Jordan National Agricultural Research Center provided three six-row barley genotypes (Acsad 176, Athroh, and Rum) used in this study, generally grown as feed crops in Middle Eastern countries. Tested genotypes are mainly characterized as the following: Acsad176 (released 1987) is medium in height and produces short spikes; under favorable conditions, it produces vigorous vegetative growth. Athroh (released 2004) is characterized by a medium height and long spikes, long coleoptile, produces vigorous vegetative growth and fresh weight. Rum (released 1986) is characterized by producing many tillers that have short spikes, medium height, early to medium flowering, fast filling, and large grain size. It is adapted and strongly tolerant to drought stress in semi-arid regions of the Middle East. Before treatments, freshly harvested seeds were surface sterilized by suspending seeds in 100% bleach (v/v, 6% sodium hypochlorite) for 5 minutes, followed by washing them five times with sterile distilled water. The experiments were conducted in a controlled environment using a substrate consisting of 50% peat moss and 50% perlite and varying concentrations of sodium chloride (NaCl) (0, 25, 50, 75, 100, and 200 mM), Mannitol (0, 50, 100, 150, 200 and, 300 mM), and Sorbitol (0, 50, 100, 150, 200 and, 300 mM) these concentrations were chosen based on previously published studies [24, 25]. Seedlings were grown in a growth chamber at a constant 25 ºC with continuous illumination (40 mol m^-2 s^-1) from cool white fluorescent lamps. The three barely genotypes (Acsad 176, Athroh, and Rum) used in this study are cultivated cultivars in Jordan and Middle Eastern countries. All experimental work and plant materials collection were performed to comply with relevant institutional, national, and international guidelines and legislation.

Three separate sets of ten surface-sterilized Acsad 176, Athroh, and Rum seeds were placed on two filter papers in each petri dish saturated with different NaCl concentrations for the seed sensitivity assay (0, 25, 50, 75, 100 and 200 mM), separately, mannitol (0, 50, 100, 150, 200, and 300 mM) and sorbitol (0, 50, 100, 150, 200, and 300 mM) were used. Ten days after the seeds were germinated, we recorded the number of radical sprouts that had emerged. The germination rate was determined and compared to that of untreated seeds to assess the influence of NaCl, mannitol, and sorbitol on seed germination. Every measurement was performed in triplicate.

For each treated seedling from the three barley genotypes, three parameters were measured: seedling length (cm), shoot fresh, and dry weight (g). After 10 days, Acsad 176, Athroh, and Rum were compared to non-treated seedlings to determine plant growth. The seedling's length was measured from the emerging plant at the soil level to the shoot tip using a ruler. Shoot fresh weight (g) was obtained by weighing the shoot of each seedling and shoot dry weight (g) after drying the shoot in the oven at 70 ºC for 24 hours.

The chlorophyll contents (a and b) were determined using a modification of the protocol published earlier [26]. To generate chlorophyll extracts, 250 mg of fresh leaves were combined with 25 mL of 90% ice-cold acetone in a tissue homogenizer. The combination was centrifuged for five minutes at 14,000 rpm (Ace Industries Pvt. Ltd. India). The supernatant was then moved to a separate container for chlorophyll analysis. Using the microplate reader, the extract's absorption was determined at frequencies of 664 nm and 647 nm (Multiskan FC, Thermo-Fisher Scientific, Finland). Then, to calculate the total amount of chlorophyll, we calculated the amounts of chlorophyll a and b using the coefficients of the presented equation. Each measurement was carried out three times.

Three separate sets of two-week-old seedlings of each barely genotype (Acsad 176, Athroh, and Rum) were grown in synthetic soil and irrigated every day for ten days with 25, 50, 75, 100, and 200 mM NaCl; 50, 100, 150, 200, and 300 mM mannitol, and 50, 100, 150, 200, and 300 mM sorbitol [23-37] separately. After 10 days, the shoots were collected to determine the malondialdehyde (MDA) amount. Malondialdehyde is used as a reference for reactive oxygen species in seedling shoots using the thiobarbiturate reactive substances (TBARS assay) [27]. Using mortar and pestle, 250 mg of tissue was crushed, and then 0.25 mL of 0.5% (w/v) thiobarbituric acid and 0.25 mL of 175 mM NaCl in 50 mM Tris-HCl at pH 8 were added. For 25 minutes, tubes were preheated to 90 °C. The substance was centrifuged for 20 minutes at 15,000 rpm to collect the supernatant. At 532 nm, the absorbance of the supernatant was measured using the microplate reader (Multiskan FC, Thermo-Fisher Scientific, Finland). An MDA standard curve's nmol/mg FW readings were used to determine the MDA concentrations. For each treatment, an average of three replicate sets was determined.

Three different sets of two-week-old Acsad 176, Athroh, and Rum genotype seedlings were grown in synthetic growth soil and irrigated every day with different concentrations of NaCl, Mannitol, and Sorbitol (see Seed germination rate under salinity and osmotic stress sub-title). The shoots were harvested for GABA level analysis after 10 days. GABA metabolites were extracted according to Zhang and Bown [28] with the following modification:

Shoot tissues (500 mg) of fourteen-day-old seedlings of each genotype treated by NaCl, mannitol, and sorbitol separately were grounded with a mini pestle and mortar and placed in 1.5 mL microcentrifuge tubes. 0.4 mL of methanol was added to each tube, and the samples were mixed for 10 minutes. Liquid from the samples was removed by regular evaporation overnight (tubes were kept open to allow methanol evaporation). Then, 0.5 mL of 70 mM lanthanum chloride was added to each tube. The tubes were mixed for 15 minutes and subsequently centrifuged at 10 000× g for 5 min. The supernatant was removed to new tubes and mixed with 0.16 mL of 1 M potassium hydroxide (KOH). The tubes were mixed for 10 min and then centrifuged at 10 000× g for 5 min. The supernatant containing metabolite was transferred to a new tube to measure the GABA level.

Zhang and Bown’s [28] method was used to quantify GABA levels with the following modifications: the reaction mixture contained 50 µL of sample extract, 14 μL of 4 mM NADP+, 19 μL of 0.5 M potassium pyrophosphate at pH 8.6. 10 µL of GABASE (2 U μL-1) (GABASE enzyme powder was suspended in 0.1 M potassium pyrophosphate at pH 7.2 containing 12.5% glycerol and 5 mM β-mercaptoethanol) and 10 μL of α-ketoglutarate. Change in absorbance at 340 nm was recorded after 90 min incubation at 25°C using the microplate reader (Multiskan FC, Thermo-Fisher Scientific, Ratastie, Finland). The content of GABA was determined using the NADPH standard curve. For each treatment, three replicates were conducted.

Total RNA from fresh shoot samples was extracted using the Qiagen RNeasy Plant Mini reagent (Mini-prep, easy-spinTM IIP, Intron Biotechnology, Korea) according to the manufacturer's instructions. Total RNA was suspended in RNase-free water. RNA amounts were determined using a nanodrop spectrophotometer (ND-1000) and their absorbance at A260. The integrity of the RNA was evaluated and imaged using a UV trans-illuminator detection technique after electrophoresis, ethidium bromide staining, and RNA separation on a 1.5% (w/v) agarose gel (UVP BioDoc-It Imaging System. UniGreenScheme, UK).

According to the experiment, reverse transcriptase-PCR (RT-PCR; Intron Biotechnology, Korea) measurement of steady-state mRNA levels in all barley samples was performed using gene-specific primers for the GAD enzyme gene (reverse primer 5'-CGG TTC TGG AGC TCG GTG GTG AC -3' and forward primer 5'-TGC CGG AGA ACT CGA TCC CCA AG -3'). According to Xu et al. [29], all sequenced GAD genes coming from different plants share a considerable portion of identity. In a one-step RT-PCR procedure, the GAD gene-specific primer pairs developed by Mazzucotelli et al. [30] were applied. SuperScriptTM III, One-Step RT-PCR instrument with platinum® TaqDNA polymerase was used according to the manufacturer's instructions (Intron Biotechnology) as the following: one cycle of reverse transcription reaction (45°C for 30 min) and denaturation of RNA:cDNA hybrid (94°C for 5 min) followed by three step cycling (denaturation (94°C for 30 s), annealing (56°C for 40 s), extension (72°C for 1 min) for 40 cycles then final extension (72°C for 5 min) for one cycle. RT-PCR. On 2% agarose gels, RT-PCR amplified products were sorted and stained with ethidium bromide. For all barley genotypes, the GAD gene expression level was determined by Al-Quraan et al. [31]. To guarantee constant RNA yields, each reaction tube also held an internal standard made up of a primer pair for the 18S RNA (forward primer 5’-CCA CCC ATA GAA TCA AGA AAG AG-3’and reverse primer 5’-GCA AAT TAC CCA ATC CTGAC-3’) as an internal control in each tube was used to normalize the abundance of transcribed GAD RN as outlined by Jarošová and Kundu [32]. Individual cDNA amplicons' levels of RNA expression were measured using the UVP BioDoc-It Imaging System (UniGreenScheme, UK) to determine the fluorescence. The fluorescence value of each DNA band was calculated after the background fluorescence was eliminated from it. To evaluate the amounts of GAD gene expression that happened as a consequence of treatments with NaCl, Mannitol, and Sorbitol, the data were calculated as a Log2 fold change in all three barley genotypes under all treatments.

Acsad 176, Athroh, and Rum genotype seedlings were grown in a soil substrate and watered, as previously mentioned, when they were two weeks old. On day 10 of watering, plant shoots were harvested for protein extraction and quantification using a SMART BCA Protein Assay Kit (Intron Biotechnology, Korea) in agreement with the directions given by the producer, with the necessary modifications made one mL of the working solution was added after 0.05 mL of each treated sample had been collected, and the combination was blended entirely. The samples were then permitted to settle to room temperature after 30 minutes of incubation at 37 °C. The samples' absorbance at 562 nm was measured with a microplate reader (Multiskan FC, Thermo-Fisher Scientific, Finland), and the protein concentrations of each sample were calculated using the BSA reference curve.

Acsad 176, Athroh, and Rum genotypes' two-week-old seedlings were grown in an artificial growth medium and watered as previously described. The shoots of the plants were collected on the 10^th day after they were irrigated and then used following Gerhardt [33] with the following modifications: After obtaining 0.05 mL of each treated shoot sample and chilling them on ice, mixing all of the tubes with 0.1 mL of chilled 75% H₂SO₄, adding 0.2 mL of chilled anthrone solution to each tube, and then placing all of the COD (Chemical Oxygen Demand) tubes in a water bath heated to 100 °C for 15 minutes, the tubes were removed and left to cool at room temperature. Plotting the absorbance recorded at 578 nm using the microplate reader (Multiskan FC, Thermo-Fisher Scientific, Finland) against a glucose standard curve allowed us to estimate the total quantity of carbohydrates in the samples.

This study's experiments were all completely randomized (CRD). Every assay was performed in three replicates. Calculations were made to determine the mean and standard deviation (SD) for each trait of every experiment. The findings were reported using mean and standard deviation. One-way analysis of variance (ANOVA), normality tests, and multiple comparison tests using the Least Significant Difference (LSD) were used to analyze the data. The SPSS version 16.0 software was used for all statistical analyses. P-values equal to or less than 0.05 were considered significant in all data analyses, and P-values equal to or less than 0.01 were considered highly significant.

Under salinity and osmotic stress, germination rates were measured in Acsad 176, Athroh, and Rum (Table 1). In the control treatment, the germination percentage was 100% in Acsad 176, 97% in Athroh, and 93% in Rum. The germination percentages of Acsad 176, Athroh, and Rum decreased significantly with a negative correlation within genotype when different levels of sorbitol, NaCl, and mannitol were increased, respectively (Table 1). Athroh germination was decreased under osmotic stress (mannitol) (P = 0.022), with the highest germination reduction (30%) under 300 mM mannitol compared to control. According to the data, Athroh showed a 20% decrease in germination when exposed to 300 mM sorbitol (P = 0.002) and a 27% reduction under 200 mM NaCl (P = 0.005). Under salinity (NaCl) and osmotic stress (mannitol and sorbitol), Acsad 176 showed a modest reduction in germination where the reduction under 300 mM sorbitol (P = 0.034) reached 15% and 20% reduction under 300 mM mannitol (P = 0.002) and 200 mM NaCl stress (P = 0.028). According to our findings, Rum is more responsive to sorbitol treatment compared to other treatments (NaCl and mannitol). Rum germination was reduced to 30% under 300 mM sorbitol, 23% under 200 mM NaCl, and 17% under 300 mM mannitol.

The three barley genotypes showed no significant differences in seedling length under osmotic and salt stresses, except Acsad 176 had a significant reduction in seedling length with increasing concentrations of mannitol (P = 0.012) and sorbitol (P = 0.005) (Table 2). These results might be due to the short duration of treatment (10 days), which might not be enough to have an observed significant effect on seedling length.

Barley genotypes showed no significant difference in fresh weight under salt and osmotic stresses. Acsad 176, Athroh, and Rum, NaCl (P = 0.432, 0.083, and 0.124), mannitol (P = 0.162, 0.487, and 0.026), and sorbitol (P = 0.188, 0.18, and 0.265), respectively, but there is a negative correlation of fresh weight and increasing stress levels in the three barley genotypes (Table 3).

Salt and osmotic stress decreased plant dry weight in Acsad 176, Athroh, and Rum compared with the control treatment (Table 4). In the control treatment, the dry weight of plant was 0.29 g in Acsad 176, 0.28 g in Athroh, and 0.29 g in Rum. The dry weight under NaCl treatment of Acsad 176, Athroh, and Rum were negatively correlated with increasing stresses of NaCl (r = -0.713, -0.652, and -0.641), mannitol (r = -0.706, -0.66, and -0.809), and sorbitol (r = -0.714, -0.627, and -0.683), respectively.

In all barley genotypes studied, chlorophyll a and b content significantly decreased when exposed to salt and osmotic pressure (Tables 5 and 6). Athroh showed a significant decrease of chlorophyll a content under mannitol treatment (P = 0.011), which was correlated with a low level of GABA accumulation under 300 mM mannitol, indicating the lethal effect of high mannitol level on Athroh genotype. Acsad 176 showed a high chlorophyll-a level under NaCl stress (P = 0.009), which was associated with high GABA accumulation under 200 Mm NaCl treatment. Furthermore, Rum revealed a relation between GABA levels and chlorophyll content under 300 mM mannitol treatment (P = 0.046).

The amount of oxidative damage, reactive oxygen species (ROS), and lipid peroxidation in barley seedlings under the investigated conditions is reflected in the outcomes of our investigation, which were determined based on the level of malondialdehyde (MDA) accumulation. In the barley seedlings of the Acsad 176, Athroh, and Rum genotypes, the concentrations of MDA increased with increasing amounts of NaCl (r = 0.889, 0.965, and 0.98), mannitol (r = 0.976, 0.944, and 0.64), and sorbitol (r = 0.974, 0.923, and 0.935), respectively. The data show that Acsad 176 had the least MDA accumulation under salt stress (NaCl) compared with other genotypes (P = 0.019) (Fig. 1a), which might indicate a low ROS level in Acsad 176 tissue under NaCl treatments due to physiological adaptation by Acsad 176 genotype to salt stress. Athroh showed the highest MDA accumulation under 300 mM of mannitol (P = 0.005), which is an indicator of oxidative damage that is created in response to osmotic stress (mannitol) (Fig. 1b). Rum, on the other hand, demonstrated the highest MDA accumulation during sorbitol-induced osmotic stress (P = 0.006) (Fig. 1c).

Fig. (1). Malondialdehyde (MDA) level (nmol/mg FW) in 14 days old seedlings of three barley (Acsad 176, Athroh, and Rum) irrigated with five different NaCl concentrations mM (A), five different mannitol concentrations mM (B), five different sorbitol concentrations mM (C). Columns with different letter scripts are statistically different (P ≤ 0.05). (*) P ≤ 0.05, (**) P ≤ 0.01.

GABA levels significantly increased and positively correlated in the three barley genotypes as the levels of NaCl (r = 0.876, 0.967, and 0.964), mannitol (r = 0.89, 0.79, and 0.872), and sorbitol (r = 0.879, 0.918, and 0.615), respectively (Fig. 2A). Athroh showed a significant increase in GABA metabolite accumulation under 300 mM sorbitol (P = 0.010) compared to the control. No significant increase of GABA accumulation in Athroh genotype under mannitol treatments (P = 0.061) (Fig. 2B), which indicates that Athroh tolerates osmotic stress-induced via sorbitol possibly by the synthesis of GABA rather more than osmotic stress caused on by treatment with mannitol (Fig. 2C). The Acsad 176 genotype showed high GABA accumulation under 200 mM NaCl treatment (P = 0.022) (Fig. 2A), which indicates a higher tolerance of Acsad 176 genotype to salt stress compared to other barley genotypes. Under 300 mM mannitol, there was a highly significant increase in GABA accumulation in Rum (P = 0.024). This suggests that, compared to treatments with NaCl and sorbitol, Rum is more tolerant of high concentrations of mannitol due to highly significant GABA accumulation (Fig. 2).

Fig. (2). GABA level (nmol/mg FW) in 14 days old seedlings of three barley (Acsad 176, Athroh, and Rum) irrigated with five different NaCl concentrations mM (A), five different mannitol concentrations mM (B), five different sorbitol concentrations mM (C). Columns with different letter scripts are statistically different (P ≤ 0.05). (*) P ≤ 0.05, (**) P ≤ 0.01.

Regarding seed germination, the level of GAD RNA transcription correlates with the response to salt and osmotic treatments (Table 1). This study demonstrated MDA accu- mulation (Fig. 1) and GABA accumulation (Fig. 2). Under salt and osmotic stress, the transcription level of GAD significantly increased (P ≤ 0.05) in all treated barley genotypes tested in this investigation (Acsad 176, Athroh, and Rum) (Fig. 3). The GAD improves decarboxylation of glutamate to CO₂ and GABA. Rum genotypes subjected to osmotic stress (mannitol) had significant levels of GAD expression (Fig. 3B), which was consistent with the high amount of GABA accumulation (mannitol). Acsad 176 genotype showed high GAD expression under NaCl stress (Fig. 3A), consistent with the high level of GABA accumulation under the same conditions. GAD expression in Athroh, on the other hand, was lower under mannitol because GABA accumulation and germination were low under the same conditions. This suggests that mannitol has a negative effect on Athroh shoot tissue, in contrast to high GAD expression under NaCl and sorbitol treatments (Fig. 3C). Rum genotype responded to salt and osmotic stresses with moderately significant GAD expression and GABA accumulation (Fig. 3). Under mannitol, the highest GAD expression was found (Fig. 3B). This demonstrates that the GABA shunt was activated in response to stressors based on metabolite production and GAD expression (salt and osmotic).

Protein content demonstrated a significant difference among Acsad 176, Athroh, and Rum responses to stresses (salt and osmotic) (Fig. 4). The protein concentration in Rum was the highest under 50 mM NaCl treatment (Fig. 4A). Rum showed no significant difference in protein level under mannitol stress (P = 0.378) (Fig. 4B), which is correlated with high GABA accumulation in response to mannitol. This result indicated that mannitol treatments did not affect Rum's protein content. The protein level in Rum significantly decreased under sorbitol (P = 0.034) treatment (Fig. 4C). This observation could be attributed to the deadly effect of high-concentration NaCl and sorbitol treatments on barley. According to protein accumulation, Rum genotype is more tolerant to high mannitol concentrations than Acsad 176 and Athroh (Fig. 4). Our data showed the highest protein content in Athroh compared to the control treatment. Athroh’s protein content was significantly reduced by mannitol treatment (P = 0.045), which was negatively correlated with low GABA accumulation under osmotic stress (mannitol) (r = -0.785). This finding suggested that Athroh is less tolerant to high mannitol concentrations than NaCl and sorbitol through protein accumulation under stress treatments. Protein content in Acsad 176 did not differ significantly in response to NaCl stress (P = 0.356). On the other hand, Acsad 176 was strongly affected under mannitol stress, which was observed by reduced protein content (r= -0.921, P = 0.009).

Fig. (3). GAD RNA transcription level (GAD log2 fold expression) in 14 days old seedling of three cultivated barley (Acsad 176, Athroh, and Rum) irrigated with different NaCl (A), mannitol (B), and sorbitol (C) concentrations (mM). Columns with different letter scripts are statistically different (P ≤ 0.05). (*) P ≤ 0.05, (**) P ≤ 0.01.

Fig. (4). Total protein content levels (µg/mL FW) in 14 days old seedling shoot tissues of three cultivated barley (Acsad 176, Athroh, and Rum) irrigated with different NaCl (A), mannitol (B), and sorbitol (C) concentrations (mM). Columns with different letter scripts are statistically different (P ≤ 0.05). (*) P ≤ 0.05, (**) P ≤ 0.01.

Carbohydrate content in Acsad 176, Athroh, and Rum was significantly decreased under salt and osmotic stress (Fig. 5). Acsad 176 showed a significant decrease in carbohydrate content under salt treatment (NaCl) (P = 0.023) compared with mannitol (P = 0.048) and sorbitol stress (P = 0.02). According to the findings of this study, Acsad 176 may adapt to salt stress (NaCl) by accumulating carbohydrates more than Athroh and Rum genotypes (Fig. 5A). Carbohydrate concentration in Rum was decreased significantly under osmotic stress (sorbitol) (P = 0.049) (Fig. 5C), which was correlated with low GABA accumulation in Rum tissue under sorbitol treatment. Athroh genotype showed a significant decrease under osmotic stress (mannitol) (P = 0.021) (r= -0.726) (Fig. 5B).

Fig. (5). Carbohydrates content levels (µg/mL FW) in 14 days old seedling shoot tissues of three cultivated barley (Acsad 176, Athroh, and Rum) irrigated with different NaCl (A), mannitol (B), and sorbitol (C) concentrations (mM). Columns with different letter scripts are statistically different (P ≤ 0.05). (*) P ≤ 0.05, (**) P ≤ 0.01.