh and dry weights of the roots and leafy stems of the plants; plant height; leaf length; chlorophyll and carotene contents; total protein, proline and MDA accumulation; and antioxidant enzyme activities were also analyzed. Concentrations of both sodium and potassium chloride salts above 100 mM cause high levels of stress in hulled wheat. When sodium and potassium chloride salts are given together, this stress decreases by approximately 20–30%, and when plants are supplemented with exogenous glycine-betaine, almost all the negative effects of stress disappear. For successful plant growth in saline/sodic environments, it seems that both the type of salt and the type of plant grown in the salty environment are important rather than the amount of salt in the environment. Specifically, in soils where salinity is caused by sodium, ancient hulled wheat is grown rather than modern cultivated wheat, and if potassium-based fertilizers are applied to plants in addition to nitrogen-based fertilizers, the plant can control the ingress of salt into the cell and cope with salinity stress by maintaining the intracellular K+/Na + balance.

Agronomy

Salt stress

Glycine-betaine

ROS

Hulled wheat

Antioxidant

Soil is a nonrenewable resource that cannot be recovered once lost. Soil salinization is the second major factor causing soil degradation after soil erosion. Soil salinization severely restricts plant growth and development, affects crop yield, and has caused a decrease in agricultural activities for more than 10,000 years (Yang and Guo 2018; Shahid et al. 2018). Soil salinity is dynamic and spans more than 100 countries globally; no continent is completely free of salinity. Salinization causes the loss of approximately 2000 hectares of arable land per day worldwide, as well as a 10–25% loss in crop yield, and at advanced levels, salinization can desertify the soil and cause complete crop loss (Shahid et al. 2018).

Soil salinity is a measure of the concentration of all soluble salts in soil water. The main soluble mineral salts are sodium (Na⁺), calcium (Ca²⁺), magnesium (Mg²⁺), potassium (K⁺), chloride (Cl⁻), sulfate (SO₄ ^− 2), bicarbonate (HCO₃⁻), carbonate (CO₃ ^− 2), and nitrate (NO₃⁻) (Tanji 1990). The misuse of fertilizers, excessive irrigation, and industrial pollution are the major causes of widespread soil salinity and pose serious threats to agricultural productivity and food security for both humans and animals (Ouhibi et al. 2014; Hoque et al. 2022). Salinization of soils can affect ecosystems such that they can no longer serve as “environmental stewards” to their full potential. The development of crops that can grow normally in saline soils is fundamental to solving this problem. High Na⁺ and Cl⁻ concentrations in soil cause both osmotic and ionic stress, which reduces the water and nutrient uptake capacity of plants (Mahajan et al. 2008; Ismail et al. 2014, 2020).

Wheat species that have remained unchanged for more than a hundred years are called ancient wheat (Dinu et al. 2018). Most ancient wheat plants have a hull that must be separated from the grain in the mill; therefore, these plants are also known as hulls (Longin et al. 2015). The most common ancient wheat species include Einkorn-Einkorn (Triticum monococcum L. and Triticum boeoticum Boiss), emmer (Triticum turgidum L. spp. dicoccum Schrank), and spelt (Triticum aestivum spp. spelta) wheat plants (Temizgul et al. 2024). T. monococcum, one of the first plants in the Fertile Crescent, was bred from T. boeoticum (Dvorak et al. 1998). Einkorn is a diploid wheat plant that is currently grown in limited regions around the world (Arzani and Ashraf 2017). Emmer (T. turgidum L. spp. dicoccum Schrank) is a tetraploid wheat plant that is a domesticated form of T. turgidum spp. dicoccoides (wild emmer wheat) (Dhanavath and Prasada Rao 2017). Spelt wheat (Triticum aestivum ssp. spelta) probably developed as a result of natural hybridization with two sets of chromosomes (genome DD) between goat grass species (Aegilops squarrosa) and emmer wheat (Lacko-Bartosova and Otepka 2001). Therefore, spelt wheat (T. aestivum subsp. spelta) is a hexaploid wheat and is the ancestor of common free-threshing wheat (Schober et al. 2006). Einkorn (T. monococcum and T. boeoticum), emmer (T. dicoccum), and red wheat (T. speltoides) form a bridge between modern bread, durum wheat, and wild wheat and represent the four main types of hulled wheat produced.

Hulled wheat plants are typical examples of an underutilized plant species. These species are highly important in terms of food security and local cultural value, but they are relatively unknown and undervalued in commercial production. Often neglected by researchers and policy makers, many underutilized species are in danger of extinction for various reasons, ranging from agricultural and genetic factors to economic and cultural factors. Today, emmer wheat covers less than 1% of the world’s wheat area (Zaharieva et al. 2010). Previous studies have shown that emmer wheat can be grown as a suitable crop in marginal areas using organic agriculture (Konvalina and Moudrý 2007).

With the development of lifestyles, natural resources are being exploited, and degradation of the natural environment; salt stress; drought stress; waterlogging; high and low temperatures; excessive and low light intensity; radiation stress; ozone; metal and metalloid toxicity; and other organic or inorganic pollutants can cause various abiotic stress types. Environmental excess and insensitivity reduce facility efficiency, while the population is constantly rising; therefore, the need for food increases daily. To meet this increasing demand, new arable products and higher yields per unit area are needed, as well as the use of arable lands at the highest possible efficiency under adverse conditions.

In light of all this, in this study, we tried to predict to what extent four different types of ancient hulled wheat from Turkey, the homeland of wheat, can be processed in saline soils based on their antioxidant responses to different salt stresses. We also examined the role of exogenous glycine-betaine application in coping with salt stress. The enzymatic and non-enzymatic responses of the roots and leafy stems of the hulled wheat varieties to salt stress were analyzed separately.

2.1. Plant cultivation and stress management practices

In this study, four different ancient hulled wheat varieties (T. monococcum L., T. boeoticum Boiss, T. dicoccum Schrank and T. speltoides) were used as materials. The experiments were carried out in culture containers (15 cm wide × 6 cm deep) in an air-conditioning cabin under 25 ± 1°C, 70 ± 5% humidity and 14/10 light (100 µmol m^− 2s^− 1)/dark conditions. Before being placed in culture containers, the seeds were washed briefly with liquid soap and then subjected to surface sterilization with ethyl alcohol for 5 min and 5% calcium hypochlorite solution for 3 min. After the ethyl alcohol and hypochlorite applications, the seeds were washed with plenty of sterile distilled water at least 3 times. Seeds were planted in culture pots (20 pieces each). The plants were grown without any stress for 10 days, and salt stress was applied for the next 15 days. Control plants were grown with only Hoagland (Hoagland and Arnon 1950) solution for 25 days, and hoagland was frequently added to prevent the plants from becoming dehydrated. The solutions in the culture dishes were renewed daily.

The salt stress conditions used were NaCl (30-50-100-150-200 mM), KCl (30-50-100-150-200 mM), NaCl + KCl (50–50 mM and 100–100 mM), NaCl + GB (150 mM NaCl + 500 µM GB and 200 mM NaCl + 500 µM GB), KCl + GB (150 mM KCl + 500 µM GB and 200 mM KCl + 500 µM GB), and NaCl + KCl + GB (50 mM NaCl + 50 mM KCl + 500 µM GB and 100 mM NaCl + 100 mM KCl + 500 µM GB). Following the 15-day stress treatment, the roots and leaves of the plants were quickly harvested on ice molds, cut into small pieces, blended, weighed on a 0.5 and 0.25 g scale, placed in aluminum foil, and frozen in liquid nitrogen at 20°C until use. The experiments were carried out with three repetitions.

2.2. Measuring the effects of salt stress on plant growth (fresh weight, dry weight, leaf length, total plant height)

Following 15 days of stress application, the fresh and dry weights of the roots and leafy stems of all the wheat plants were weighed separately on an analytical balance, and the total plant height and length of the first leaf emerging from the stem were measured using a ruler. Plant height and leaf length were measured for each plant in the culture container, and the average plant height and length were calculated. Before the fresh weight was measured, the roots of the plants removed from the culture containers were washed with pure water to remove salt and subsequently dried with a towel. The roots and stems were separated from each other with the help of scissors, weighed on a scale, and recorded as fresh weight. For dry weight measurements, the samples were dried for 48 h in an oven set at 65°C, weighed on a scale, and recorded as dry weight.

2.3. Preparation of crude enzyme extracts and determination of protein concentrations

For the crude enzyme extracts (except for APX), the method of Yilmaz et al. (2017) was used with minor modifications. A tissue sample (0.5 g) was crushed using a prechilled mortar and pestle on ice in 2 ml of 0.1 M potassium phosphate buffer (pH 7.5) containing 1% polyvinylpyrrolidone (PVPP), 0.1% EDTA, and 15,000 × g. The supernatant was collected by centrifugation at + 4°C for 20 min and labeled the crude enzyme extract. For APX, 0.5 g of fresh tissue was ground in liquid nitrogen and suspended in 2 ml of buffer consisting of 50 mM Tris-HCl (pH 7.2), 2% PVP, 1 mM Na₂EDTA, and 2 mM ascorbate (Akbulut and Çakır 2010). Crude enzyme extracts were stored at 20°C until use. The protein concentrations were determined spectrophotometrically at 595 nm using the Bradford (1976) method. Bovine serum albumin (BSA) fraction V was used as the standard. The results were recorded as µg ml^− 1 protein.

2.4. Determination of the chlorophyll and carotene contents

The chlorophyll and carotene contents of the leaves were determined according to the methods of Yilmaz et al. (2020). Briefly, 100 mg of fresh leaf tissue was taken into a 50 ml test tube, 7 ml of dimethyl sulfoxide (DMSO) was added, and the mixture was kept in a water bath at 65°C until the color disappeared. After the color disappeared, the liquid part was transferred to a new tube, and the total volume was adjusted to 10 ml. The optical densities of the samples were read against DMSO at 647, 663, and 470 nm (with a UV-1800 Shimadzu Spectrophotometer, Shimadzu Corporation, Kyoto, Japan). The amount of chlorophyll was determined as mg gr^− 1 fresh weight (fw). Chlorophyll and carotene contents were calculated using the formulas below.

$$Chl a\left(\frac{mg gr}{gr}fw\right)=\left(\text{12,25}*A663\right)-\left(\text{2,79}*A647\right)$$

$$Chl b\left(\frac{mg}{gr}fw\right)=\left(\text{21,50}*A647\right)-\left(\text{5,1}*A663\right)$$

$$Total Chl\left(\frac{mg}{gr}fw\right)=\left(\text{7,15}*A663\right)+\left(\text{18,71}*A647\right)$$

$$Carotene\left(\frac{mg}{gr}fw\right)=\frac{\left(1000*A470\right)-\left(\text{1,82}-Chl a\right)-\left(\text{85,02}*Chl b\right)}{198}$$

2.5. Determination of enzyme activity

2.5.1. CAT activity (EC 1.11.1.6)

The method of Duman et al. (2011) was used with minor modifications to determine the catalase activity of the samples. Briefly, 20 mM sodium hydrogen phosphate (NaHPO₄) buffer (pH 7.5), 15 mM hydrogen peroxide (H₂O₂) and 50 µl of crude enzyme extract were used. Measurements were performed on a Shimadzu UV-1800 model cooled spectrophotometer (Shimadzu Corporation, Kyoto-Japan) using a quartz cuvette with a cuvette volume of 2 ml. Buffer without the enzyme extract was used as a blank. The reaction started with the addition of H₂O₂ to the cuvette, and a decrease in absorbance was observed for 3 min at 25°C. The molar absorption coefficient (Ɛ) of H₂O₂ for catalase is 40 mM⁻1 cm^− 1. The specific activity of the samples was calculated using the following formula. The results were expressed as mg^− 1 protein.

$$SA \left(\frac{unite}{mg}protein\right)=\frac{\varDelta \text{A}\text{b}\text{s}/\text{m}\text{i}\text{n}}{40 }*\frac{\text{c}\text{r}\text{u}\text{d}\text{e} \text{e}\text{n}\text{z}\text{y}\text{m}\text{e} \text{v}\text{o}\text{l}}{\text{c}\text{u}\text{v}\text{e}\text{t}\text{t}\text{e} \text{v}\text{o}\text{l} }*\frac{1}{\text{p}\text{r}\text{o}\text{t} \text{c}\text{o}\text{n}\text{s} }*1000$$

2.5.2. Superoxide dismutase (SOD) (EC 1.15.1.1) activity

To determine the superoxide dismutase activity of the samples, the method of Duman et al. (2011) was used with minor modifications. Briefly, a reaction mixture containing 20 mM sodium phosphate buffer (pH 7.4), 0.1 mM EDTA, 10 mM methionine, 0.1 mM NBT, and 0.005 mM riboflavin was prepared in a lightproof amber bottle. Then, an SOD solution containing 0.1 mg ml^− 1 SOD was prepared, and standard tubes containing 10–500 ng ml^− 1 SOD were prepared from this solution. Three milliliters of the prepared reaction mixture was added to screw-capped glass tubes, and 20 µl of crude enzyme extract was added to each tube, after which the mixture was vortexed. The reaction mixture was added to the standards and the samples (3 ml each) and vortexed. Two additional tubes (a light control and a blank tube) containing only the reaction mixture were prepared along with the sample and standards. One of these two tubes was covered with aluminum foil to prevent light exposure before starting the studies, and the other was exposed to light along with the samples. Samples and standards were kept under a fluorescent lamp (150 µmol m^− 2s^− 1) at a distance of 20 cm from the lamp for 15 min. At the end of the period, samples and standards were collected and read against the blank in a spectrophotometer at 560 nm. The studies were conducted three times in duplicate. The % inhibition values of the samples were used in the SOD activity calculation. One SOD unit is the amount of enzyme that provides 50% inhibition. The percentage inhibition value was calculated according to the formula below.

$$Inh\%=\frac{\text{L}\text{i}\text{g}\text{h}\text{t} \text{C}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l} \text{A}\text{b}\text{s}.-\text{S}\text{a}\text{m}\text{p}\text{l}\text{e} \text{A}\text{b}\text{s}}{\text{L}\text{i}\text{g}\text{h}\text{t} \text{C}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l} \text{A}\text{b}\text{s}. }*100$$

A logarithmic graph was generated for the enzyme concentrations versus percentage inhibition. A new graph was generated by taking the logarithms of the standard SOD enzyme concentrations used in the graph and using the percent inhibition values exactly. The SOD concentrations of the samples were determined using the equation of the line obtained in the graph. The results were calculated as units of mg^− 1 protein.

2.5.3. Ascorbate peroxidase (APX) (EC 1.11.1.11) activity

To determine the APX activity of the samples, the method of Akbulut and Çakır (2010) was used with minor modifications. The reaction mixture contained 50 mM potassium phosphate buffer (pH 7.0), 0.20 mM ascorbate, 10 mM H₂O₂, and the enzyme extract (50 µl) in a final volume of 1 ml. The reaction was initiated by the addition of H₂O₂. The decrease in the ascorbate concentration was recorded at 290 nm for 3 min. Buffer was used as a blank, and the reaction was performed in a quartz cuvette at 25°C. Enzyme activity was calculated using the following formula as the amount of H₂O₂ consumed using the extinction coefficient (Ɛ) of H₂O₂ (2.8 mM^− 1 cm^− 1 at 240 nm): The results were expressed as units of mg^− 1 protein.

$$SA \left(\frac{unite}{mg}protein\right)=\frac{\varDelta \text{A}\text{b}\text{s}/\text{m}\text{i}\text{n}}{\text{2,8}}*\frac{\text{c}\text{r}\text{u}\text{d}\text{e} \text{e}\text{n}\text{z}\text{y}\text{m}\text{e} \text{v}\text{o}\text{l}}{\text{c}\text{u}\text{v}\text{e}\text{t}\text{t}\text{e} \text{v}\text{o}\text{l} }*\frac{1}{\text{p}\text{r}\text{o}\text{t} \text{c}\text{o}\text{n}\text{s} }*1000$$

2.5.4. Glutathione reductase (GR) (EC 1.6.4.2) activity

GR activity was measured according to the methods of Misra and Gupta (2006). The reaction was initiated by adding 50 µl of crude enzyme extract to 100 mM potassium phosphate (KHPO₄, pH 7.5) buffer supplemented with 0.1 mM Na₂EDTA, 0.1 mM nicotinamide adenine dinucleotide phosphate (NADPH), 1 mM oxidized glutathione (GSSG), and 2 ml of cuvette. A decrease in absorbance was observed for 5 min at 25°C. Buffer was used as a blank, and the reaction was performed in a quartz cuvette. The molar absorption coefficient (Ɛ) of NADPH at 340 nm was 6.2 mM⁻1 cm^− 1. The specific activity of the samples was calculated using the following formula. The results were expressed as mg^− 1 protein.

$$SA \left(\frac{unite}{mg}protein\right)=\frac{\varDelta \text{A}\text{b}\text{s}/\text{m}\text{i}\text{n}}{\text{6,2} }*\frac{\text{c}\text{r}\text{u}\text{d}\text{e} \text{e}\text{n}\text{z}\text{y}\text{m}\text{e} \text{v}\text{o}\text{l}}{\text{c}\text{u}\text{v}\text{e}\text{t}\text{t}\text{e} \text{v}\text{o}\text{l} }*\frac{1}{\text{p}\text{r}\text{o}\text{t} \text{c}\text{o}\text{n}\text{s} }*1000$$

2.5.5. Glutathione S-transferase (GST) (EC 2.5.1.18) activity

GST activity was determined according to the methods of Yilmaz et al. (2020). The reaction mixture was 100 mM potassium containing 0.1 mM EDTA, 0.1 mM NADPH, 1 mM glutathione (GSH), and 1 mM 1-chloro-2,4-dinitrobenzene (CDNB). The reaction was performed in phosphate (KHPO₄, pH 7.5) buffer. After adding 50 µl of crude enzyme extract to the reaction medium, the nonspecific activity was allowed to increase for 5 min, after which the change in absorbance was observed for 5 min at 340 nm in a 2 ml quartz cuvette at 25°C. The molar absorption coefficient (Ɛ) of NADPH at 340 nm was 6.2 mM⁻1 cm^− 1. The specific activity of the samples was calculated using the following formula. The results were expressed as mg^− 1 protein.

2.6. Lipid peroxidation (LPO) (MDA)

The effect of salt application on lipid peroxidation was determined by evaluating the thiobarbituric acid reactive substances (TBARS) content in the tissues. TBARS are formed as a byproduct of lipid peroxidation (i.e., as degradation products of fats) and can be detected by TBARS analysis using thiobarbituric acid as the reagent. Reactive oxygen species (ROS) are difficult to measure directly because they have an extremely short half-life. Instead, attempts have been made to estimate the degree of damage to membranes by measuring various products of damage produced by oxidative stress, such as TBARS (Jardine et al. 2002). TBARS analysis measures malondialdehyde (MDA) present in samples as well as malondialdehyde produced from lipid hydroperoxides by the hydrolytic conditions of the reaction [Janero 1990].

The method of Madhava and Sresty (2000) was used with minor modifications to determine the lipid peroxidation of the samples. Briefly, after 0.5 g of fresh tissue was crushed in 5 ml of 0.1% trichloroacetic acid (TCA) solution, the homogenate was centrifuged at 12,000 × g for 5 min. For every 1 ml of supernatant, 4 ml of 20% TCA solution containing 0.5% 2-thiobarbituric acid (TBA) was added. The mixture was placed in screw-capped centrifuge tubes and incubated in boiling water for 30 min, followed by rapid cooling under tap water. After the tubes were centrifuged at 12,000 × g for 15 min, the OD532 and OD600 were measured with a Shimadzu UV-1800 spectrophotometer. A 20% TCA solution containing 0.5% TBA was used as a blank for reading. Calculations were performed by subtracting the OD₆₀₀ (background correction) from the OD₅₃₂. The molar absorption coefficient (Ɛ) for MDA was 155 mM⁻1 cm^− 1. The results were determined as TBARS (nmolgr^− 1 fw).

2.7. Determination of the proline concentration

The amount of proline in the samples was determined according to the methods of Temizgul et al. (2016). Fresh tissue (0.25 g) was crushed on ice with a precooled mortar and pestle in 5 ml of 3% sulphosalicylic acid and centrifuged at 5,000 × g for 10 min at + 4°C. Two milliliters of supernatant was taken into screw cap glass tubes, 2 ml of acid ninhydrin was added, and the tubes were vortexed immediately. After adding 2 ml of 96% acetic acid and 1 ml of 3% sulphosalicylic acid to this mixture, the mixture was vortexed again, and the tubes were left to boil in boiling water for 1 h. Then, the tubes were rapidly cooled under tap water, and 4 ml of toluene was added and mixed by vortexing thoroughly. The tubes were read against toluene at OD₅₂₀ nm after being left in the dark for 1–2 hours for the toluene to absorb the color. In addition to the samples, standards containing 0.01 µM-1.5 mM proline (10 standards) were subjected to the same processes, and the equation of the line obtained from this standard was used to calculate the amount of proline in the samples. The amount of proline in the samples was calculated as nmolgr^− 1 fw.

2.8. Statistical evaluation of the results

Statistical analyses of the data obtained from various analyses and measurements were performed to test the main effects and interactions between the factors examined in this study (salt doses, wheat varieties, plant parts) using one-way (with SPSS 28.0), two-way (with SPSS 28.0) and three-way analysis of variance (ANOVA) (GraphPad Prism version 10.0.0). Three-way analysis of variance (ANOVA) was performed for salt dose (19 doses), plant part (root and leafy stem), and wheat variety (T. monococcum; T. dicoccum; T. speltoides; and T. boeoticum). Multiple comparisons were also made using Duncan’s multiple range test, the least significant difference test (LSD), and the Tukey test at the P ≤ 0.05 significance level to test the significance between different levels of the factors. All the experiments were repeated three times with three replicates. The values are expressed as the means ± SDs. Genotype biplot charts were created to visually visualize the relationships between the applications and the examined features. Biplot analysis was performed using Genstat 12.0 statistical software. Genotype–trait (GT) biplot plots were created to determine which genotype(s) stood out with which trait(s) (Yan, 2014). Different lowercase letters (such as a, b, c, d, e) on the tables and figures indicate statistical significance between the averages.

3.1. Effects of salt stress on plant growth

3.1.1. Effect of salt stress on the fresh weight of plant roots and leafy stems

In hulled wheat varieties under salt stress, decreases in fresh weight were observed in parallel with increasing salt doses in the roots (from 5–30%) (Table S1.1). In particular, after the application of 200 mM NaCl or 200 mM KCl, significant decreases in plant fresh weight were observed (30.41% and 30.21%, respectively). However, increases in root biomass on a fresh weight basis were also observed under salt stress, and the greatest increase was obtained from the application of 100 mM NaCl + 100 mM KCl + 0.5 mM GB (13%) (Table 1). This was followed by 50 mM NaCl + 50 mM KCl + 0.5 mM GB (8.8%), 30 mM KCl (5.8%), and 30 mM NaCl (3.98%). Although individual applications of sodium and potassium chloride cause a general decrease in the fresh weight of wheat roots, when sodium and potassium chloride salts are applied together, this decrease in the fresh weight of the roots is approximately 50% (Table 1). Similarly, GB application reduced the decrease in fresh weight due to salt stress by approximately 50%, and the application of glycine-betaine in combination with potassium chloride reduced the decrease in fresh weight of the roots by approximately 70%. While sodium chloride and potassium chloride salts reduce the root biomass of wheat when applied individually, the damage caused when these two salts are applied together decreases, and if additional glycine-betaine support is externally administered (0.5 mM), the root biomass of wheat increases (Table 1).

Table 1

Effects of salt application on plant growth and the antioxidant defense system in roots (all wheat varieties were evaluated together)
Salt Applications	FW(gr)	DW(gr)	Dw/Fw (%)	TP	SOD	CAT	GR	GST	APX	PRO	MDA
Control	4,17 ± 0,26^b	0,58 ± 0,07^c	13,91^gh	316,33 ± 27,04^a	0,972 ± 0,11^de	0,016 ± 0,01^a	0,089 ± 0,03^a	0,106 ± 0,03^b	0,279 ± 0,03^a	68,99 ± 11,70^a	19,38 ± 2,16^a
30 mM NaCl	4,33 ± 0,11^b	0,65 ± 0,6^d	15,01^h	372,33 ± 36,96^c	1,047 ± 0,08^h	0,040 ± 0,01^d	0,155 ± 0,05^e	0,116 ± 0,02^bc	0,328 ± 0,02^c	275,75 ± 68,69^d	45,67 ± 7,55^b
50 mM NaCl	3,96 ± 0,12^b	0,48 ± 0,03^c	12,12^g	392,45 ± 24,45^d	1,013 ± 0,07^f	0,046 ± 0,01^e	0,207 ± 0,07^h	0,142 ± 0,02^d	0,361 ± 0,02^e	334,64 ± 60,98^e	62,27 ± 9,87^c
100 mM NaCl	3,63 ± 0,09^ab	0,26 ± 0,03^b	7,16^ef	391,75 ± 31,26^d	1,016 ± 0,09^fg	0,040 ± 0,01^d	0,221 ± 0,09ⁱ	0,133 ± 0,02^cd	0,323 ± 0,01^c	442,25 ± 48,18^g	179,50 ± 22,73^e
150 mM NaCl	3,32 ± 0,10^ab	0,10 ± 0,01^a	3,01b^c	373,75 ± 40,37^c	0,941 ± 0,06^c	0,032 ± 0,01^c	0,174 ± 0,07^f	0,181 ± 0,09^e	0,299 ± 0,01^ab	257,00 ± 17,68^c	283,00 ± 31,27^fg
200 mM NaCl	2,90 ± 0,05^a	0,10 ± 0,02^a	3,45^c	328,75 ± 21,76^b	0,855 ± 0,05^a	0,023 ± 0,01^ab	0,103 ± 0,03^b	0,086 ± 0,01^a	0,271 ± 0,01^a	144,92 ± 13,40^b	379,17 ± 23,74^h
30 mM KCl	4,41 ± 0,6^b	0,59 ± 0,06^c	13,38^g	442,75 ± 25,24^f	0,903 ± 0,03^b	0,031 ± 0,01^bc	0,141 ± 0,05^d	0,108 ± 0,01^b	0,318 ± 0,02^bc	261,58 ± 27,28^c	40,42 ± 3,82^b
50 mM KCl	4,08 ± 0,09^b	0,53 ± 0,06^c	12,99^g	491,33 ± 18,40^h	1,012 ± 0,11^f	0,038 ± 0,02^cd	0,185 ± 0,06^fg	0,127 ± 0,02^c	0,336 ± 0,03^cd	418,00 ± 47,61^g	58,75 ± 8,48^bc
100 mM KCl	3,64 ± 0,5^ab	0,30 ± 0,06^b	8,24^f	469,00 ± 10,73^g	1,028 ± 0,13^g	0,031 ± 0,02^bc	0,170 ± 0,06^ef	0,120 ± 0,02^c	0,312 ± 0,03^b	511,42 ± 44,04ⁱ	173,50 ± 20,72^e
150 mM KCl	3,34 ± 0,09^ab	0,13 ± 0,02^a	3,89^c	450,25 ± 12,14^f	0,965 ± 0,09^d	0,027 ± 0,01^b	0,148 ± 0,04^de	0,110 ± 0,02^b	0,306 ± 0,02^b	372,83 ± 20,86^f	292,00 ± 42,23^g
200 mM KCl	2,91 ± 0,07^a	0,04 ± 0,01^a	1,38^a	410,17 ± 19,66^e	0,887 ± 0,07^b	0,025 ± 0,01^b	0,109 ± 0,04^b	0,097 ± 0,01^ab	0,276 ± 0,03^a	141,08 ± 8,21^b	385,08 ± 26,96^h
50 mM NaCl + 50 mM KCl	3,88 ± 0,09^ab	0,25 ± 0,04^b	6,44^e	530,17 ± 31,52^j	1,179 ± 0,15ⁱ	0,036 ± 0,01^cd	0,210 ± 0,07^h	0,144 ± 0,02^d	0,326 ± 0,01^c	615,67 ± 32,00^k	54,58 ± 6,61b^c
100 mM NaCl + 100 mM KCl	3,59 ± 0,09^ab	0,54 ± 0,09^c	15,04^h	489,67 ± 14,09^h	0,892 ± 0,07^b	0,027 ± 0,01^b	0,139 ± 0,06^d	0,094 ± 0,01^ab	0,295 ± 0,03^ab	592,33 ± 27,29^j	172,17 ± 22,04^e
150 mM NaCl + 500 µM GB	3,76 ± 0,13^ab	0,17 ± 0,02^ab	4,52^d	534,08 ± 22,44^j	0,983 ± 0,06^e	0,039 ± 0,01^d	0,193 ± 0,08^g	0,115 ± 0,01^bc	0,341 ± 0,02^d	411,17 ± 28,00^g	259,00 ± 25,19^f
200 mM NaCl + 500 µM GB	3,21 ± 0,15^a	0,15 ± 0,02^a	4,67^d	506,92 ± 12,43ⁱ	0,904 ± 0,04^b	0,061 ± 0,11^f	0,123 ± 0,03^c	0,107 ± 0,01^b	0,317 ± 0,03^bc	266,33 ± 26,77^cd	317,00 ± 14,35^g
150 mM KCl + 500 µM GB	4,18 ± 0,14^b	0,24 ± 0,03^b	5,74^de	546,00 ± 12,27^k	1,013 ± 0,09^f	0,034 ± 0,02^c	0,187 ± 0,04^g	0,141 ± 0,02^d	0,431 ± 0,05^h	463,42 ± 31,60^h	236,08 ± 21,38^f
200 mM KCl + 500 µM GB	3,61 ± 0,14^ab	0,09 ± 0,02^a	2,49^b	609,17 ± 14,84^l	0,964 ± 0,08^d	0,035 ± 0,01^c	0,139 ± 0,05^d	0,121 ± 0,01^c	0,382 ± 0,01^ef	245,92 ± 18,69^c	299,92 ± 28,60^g
50 mM NaCl + 50 mM KCl + 500 µM GB	4,74 ± 0,23^b	0,40 ± 0,02^bc	8,44^f	667,92 ± 15,38^m	1,252 ± 0,16^j	0,046 ± 0,01^e	0,260 ± 0,07^j	0,179 ± 0,02^e	0,416 ± 0,02^g	748,42 ± 54,23^l	40,58 ± 1,83^b
100 mM NaCl + 100 mM KCl + 500 µM GB	4,53 ± 0,28^b	0,77 ± 0,03^d	17,00ⁱ	597,25 ± 10,42^l	0,897 ± 0,27^b	0,041 ± 0,01^de	0,160 ± 0,07^e	0,128 ± 0,01^cd	0,373 ± 0,01^e	772,67 ± 42,89^m	103,33 ± 7,88^d
Total	3,80 ± 0,72	0,34 ± 0,04	8,36	469,48 ± 28,44	0,985 ± 0,14	0,035 ± 0,03	0,164 ± 0,07	0,124 ± 0,07	0,331 ± 0,05	386,76 ± 196,32	179,57 ± 123,35

FW: fresh weight; DW: dry weight; TP: total protein; GB: glycine-betaine; *differences in the letters indicate statistical significance at the 5% level in the columns.

With respect to the individual salt stress treatments applied to the wheat stems, unlike those applied to the roots, an increase in stem fresh weight was observed at salt concentrations up to 150 mM (although the extent of increase decreased with increasing dose) (Table S1.2). Significant decreases were observed with the application of 200 mM sodium chloride or potassium chloride (14.63% and 10.02%, respectively) (Table 2). When glycine-betaine was applied in combination with sodium or potassium chloride (50 mM NaCl + 50 mM KCl + 0.5 mM GB and 100 mM NaCl + 100 mM KCl + 0.5 mM GB), this decrease decreased by 80% and 54%, respectively. When sodium and potassium chloride salts were applied in combination with GB support, the fresh weight of the trunks increased by approximately 20% compared to that of the control (Table 2).

Table 2

Effect of salt application on plant growth in leafy stems (all wheat varieties were evaluated together)
Individual Salt Applications	N	Fresh weight (gr)	Dry weight (gr)	Dw/Fw (%)	Plant height (cm)	Leaf height (cm)	Chl a (mg/gr fw)	Chl b (mg/gr fw)	Chl a/b	Total Chl (mg/gr fw)	Carotene (mg/gr fw)
Control	24	29,28 ± 0,55^h	3,68 ± 0,09^g	12,57^b	25,17 ± 0,47^h	19,75 ± 0,33^hi	2,18 ± 0,03^g	0,89 ± 0,01^bcd	2,45^de	3,08 ± 0,03^fg	0,29 ± 0,03^l
30 mM NaCl	24	32,11 ± 0,09^d	4,32 ± 0,09^c	13,45^c	29,75 ± 0,45^c	20,50 ± 0,33^fgh	2,77 ± 0,02^cd	0,97 ± 0,01^abc	2,86^ef	3,74 ± 0,02^c	0,36 ± 0,03^k
50 mM NaCl	24	33,15 ± 0,22^bc	4,52 ± 0,07^c	13,64^c	30,915 ± 0,41^b	20,42 ± 0,39^fgh	2,96 ± 0,01^b	1,03 ± 0,01^a	2,87^ef	3,99 ± 0,02^b	0,46 ± 0,03ⁱ
100 mM NaCl	24	31,28 ± 0,30^fg	4,28 ± 0,08^def	13,68^c	29,25 ± 0,33^cd	18,75 ± 0,33^j	2,00 ± 0,01^h	0,98 ± 0,01^ab	2,04^d	2,98 ± 0,02^g	0,47 ± 0,03ⁱ
150 mM NaCl	24	30,27 ± 0,23^h	3,90 ± 0,10^h	12,88^b	27,50 ± 0,33^f	20,84 ± 0,33^efg	1,39 ± 0,02^jk	0,94 ± 0,01^abc	1,48^b	2,41 ± 0,02^ij	0,45 ± 0,03^j
200 mM NaCl	24	24,99 ± 0,34^k	3,48 ± 0,07ⁱ	13,93^c	23,75 ± 0,33ⁱ	19,75 ± 0,33^hi	1,21 ± 0,02^l	0,90 ± 0,02^bcd	1,34^a	2,11 ± 0,02^k	0,44 ± 0,03^j
30 mM KCl	24	32,30 ± 0,19^cd	3,83 ± 0,20^efg	11,86^a	27,25 ± 0,33^f	21,42 ± 0,39^de	2,51 ± 0,01^e	0,92 ± 0,01^abcd	2,73^e	3,43 ± 0,02^d	0,35 ± 0,03^k
50 mM KCl	24	33,35 ± 0,21^b	4,20 ± 0,06^d	12,59^b	27,67 ± 0,33^ef	21,17 ± 0,33^def	2,89 ± 0,01^b	0,96 ± 0,02^abc	3,01^ef	3,86 ± 0,02^bc	0,42 ± 0,05^j
100 mM KCl	24	31,83 ± 0,35^ef	4,33 ± 0,11^de	13,60^c	25,92 ± 0,33^gh	20,09 ± 0,33^gh	2,26 ± 0,02^fg	0,96 ± 0,01^abc	2,35^de	3,21 ± 0,02^ef	0,52 ± 0,03^g
150 mM KCl	24	30,37 ± 0,27^h	3,76 ± 0,09^h	12,38^b	24,09 ± 0,41ⁱ	17,42 ± 0,25^k	1,95 ± 0,01^h	0,84 ± 0,01^cde	2,32^de	2,80 ± 0,01^h	0,51 ± 0,04^h
200 mM KCl	24	26,34 ± 0,36^j	3,43 ± 0,09ⁱ	13,02^c	21,92 ± 0,39^j	16,33 ± 0,33^l	1,76 ± 0,01ⁱ	0,75 ± 0,02^e	2,35^de	2,51 ± 0,02ⁱ	0,50 ± 0,05^h
50 mM NaCl + 50 mM KCl	24	33,20 ± 0,37^bc	4,38 ± 0,06^de	13,19^c	28,42 ± 0,39^de	21,83 ± 0,39^cd	2,87 ± 0,02^bc	0,88 ± 0,09^bcd	3,26^f	3,75 ± 0,11^c	0,55 ± 0,02^f
100 mM NaCl + 100 mM KCl	24	30,77 ± 0,22^g	3,81 ± 0,13^fg	12,38^b	23,49 ± 0,25ⁱ	19,17 ± 0,33^ij	2,32 ± 0,01^f	0,90 ± 0,02^bcd	2,58^e	3,22 ± 0,03^e	0,59 ± 0,02^d
150 mM NaCl + 500 µM GB	24	30,72 ± 0,41^g	4,88 ± 0,12^c	15,89^d	30,58 ± 0,45^b	24,17 ± 0,39^b	1,49 ± 0,02^j	0,97 ± 0,01^abc	1,54^c	2,47 ± 0,02^ij	0,55 ± 0,02^f
200 mM NaCl + 500 µM GB	24	25,78 ± 0,45^j	4,45 ± 0,26^de	17,26^f	28,75 ± 0,33^d	22,67 ± 0,45^c	1,38 ± 0,01^k	0,95 ± 0,01^abc	1,45^b	2,33 ± 0,02^j	0,57 ± 0,02^e
150 mM KCl + 500 µM GB	24	31,13 ± 0,61^ef	4,72 ± 0,30^c	15,16^d	27,67 ± 0,45^ef	23,00 ± 0,39^c	2,17 ± 0,01^g	0,89 ± 0,01^bcd	2,44^de	3,06 ± 0,01^g	0,65 ± 0,02^c
200 mM KCl + 500 µM GB	24	26,96 ± 0,68ⁱ	4,38 ± 0,15^efg	16,25^e	26,25 ± 0,45^g	22,34 ± 0,41^c	1,96 ± 0,01^h	0,81 ± 0,01^de	2,42^de	2,77 ± 0,01^h	0,64 ± 0,02^c
50 mM NaCl + 50 mM KCl + 500 µM GB	24	28,69 ± 0,55^a	5,32 ± 0,23^a	18,54^g	32,75 ± 0,45^a	25,42 ± 0,51^a	3,37 ± 0,01^a	1,04 ± 0,01^a	3,24^f	4,41 ± 0,02^a	0,73 ± 0,02^b
100 mM NaCl + 100 mM KCl + 500 µM GB	24	31,27 ± 0,76^e	4,73 ± 0,17^b	15,13^d	28,92 ± 0,45^cd	23,92 ± 0,53^b	2,72 ± 0,01^d	1,03 ± 0,01^a	2,64^e	3,77 ± 0,01^c	0,74 ± 0,02^a

*Differences in the letters indicate statistical significance at the 5% level in the columns.

Among the wheat varieties, the highest fresh weight was observed for the T. dicoccum variety (199.83 g), followed by T. monococcum (196.59 g) and T. boeoticum (147.17 g). The lowest fresh weight was observed for the T. speltoides variety (141.58 g) (Table S1.1-3).

3.1.2. Effect of salt stress on the dry weight of plant roots and leafy stems

Due to the increased salt accumulation in the roots, the decrease in dry weight in the plant stem biomass was much greater than that in the roots, reaching approximately 94% after 200 mM salt was applied. However, when salt stress was applied in combination with GB, the decrease in biomass was compensated for by approximately 68% (Table 1, Table S1.1, and Fig. 1).

Although an increase of 2–44% in dry weight was observed due to the increase in salt dose in the wheat stems (except for those treated with 200 mM salt), decreases of 5.65% and 6.76% were observed with the application of 200 mM sodium chloride and 200 mM potassium chloride, respectively. The application of 50 mM NaCl + 50 mM KCl + 0.5 mM GB resulted in the highest trunk dry weight (44.37%) (Tables 2 and 3). Among the wheat varieties, the highest dry weight was observed for the T. dicoccum variety (26.71 g), followed by T. monococcum (24.91 g) and T. boeoticum (20.67 g). The lowest fresh weight (18.99 g) was observed for the T. speltoides variety (Table S1.1-3).

3.1.3. Effect of salt stress on leaf length

Applied salt doses provide an approximately 9% increase in leaf length up to 100 mM in wheat varieties, and salt doses above 100 mM cause a decrease in leaf length of 5.0–17%. However, when high doses of sodium chloride and potassium chloride (150 and 200 mM) were supplemented with GB, an increase in leaf length was observed. This increase reached 22.38% in the 150 mM KCl + 0.5 mM GB treatment and 21.11% in the 100 mM NaCl + 100 mM KCl + 0.5 mM GB treatment (Table 2).

Among the wheat varieties, the longest leaf length was observed for T. dicoccum (23.28 cm), followed by T. monococcum (20.96 cm) and T. boeoticum (20.26 cm). The shortest leaf length (19.34 cm) was observed for the T. speltoides variety (Table 5). Changes in the leaf lengths of the hulled wheat varieties depending on the applied salt dose are given in Tables S1.2 and S1.3 in the supplementary material.

3.1.4. Effect of salt stress on total plant height

The changes in plant height in response to increasing salt doses in the wheat varieties paralleled the changes observed in leaf length. In general. Up to 150 mM salt was applied, the increase in plant height varied from approximately 3–23%, while 150 and 200 mM salt were applied to increase the total decrease in plant height (200 mM NaCl). 5.64%; 200 mM KCl. 12.91%) (Table 2, S1.2 and S1.3). Similarly, the combined application of salt stress and glycine-betaine increased the plant height by approximately 30% (Table 2, S1.2-3). Among the wheat varieties, the tallest total plant height was observed for the T. dicoccum variety (32.94 cm), followed by those for T. monococcum (30.44 cm) and T. boeoticum (23.94 cm). The lowest plant height was observed for the T. speltoides variety, at 22.12 cm (Table 5). Changes in the plant height of the hulled wheat varieties depending on the applied salt dose are given in Tables S1.2 and S1.3 in the supplementary material.

3.1.5. Effects of salt stress on the chlorophyll (chl) a, b, and total chlorophyll and carotene contents

After 30 and 50 mM sodium and potassium chloride were applied, the chl_a content increased by approximately 35%, the chl_a content decreased due to the increase in salt dose, and after 200 mM, the decrease reached 45%. When individual sodium or potassium chloride was applied in combination with GB, the decrease in chl_a content due to stress continued to decrease (a decrease of 10% compared to that of the control). When combined with GB, the chl_a content increased by approximately 30% compared with that in the control (Table 2, S1.2-3).

Increasing NaCl doses caused a 1–15% increase in chl_b content, unlike chl_a. With KCl application, this increase occurred at concentrations up to 100 mM, and KCl application at 150 mM or above reduced the chl_b content by approximately 6–16%. However, when sodium chloride and potassium chloride were combined with GB, the chl_b content increased by approximately 16% compared with that of the control (Table 2, S1.2-3).

The chlorophyll a/b ratios decreased by approximately 20–40% at salt doses of 100 mM and above. A 30% increase was observed in response to the 50 mM KCl treatment. Although an increase of 33% in the 50 mM NaCl + 50 mM KCl treatment group and a 32% increase in the 50 mM NaCl + 50 mM KCl + 0.5 mM GB treatment group were obtained, there was no change in the 100 mM NaCl + 100 mM KCl + 0.5 mM glycine-betaine treatment group compared to the control group (Table 2, S1.2-3).

Although there was an increase of approximately 20–30% in the total chlorophyll content up to 100 mM salt application, a decrease of approximately 10–30% was observed for both individual and combined salt applications of 100 mM and above. Compared with other salt applications, glycine-betaine-supported combined salt applications increase the total chlorophyll content by approximately 20–30%, unlike what is observed in other salt applications (Table 2, S1.2-3).

With increasing salt dose, the carotene content in wheat generally increases. This increase reached 100% in combined salt applications and up to 255% in glycine-betaine-supported combined salt applications (Table 2, S1.2-3).

Among the wheat varieties, the highest chl_a, chl_b, total chlorophyll and carotene contents were observed in T. boeoticum (2.26; 0.96; 3.24 and 0.66 mgg^− 1 fw, respectively), followed by T. speltoides (2.24; 0.94; 3.18 and 0.62 mgg-1 fw, respectively) and T. monococcum (2.22; 0.90; 3.12 and 0.42 mgg^− 1 fw, respectively), and were observed in T. dicoccum (2.18; 0.90; 3.10 and 0.38 mgg^− 1 fw, respectively) (Tables 5 and 6, Table S1.8-11). The highest chlorophyll a/b ratio was obtained for the T. monococcum variety, with a value of 2.47.

3.2. Effect of salt stress on the total protein content

Compared with those in the control treatment, the wheat varieties in the treatment groups showed varying levels of increase in protein content depending on the salt dose. 3–24% under NaCl application; 29–55% under KCl application, 55–68% under combined salt application, 60–90% under GB-supplemented salt application and 90–111% under GB-supplemented combined salt application were detected (Table 1, Table 3–4).

3.3. Effects of salt stress on antioxidant enzyme activity in plant roots and leafy stems

The enzymatic and non-enzymatic defense responses of hulled wheat plants to salt stress are shown in Table 1, Tables 3–6 and S1.4-7, and the changes in the percentages of these enzymes compared with those in the control are shown in S1.8-11.

Table 3

Effects of salt application on the enzymatic and nonenzymatic antioxidant defense systems in leafy stems (all wheat varieties were evaluated together)
Salt Applications	Total protein	SOD	CAT	GR	GST	APX	PROLINE	MDA
Control	450,17 ± 32,23^b	0,808 ± 0,06^b	0,014 ± 0,01^a	0,092 ± 0,02^a	0,095 ± 0,02^b	0,300 ± 0,02^b	63,63 ± 10,43^a	17,31 ± 1,14^a
30 mM NaCl	476,17 ± 38,04^cd	0,931 ± 0,10^f	0,050 ± 0,09^d	0,122 ± 0,03^bc	0,115 ± 0,01^c	0,333 ± 0,02^cd	259,58 ± 30,37^de	34,25 ± 5,21^ab
50 mM NaCl	502,08 ± 32,38^e	0,850 ± 0,02^cd	0,029 ± 0,01^c	0,190 ± 0,17^e	0,150 ± 0,02^e	0,350 ± 0,02^d	311,25 ± 41,93^f	40,25 ± 6,18^b
100 mM NaCl	522,08 ± 27,56^f	0,835 ± 0,05^c	0,026 ± 0,01b^c	0,145 ± 0,05^c	0,128 ± 0,02^cd	0,327 ± 0,02^cd	385,75 ± 33,30^h	158,00 ± 25,17^d
150 mM NaCl	526,33 ± 28,24^f	0,832 ± 0,05^c	0,020 ± 0,01^b	0,119 ± 0,03^b	0,112 ± 0,01^c	0,302 ± 0,03^bc	279,75 ± 32,43^e	242,50 ± 82,30f
200 mM NaCl	461,00 ± 27,28^c	0,813 ± 0,06^b	0,017 ± 0,01^ab	0,094 ± 0,02^a	0,080 ± 0,01^a	0,279 ± 0,02^a	117,75 ± 29,58^b	394,67 ± 23,06ⁱ
30 mM KCl	467,17 ± 18,10^c	0,795 ± 0,02b	0,016 ± 0,01^a	0,110 ± 0,03^b	0,093 ± 0,01^ab	0,304 ± 0,02b^c	251,33 ± 22,55^d	32,92 ± 1,78^ab
50 mM KCl	521,92 ± 14,03^f	0,802 ± 0,22^b	0,020 ± 0,01^b	0,133 ± 0,04^bc	0,106 ± 0,01^bc	0,325 ± 0,02^c	388,50 ± 22,04^h	45,83 ± 8,87^b
100 mM KCl	509,00 ± 20,04^e	0,904 ± 0,11^e	0,018 ± 0,01a^b	0,119 ± 0,03^b	0,097 ± 0,01^b	0,322 ± 0,02^c	486,58 ± 22,94^k	152,08 ± 18,80^d
150 mM KCl	473,08 ± 17,46^cd	0,874 ± 0,10^d	0,020 ± 0,02^b	0,104 ± 0,02^ab	0,089 ± 0,01^ab	0,307 ± 0,01^b	379,17 ± 23,05^gh	355,75 ± 27,34^gh
200 mM KCl	404,00 ± 25,08^a	0,758 ± 0,06^a	0,012 ± 0,01^a	0,084 ± 0,01^a	0,082 ± 0,01^a	0,277 ± 0,02^a	117,67 ± 8,27^b	369,08 ± 22,36^h
50 mM NaCl + 50 mM KCl	563,42 ± 15,92^h	0,995 ± 0,11^g	0,021 ± 0,01^b	0,155 ± 0,04^d	0,118 ± 0,01^c	0,347 ± 0,02^d	606,33 ± 32,26^m	47,75 ± 9,03^b
100 mM NaCl + 100 mM KCl	488,83 ± 22,67^d	0,813 ± 0,10^b	0,014 ± 0,01^a	0,089 ± 0,02^a	0,087 ± 0,01^ab	0,304 ± 0,03^bc	451,67 ± 49,49^j	157,67 ± 17,31^d
150 mM NaCl + 500 µM GB	553,83 ± 21,20^h	0,888 ± 0,06^de	0,028 ± 0,01^c	0,153 ± 0,03^cd	0,128 ± 0,01^cd	0,326 ± 0,02^c	417,83 ± 34,86ⁱ	250,08 ± 13,07^f
200 mM NaCl + 500 µM GB	538,25 ± 15,20^g	0,887 ± 0,05^de	0,023 ± 0,01^bc	0,113 ± 0,03^b	0,109 ± 0,01^bc	0,291 ± 0,02^ab	247,50 ± 26,57^d	325,42 ± 18,01^g
150 mM KCl + 500 µM GB	596,17 ± 6,71ⁱ	0,934 ± 0,10^f	0,024 ± 0,01^bc	0,143 ± 0,02^c	0,136 ± 0,03^d	0,320 ± 0,03^c	437,83 ± 22,51^ij	256,33 ± 11,58^f
200 mM KCl + 500 µM GB	608,42 ± 23,65^j	0,822 ± 0,07^c	0,019 ± 0,01^ab	0,105 ± 0,02^ab	0,125 ± 0,02^cd	0,296 ± 0,02^b	210,33 ± 7,89^c	285,00 ± 13,08^f
50 mM NaCl + 50 mM KCl + 500 µM GB	661,33 ± 23,03^k	1,058 ± 0,10^h	0,029 ± 0,01^c	0,213 ± 0,06^e	0,149 ± 0,02^e	0,416 ± 0,03^e	741,92 ± 45,99ⁿ	27,83 ± 1,75^ab
100 mM NaCl + 100 mM KCl + 500 µM GB	604,58 ± 13,10^j	0,881 ± 0,09^de	0,023 ± 0,01b^c	0,116 ± 0,02^b	0,138 ± 0,01^d	0,339 ± 0,02^d	569,25 ± 41,12^l	97,33 ± 7,97^c
Total	522,52 ± 66,54	0,867 ± 0,11	0,022 ± 0,02	0,126 ± 0,06	0,112 ± 0,03	0,319 ± 0,04	353,88 ± 173,97	173,016 ± 131,03

*Differences in the letters indicate statistical significance at the 5% level in the columns.

3.3.1. Effect on SOD activity

Considering the whole plant resistance to salt application, SOD activity increased the most in T. monococcum (19.30%) compared with that in the control. The lowest increase was observed for T. dicoccum (0.36%) (Table 6, Fig. 3a-b). An increase in SOD enzyme activity was also observed with increasing salt dose (Table 4). SOD enzyme activity in the roots of hulled wheat was significantly greater than that in the stems (the average SOD activity in the roots was 0.985 ± 0.14 unite mg^− 1 fw, and that in the stems was 0.867 ± 0.11 unite mg^− 1 fw, p ≤ 0.01) (Tables 1 and 3). The highest SOD activity in both the roots and stems was obtained with the application of 50 mM NaCl + 50 mM KCl + 0.5 mM GB (1,252 ± 0.16 unite mg^− 1 fw in the roots and 1,058 ± 0.10 unite mg^− 1 fw in the stems).

Table 4

Effects of salt application on the enzymatic and nonenzymatic antioxidant defense systems throughout the plant (all wheat varieties were evaluated together)
Salt Applications	Total protein	SOD	CAT	GR	GST	APX	PROLINE	MDA
Control	383,25 ± 74,29^a	0,89 ± 0,12^cde	0,01 ± 0,00^a	0,09 ± 0,03^a	0,10 ± 0,03^abc	0,29 ± 0,02^b	66,31 ± 11,18^a	18,35 ± 2,00^a
30 mM NaCl	424,25 ± 64,48^d	0,99 ± 0,11^g	0,05 ± 0,04^d	0,14 ± 0,04^defg	0,12 ± 0,01^abc	0,33 ± 0,02^fg	267,67 ± 52,59^e	39,96 ± 8,61^b
30 mM KCl	454,96 ± 24,84^ef	0,85 ± 0,06^abc	0,02 ± 0,01^abc	0,13 ± 0,04^cdef	0,10 ± 0,01^abc	0,31 ± 0,02^de	256,46 ± 25,03^d	36,67 ± 4,82^b
50 mM NaCl	449,65 ± 62,70^e	0,93 ± 0,10^ef	0,04 ± 0,01^bcd	0,20 ± 0,13^k	0,15 ± 0,02^cd	0,36 ± 0,02ⁱ	322,43 ± 52,08^f	50,78 ± 13,78^c
50 mM KCl	506,63 ± 22,36^h	0,91 ± 0,20^de	0,03 ± 0,01^abcd	0,16 ± 0,06^ghi	0,12 ± 0,02^abc	0,33 ± 0,02^fg	403,25 ± 39,29^h	52,29 ± 10,75^c
100 mM NaCl	456,92 ± 72,54^ef	0,93 ± 0,12^ef	0,03 ± 0,01^abcd	0,18 ± 0,08^jk	0,13 ± 0,02^bcd	0,33 ± 0,02^f	414,00 ± 49,73ⁱ	168,75 ± 25,90^e
100 mM KCl	489,00 ± 25,78^g	0,97 ± 0,13^fg	0,03 ± 0,01^abcd	0,15 ± 0,05^efgh	0,11 ± 0,02^abc	0,32 ± 0,02^e	499,00 ± 36,60^k	162,79 ± 22,23^e
150 mM NaCl	450,04 ± 85,06^e	0,89 ± 0,08^bcde	0,03 ± 0,01^abcd	0,15 ± 0,06^fgh	0,15 ± 0,03^cd	0,30 ± 0,02^c	268,38 ± 28,06^e	262,75 ± 64,31^h
150 mM KCl	461,67 ± 18,77^f	0,92 ± 0,10^ef	0,02 ± 0,01^abc	0,13 ± 0,04^cdef	0,10 ± 0,02^abc	0,31 ± 0,02^cd	376,00 ± 21,74^g	323,88 ± 47,65^j
200 mM NaCl	394,88 ± 71,73^b	0,83 ± 0,06^ab	0,02 ± 0,01^ab	0,10 ± 0,03^ab	0,08 ± 0,01^a	0,28 ± 0,02^a	131,33 ± 26,40^b	386,92 ± 24,22^l
200 mM KCl	407,08 ± 22,26^c	0,82 ± 0,09^a	0,02 ± 0,01^ab	0,10 ± 0,03^ab	0,09 ± 0,01^ab	0,28 ± 0,03^a	129,38 ± 14,42^b	377,08 ± 25,57^k
50 mM NaCl + 50 mM KCl	546,79 ± 29,75^j	1,09 ± 0,16^h	0,03 ± 0,01^abcd	0,18 ± 0,06^jk	0,13 ± 0,02^bcd	0,34 ± 0,02^gh	611,00 ± 31,78^m	51,17 ± 8,49^c
100 mM NaCl + 100 mM KCl	489,25 ± 18,47^g	0,85 ± 0,09^abcd	0,02 ± 0,01^abc	0,11 ± 0,05^bc	0,09 ± 0,01^ab	0,30 ± 0,03^c	522,00 ± 81,79^l	164,92 ± 20,75^e
150 mM NaCl + 500 µM GB	543,96 ± 23,61^j	0,94 ± 0,08^efg	0,04 ± 0,01^abcd	0,17 ± 0,06^ij	0,12 ± 0,01^abcd	0,33 ± 0,02^gh	414,50 ± 31,11ⁱ	254,54 ± 20,15^g
150 mM KCl + 500 µM GB	571,08 ± 27,39^k	0,97 ± 0,10^fg	0,03 ± 0,01^abcd	0,17 ± 0,04^hij	0,14 ± 0,02^cd	0,38 ± 0,07^j	450,63 ± 29,84^j	246,21 ± 19,74^f
200 mM NaCl + 500 µM GB	522,58 ± 20,99ⁱ	0,90 ± 0,05^cde	0,04 ± 0,02^cd	0,12 ± 0,03^bcd	0,11 ± 0,01^abc	0,30 ± 0,03^cd	256,92 ± 27,80^d	321,21 ± 16,50^j
200 mM KCl + 500 µM GB	608,79 ± 19,32^m	0,89 ± 0,10^cde	0,03 ± 0,01^abcd	0,12 ± 0,04^cde	0,12 ± 0,01^abcd	0,34 ± 0,05^h	228,13 ± 22,96^c	292,46 ± 23,05ⁱ
50 mM NaCl + 50 mM KCl + 500 µM GB	664,63 ± 19,44ⁿ	1,16 ± 0,17ⁱ	0,04 ± 0,01^bcd	0,24 ± 0,07^l	0,16 ± 0,02^d	0,42 ± 0,02^k	745,17 ± 49,29^o	34,21 ± 6,74^b
100 mM NaCl + 100 mM KCl + 500 µM GB	600,92 ± 12,17^l	0,89 ± 0,20^cde	0,03 ± 0,01^abcd	0,14 ± 0,06^defg	0,13 ± 0,01^bcd	0,36 ± 0,02ⁱ	670,96 ± 111,73ⁿ	100,33 ± 8,33^d
Total	496,22 ± 87,30	0,93 ± 0,14	0,03 ± 0,02	0,15 ± 0,07	0,12 ± 0,05	0,33 ± 0,04	370,29 ± 185,99	176,34 ± 127,16

*Differences in the letters indicate statistical significance at the 5% level in the columns.

3.3.2. Effect on CAT activity

Catalase activity in hulled wheat increased between 112% and 231% compared to that in the control. The highest catalase activity was detected in the T. boeoticum and T. speltoides varieties (231% and 228%, respectively), and the lowest activity was detected in the T. monococcum variety (112.9%) (Table 6, Fig. 3c-d). At all the salt concentrations applied, the catalase activity in both the roots and stems was greater than that in the control (Table 1, 3–4; S1.4-12). The highest catalase activity was detected in the stems (0.050 ± 0.09 unite mg^− 1 fw) after 30 mM NaCl application and in the roots (0.061 ± 0.11 unite mg^− 1 fw) after 200 mM NaCl + 0.5 mM GB application (Tables 1 and 3).

3.3.3. Effect on GR activity

GR activity in hulled wheat was similar to that of catalase. Compared with that in the control treatment, the highest increase in GR activity was observed in T. boeoticum (195.58%), and the lowest increase in activity was observed in T. monococcum (76.2%) (Table 6, Fig. 3e-f). Although GR activity generally increased at various rates depending on the applied salt dose, a slight decrease in GR activity was observed in the trunk compared with that in the control for both the 200 mM KCl and 100 mM NaCl + 100 mM KCl treatments (8.7% and 3.26%, respectively) (Table 3). The highest GR activity in both the stems and roots was obtained from the combined treatment of 50 mM NaCl + 50 mM KCl + 0.5 mM GB (0.260 ± 0.07 U unite mg^− 1 fw in the roots; 0.213 ± 0.06 unite mg^− 1 fw in the stems) (Table 1 and Table 3–4). Changes in GR activity in the roots and leafy stems of the hulled wheat varieties compared with that in the control, depending on the applied salt dose, are given in the supplementary material Table S1.8-11.

3.3.4. Effect on the GST activity

T. boeoticum, the hulled wheat variety with the highest SOD, CAT, and GR activity, had the lowest activity level (0.97%) in terms of GST activity (Table 6, Fig. 3g-h). Among the hulled wheat plants, T. dicoccum had the highest GST activity (115.6%). Among the applied salt doses, the highest GST activity in the roots was obtained at 150 mM NaCl (0.181 ± 0.09), whereas at 200 mM NaCl, the GST accumulation decreased sharply (0.080 ± 0.01 unite mg^− 1 fw) (Table 1). In the body, the highest GST activity was obtained from the 50 mM NaCl and 50 mM NaCl + 50 mM KCl + 0.5 mM GB treatments (0.150 ± 0.02 unite mg^− 1 fw and 0.149 ± 0.02 unite mg^− 1 fw, respectively) (Table 3–4). The changes in GST activity in the roots and leafy stems of hulled wheat varieties in response to the applied salt dose compared with that in the control are shown in supplementary material tables S1.8-11.

3.3.5. Effect on APX activity

Ascorbate peroxidase (APX), which is a non-enzymatic antioxidant, exhibited the greatest increase in activity in T. dicoccum (33.96%) under salt stress among the hulled wheat plants (Table 6, Fig. 3.i-j). T. speltoides had the least increase in APX activity (19.70%). Among the salt doses applied to the roots of hulled wheat, 150 mM KCl had the highest APX accumulation (0.431 ± 0.05 unite mg^− 1 fw), while 200 mM NaCl and 200 mM KCl had the lowest APX accumulation (0,271 ± 0.01 unite mg^− 1 fw and 0,276 ± 0,03 U unite mg^− 1 fw, respectively). In the body, the 50 mM NaCl + 50 mM KCl + 0.5 mM GB application had the highest APX accumulation (0.416 ± 0.03 unite mg^− 1 fw), while the 200 mM NaCl and 200 mM KCl applications had the lowest APX accumulation (0,277 ± 0,02 unite mg^− 1 fw and 0,279 ± 0,02 unite mg^− 1 fw, respectively) (Tables 3 and 4). The changes in APX activity in the roots and leafy stems of the hulled wheat varieties compared to those in the control, depending on the applied salt dose, are shown in Table 5 and supplementary material Table S1.8-11.

3.4. Effect on proline accumulation

The content of proline, a non-enzymatic oxidant, increased significantly in the hulled wheat varieties due to salt stress. Among the hulled wheat plants, T. monococcum had the highest percentage increase in proline content compared with that in the control at a rate of 1099.85%, and the lowest increase was observed in T. speltoides at a rate of 842.15% (Table 6). While the highest proline accumulation in roots was obtained from plants supported with GB combined with 50 or 100 mM sodium and potassium chloride (748.42 ± 54.23 unite mg^− 1 fw and 772.67 ± 42.89 unite mg^− 1 fw, respectively), the lowest proline accumulation was obtained from plants treated with 200 mM NaCl or KCl (144.92 ± 13.40 unite mg^− 1 fw and 141.08 ± 8.21 unite mg^− 1 fw, respectively) (Table 1). Similarly, in the roots, the greatest proline accumulation in the stem was obtained from the GB-supplemented 50 mM NaCl + 50 mM KCl combined application (741.92 ± 45.99 unite mg^− 1 fw) and 50 mM NaCl + 50 mM KCl combined application (606, 33 ± 32.36 unite mg^− 1 fw). The lowest proline accumulation in the stem was observed in the 200 mM KCl and 200 mM NaCl treatment groups (117.67 ± 8.27 unite mg^− 1 fw and 117.75 ± 29.58 unite mg^− 1 fw, respectively) (Table 3–4). The changes in proline accumulation in the roots and leafy stems of the hulled wheat varieties in response to the applied salt dose compared with that in the control are shown in Table 5 and supplementary material Table S1.8-11.

3.5. Effect on lipid peroxidation (LPO) (MDA)

Table 5

Effects of salt application on plant growth and antioxidant defense in hulled wheat
Wheats	N	FW	DW	PH	LL	Chl a	Chl b	Chl a/b	TC	Carotene
Control	12	16,72^b	2,13^c	25,17^c	19,75^cd	2,18^c	0,89^a	2,45^bc	3,08^c	0,29^a
T. monococcum	114	19,66^c	2,49^d	30,44^d	20,96^b	2,22^bc	0,90^b	2,47^c	3,12^c	0,42^d
T. dicoccum	114	19,98^c	2,67^e	32,94^e	23,28^a	2,18^c	0,90^b	2,42^b	3,10^c	0,38^e
T. speltoides	114	14,16^a	1,90^a	22,12^a	19,34^d	2,24^ab	0,94^a	2,38^ab	3,18^b	0,62^c
T. boeoticum	114	14,72^a	2,07^b	23,94^b	20,26^c	2,26^a	0,96^a	2,35^a	3,24^a	0,66^b
Wheats	N	Total protein		SOD	CAT	GR	GST	APX	PRO	MDA
Control	12	383,25^a		0,89^b	0,01^a	0,09^a	0,10^a	0,29^a	66,31^a	18,35^a
T. monococcum	114	508,26^c		0,873^b	0,021^b	0,106^b	0,097^a	0,320^b	332,55^b	166,19^c
T. dicoccum	114	513,75^d		0,847^a	0,020^b	0,104^b	0,116^b	0,299^a	362,71^c	157,84^b
T. speltoides	114	480,14^b		1,015^d	0,037^c	0,194^d	0,140^c	0,348^d	400,55^e	194,34^e
T. boeoticum	114	482,62^b		0,971^c	0,037^c	0,177^c	0,121^b	0,333^c	385,48^d	187,09^d

*Differences in the letters indicate statistical significance at the 5% level in the columns.

The LPO content resulting from salt stress was approximately 18 times greater in the hulled wheat plants than in the control plants. The lowest increase was observed in T. monococcum (1751.75%), and the highest increase was observed in T. boeoticum (1885.45%) (Table 6). The greatest LPO accumulation in roots was caused by the 200 mM KCl and 200 mM NaCl applications (385.08 ± 26.96 nmol g^− 1 fw and 379.17 ± 23.74 nmol g^− 1 fw, respectively). The lowest LPO accumulation in roots was obtained in the 50 mM KCl and GB supplemented with 50 mM salt (40.42 ± 3.52 nmol g^− 1 fw and 40.58 ± 1.83 nmol g^− 1 fw, respectively) treatments (Table 1). The highest LPO accumulation in the body was again caused by the 200 mM salt application (NaCl. 394.67 ± 23.06 nmol g^− 1 fw and KCl. 369.08 ± 22.36 nmol g^− 1 fw). The least LPO accumulation in the stem was obtained from GB supplemented with 50 mM salt, which was also observed in the roots (27.83 ± 1.75 nmol g^− 1 fw) (Table 3–4). The changes in MDA accumulation in the roots and leafy stems of the hulled wheat varieties compared with those in the control, depending on the applied salt dose, are shown in Table 5 and supplementary material Table S1.8-11.

Table 6

Percent changes in the responses of spelled wheat to salt stress compared with those of the control
Wheats	SOD	CAT	GR	GST	APX	Proline	MDA	Carotene	Fresh weight	Dry weight	Total length	Leaf length
T. monococcum	19,3	112,90	76,20	54,45	24,80	1099,85	1751,75	51,85	14,74	22,71	21,35	1,52
T. dicoccum	0,36	125,68	85,56	115,6	33,96	1079,46	1863,06	82,68	3,19	20,51	9,10	7,95
T. speltoides	4,34	228,59	126,88	12,50	19,70	842,15	1830,44	93,77	1,16	4,79	3,91	5,76
T. boeoticum	14,02	231,37	195,58	0,97	29,81	918,78	1885,45	111,28	-3,45	12,75	1,25	11,11

*Differences in the letters indicate statistical significance at the 5% level in the columns.

3.6 Statistical data analysis results

The statistical analysis results of the study (multiple comparisons, tests of between-subject effects, Levene's test and multivariate MANOVA) are given in supplementary material 2. The GT biplot explained 66.1% of the variation in the T. boeoticum stem. In the present study, MDA, DW/FW, DW, TP, LH, GST, APX, PRO, and TCHL were measured. CHLA, FW, and CHL A/B were among the features with high discrimination power. SOD, CAT and Chl_b were features with low discrimination power. In addition, GR, PRO and APX; Chl_b; SOD; DW; PT; CAR; PH; and LH had positive relationships with the T. boeoticum stem. A negative relationship was observed between MDA and both Chl a/b and FW. T14 (200 mM NaCl + 0.5 mM GB) and T13 (150 mM NaCl + 0.5 mM GB) were used in combination with DF/FW; T18 (100 mm NaCl + 100 mM KCl + 0.5 mM GB) were used in combination with SOD and Chl_b; T17 (50 mm NaCl + 50 mM KCl + 0.5 mM GB) was used in combination with GST, PRO and GR; T11 (50 mM NaCl + 50 mM KCl) was used in combination with Chl a/b and FW; and T5 (200 mM NaCl) was used in combination with MDA. For T. boeoticum roots, the GT biplot explained 72% of the variation. Among the examined features, a negative relationship was observed between MDA and DW and between DW and FW, and a positive relationship was observed between APX and CAT and between GP and CAT. GT biplot analysis of T. dicoccum stems explained 75% of the variation. A positive relationship was observed between CAR and DW/FW; between DW and GST; between GR, CAT, LH and PRO; between SOD and APX; and between Chl a/b and carotene in the examined features. In the T. monococcum stem, the GT biplot analysis explained 73.1% of the variation. Among the features examined in the present study, the discrimination power of the CAT concentration in T. monococcum stems was low; the discrimination power of the carotene, LH, FW and Chl_b features was moderate; and the discrimination power of the other features was very high. GT biplot analysis explained 70.6% of the variation in terms of the traits examined in T. speltoides stems. Among the features included in the study, the discriminatory power of SOD, Chl_b, LH and carotene was found to be intermediate, while the discriminative power of the other features was found to be quite high. The control application was located in an area far from other applications and the examined features (Fig. 8).

4.1. Effects of Salt Stress on Plant Development

4.1.1. Effect on Plant Biomass

Salt stress causes ion toxicity and disruption of the nutritional balance in plants, causing the physiological processes of the plant to deteriorate and the amount of product to decrease substantially (Taha et al. 2021). In addition, salt stress triggers oxidative stress in plants by disrupting enzymatic activities, photosynthesis, membrane structure and integrity, ionic homeostasis, hormonal balance, and water and nutrient uptake (Hussain et al. 2021; Ibrahimova et al. 2021). Guo et al. (2015) and Zou et al. (2016) observed a decrease in root and shoot length and dry weight in wheat plants compared with those in control plants under 100 mM salt stress. In our study, in hulled wheat varieties under salt stress, decreases in fresh weight were observed in parallel with increasing salt doses in the roots (from 5–30%). In particular, after the application of 200 mM NaCl or KCl, significant decreases in the fresh weight of plant roots were observed (30.41% and 30.21%, respectively). Excessive Na⁺, K⁺ and Cl⁻ ions in plants prevent the uptake of essential nutrients from the soil, which changes plant processes. Guo et al. (2015) reported a decrease in K⁺, Ca²⁺ and Zn^+ 2 uptake and an increase in Na⁺ and Cl⁻ uptake in salt-sensitive wheat. In our study, the observation of severe decreases in the fresh and dry weights of the plants in parallel with increasing salt concentrations showed that the high amounts of sodium, potassium and chloride ions passing into the plant cells disrupted the ion balance in the cells, causing nutritional deficiencies and cellular moisture loss (Table 1–2, Fig. 1).

Fortmeier and Schubert (1995) reported that high sodium concentrations in plants interfere with K⁺ accumulation and stomatal regulation. However, increasing the Na⁺ concentration in plant vacuoles through the tonoplast pathway driven by the proton gradient is also considered a critical strategy against salinity. It was previously reported by Neubert (2005) that plants develop a resistance mechanism against such ions by saving their basic organelles, such as the cytosol, from excess sodium. In our study, compared with the control group, Individual applications of 200 mM NaCl or KCl caused approximately 90% weight loss in fresh and dry weights in roots and stems, whereas the combined application of 100 mM NaCl + 100 mM KCl caused approximately 5% weight loss (Table 1–2, Table S1.1-3, Fig. 1). Taken together, these findings show that the type of factors that create salt stress (such as sodium-based or potassium-based factors) is important for plant development. The plant can protect itself against NaCl or KCl stress when there can be simultaneous K⁺ inflow from the external environment to ensure an intracellular K⁺/Na⁺ balance in parallel with Na⁺ entry into plant plasma membranes. In fact, we predict that if potassium is applied as fertilizer to plants grown on lands with high sodium-based salt contents, the plant will be less affected by salt stress.

Plants accumulate Na⁺ ions in the vacuoles of roots via the tonoplast pathway to reduce sodium transport from the roots to the stem and leaves [Neubert 2005]. When optimizing the K⁺ uptake rate, plants not only restrict Na⁺ entry but also benefit from sodium removal from the cell under salt stress. Wakeel (2011) reported that this mechanism helps maintain the K⁺/Na⁺ ratio in the cytosol and ensures the survival of plants under saline conditions. In our study, the increase in stem dry and fresh weight by approximately 20% and plant height by 30% in the combined application of 100 mM NaCl + 100 mM KCl indicated that sodium and potassium salts entering the plant cell from the external environment were transported to the stem instead of being stored in the root. Due to the increased salt accumulation in the roots, the decrease in plant stem biomass was much greater than that in the roots, reaching approximately 94% after 200 mM salt was applied. Although 200 mM salt application reduced plant height by approximately 5–12%, biomass loss was compensated for by approximately 68% in the presence of glycine-betaine combined with salt, and the plants were approximately 30% taller than the control plants were (Table 2; S1.2–3; Fig. 2b).

Salinity stress has polygenic effects controlled by multiple genes. Na⁺ release and K⁺ uptake, maintenance of the optimum K⁺/Na⁺ ratio, osmotic regulation, and regulation of antioxidant enzyme activities are vital for plants under salt stress (Rahman et al. 2016). In addition to traditional culture techniques, many techniques, such as screening and selecting suitable genotypes and transferring desired genes to plants, are used to increase the amount of product produced from plants under salt stress, but these processes are very costly and take a long time (Hassan et al. 2018). Under these conditions, techniques such as the application of osmoprotectants (such as glycine-betaine and proline), seed coating, nutrient management, and hormone application (auxin, gibberellic acid and brassinosteroids, etc.) to manage salt stress can offer promising results, as previously reported by Hasanuzzaman (2017a). In the present study, sodium or potassium salts applied individually, especially at salt doses of 100 mM, created significant stress in all the hulled wheat plants (in parallel with the increase in salt dose) and halted the development of the wheat plants (Table 1–2; Fig. 1). However, it seems that the stress caused by salt can be largely controlled when exogenous glycine-betaine is applied as an osmoprotectant in addition to individual salt doses. However, when glycine-betaine is applied in combination with sodium and potassium chloride salts, wheat plants can cope with salt stress much better and maintain their vitality almost as if there was no stress. This situation shows that to cultivate wheat efficiently in sodic/saline soils, an osmoprotectant (such as glycine-betaine) should be added to the plant growth medium (starting from seed planting), and potassium-based fertilizers should be used.

4.1.2. Effects on Chlorophyll a, b, and Total Chlorophyll and Carotene Contents

For a plant to survive, it must have suitable environmental conditions and optimum photosynthetic activity (Badawy et al. 2021). Photosynthesis is blocked due to ion accumulation (Na⁺, K⁺ and Cl⁻) in chloroplasts and a decrease in plant water potential due to high salt stress (Hasanuzzaman 2013). Guo et al. [2015] examined the physiological aspects of wheat plants under saline conditions and reported that salinity stress leads to stomatal closure, induces less CO₂ absorption, and reduces the transpiration rate. However, it has been reported that high salt stress significantly reduces the amount of photosynthetic pigments in the chloroplast, which in turn significantly reduces photosynthetic efficiency and productivity. In this study, we observed a decrease of approximately 45% in the chl_a content, especially at 200 mM salt, due to increasing salt doses. When 0.5 mM glycine-betaine is added to the growing medium as an osmoprotectant in addition to sodium or potassium salts, the chl_a content improves by approximately 10%. Compared with those of plants subjected to individual salt stress (NaCl or KCl), when these salts were applied in combination with GB support, the chl_a content increased by approximately 30% compared with that of the control (Table 2; Table S1.2-3; Fig. 4c). Taken together, these findings show that exogenous GB application significantly improved the photosynthetic activity of hulled wheat plants.

Salinity stress can lead to increased ionic toxicity, decreased leaf growth, reduced carboxylation, decreased photosynthesis, and premature leaf abscission. In addition, salinity stress reduces the effectiveness of PS-II, stomatal conductance, intercellular CO₂, and electron transport; all of these factors contribute to a decrease in photosynthesis (Seleiman et al. 2022). Sarker and Oba (2019) stated that when NaCl or KCl is applied alone as salt stress, the sodium or potassium ion concentration in the environment disrupts the K⁺/Na⁺ ion balance, especially in plant root cells, and the ions accumulated here are transmitted to the stem and leaves through the xylem. These ions transferred to leaves not only create ion toxicity but also trigger the production of ROS through the active energy released through sunlight and CO₂ carboxylation used in photosynthesis, which causes damage to plant photosystems (PS-I and PS-II). Due to the damage caused by PS-I, the chl_a content in the leaves decreased, which changed the chl a/b ratio, reducing the effectiveness of PS-II. Under high salt concentrations, a decrease in the number of stomata and an increase in the number of closed stomata leads to a decrease in CO₂ absorption, further inhibiting photosynthesis (Charfeddine et al. 2019; Levy et al. 2013). In our study, 150 and 200 mM individual salt applications caused a significant decrease in the chlorophyll a content of wheat and, accordingly, the chlorophyll a/b ratio (Table 2). Taken together, these findings show that a high-salt environment negatively affects the activity of photosystem I. However, the combined application of salt (especially when supplemented with glycine and betaine) appears to play an important role in restoring photosynthetic activity. These findings showed that maintaining the intracellular K⁺/Na⁺ ion balance directly affects photosynthetic efficiency.

The ultrastructure of chloroplasts is also affected by salt stress. Salt stress significantly inhibits photosystem II, which is crucial for light energy conversion and photosynthetic efficiency (Kolomeichuk et al. 2020). In wheat (T. aestivum L), the granum thylakoids of chloroplasts have been reported in previous studies to be loosely arranged in a thin spindle shape under 200 mM NaCl compared with those under non-stress conditions (Zhu et al. 2021). Increasing the number of chloroplasts is a strategy developed by halophytes to cope with salinity stress (Bose et al. 2017). The significant decrease (approximately 16–20%) in the chl_b content we analyzed in this study at 150–200 mM salt is an indication that PS-II is strongly damaged in hulled wheat due to salt stress. However, when sodium chloride and potassium chloride salts were applied in combination with glycine-betaine instead of individually, an increase of approximately 16% in the chl_b content was observed in the hulled wheat plants (Table 2; S1.2-3). This finding encouraged us to consider whether glycine-betaine plays a protective role in chlorophyll biosynthesis in wheat.

Insufficient energy in the photosynthetic process during salt stress reduces molecular oxygen and results in the production of large amounts of ROS, including H₂O₂, O₂, ¹O₂, and OH● (Hasanuzzaman et al. 2017b; Singh et al. 2019). Moreover, plant cells must constantly resist oxidation of their vital cellular components due to the presence of 21% molecular O₂ in the atmosphere; this situation is further complicated by the overproduction of light-induced ROS during photosynthesis (Zhu et al. 2016). Although low-level ROS play a signaling role, excessive ROS production is harmful to cells; therefore, ROS production should be regulated to maintain redox homeostasis (Hasanuzzaman et al. 2018; Nahar et al. 2017). As shown in our study, exogenously administered glycine-betaine in combination with potassium ions strongly contributed to the control of ROS released during stress.

Carotene accumulation in hulled wheat also increased with increasing doses of salt stress (Table 2; Table 5–6; S1.2-3, S1.8-12; Fig. 2c, Fig. 4c). The accumulation of carotene, a non-enzymatic antioxidant, is especially important for protecting the photosystem from the harmful effects of ROS. The increase in carotene content can reach up to 100% when glycine-betaine-supported sodium or potassium chloride salt is applied, and when sodium and potassium chloride glycine-betaine are combined, this increase can reach up to 150% (Table S1.10-11). Among the hulled wheat plants, the highest chl a, b, total chl. and carotene contents were observed in the T. boeoticum variety (Table 6; Fig. 2a). Because T. boeoticum, one of the oldest ancestors of modern wheat, is resistant to salt stress, this species is promising for the more efficient agricultural use of saline/sodic soils of ancestral wheat. According to the data we obtained from this study, hulled wheat can maintain its photosynthetic activities up to 100 mM sodium or potassium salt stress and is characterized by a significant decrease in total chlorophyll content due to damage to the photosystems in salt applications above 100 mM. However, when sodium and potassium salts are applied in combination with glycine-betaine support, plants not only maintain their photosynthetic activity even under 200 mM salt stress but also exhibit an increase of approximately 20–30% in their total chlorophyll content (Fig. 2c, and 4c).

4.2. Effect of Salt Stress on Protein Concentrations

To survive against ionic, oxidative, and osmotic stress, plants produce numerous osmoprotectants (proline, glycine-betaine, dimethylsulfoniopropionate (DMSP), trehalose, etc.) and many other unidentified proteins. Common osmotic response pathways (both long-term and short-term) trigger the biosynthesis and accumulation of compatible osmolytes that can stabilize proteins, cellular structures, and morphology and restore osmotic potential in cells. Sayed (2011) reported that, compared with salt-sensitive varieties, salt-tolerant bean plants have a lower protein content and greater proline and amino acid content. Compatible osmolytes have been reported in previous studies to prevent water loss to resist short-term osmotic stress and increase cellular turgor and cellular expansion to cope with long-term osmotic stress (Yang and Guo 2018; Apse and Blumwald 2002; Blumwald 2003). It is also known that many compatible osmolytes that are biosynthesized under salt stress also accumulate under other stresses, such as drought and cold stress, and that their biosynthesis is partially species- and tissue-specific (Yang and Guo 2018; Parvanova et al. 2004; Yancey 2005; Pirzad et al. 2011; Sailaja et al. 2014). Many studies have shown that salt and metal stress can be alleviated in plants by the application of exogenous osmoprotectants (Hasanuzzaman et al. 2014; Aamer et al. 2018; Dustgeer et al. 2021). In the present study, increases in protein content between 3 and 111% were detected with increasing doses of salt stress. In particular, after GB-supported combined salt application, an approximately 111% increase in the total protein content was observed (Tables 1, 3 and 4; Table S1.4-7). This situation ensures that both the combined application of sodium and potassium (60–90% protein increase) and exogenous GB application (90–111% protein increase) trigger the production of both enzymatic and non-enzymatic antioxidants in hulled wheat, resulting in effective defense against ROS.

4.3. Effects of Salt Stress on the Enzymatic and Non-enzymatic Antioxidant Defense Systems

Plants have an antioxidant defense system in which enzymatic and non-enzymatic antioxidants are present in their cellular organelles to scavenge different ROS. When ROS production exceeds the scavenging ability of the antioxidant system, oxidative damage occurs. The antioxidant defense system consists of enzymatic [superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), glutathione reductase (GR), glutathione S-transferase (GST), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), glutathione peroxidase (GPX) and peroxiredoxin (PRX)] and non-enzymatic [ascorbate (AsA), glutathione (GSH), carotenoids, alkaloids, tocopherols, flavonoids, non-protein amino acids and phenolic compounds] (Hassan et al. 2018; Guo et al. 2015; Zou et al. 2016; McCord 2000). The AsA-GSH cycle consists of ASA, GSH, and four antioxidant enzymes (APX, DHAR, GR, and MDHAR), which play important roles in regulating ROS homeostasis by detoxifying H₂O₂ (Tamaki et al. 2021). Carotenoids, flavonoids, and phenolic acids help regulate ROS homeostasis by scavenging free radicals (Hussain et al. 2019; Agati et al. 2012; Liu et al. 2014; Di Ferdinando et al. 2012). All these components play regulatory roles in helping plants cope with oxidative stress caused by salt stress and transmit stress signals by controlling ROS homeostasis. Figure 5 shows that the antioxidative defense system developed in plants under salt stress. Although the SOS pathway has been reported to play a role in potassium uptake as well as sodium uptake and is crucial for the regulation of K+/Na + homeostasis in plants (Zhu 2016), it remains unclear how plants actively regulate potassium uptake, including whether it directly regulates potassium uptake.

Many studies have shown that the antioxidant defense system controls oxidative damage during biotic and abiotic stress in plants (Munns and Tester 2008; Hassan et al. 2020; Al-Ashkar et al. 2019; Hussain et al. 2021; Ibrahimova et al. 2021; Chinnusamy et al. 2005; Mittler 2002; Bose et al. 2014; Meneguzzo et al. 1999). A close relationship between antioxidants and salinity tolerance in wheat species was previously reported by Meneguzzo et al. (1999). Sreenivasulu et al. (2000) reported that plants increased the activities of antioxidant enzymes under salt stress. Athar et al. (2007) reported that the K⁺/Na⁺ ratio decreased in sensitive and tolerant wheat varieties under high salt stress (150 mM NaCl). They observed that growth and photosynthetic activity decreased. However, they also reported that tolerant wheat varieties increased endogenous AsA production and CAT activity to cope with salt stress. It has also been reported that salt-tolerant plants resist salinity by releasing sodium ions from their leaves through increases in SOD, APX, and CAT activities as well as photosynthetic activity through AsA (Athar et al. 2009). In our study, SOD enzyme activity increased in hulled wheat varieties with increasing salt dose, and the SOD activity in the roots was much greater than that in the stem (0.867 ± 0.11 units mg^− 1 fw in the stem, 0.985 ± 0.14 units mg^− 1 fw in the root) (Fig. 3a-b, Fig. 6; Table 1, 3–4). Moreover, among the salt doses applied, 50 mM NaCl + 50 mM KCl + 0.5 mM GB had the greatest increase in SOD activity in both the roots and stems (1,252 ± 0.16 unitsmg^− 1 fw in the roots, 1,058 ± 0.10 unitsmg^− 1 fw in the stems). Among the hulled wheat plants, the highest increase in SOD activity was detected in T. boeoticum (18.27%), and the lowest increase was detected in T. dicoccum (8.17%). The 15% increase in SOD activity in the T. boeoticum and T. monococcum varieties compared with that in the control, even under 150 mM salt stress, suggested that these varieties are more resistant to high salinity than are the other varieties (Table 5). In these species, SOD enzyme activity increased by 75% in the group treated with glycine-betaine supplemented with salt compared with the control group (Table 5–6, Fig. 3a-b).

Tetraploid wheat is relatively more sensitive to salt than bread wheat is (Munns et al. 2000). This is due to the decreased accumulation of K⁺ ions in the leaves of bread wheat; these ions are expressed as Kna1 loci and are controlled by chromosome 4D (Dubcovsky et al. 1996; Gorham et al. 1990). In our study, T. dicoccum had lower values in terms of the activity of all the enzymatic and non-enzymatic antioxidants (Table 5–6). Studies have shown that HKT genes play a role in sodium ion exclusion under salinity stress. According to these studies, Yang et al. (2014) reported that TaHKT1;5-D changes the transcriptional programing of Aegilops tauschii under salt stress, Byrt et al. (2014) reported that no change could be observed in hexaploid wheat, and Zhao et al. (2014) and Wang et al. (2020) reported that the function of this gene was significantly reduced in the JN177 hexaploid wheat variety. These contradictory results raise some questions regarding whether the TaHKT1;5-D response is tissue specific or based on HKT genes. In our study, the activity of T. speltoides, a hexaploid wheat plant, was second highest after that of T. dicoccum, and these findings support the findings of Byrt et al. (2014) under salt stress (Fig. 3. ).

Jabeen et al. (2020) reported an increase in H₂O₂ and O₂⁻ concentrations in wheat under 300 mM salt stress, and the plant increased SOD, POD, CAT, and proline activities to increase salt tolerance. Zeeshan et al. (2020) reported that 100 mM salt stress caused ROS and MDA accumulation, and POD, CAT, APX, and GR activities significantly increased to reduce the effects of salt stress. Dong et al. (2017) reported that 120 mM salinity stress induced oxidative and osmotic stresses, and POD, SOD, and CAT activities increased significantly to reduce the effects of salt-induced damage. Ahanger et al. (2019) reported that 100 mM salt stress caused the accumulation of hydrogen peroxide and superoxide, and CAT, SOD, and APX activities were significantly upregulated to scavenge ROS. Mandhania et al. (2006) reported that 10 dSm^− 1 salt stress induces ROS accumulation and increases MDA content, while CAT and APX activities are upregulated to counteract the effects of oxidative stress. According to the data we obtained in our study, compared with that in the control, the CAT activity in hulled wheat under salt stress was 112–231%; SOD activity was increased by 1–19%, GR activity was increased by 76–196%, GST activity was increased by 1-116%, and APX activity was increased by 20–34% (Table 6; Figs. 3 and 6). In terms of SOD, CAT, and GR activities, T. boeoticum and T. speltoides presented the greatest increase in activity (Fig. 2a). Interestingly, GST and APX activities were highest in the T. dicoccum variety, although all the other antioxidant enzyme activities were lowest (Table 6, Fig. 3). These findings suggested that the main antioxidant defense enzymes in T. dicoccum are GST and APX. In T. boeoticum, the activity of enzymes, especially CAT and GR, increased to 300% in response to combined salt application and glycine-betaine combined salt application. These findings suggested that HKT genes play an active role in maintaining the K⁺/Na⁺ ion balance in T. boeoticum, as suggested by Yang et al. (2014), Zhao et al. (2014), and Wang et al. (2020).

4.4. Effects of salt stress on proline accumulation

When plants face osmotic stress, they undergo osmoregulation and accumulate sugars, polyols, amino acids, and quaternary ammonium compounds to reduce the negative effects of stress (Farooq et al. 2015). Osmoregulation is responsible for triggering defense mechanisms against antioxidant species to regulate plant‒water relationships (Bose et al. 2014). Proline, an osmoprotectant, helps in osmotic adjustment as well as detoxification of ROS and strengthening of the PS-II structure (Szabados and Savouré 2010). It has been previously reported that the activities of various antioxidant enzymes (SOD, APX, and CAT) increase in response to exogenously applied GB, which significantly improves the salinity tolerance of wheat (Raza et al. 2006). In our study, increases in proline accumulation in hulled wheat plants (2–10 times compared to the control) were observed with increasing salt dose (Table 6; Table S1.8-11, Fig. 7). In another study, it was reported that GB application (10 and 30 mM) increased germination and calcium and chlorophyll contents in shoots and leaves and improved salinity tolerance (Akhter et al. 2007). Similarly, exogenous proline application (60 ppm) was reported to down regulate malondialdehyde (MDA) levels and improve salinity tolerance in wheat (Hendawey 2015). In a study conducted by Rao et al. (2013), increased Pro and GB production was reported to reduce the harmful effects of salt stress by activating antioxidant enzymes. It has also been reported that exogenous osmoprotectant applications increase proline and potassium accumulation and improve the K⁺/Na⁺ ratio, thereby stabilizing protein and lipid structures (Duman et al. 2010). As a result, hormone and osmoprotectant applications improve antioxidant activities, photosynthetic efficiency, and membrane stability and provide significant recovery under salinity stress by detoxifying ROS. In the present study, the highest proline accumulation was obtained in the 50 mM NaCl + 50 mM KCl + 0.5 mM GB treatment group. However, we observed that when 50 and 100 mM combined sodium and potassium chloride were supplemented with 0.5 mM GB, all the antioxidant enzymes increased by 10–300%, especially SOD, CAT and APX (Table 1, 3–4; Table S1.5, 7, 10 and, 11; Fig. 2, 3 and, 4b,d). These results provide evidence that proline accumulation increases under salt stress. In addition, exogenous GB application supported this increase in proline content and provided great support for plants to cope with ROS caused by salt stress (Fig. 4a and, 7; Table 5–6).

4.5. Effects of salt stress on lipid peroxidation (LPO; MDA)

In a study conducted by Hasanuzzaman et al. (2011), it was observed that salt-sensitive wheat varieties grown under saline conditions had more H₂O₂ and lipid peroxidation than salt-tolerant varieties. Zou et al. (2016) reported that the level of malondialdehyde (MDA) in wheat plants exposed to 100 mM NaCl salt for 5 and 10 days increased by up to 35% and 68%, respectively. In our study, with increasing salt dose, 30 mM NaCl increased MDA accumulation 2-fold, 50 mM 3–4, 100 mM 15–17, 150 mM 27–29 and 200 mM NaCl 39-42-fold compared with the control. No significant difference was detected between the effects of sodium chloride application and potassium chloride application on MDA accumulation (p ≥ 0.784; suppl. mat. S2). However, when sodium and potassium salts were applied together (50 mM NaCl + 50 mM KCl; 100 mM salt in total), the MDA accumulation was approximately 5 times lower than that resulting from the individual applications (Table 1, Table 3–4; Table S1.8-11; Fig. 2d; Fig. 4a, Fig. 7). This shows that the degree of membrane damage caused by salt stress depends on the type of salt rather than its concentration and that the damage is much lower than expected, especially in cases where the K⁺/Na⁺ balance in the membranes can be maintained. Previous studies have reported that ionic homeostasis is a key process that regulates ion flow to create low Na⁺ and high K⁺ concentrations (Hasegawa et al. 2000; Farooq et al. 2015). The regulation of intracellular Na⁺ and K⁺ ions (homeostasis) depends on the performance of various enzymes in the cytosol and maintains membrane potential and cell volume. The Na⁺ and K⁺ concentrations in the cytosol must be maintained at a balance. Plants excrete excess salt through primary and secondary active transport and accumulate these positively charged ions in plasma and tonoplast to maintain homeostasis during salt stress (Hasegawa et al. 2000). Cordovilla et al. (1995) reported that various K⁺ genes were up- and down regulated by salt stress. The vacuole is also compartmentalized to protect the cytosol from the harmful effects of excess Na⁺ ions (Farooq et al. 2015). Previous studies have shown that plants use some affinity-based transporters found in biological membranes (related to the K⁺/Na⁺ balance) for K⁺ uptake (Blumwald 2000; Amtmann and Sanders 1998).

4.6. Data analysis and biplot evaluation

When the responses of hulled wheat to salt stress were compared statistically, there was no difference between the varieties in terms of CAT or GR enzyme activity (p ≤ 0.97 and p ≤ 0.68, respectively) (Table S2.1). There was no significant difference between the roots and stems of wheat (wheats*sections) in terms of protein concentration (p ≤ 0.980) or SOD (p ≤ 0.254) or GST (p ≤ 0.193) enzyme activity. According to the antioxidant responses (wheats*doses) of wheat to different salt doses, there was no significant difference in the activities of the enzymes CAT (p ≤ 0.165) or GST (p ≤ 0.310). There was no difference in the antioxidant enzyme responses of plant parts (Sections*Doses) to salt dose between CAT (p ≤ 0.420) and GST (p ≤ 0.532). When all the variables were evaluated together (Wheats*Sections*Doses), no significant differences were detected in the activities of the CAT (p ≤ 0.414), GR (p ≤ 0.131) or GST (p ≤ 0.383) enzymes (Table S2.2). According to the results of Lewene’s test, there were significant differences in the activities of all the antioxidant enzymes (p ≤ 0.001) (Table S2.3). According to the MANOVA-Multivariate test, there were significant differences in enzyme activity depending on the application (Wheats*Sections*Doses) (Pillai's Trace, V: 2.566, F: 2.65, p ≤ 0.001; Wilks' Lambda, V: 0.030, F: 3.040, p ≤ 0.001; Hotelling's Trace, V: 5.237, F: 3.567, p ≤ 0.001; and Roy's Largest Root, V: 2.272, F: 12.746, and p ≤ 0.001) (Table S2.4).

A study was considered safe if the GT biplot analysis explained more than 50% of the variation (Akcura et al. 2011). The rates obtained in the study were well above this value (> 65). Biplot analysis can be applied equally to all genotypes by entering bidirectional data such as genotype-trait data by the user (Kaplan et al. 2014). Genotypes can be screened in terms of desired characteristics via biplot analysis (Yan and Tinker 2006). Biplot analysis also visually revealed the relationships between the examined features. According to biplot analyses, APX, GR, GST and SOD in T. monococcum; SOD and CAT in T. dicoccum; and only SOD in T. speltoides and T. boeoticum are at the forefront in combating salt stress in the roots of hulled wheat. (Fig. 8). In the stems of hulled wheat, GR, GST, and SOD in T. monococcum; SOD and APX in T. dicoccum; GR in T. speltoides; and the GST antioxidant enzyme in T. boeoticum, unlike in the roots, reacted first (Fig. 8). In addition, GB-supported combined with salt application increased the activities of antioxidant enzymes.

The development of salt-tolerant wheat varieties via appropriate agronomic practices or the introduction of ancestral hulled wheat varieties, which are likely to be naturally salt resistant, into breeding programs can help improve crop production under salt stress conditions. In Turkey, which is the gene center of wheat, there is a high probability that salt-tolerant varieties can result in higher crop yields than modern breeding wheat in sodic/saline soils rather than higher crop yields than existing varieties under normal growing conditions. It is highly important for food safety to use these varieties in agricultural lands with increasing salinity. According to the results of our study, it is more appropriate to apply potassium fertilizers to plants than nitrogenous fertilizers in soils with high salinity because, under these conditions, the plant's ability to combat salt stress seriously increases, and its biological activities normalize.

Wheat is the most popular and consumed grain product in the world. However, salinity stress poses a major threat to global wheat production and food and nutritional security. The hulled wheat we work with is highly important because it is the oldest ancestor of today’s modern wheat and was obtained from a genetic source. Because modern wheat plants are included in breeding programs for certain traits (especially yield and nutritional quality) and most are grown under optimum conditions, some of the naturally inherent genetic characteristics of wheat (salinity resistance, disease resistance, etc.) may disappear over time. It is necessary to evaluate wild relatives of these plants, introduce wheat plants with strong salinity tolerance and reintroduce them into agriculture. However, our study revealed that exogenous applications of osmoprotectants, phytohormones, and nutrients in addition to seed preparation (such as seed coating) can also help improve salt tolerance in wheat. All these efforts will help alleviate the negative effects of salinity stress on wheat crops, and salinity stress is predicted to contribute to increased wheat productivity and food security.

In addition, in recent years, producer and consumer interest in the use of whole wheat flours, which are called ancient grains instead of refined grains and can offer health benefits through bioactive compounds, has been increasing. Ancient hulled wheat species can offer a healthier and better nutritional profile than modern wheat plants can, and individuals with gluten intolerance, which is rapidly becoming widespread today, can better tolerate the products obtained from them. The use of today's ancient hulled wheat in bringing salty/sodic areas into agriculture will not only increase food security but also contribute to the supply of hulled flour needed by today's market.

Food security is currently a global threat worldwide, where approximately 65% of the land is considered infertile and approximately 90% is in danger of salinity/sodicity due to global climate change and various human factors. However, EU policies, especially the current EU Common Agricultural Policies (CAP), the European Green Deal (EGD), the EU Soil Strategy 2030, and the EU Biodiversity Strategy 2030, do not address this issue much, and legal arguments and regulations specifically addressing salt-affected soils are available only for a few EU countries (FAO 2023). The EU Policy Landscape includes salt-affected soils in only two of the 10 CAP main targets (European Commission 2023). Additionally, the EGD and Farm to Fork (F2F) strategies make almost no mention of salt-affected soils or saline agriculture (European Commission 2019). Therefore, there is a policy gap at the EU level, and there is a dire need for them to take an active role in tackling this global problem by incorporating policies that adapt to salinity or support broader mitigation measures into EU-level policies.

Funding

This study was supported financially by the Scientific Research Project Unit of Erciyes University (Project Code: FOA-2015-6008).

Declaration of competing interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

All the data generated or analyzed during this study are included in this published article (and its supplementary information files).

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Antioxidant defense responses of hulled wheat varieties to the addition of sodium and potassium salts and exogenous glycine-betaine, and evaluation of the usability of these hulled wheats in the remediation of saline soils

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Abstract

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1. INTRODUCTION

2. MATERIALS AND METHODS

2.1. Plant cultivation and stress management practices

2.3. Preparation of crude enzyme extracts and determination of protein concentrations

2.4. Determination of the chlorophyll and carotene contents

2.5. Determination of enzyme activity

2.5.1. CAT activity (EC 1.11.1.6)

2.5.2. Superoxide dismutase (SOD) (EC 1.15.1.1) activity

2.5.3. Ascorbate peroxidase (APX) (EC 1.11.1.11) activity

2.5.4. Glutathione reductase (GR) (EC 1.6.4.2) activity

2.5.5. Glutathione S-transferase (GST) (EC 2.5.1.18) activity

2.6. Lipid peroxidation (LPO) (MDA)

2.7. Determination of the proline concentration

2.8. Statistical evaluation of the results

3. RESULTS

3.1. Effects of salt stress on plant growth

3.1.1. Effect of salt stress on the fresh weight of plant roots and leafy stems

3.1.2. Effect of salt stress on the dry weight of plant roots and leafy stems

3.1.3. Effect of salt stress on leaf length

3.1.4. Effect of salt stress on total plant height

3.2. Effect of salt stress on the total protein content

3.3. Effects of salt stress on antioxidant enzyme activity in plant roots and leafy stems

3.3.1. Effect on SOD activity

3.3.2. Effect on CAT activity

3.3.3. Effect on GR activity

3.3.4. Effect on the GST activity

3.3.5. Effect on APX activity

3.4. Effect on proline accumulation

3.5. Effect on lipid peroxidation (LPO) (MDA)

3.6 Statistical data analysis results

4. DISCUSSION

4.1. Effects of Salt Stress on Plant Development

4.1.1. Effect on Plant Biomass

4.1.2. Effects on Chlorophyll a, b, and Total Chlorophyll and Carotene Contents

4.2. Effect of Salt Stress on Protein Concentrations

4.3. Effects of Salt Stress on the Enzymatic and Non-enzymatic Antioxidant Defense Systems

4.4. Effects of salt stress on proline accumulation

4.5. Effects of salt stress on lipid peroxidation (LPO; MDA)

4.6. Data analysis and biplot evaluation

5. Conclusions AND EXPECTATIONS

Declarations

References

Additional Declarations

Supplementary Files

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