GABA metabolism and ROS accumulation in barley (Hordeum vulgare L.) seedlings under the effect of salt and osmotic stress

doi:10.21203/rs.3.rs-2480355/v1

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GABA metabolism and ROS accumulation in barley (Hordeum vulgare L.) seedlings under the effect of salt and osmotic stress

https://doi.org/10.21203/rs.3.rs-2480355/v1

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GABA is a secondary metabolite that protects against oxidative damage, regulates nitrogen metabolism, and controls cytosolic pH in response to abiotic and biotic stress. This study examines the role of GABA shunt pathway response to salt and osmotic stress in three barley genotypes ("Acsad 176", "Athroh", and "Rum") interm of oxidative damage, seed germination and seedling growth as measured by glutamate decarboxylase gene (GAD) expression malondialdehyde accumulation (MDA), total proteins, total carbohydrates level, and GABA accumulation level. As Mannitol, NaCl, and sorbitol concentrations increased, seed germination decreased for all genotypes. All genotypes seedling MDA concentrations increased with increasing NaCl, mannitol, and sorbitol concentrations. Acsad 176 showed high GABA accumulation under NaCl treatment. Rum's GABA accumulation under mannitol testament increased significantly. All salt and osmotic treatments decreased chlorophyll a and b and carbohydrate content. At the same time it increased GAD transcription in all barley genotypes. Salt and osmotic stresses affected protein content in all genotypes. Acsad 176 may adapt to NaCl stress by accumulating carbohydrates more than Athroh and Rum. GABA shunt is a crucial signaling and metabolic pathway that facilitates barley's adaptation to salt and osmotic stress. In high salt and osmotic soil, Acsad 176 is the recommended genotype.

Biological sciences/Plant sciences

Biological sciences/Plant sciences/Natural variation in plants

GABA

GAD

Hordeum vulgare

Metabolism

ROS

Salt Stress

Osmotic Stress

One of the most widely used cereal grains, barley (Hordeum vulgare L.) is grown all over the globe. In addition, barley is highly resistant to a variety of abiotic challenges, including water scarcity, salt, and high temperatures, which are critical factors for crop production¹. Middle east countries are known for its mild wet winters and dry hot summers². where barley production varies from year to year depending on the annual rainfall (~ 30–100 mm in the steppe desert to 600 mm in the highlands), and its distribution during the growing season (October to March) which varies from region to region³. In the middle east countries, barley yields are mostly affected by the aforementioned fluctuations in precipitation over the period of the season⁴.The genotype (G), environmental (E), and G by E effects all contribute to the plant phenotype. Seed germination and growth, among other plant-growth stages, is profoundly affected by environmental stressors⁵ .About half of the world's irrigated areas are subject to salinity stress, which inhibits growth and reproduction⁶. Plant species, growth stage, and exposure time all affect how salinity affects a plant⁷.

One of the environmental stresses caused by salinity, drought, or extreme heat is osmotic stress⁸.Water loss or solute accumulation increase osmotic stress by impairing water absorption and transport from soil solution to root cells⁹. Osmotic stress causes significant changes in plant growth, productivity, changes in cell division and elongation, and many other physiological parameters¹⁰. Several mechanisms allow plants to respond to osmotic stress. One of these is the accumulation of the phytohormone abscisic acid (ABA)¹¹. Salinity stress has a negative impact on the number of tillers, spike length, spikelet count per spike, plant yield and biomass, root and shoot lengths, germination percentages, and fresh and dry weights¹². Barley, unlike wheat and other cereal crops, can provide a consistent and profitable harvest even in less-than-ideal environments. Barley is well-known for its ability to produce a large number of tillers, which cover the soil's surface and limit the quantity of water lost through evaporation¹³. This could explain why barley is more resistant to drought than maize, wheat, and rice.

According to research^14,15, there is a short cut for GABA metabolism known as the GABA shunt that bypasses two phases of the Tricarboxylic acid (TCA) cycle. Mitochondrial GABA transaminase (GABA-T) enzymes, glutamate decarboxylase (GAD), and succinic semialdehyde dehydrogenase (SSADH) are all involved in the GABA shunt¹⁶. Many studies suggests that GABA may play a role in the regulation of cytosolic pH, the modification of nitrogen and carbon metabolism, and the provision of protection against oxidative stress^17,18. Under oxidative stress and other abiotic conditions, it was expected that GABA breakdown would decrease the amount of reactive oxygen that accumulated¹⁷. Plants that are subjected to biotic and abiotic stresses are rapidly accumulate GABA^17,19−21. Insects crawling on leaves raise GABA levels²². Wheat under stress (salt and osmotic) had significantly higher GABA levels, according to²³. They investigated how three barley genotypes responded to salt and osmotic stress induced by sorbitol, mannitol, and NaCl treatments. These effects included GABA accumulation level, oxidative damage, carbohydrates and proteins levels, seed germination, seedling growth, and characterization of glutamate decarboxylase gene (GAD) expression.

Plant materials and growth conditions

Jordan National Agricultural Research Center provided three six-row barley genotypes (Acsad 176, Athroh, and Rum) used in this study which are normally grown in the middle east counntries. Tested genotypes are mainly characterized as the following: Acsad176 (released 1987) is a medium height and produces short spikes, under good conditions it produces vigorous vegetative growth, Athroh (released 2004) is characterized by a medium in height and has long spikes, long coleoptile, produces vigorous vegetative growth and fresh weight. mainly grown in semi-arid regions and Rum (released 1986) is characterized by the production of the large number of tillers has short spikes, medium height, early to medium flowering, with fast filling, and large grain size. Adapted and strong tolerant to drought stress in semi-arid regions of the middle east. The experiments were conducted in a controlled environment using a substrate consisting of 50% peatmoss and 50% perlite and varying oncentrations 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). Seedlings were kept in a constant 25 ºC environment with continuous illumination (40 mol m^− 2 s^− 1) from cool white fluorescent lamps. The three barely genotypes (Acsad 176, Athroh, and Rum) that used in this study are cultivated cultivars in Jordan as well as in middle east countries and all experimental work and plant materials collection were performed to complies with relevant institutional, national, and international guidelines and legislation.

Seed sensitivity to salt and osmotic stress

Three separate sets of ten surface-sterilized Acsad 176, Athroh, and Rum seeds were planted 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. To assess the influence of NaCl, mannitol, and sorbitol on seed germination, the germination rate was determined and compared to that of untreated seeds. Every measurement was performed in triplicate.

Determination of seedling growth

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

Extraction and determination of chlorophyll

The determination of the chlorophyll contents (a and b) followed a modification of²⁴. 250 mg of fresh leaves were blended with 25 ml of 90% ice-cold acetone in a tissue homogenizer to create chlorophyll extracts. The mixture was then centrifuged at 14,000 rpm for five minutes. After that, the supernatant was transferred to a different tube for chlorophyll analysis. The extract's absorbance was measured at 664 nm and 647 nm wavelengths. Then, using the coefficients of the displayed equation, we estimated chlorophyll a and b to arrive at the total amount of chlorophyll. Each measurement was carried out three times.

Evaluation of Oxidative Damage

Two-week-old Acsad 176, Athroh, and Rum genotype seedlings were grown on synthetic growth soil and irrigated every day for ten days with mannitol and sorbitol at different concentrations (0, 50, 100, 150, 200, and 300 mM), as well as NaCl at different concentrations (0, 25, 50, 75, and 200 mM). After 10 days, the shoots were collected to determine the amount of malondialdehyde (MDA). Malondialdehyde is used as a reference for reactive oxygen species in seedling shoots using²⁵. thiobarbiturate reactive substances (TBARS) assay.250 mg of tissue were crushed in a mortar and pestle, and 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 afterward. Tubes were warmed to 90 degrees Celsius for 25 minutes. The material was centrifuged at 15000 rpm for 20 minutes before the supernatant was collected. At 532 nm, the supernatant's absorbance was gauged. MDA concentrations were calculated using nmol/mg FW values that were obtained from an MDA standard curve. For each treatment, an average of three replicate sets was determined.

Extraction and identification of GABA metabolites

Three different sets of two-week-old Acsad 176, Athroh, and Rum genotype seedlings were grown on synthetic growth soil and irrigated every day with NaCl in a range of concentrations (0, 25, 50, 75, 100, and 200 mM), Mannitol in a range of concentrations (0, 50, 100, 150, 200, and 300 mM), and Sorbitol in a range of concentrations (0, 50, 100, 150, 200, and 300 mM). The shoots were harvested for GABA level analysis after 10 days. [26] state that the following modifications were made to the GABA extraction process: Following a 10-day salt and osmotic treatment, shoot tissues (500 mg) of fourteen days old seedlings of each genotype were harvested and separately crushed in a pestle and mortar before being collected in 1.5 ml micro-centrifuge tubes. Each tube received 400 L of methanol, which was added and agitated for 10 minutes. We evaporated the samples over night to get rid of any liquid. 500 L of 70 mM lanthanum chloride were added to each tube, and the mixture was then stirred for 15 minutes before being centrifuged at a speed of 15000 rpm for 5 minutes. The supernatant was transferred into fresh tubes, combined for 10 minutes with 160 l of 1 M potassium hydroxide and centrifuged for 5 minutes at a speed of 15000 rpm. The metabolite-containing supernatant was moved to a separate tube in order to measure the GABA level.

GABA (γ-Aminobutyric acid)

The Zhang and Bown (1997)²⁶. method was used to quantify GABA levels, but with the following modifications: Sample extract in 50 liters, 4 mM NADP + in 14 liters, 0.5 M potassium pyrophosphate in 19 liters, pH 8.6, and 2 u/L in 10 liters. In the procedure, 10 L of -ketoglutarate and GABASE enzyme (GABAZE enzyme suspended in 0.1 M potassium pyrophosphate, pH 7.2, containing 12.5% Glycerol and 5 mM -mercaptoethanol) were utilized. After 90 minutes of incubation at 25°C, the change in absorbance at 340 nm due to the addition of -ketoglutarate was evaluated using nanodrop spectrophotometry (ND-1000). NADPH standard curves and nmol/mgFW were used to calculate the GABA concentration. For each treatment, three replicates were conducted.

RNA extraction

Following the manufacturer's recommendations, total RNA was isolated from fresh samples using the Qiagen RNeasy Plant Mini kit. Total RNA was suspended in RNase-free water. Using a nanodrop spectrophotometer (ND-1000) and their absorbance at A260, RNA quantities were calculated. After electrophoresis, ethidium bromide staining, and RNA separation on a 1.5% (w/v) agarose gel, the integrity of the RNA was assessed and photographed using a UV trans-illuminator detection method.

Reverse Transcriptase-PCR Measurement of GAD mRNA Expression

According to the experiment, reverse transcriptase-PCR (RT-PCR) 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 the findings of²⁷, who discovered a significant proportion of similarity in all sequenced GAD genes originating from various plants. The GAD gene-specific primer pairs developed by²⁸ were used in a one-step RT-PCR process. A SuperScriptTM III, One-Step RT-PCR system with platinum® TaqDNA polymerase was used, per the manufacturer's recommendations. The ethidium bromide-stained separation of RT-PCR amplification products on 1% agarose gels. The GAD gene expression level was calculated for all barley genotypes by²⁹. Each reaction tube also contained an internal standard consisting of a primer pair for the CBP20 protein, as described by³⁰, to ensure consistent RNA yields. RNA expression levels were measured to determine the fluorescence of individual cDNA amplicons. The fluorescence of the background decreased the value of the fluorescence that was measured for each DNA band. After treatments with NaCl, Mannitol, and Sorbitol, the data were computed as a Log2 fold change in order to compare the levels of GAD gene expression that occurred as a result of those treatments.

Extraction and determination of proteins

Acsad 176, Athroh, and Rum genotype seedlings were grown in an artificial growth medium and watered as previously mentioned when they were two weeks old. On day 10 of watering, plant shoots were collected in order to be used for protein extraction and quantification, both of which were carried out with the assistance of a SMART BCA Protein Assay Kit (Intron Biotechnology, Korea) in accordance with the instructions provided by the manufacturer, with the the relevant adjustments that were made : After collecting 0.05 milliliters from each treated sample, one milliliter of the working solution was added, and the mixture was thoroughly combined. Following that, the samples were incubated at 37 degrees Celsius for 30 minutes before being allowed to cool to room temperature. After measuring the samples' absorbance at 562 nm, the BSA standard curve was utilized in order to determine the protein concentrations of each sample.

Estimation and extraction of total carbohydrates

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 tenth day after they were watered and then used in accordance with³¹, with the following modifications: After obtaining 0.05 milliliters of each treated shoot sample and placing them on ice to chill, adding 0.1 milliliters of chilled 75% H2SO4 and mixing all of the tubes, adding 0.2 milliliters of chilled anthron solution to each tube, and then placing all of the COD tubes in a water bath heated to 100 degrees Celsius for 15 minutes, the tubes were taken out and allowed to cool at room temperature. Estimating the amount of total carbs in samples was accomplished by plotting the absorbance measured at 578 nm against a glucose standard curve.

Data analysis and experimental design

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 that were equal or less than 0.05 were regarded as significant in all of the analyses of the data, and P-values that were equal or less than 0.01 were regarded as highly significant.

Sensitivity of barley seed germination to osmotic stress and salt

Under salt 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 showed an observable significant decreased in germination under osmotic stress (mannitol) (P = 0.022) and was decreased up to 30% under 300 mM mannitol compared with control. According to the data, Athroh showed a 20% decrease in germination when exposed to 300 mM sorbitol (P = 0.002) and 27% reduction under 200 mM NaCl (P = 0.005). Under salt (NaCl) and osmotic stress, Acsad 176 showed a modest reduction in germination (mannitol and sorbitol), where the reduction under 300 mM sorbitol (P = 0.034) reached to 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 than to treatments with NaCl and mannitol. Rum germination reduced up to 30% under 300 mM sorbitol, 23% under 200 mM NaCl, and 17% under 300 mM mannitol.

Table 1

Germination percentages of three barley (Acsad 176, Athroh, and Rum) treated with different concentrations of sodium chloride, mannitol and sorbitol. After 10 days, the radicle emergences from germinating seeds were evaluated. (*) P ≤ 0.05, (**) P ≤ 0.01, and r = correlation coefficient.
[mM]	Acsad 176	Athroh	Rum
Control	100.00	96.67	93.33
NaCl 25	100.00	93.33	90.00
NaCl 50	100.00	96.67	80.00
NaCl 75	96.67	83.33	76.67
NaCl 100	86.67	76.67	76.67
NaCl 200	86.67	66.67	70.00
P	0.028*	0.005**	0.013*
r	-0.859	-0.944	-0.904
Mannitol 50	100.00	93.33	93.33
Mannitol 100	93.33	86.67	90.00
Mannitol 150	86.67	70.00	90.00
Mannitol 200	83.33	66.67	86.67
Mannitol 300	80.00	63.33	76.67
P	0.002**	0.022*	0.001**
r	-0.963	-0.877	-0.983
Sorbitol 50	100.00	93.33	90.00
Sorbitol 100	100.00	86.67	90.00
Sorbitol 150	93.33	83.33	80.00
Sorbitol 200	90.00	83.33	73.33
Sorbitol 300	90.00	73.33	63.33
P	0.034*	0.002**	0.002**
r	-0.845	-0.968	-0.969

Barley seedlings growth under salt and osmotic stress

The three barley genotypes showed no significant differences for seedling length under osmotic and salt stresses, expect Acsad 176 had significantly 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 short duration of treatment (10 day), which might not be enough to have an observed significant effect on seedling length.

Table 2

After 10 days, Acsad 176, Athroh, and Rum barley seedlings were supplemented with different concentrations of sodium chloride, mannitol and sorbitol. *(*) P ≤ 0.05, (**) P < 0.01, r* = *correlation coefficient.*
[mM]	Acsad 176	Athroh	Rum
Control	19.33	19.33	18.67
NaCl 25	19.00	18.67	18.00
NaCl 50	18.67	18.67	17.67
NaCl 75	17.67	19.67	17.33
NaCl 100	18.00	18.33	18.00
NaCl 200	19.00	18.33	18.00
P	0.73	0.294	0.61
r	-0.182	-0.517	-0.266
Mannitol 50	18.33	16.33	17.67
Mannitol 100	17.67	18.67	18.00
Mannitol 150	18.67	18.00	16.67
Mannitol 200	17.33	17.33	17.33
Mannitol 300	16.00	18.00	17.67
P	0.012*	0.798	0.446
r	-0.908	-0.136	-0.389
Sorbitol 50	19.00	19.00	17.67
Sorbitol 100	18.00	18.00	17.00
Sorbitol 150	18.00	17.00	18.33
Sorbitol 200	17.33	16.67	18.00
Sorbitol 300	16.67	18.00	18.67
P	0.005**	0.322	0.526
r	-0.945	-0.492	0.328

Barley genotypes showed no significant difference of 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 negatively correlation of fresh weight and increasing stress levels in the three barley genotypes (Table 3).

Table 3

Fresh weight (grams) of 10 days old seedling shoot tissues of three barley (Acsad 176, Athroh, and Rum) subjected to different concentrations of NaCl, mannitol, and sorbitol; individually. *(*) P ≤ 0.05, (**) P ≤ 0.01, r* = *correlation coefficient.*
[mM]	Acsad 176	Athroh	Rum
Control	0.64	0.49	0.75
NaCl 25	0.55	0.49	0.92
NaCl 50	0.55	0.35	1.06
NaCl 75	0.76	0.4	0.62
NaCl 100	0.47	0.4	0.78
NaCl 200	0.5	0.34	0.45
P	0.432	0.083	0.124
r	-0.4	-0.755	-0.696
Mannitol 50	0.73	0.75	0.99
Mannitol 100	0.97	0.8	0.73
Mannitol 150	0.93	0.66	0.46
Mannitol 200	0.52	0.63	0.35
Mannitol 300	0.3	0.5	0.21
P	0.162	0.487	0.026*
r	-0.651	-0.357	-0.865
Sorbitol 50	0.77	0.43	0.72
Sorbitol 100	0.72	0.76	0.51
Sorbitol 150	0.72	0.76	0.32
Sorbitol 200	0.8	0.71	0.52
Sorbitol 300	0.46	0.51	0.49
P	0.188	0.88	0.265
r	-0.621	-0.18	-0.544

Salt and osmotic stress decreased plant dry weight in Acsad 176, Athroh, and Rum compared with 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.

Table 4

Dry weight (grams) of 10 days old seedlings shoot tissues of three barley (Acsad 176, Athroh, and Rum) exposed to different NaCl, mannitol, and sorbitol concentrations; individually. (*) P ≤ 0.05, (**) P ≤ 0.01, r = correlation coefficient.
[mM]	Acsad 176	Athroh	Rum
Control	0.29	0.28	0.29
NaCl 25	0.16	0.14	0.16
NaCl 50	0.13	0.14	0.13
NaCl 75	0.13	0.12	0.13
NaCl 100	0.1	0.12	0.12
NaCl 200	0.1	0.11	0.12
P	0.112	0.161	0.17
r	-0.713	-0.652	-0.641
Mannitol 50	0.16	0.13	0.2
Mannitol 100	0.14	0.12	0.16
Mannitol 150	0.14	0.1	0.14
Mannitol 200	0.14	0.09	0.13
Mannitol 300	0.11	0.09	0.11
P	0.177	0.154	0.051
r	-0.706	-0.66	-0.809
Sorbitol 50	0.15	0.15	0.14
Sorbitol 100	0.14	0.1	0.13
Sorbitol 150	0.13	0.1	0.1
Sorbitol 200	0.12	0.1	0.083
Sorbitol 300	0.1	0.1	0.09
P	0.111	0.183	0.135
r	-0.714	-0.627	-0.683

Response of chlorophyll to osmotic and salt stress

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 low level of GABA accumulation under 300 mM mannitol, indicating the lethal effect of high mannitol level on Athroh genotype. Acsad 176 showed high chlorophyll-a level under NaCl stress (P = 0.009), which was associated with high GABA accumulation in the presence of 200 Mm NaCl Furthermore, Rum revealed a relation between GABA levels and chlorophyll a content in the presence of 300 mM mannitol (P = 0.046).

Table 5

Chlorophyll a level (nm/mg FW) of 10 days old seedlings shoot tissues of three barley (Acsad 176, Athroh, and Rum) treated to different concentrations of NaCl, mannitol, and sorbitol; independently. (*) P ≤ 0.05, (**) P ≤ 0.01, r = correlation coefficient.
[mM]	Acsad 176	Athroh	Rum
Control	8.82	7.27	8.52
NaCl 25	8.83	6.35	8.04
NaCl 50	8.73	6.12	7.83
NaCl 75	7.91	5.84	7.69
NaCl 100	7.42	5.07	7.15
NaCl 200	7.07	5.11	6.22
P	0.009**	0.029*	0.001**
r	-0.92	-0.857	-0.991
Mannitol 50	9.00	3.28	8.33
Mannitol 100	8.36	2.59	7.91
Mannitol 150	8.03	2.10	7.54
Mannitol 200	7.32	1.55	6.56
Mannitol 300	7.40	1.37	6.91
P	0.031*	0.011*	0.046*
r	-0.853	-0.727	-0.819
Sorbitol 50	9.43	6.97	5.39
Sorbitol 100	8.28	6.62	4.80
Sorbitol 150	7.87	6.06	4.54
Sorbitol 200	7.33	5.71	4.30
Sorbitol 300	6.65	5.50	3.26
P	0.011*	0.011*	0.049*
r	-0.915	-0.912	-0.807

Table 6

Chlorophyll b level (nm/mg FW) of 10 days old seedlings shoot tissues of three barley (Acsad 176, Athroh, and Rum) treated to different concentrations of NaCl, mannitol, and sorbitol; independently. (*) P ≤ 0.05, (**) P ≤ 0.01, r = correlation coefficient
[mM]	Acsad 176	Athroh	Rum
Control	4.63	4.71	5.4
NaCl 25	4.96	3.99	5.10
NaCl 50	3.83	3.14	3.20
NaCl 75	3.24	2.59	2.29
NaCl 100	2.88	1.73	1.91
NaCl 200	2.15	0.96	1.12
P	0.008**	0.004**	0.016*
r	-0.927	-0.948	-0.893
Mannitol 50	3.89	2.88	5.66
Mannitol 100	3.54	2.33	4.52
Mannitol 150	2.3	1.147	3.39
Mannitol 200	1.26	0.87	2.04
Mannitol 300	0.98	0.57	1.51
P	0.013*	0.038*	0.01**
r	-0.907	-0.837	-0.917
Sorbitol 50	3.73	4.03	3.77
Sorbitol 100	2.75	3.65	2.64
Sorbitol 150	2.48	2.89	1.35
Sorbitol 200	1.35	1.27	0.85
Sorbitol 300	0.67	0.98	0.59
P	0.006**	0.01**	0.034*
r	-0.937	-0.919	-0.846

Salt and osmotic stress cause oxidative damage in barley seedlings.

The amount of oxidative damage, reactive oxygen species (ROS), and lipid peroxidation in barley seedlings under the investigated conditions is reflected based on the outcomes of our investigation which were determined on the basis of 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 to 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).

3.5. GABA levels in barley seedlings under salt and osmotic stress

GABA level 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 of 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). 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 300mM mannitol, there was a highly significant increase in GABA accumulation in Rum (P = 0.024). This suggests that, in comparison to treatments with NaCl and sorbitol, Rum is more tolerant of high concentrations of mannitol due to highly significant GABA accumulation (Fig. 2).

3.6. GAD mRNA transcripts in barley under osmotic and salt stress

Regarding seed germination, the level of GAD RNA transcription correlates with the response to salt and osmotic treatments (Table 1), This study demonstrated MDA accumulation (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). Decarboxylation of glutamate to CO2 and GABA is improved by the GAD¹⁷. Rum genotypes subjected to osmotic stress (mannitol) had significant levels of GAD expression (Fig. 4b), which was consistent with the high amount of GABA accumulation (mannitol). Acsad 176 genotype showed high GAD expression under NaCl stress (Fig. 4a), 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. 4c). Rum genotype responded to salt and osmotic stresses with moderately significant GAD expression and GABA accumulation (Fig. 4). Under mannitol, the highest GAD expression was found (Fig. 4b). This demonstrates that the GABA shunt was activated in response to stressors based on metabolite production and GAD expression (salt and osmotic).

Barley protein level under salt and osmotic stress

Protein content demonstrated a significant difference in among Acsad 176, Athroh, and Rum responses to stresses (salt and osmotic) (Fig. 5). The protein concentration in Rum was the highest under 50 mM NaCl treatment (Fig. 5a). Rum showed no significant difference in protein level under mannitol stress (P = 0.378) (Fig. 5b) which is correlated with high GABA accumulation in response to mannitol. This result indicated that mannitol treatments had no effect on Rum's protein content. The protein level in Rum significantly decreased under sorbitol (P = 0.034) treatment (Fig. 5c). 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 both Acsad 176 and Athroh (Fig. 5). Our data showed the highest protein content in Athroh compared with 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 concentration than both 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 other hand, Acsad 176 was strongly affected under mannitol stress that was observed by reduced protein content (r= -0.921, P = 0.009).

Carbohydrates level under salt and osmotic stress in barley

Carbohydrate content in Acsad 176, Athroh, and Rum were significantly decreased under salt and osmotic stress (Fig. 6). Acsad 176 showed a significant decrease of carbohydrates 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. 6a). Carbohydrates concentration in Rum was decreased significantly under osmotic stress (sorbitol) (P = 0.049) (Fig. 6c), which was correlated with low GABA accumulation in Rum tissue under sorbitol treatment. Athroh genotype showed significant decrease under osmotic stress (mannitol) (P = 0.021) (r= -0.726) (Fig. 6b).

Environmental stresses harm plant development and growth by causing physiological and oxidative damage to plants systems³². Numerous plant species had demonstrated reduced growth when exposed to salt stress³³. Several studies have found that abiotic stresses cause serious harm to the growth of crops and lead to yield reduction¹³. The growth of roots and shoots and roots in salt-sensitive plants were significantly reduced under external osmotic stress solution³⁴. As opposed to that, ³³ discovered that sorbitol improved wheat germination quality and quantity, whereas ³⁵ confirmed that sorbitol causes a decrease in maize seedling growth parameters due to osmotic stress. One of the components of seeds required for germination is sorbitol, which are capable of being taken up by plant cells³⁶. ³⁵ also pointed, that the number of seedlings reduced when mannitol level increased, despite the fact that mannitol, like sorbitol, is a suitable solute, but it is not one of the seed components necessary for germination. ³⁷ investigated the effects of salt, osmotic, cold, and heat shocks on two genotypes of Jordanian lentil (Lens culinaris Medik) called Jordan 1 and Jordan 2. They observed that these stresses had a direct influence on seed viability and photosynthetic pigments. Osmotic and salt stress significantly reduced seed germination in 5 Jordanian wheat genotypes²³. Furthermore, ²¹ discovered seed sensitivity and decreased seed germination in various wheat and barely cutlivars exposed to different NaCl treatments. For seedlings growth under salt and osmotic treatments; our results were in disagreement with ³⁸ who found the difference in seedling length among three tomato genotypes exposed to salinity stress, where the reduction reached up to 12% compared with control. The fresh weights data were in direct opposition to the findings that were published by ³⁹, who said that the fresh weight of lettuce (Lactuca sativa L.) could be enhanced by as much as 28% when it was subjected to salt stress. Despite the fact that numerous research have shown a beneficial relationship between salinity and fresh weight, there are also results showing a detrimental relationship between salt stress and fresh weight. These include a study by ⁴⁰ on radish plants Raphanus sativus L., and a study by ⁴¹ on Brassica campestris L. Salt stress, as established by a large number of studies, causes a reduction in the dry weight of the shoot system. The exact amount by which this reduction occurs varies according to salinity level, kind of salt present, and crop genotype⁴². Other studies have found different outcomes, including the one conducted by ⁴². These researchers found that the application of 200 mM of sodium chloride to sea beet (Beta maritime L.) and fodder beet (Beta vulgaris L.) significantly increased the dry weight of the shoot system for both beet species. Our findings, on the other hand, found that the sodium chloride treatment had the opposite effect.

The rate of photosynthesis and how much dry matter is produced by plants under stress can be estimated from their total chlorophyll content. Researchers discovered that the content of chlorophyll is significantly correlated positively with grain yield⁴³. A reduced chlorophyll content will result in a decreased rate of photosynthesis, which will then lead. to a lower production of dry mater and a lower crop yield. Environmental stress is connected with a reduction in chlorophyll, and the change in total chlorophyll is an useful predictor of the influence that stress has on the development of crops⁴⁴. ⁴⁵ identified tolerant genotypes with regard to more leaf chlorophyll retention under stress conditions. Wheat genotypes exposed to long-term soil salinity (NaCl), caused impaired and deteriorated effects on membrane stability index, carotenoids, proline, chlorophyll, soluble sugar, superoxide dismutase and biomass and grain yield⁴⁶. Chlorophyll biosynthesis was reduced in Zea mays L. under low temperature due to damaged development of thylakoid membranes⁴⁷. ⁴⁸ reported that a reduction of chlorophyll contents when the plant is under salt and osmotic stress through enhancing the activity of the chlorophyllase enzyme, which is responsible for the degradation of chlorophyll pigments. Abiotic stress activated the deterioration of chloroplast structure and reduced photosynthesis rate.

Due to membrane lipid peroxidation, the amount of MDA accumulation in plants tissues under stress increased²⁵. This accumulation of MDA under salt and osmotic stress is consistent with the findings of other investigations that found a significant concentration of MDA accumulating in wheat shoots under conditions in which they were subjected to osmotic and salt stress²³. Variations in MDA concentrations were also found in rice (Oryza sativa L)⁴⁹. Under salt stress, lipid peroxidation in both shoots and roots tissues increased in barley genotypes. In addition ⁵⁰ described that, lipid peroxidation increased in two beet species under salt stress. It was discovered by ³⁶ that individual molecules of sorbitol can be taken up by plant cells. As a result, the three genotypes of barley (Acsad 176, Athroh, and Rum) were not impacted by the treatment with 50 mM sorbitol, as demonstrated in Fig. 1C. Alfalfa (Medicago sativa L.) plants showed high accumulation of MDA under stress (different concentrations of NaCl)⁵¹. At several growth stages, maize genotypes demonstrated a growth loss when exposed to salt stress⁵². Moreover, ⁵³ desocvered that differing saline systems in hydroponic culture resulted in a growth reduction of the rice genotype⁵⁴ informed that MDA levels increased in Arabidopsis thaliana seedlings shoot and root tissues under hydrogen peroxide and paraquat treatments. ⁵⁵ informed that barley genotypes indicated a remarkable increase in MDA levels after fungal infection, where the MDA level started to rise at 4 days after infection and reached 185% increase when compared with uninfected control plants after 6 days.

More than half a century ago, the gamma-aminobutyric acid (GABA) shunt was discovered in plants for the first time in the tuber of the potato plant⁵⁶. The levels of GABA that are found in plant tissue have a tendency to rise in response to environmental stressors such as dryness, darkness, and hypoxia^17,18. ⁵⁷ discovered that Gamma-aminobutyric acid (GABA) was readily accumulated at the whole plant level during NaCl application in Arabidopsis thaliana. In addition, the activity of GABA transaminase (GABA-TP), which is utilized as an predector to evaluate the concentration of GABA, rapidly rose in Arabidopsis seedlings in response to treatment with 150 mM NaCl. This was the case even though the seedlings had been exposed to the same amount of salt. This was observed in reaction to the fact that the seedlings were subjected to the salt concentration⁵⁷. ²⁸ displayed that the GABA shunt metabolite highly accumulated in wheat and barley genotypes when subjected to the stresses of cold and freezing temperatures. The levels of GABA accumulation were found to be closely proportional to the amount of stress that was endured during the treatments. The accumulation of GABA is promoted when non-hardened plants are subjected to exogenous glutamate in a low temperature environment²⁸ GABA levels increase in plant tissue that is exposed to anaerobic and heat stresses⁵⁸ According to ²³, wheat genotypes were subjected to salt and osmotic stress, which resulted in an increase in the accumulation of GABA. In agreement with our findings, ⁵⁹ discovered that the only non-amino acid intermediary of the GABA shunt that increased under osmotic stress was GABA. Wheat seedlings exposed to 20% polyethylene glycol 6000 for 28 hours as an osmotic stressor experienced a considerable increase in GABA buildup⁶⁰. In addition, both cold and heat treatments raised the GABA levels in Arabidopsis calmodulin mutants⁶¹.

Numerous studies had found evidence to support the concept that GAD and GABA may be components of a signal transduction pathway that is activated in crops when they are subjected to a stressful environment⁶². Environmental stresses enhance the increase of Ca²⁺ level in the plant cells⁶³. Anoxia leads to mitochondrial release of Ca²⁺ into the cytosol under abiotic stress, wind and cold increase Ca²⁺ signaling pathways in the cytoplasm⁶⁴. All plant GADs contain calmodulin-binding domains (CaM-BDs). GADs were stimulated by Ca²⁺/CaM as a binding complex, but not by either Ca²⁺ or CaM alone⁶⁵. ⁶⁶ reported overexpression of glutamate decarboxylase in transgenic tobacco plants in response to the root-knot nematode exposure and heat shock¹⁹. Data showed that GAD expression level increased in wheat genotype seedling under NaCl, mannitol, and sorbitol treatments²³. In conformity with our findings, GAD activity significantly increased in response to a NaCl stressor, which was correlated with a rise in GAD gene expression in an Arabidopsis thaliana CMSII case. On the contrary, GABA accumulation in Arabidopsis seedlings was prevented under heat stress due to disruption of the GAD1 gene¹⁴.

This study found that under salt and osmotic stress, barley seedlings accumulated more GABA, had higher MDA levels, and had lower protein contents. The findings of this research offer substantial support for the proposition that GABA may be an Integrated and Adaptive Mechanism for the Metabolism that aids plant resistance to abiotic stress brought on by salt and osmotic treatments. Protein production in plants may respond differently to environmental stress⁶⁷. The growth, development, and yield of the crop were all negatively impacted by the presence of abiotic stresses. It has been discovered that plant stress adaptation occurs through changes in gene expression, which modify plant phenotypes. Plants showed different mechanisms at the transcriptional and the enzymatic activities levels overlapping patterns in the reactions of the adaptive mechanisms to various stresses. In agreement with our study, two cotton genotypes revealed significant decrease in soluble protein concentrations under 50 and 100 mM NaCl treatments⁶⁸. In addition, the protein concentrations of actin-depolymerizing factor, superoxide dismutase, and salt-induced protein in rice were altered as a result of drought and osmotic stress⁶⁹. Some plants excrete glycine-betaine (GB) in response to abiotic stress (salt and drought stress), where the plant cells accumulate GB for stabilizing the quaternary structure of protein complexes and membrane structures against various abiotic stresses⁷⁰. Furthermore, GB increase the ability of plant cell to save water without disturbing normal cellular function. GB accumulation in transgenic apple expressed Osmyb4 gene, which is stress regulator gene, was linked to high cold and drought tolerance⁷⁰.

Furthermore, our findings revealed a strong connection between carbohydrate metabolism and GABA accumulation. This suggests that GABA may function as a regulatory metabolic molecule in the tricarboxylic acid (TCA) cycle and C:N assimilation during adaptation to salt and osmotic treatments in barely seedlings. Many studies have shown that the carbohydrate content of the shoot system are decreased under various abiotic stresses. Also, ⁷¹ noticed a decrease of carbohydrates content in roots of flooded plants. Plants are able to switch between their primary and secondary routes of biochemical glucose metabolism when these conditions are present ⁷². Despite the fact that numerous studies have demonstrated that stress has a beneficial effect on the amount of carbohydrates in the body and the metabolism, The state of carbohydrates in reproductive structures of crops can become more unstable when there is a drought. Under the influence of the herbicide diclofop-methyl, maize was found to have a greater accumulation of carbohydrates⁷³. Oligosaccharides were also observed to be accumulated in a variety of plants when they were placed under the stress of a low temperature⁷⁴. Plants respond to environmental stresses by adjusting their nutrient uptake and metabolism¹⁸. GABA metabolism is included in the regulation of C:N equilibrium by regulating nitrate uptake, sugar level, and internal N assimilation in response to stress. This study highlighted the direct metabolic role that GABA plays in the metabolism of amino acids and sugars, as well as in the transport and storage of carbon and nitrogen, the assimilation of carbon from glutamate, and the generation of carbon and nitrogen fluxes that enter the TCA cycle. GABA accumulation was found to correlate with increased total carbohydrates and protein levels in response to salt and osmotic stress treatments.

Abiotic stress inhibits plant development and maintains the metabolic homeostasis of individual cells. Under salt and osmotic stressors, all genotypes including Acsad 176, Athroh, and Rum had a drop in fresh weight and dry weight compared to the treatment that served as the control, although the difference was not statistically significant. Acsad 176, Athroh, and Rum barley genotypes used in this research had a clear relationship between GABA accumulation, germination %, chlorophyll content, MDA level, total protein and carbohydrate contents (i.e., significant increase in GABA concentration in Rum genotypes under mannitol treatment with an increase of GAD mRNA transcription, chlorophyll content, MDA concentration, proteins and carbohydrates contents under the same treatments). The data recommends that a GABA metabolite may act as a protective molecule in the carbohydrate metabolism in conjunction with a variety of physiological responses when plants are treated with salt and osmotic stress. This hypothesis is strengthened by the fact that the metabolite is produced when crops are exposed to salt and osmotic stress. To understand more about how GABA shunt metabolic signaling works in numerous crops and more research is required to determine how it influences a diverse set of physiological processes in response to shifts in both the environment and the genes themselves. According to the outcomes of our research, the Acsad 176 barley genotype is the greatest suitable variety to be grown in soil that contains high levels of both salt and osmotic content.

Acknowledgments: The Jordan University of Science and Technology's Deanship of Research provided funding for this project under grant number [234/2016].

Author Contributions:

Zakaria: was involved in the planning of the study, was responsible for carrying out the statistical analysis, was involved in the interpretation of the results, and contributed to the writing and reviewing of the article.

Nisreen: was assisted with the methodology, participated in discussions over the data analysis, and contributed to the writing of the manuscript.

Hala: participated in the carrying out of the experimental activity, the gathering of the data, and the drafting of the initial draft of the manuscript.

Abdul Latief contributed to the writing of the manuscript and help in results interpretation

Competing interests: The authors state that there are no conflicts of interest.

Data availability: Data and material produced and/or analyzed during the present study are available from the corresponding author on reasonable request.

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GABA metabolism and ROS accumulation in barley (Hordeum vulgare L.) seedlings under the effect of salt and osmotic stress

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Abstract

Figures

Introduction

Materials And Methods

Plant materials and growth conditions

Seed sensitivity to salt and osmotic stress

Determination of seedling growth

Extraction and determination of chlorophyll

Evaluation of Oxidative Damage

Extraction and identification of GABA metabolites

GABA (γ-Aminobutyric acid)

RNA extraction

Reverse Transcriptase-PCR Measurement of GAD mRNA Expression

Extraction and determination of proteins

Estimation and extraction of total carbohydrates

Data analysis and experimental design

Results

Sensitivity of barley seed germination to osmotic stress and salt

Barley seedlings growth under salt and osmotic stress

Response of chlorophyll to osmotic and salt stress

3.5. GABA levels in barley seedlings under salt and osmotic stress

3.6. GAD mRNA transcripts in barley under osmotic and salt stress

Barley protein level under salt and osmotic stress

Carbohydrates level under salt and osmotic stress in barley

Discussion

Conclusion

Declarations

References

Additional Declarations

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