The experiment was conducted in a greenhouse environment (controlled condition) at the Department of Botany, University of Peshawar, Peshawar, 25120 (34◦15 North latitude and 71◦42 East longitudes), KP Pakistan. Temperatures typically range between 5°C and 39°C from January to February and June to July, respectively, with an average rainfall of approximately 513 mm per year. The silt-loamy soil had pH 6.9, a bulk density of 1.55 g cm⁻³, and EC 0.288 ds/m, collected from the experimental site. The seeds of local mungbean accession (Vigna radiata L.) were provided by Cereal Crop Research Institute (CCRI), Persabaq, Nowshera 24050, Pakistan. These seeds were sown in pots (15 cm in diameter) with an opening at the bottom (2 cm diameter) and filled with 2.5 kg of silt-loam soil.

In the beginning, the seedlings were watered every three hours with tap water. At the first trifoliate leaf stage (three weeks after sowing), 48 pots (2 seedlings per pot) with uniform seedlings were selected and divided into six sets for the application of saline stress (SS; NaCl and Na₂SO₄, 1:1 molar ratio) and GABA application. There were three saline stress treatments: 0-mM (control), 50-mM, and 100-mM. The remaining three groups were subjected to the same levels of saline stress but were given exogenous GABA solutions (1.5 mM, 200 ml; 100 ml GABA applied to each pot at 20- and 30-days of sowing, respectively). An initial seedlings growth experiment was conducted with GABA concentrations ranging between 0.25mM and 1.5mM ( Supplementary Table 1 ). The optimal GABA concentration was selected based on the improved growth of mungbean seedlings subjected to 50 mM saline stress. Three replicates of each treatment were conducted. We harvested the 45-day-old mung bean plants and immediately froze them in liquid nitrogen before storing them at -80°C for physiologic analysis.

The stem height and root length were measured using a measuring tape. Next fresh and dry weights of the stem, leaves, and root of the seedlings were measured using an electric balance. For dry weight determination, the plants were oven-dried at 105°C for 30 min and then dried at 75°C until constant weight.

We extracted photosynthetic chlorophyll pigments (0.1–0.3 g) from the fresh leaf samples (0.1–0.3 g) using ethanol (95%, vol/vol) following a standard method (Lichtenthaler and Buschmann, 2001). The absorbances were read at 665 nm and 649 nm using a spectrophotometer. Chlorophyll concentrations were calculated using the following equations (mg g⁻¹ FW).

We digested the leaf sample (0.05 g) with concentrated HNO₃ (3 mL). Afterwards, the extract was brought up and diluted to 15 mL with deionized water. An inductively coupled plasma-optical emission spectrometry (ICP-OES) was used to determine Na⁺ and K⁺ concentrations, following a standard method (Mostofa et al., 2015).

A standard procedure (Patterson et al., 1984) was used to determine hydrogen peroxide (H₂O₂). Using a chilled mortar, fresh leaf samples (0.2 g) were homogenized in 5 ml of trichloroacetic acid (TCA) (0.1%) in an ice bath. In the following step, the extract was centrifuged at 5000 × g for 10 min (4°C). Next, the supernatant containing the titanium reagent (50 ml of 20% titanium tetrachloride in ammonia) and titanium reagent was centrifuged at 10,000 × g for 10 min. After five washes with acetone, the precipitate was centrifuged at 10,000 g for 10 min, followed by adding 3 ml of 1 M H₂SO₄.

For the evaluation of lipid peroxidation, malondialdehyde (MDA) concentrations were determined using the thiobarbituric acid (TBA) test (Heath and Packer, 1965). We homogenized fresh leaf samples (0.5 g) in 1ml of 5% TCA and centrifuged them for 10 min at 5,000 × g (4°C). We then added 4 ml of the supernatant to two ml of 20% TCA in a separate tube and heated this mixture at 100°C for 15 min before centrifuging it at 5,000 g (10 min). The absorbance at 450, 532, and 600 nm were read using a spectrophotometer, and MDA concentration was determined using the equation below.

A chilled mortar was used to grind and homogenize fresh leaf samples in a 0.1 M phosphate buffer (pH 7.3) solution and 0.5 mM ethylenediaminetetraacetic acid (EDTA). The Homogenates were centrifuged at 12000 ×g for 10 min (at 4°C). Following this, the supernatant containing enzyme extract was used for the assays. We determined the superoxide dismutase (SOD) activity using a standard method (Giannopolitis and Ries, 1977). Approximately 0.1 mL of enzyme extract was added to a reaction mixture of 50 mM phosphate buffer (pH 7.8), 130 mM of methionine, 2.0 mM of riboflavin, and 75 mM of nitro-blue tetrazolium (NBT). The activity of SOD was determined by measuring the decline rate of nitroblue tetrazolium at a 560 nm wavelength using a spectrophotometer. During the measurement of SOD activity, one unit corresponds to the amount of enzyme required to inhibit 50% of NBT reduction at 560 nm.

Furthermore, POD activity was determined using standard methods (Wang et al., 2018) but with minor changes. An enzyme extract of 0.5 ml was mixed with 2 ml of buffer substrate (guaiacol and Na₃PO₄ pH 6.4), 24 mM H₂O₂, and 1 ml of buffer substrate. Measurement of absorbance at 460 nm was conducted twice at intervals of 1 min. Enzyme activity was calculated by increasing the absorbance of the reaction system by 0.01 up to a maximum of 1U per min, which was then converted to U/g·min⁻¹. The monitoring of H₂O₂ disappearance was used to determine CAT activity (Sabra et al., 2012). As a starting point, 50 ml of enzyme extract was dissolved in 1.5 ml of reaction mixture containing 50 mM K-phosphate buffer (pH 7.0) and 15 mM hydrogen peroxide. An absorbance measurement was conducted at 240 nm for 1 min using a spectrophotometer. The degradation of one mole of H₂O₂ per minute is equivalent to one unit of CAT. We determined the ascorbate peroxidase activity (APX) using a standard method. (Katsumi et al., 1994). Briefly, a 3 mL reaction mixture that contained 50 mM of phosphate buffer (pH 7.0), 1.0 mM of hydrogen peroxide, 0.25 mM of L-ascorbic acid, and 0.1 mL of enzyme extract was prepared. An increase in absorption, at 290 nm, was observed with a spectrophotometer following ascorbate oxidation. glutathione reductase (GR) activity was determined using a previous method (Carlberg and Mannervik, 1985). An enzyme extract was added to a reaction mixture of 50 mM Tris-HCl buffer (pH 7.5), 0.5 mM GSSG, 3 mM MgCl2, and 0.2 mM NADPH. GSSG was added to initiate the reaction. A spectrophotometer was used to measure the absorbance at 340 nm. One unit of activity corresponds to the amount of glutathione reductase capable of catalyzing the oxidation of one mol of NADPH min^−1. The Polyphenol peroxidase (PPO) activity was determined using a standard method (Cañal et al., 1988) with some modifications. We prepared a reaction mixture of 2.8 ml of 100 mM NaPi, pH 7.0, 0.1 ml of 25 mM pyrogallol, and 100 ml of enzyme extract. We maintained the mixture at 30°C for 30 min and measured the activity at 420nm after 30 minutes.

A sulfamate colorimetric method was used to determine nitrate reductase (NR) activity (Ullah et al., 2019). Briefly, fresh leaf samples (0.3 g) were homogenized in 200 mM KNO3, 5 mM EDTA, and 0.15 mM NADH in 100 mM phosphate buffer (pH 7.5), and the reaction mixture was incubated for 1 hour at 30°C. Next, the reaction mixture was centrifuged at 30,000 × g for 20 min. Next, 2 ml of sulfanilamide and N-naphthylamine reagents were added, and absorbance was read at 540 nm. Units of enzyme activity were expressed as μmol g⁻¹ protein. We determined the glutamine synthetase (GS) activity using a standard method (Ullah et al., 2019). Fresh leaf samples (0.3 g) were homogenized in 2 ml of 50 mM Tris-HCl buffer (pH 7.8; containing 0.1% TritonX-100, 15% glycerol, 1 mM EDTA, and 14 mM 2-mercaptoethanol), and incubated at 37 ^◦C for 30 min. Afterwards, the reaction mixture was centrifuged twice at 4°C for 10 min each time. Following this, 1 mL of ferric chloride reagent was added and centrifuged at 5,000°C for 10 min. At 540 nm, the absorbance was measured, and enzyme activity was expressed as μmol g⁻¹ protein. The glutamine synthase (GOGAT) activity was determined according to a standard method (Ullah et al., 2019). The Assay mixture contained100 mM K⁺-ketoglutaric acid, 20 mM L-glutamine, 3 mM NADH, and 10 mM KCl, in 25 mM Tris–HCl (pH 7.2). Next, the enzyme extract was added to initiate the reaction. An absorbance measurement at 340 nm continuously monitored the NADH oxidation. The oxidation of 1 μmol of NADH per minute was considered an enzyme unit (μmol g⁻¹ protein).

We measured the soluble sugar concentration using the anthrone method (Yemm and Willis, 1954), and the standard was glucose. The concentration of proline was determined using a standard method (107). We homogenized 0.5 grams of fresh leaf samples in 3 percent aqueous sulfosalicyclic acid and then centrifuged at 5,000 × g (10 min). Next, the filtrate (2 ml) was mixed with 2 ml of glacial acetic acid and 2 ml of acid-ninhydrin in a test tube. Afterward, the reaction mixture was boiled at 100°C for 1 hour. Toluene was used to extract the reaction mixture. We aspirated and cooled the chromophore containing toluene. Finally, a spectrometer was used to measure absorbance at 520 nm. The soluble proteins were determined using 0.3 grams of fresh leaf samples (Bradford, 1976), standard bovine serum albumin. We extracted total phenolic content from dried leaf samples (0.3) in 80% methanol using the Folin-Ciocalteu colorimetric analysis (Scalbert et al., 1989). The absorbance was measured using a spectrophotometer at 765 nm. Phenolic content was expressed as mg g⁻¹ DW, dry weight.

Three replicates of the measurements were conducted. The descriptive statistics and one-way analysis of variance (ANOVA) were conducted using SPSS version 16.0 (Chicago, IL, United States). At a significance level of p<0.05, Duncan’s multiple range tests were used to compare means. The figures were created using GraphPad Prism 8. The growth parameters, chlorophyll pigment content, osmolytes, N metabolism, mineral nutrition, H₂O₂ and MDA levels, and antioxidant enzyme activity were examined using Pearson correlation analyses (Origin Lab Corporation, Northampton, MA, USA) for further interpretation.

Mungbean plants showed obvious differences in growth performance when treated with saline stress and GABA treatments. Both salinity levels (50 mM and 100 mM) substantially reduced the growth parameters of mungbean plants. The shoot length (SL), stem fresh weight (SFW), and stem dry weight (SDW) experienced a 33.3-, 35.8- and 31.2% inhibition following 50 mM SS, while 37.9-, 59.5- and 52.7% decline was found after 100 mM SS, respectively ( Figures 1A–C ). GABA, however, improved the SL, SFW, and SDW under 50 mM (20.2-, 43.2-, and 27.6%, respectively) and 100 mM (27.2-, 29.1-, and 39.3%, respectively) compared to untreated plants. This improvement was 12.2-, 12.5-, and 23.2% when GABA was applied under controlled conditions. Similar to the stem indices, the root length (RL), root fresh weight (RFW), and root dry weight (RDW) declined by 24.4-, 44.0- and 27.1% after 50 mM SS, while following 100 mM SS, this inhibition was 38.4-, 65.1- and 56.3% as compared to the control, respectively ( Figures 1D–F ).

Nonetheless, GABA application significantly improved RL and RFW under 50 mM (18.2- and 26.3%). Further, GABA caused a non-significant increment in RL and RFW under 100 mM and RDW under both stress conditions ( Figures 1D–F ). The leaf morphology was also substantially influenced by saline stress since a 39.9-, 34.0% and 69.3-, and 55.9% inhibition was recorded in leaf fresh weight (LFW) and leaf dry weight (LDW) following the 50 mM and 100 mM SS, respectively. However, GABA significantly improved LFW, and LDW under 0 mM (29.7 and 18.4%, respectively), 50 mM (37.8 and 24.5%, respectively), and 100 mM SS (44.0-, and 10.8%, respectively), compared to untreated plants ( Figures 1G, H ).

The mungbean plants exhibited lower chlorophyll a (Chl a) and chlorophyll b (Chl b) content under salinity stress compared to the un-treated plants since a decline of 24-, 32- and 39- and 54%, following 50 mM and 100 mM, respectively ( Figures 2A, B ). Nonetheless, the supplementation of GABA to both saline levels considerably improved the Chl a and Chl b contents by 19.3-and 5-%; 19- and 33%; and 11- and 7%, respectively. Interestingly, the Chl a/b ratio showed a different behavior, and a significant increase was observed under both salinity levels ( Figure 2C ). A 13- and 34% rise was recorded under 50 Mm and 100 mM SS, respectively. GABA improved (21.6%) the Chl a/b ratio following 100 mM SS ( Figure 2C ). Carotenoid levels significantly increased at 50 mM and remained unaffected at 100 mM saline stress level. Exogenous GABA significantly increased carotenoid level under controlled condition, where it has no profound effect under salt conditions, compared to their untreated peers ( Figure 2D )

The excess Na⁺ levels strongly indicate saline/alkaline stress persistence in plants. A Similar was the case in this study since a rapid enhancement was noticed after the induction of saline stress at both levels. This increment was 1.6- and 2.3-fold following 50 mM and 100 mM SS, respectively. Although the GABA could not profoundly reduce the Na⁺ levels after 100 mM SS, however, significant inhibition was observed under 50 mM SS ( Figure 3A ).

The exposure to saline stress reduced N content, K⁺ levels, and K⁺/Na⁺ ratio. The mungbean treated with 50 mM SS exhibited 38, 21, and 51% decreases in N content, K⁺ levels, and K⁺/Na⁺ ratio, and this reduction was even greater (59, 33, and 71%) in the case of 100 mM SS, respectively ( Figures 3B, C ). Although, the exogenous application of GABA could help the mungbean plants in the considerable improvement of these parameters under 50 mM SS. Whereas the 100 mM saline-stressed plants did not display such an obvious promotion in these parameters after GABA application ( Figure 3C ).

The H₂O₂ and MDA are the major determinants of oxidative stress in plants since the production of these compounds is directly related to the increased cellular damage post oxidative stresses ( Figures 4A, B ). Similarly, in the current experiment, after the induction of oxidative stress caused by saline stress, the H₂O₂ and MDA levels were raised by 47 and 86% under 50 mM. This enhancement was 1.9- and 2.1-fold following 100 mM, respectively. Nevertheless, using exogenous GABA declined the H₂O₂ and MDA levels by 1.3- and 1.8-fold under 50 mM SS and 1.9- and 2.2-fold after100 mM SS, respectively ( Figures 4A, B ).

Salt-induced higher concentrations of H₂O₂ and MDA, the mungbean plants tremendously stimulated their antioxidant potential to scavenge excessive ROS. Briefly, the SOD, POD, APX, PPO, and GTR activities were enhanced by 1.3-, 1.3-, 1.3-, 1.16-, and 2-fold when the plants were grown under 50 mM SS, while the 100 mM SS-treated mungbean plants displayed 1.3-, 1.3-, 1.4, 1.5-, and 2.4-fold stimulation after 100 mM SS, respectively ( Figures 5A–E ). The exogenous application of GABA further improved the antioxidant activities of POD and SOD by 38 and 35% at 50 mM SS levels, while the APX, PPO, and GTR increments were 1.5-, 1.4-, and 2.2-fold after 100 mM SS, respectively ( Figures 5A–E ). Intriguingly, the CAT activity significantly increased under 50 mM (30.4%) compared to the control condition. However, GABA application significantly enhanced under 0 mM and 50 mM (17.3- and 10.5%, respectively) compared to their untreated peers. However, SS and GABA had no significant effect on CAT activity ( Figure 5F )

The nitrogen (N) concentration significantly decreased under 50 mM and 100 mM by 38.3- and 58.9%, respectively, compared to the control ( Figure 6A ). Compared to untreated peers, exogenous GABA significantly increased N concentration under 0 mM and 50 mM (19 and 31.8%, respectively) but had no significant effect under 100 mM SS. Intriguingly, the NR (32-, 33.1%), GS (15- and 51%), and GOGAT (12.5- and 65%) were significantly reduced by the induction of 50- and 100 mM SS ( Figures 6B–D ). However, GABA significantly improved NR under 0- and 50 mM SS (16- and 27-%, respectively), whereas GS under 0-, 50- and 100 mM SS (28- and 37-, and 24.2%, respectively), compared to controlled conditions. Moreover, GABA had no significant effect on GOGAT under controlled and SS conditions ( Figures 6B–D ).

The soluble sugar concentration increased under saline stress and GABA application. For instance, it increased by 29.2- and 6.5% under 50- and 100 mM SS ( Figure 7A ). Further, the GABA application enhanced soluble sugar by 30.3-, 13.3-, and 7.6% under 0-, 50-, and 100 mM SS. Interestingly, the protein content significantly improved at both GABA-treated and untreated treatments, irrespective of the salt stress. Generally, 44- and 28% of increment was observed after 50 mM of salinity stress in untreated and treated plants, respectively ( Figure 7B ). Similarly, both GABA-treated and untreated mungbean plants showed a 1.5- and 2-fold increase after 100 mM SS, respectively. Like enzymatic antioxidants, the non-enzymatic antioxidant compounds, such as proline, and phenol, were considerably influenced by the salt stress in mungbean plants ( Figures 7C, D ). When the plants were exposed to 50 mM SS, the proline, and total phenolic content, were enhanced by 88- and 36% and the GABA application further improved them by 68- and 66 4% under 50 mM SS, respectively. In addition, the mungbean plants under 100 mM salinity stress showed a 2.4- and 2- fold rise, and a further 2-and 1.9-fold increase was deployed by the GABA supplementation, respectively ( Figures 7C, D ).

The correlation analysis results displayed a significant positive relation between nitrogen related enzymes (NR, nitrate reductase; GS, glutamine synthetase; and GOGAT, glutamate synthase), chlorophyll content (chla, chlb), and biomass accumulation (fresh and dry biomass). It showed that the increasing activities of nitrogen metabolism-related enzymes prompted nitrogen uptake and result in maximum biomass production. On the other hand, antioxidant enzymes activities (POD, peroxidase; CAT, catalase; APX, ascorbate peroxidase; SOD, superoxide dismutase; PPO, polyphenol oxidases; GTR, glutathione reductase) and protein, proline, and total phenolic content (TPC) had s strong negative correlation with nitrogen related enzymes ( Figure 8 ).

Increasing soil salinity is a major global issue in sustainable agriculture as it disrupts cellular and physiological processes at all growth stages (Arif et al., 2020). Salinity-induced reduction in plant growth may be due to low uptake of mineral nutrients (Askari-Khorasgani et al., 2017), such as potassium (K⁺), as a result of excess Na⁺ accumulation (Chakraborty et al., 2016). In the present study, a similar phenomenon was observed, as increased saline stress (SS) led to impaired growth features (stem height, root length, leaf area, stem-leaf-root fresh and dry weight; Figures 1A–H ) and chlorophyll pigments (Chl a, Chl b; Figures 2A, B ) of mungbean plants, suggesting hindrance of cell division. The decline in growth and biomass is correlated negatively with increased Na⁺ levels and excessive H₂O₂ production ( Figure 8 ). Excessive H₂O₂ disrupts the plasma membrane causing ionic imbalance, inhibiting metabolic processes, eventually declining plant growth (Mittler et al., 2011; Kaya et al., 2019; Ullah et al., 2022b; Ullah et al., 2022c). In the present study, gamma-aminobutyric acid (GABA) was applied under SS to investigate whether or not it can mitigate salinity-induced adverse effects on mungbean growth and metabolism. We found that exogenous GABA relieved salt-induced reductions in plant growth features ( Figures 1A–H ). So, GABA might be an active growth regulator involved in the salinity-tolerance of mungbean plants, as has been reported in previous studies (Jin et al, 2018; Wang et al., 2015; Kalhora et al., 2018; Wu et al., 2020), which corroborated our findings. Hence, we suggest that exogenous GABA could increase the salinity-tolerance of mungbean by improving their growth under SS.

The reduction in salinity-induced inhibitions of the photosynthetic pigments primarily inhibits plant growth. During chlorophyll biosynthesis in plants, a series of reactions take place, and when any of these reactions are disrupted, chlorophyll biosynthesis is affected (Min et al., 2006; Ali et al., 2022). In our study, increased SS significantly inhibited chlorophyll (Chl a and Chl b) concentration, which could be attributed to a specific symptom of SS-induced oxidative damage (Taïbi et al., 2016) and chlorophyllase inhibition/degradation (Chakhchar et al., 2018). An overabundance of H₂O₂ in mungbean plants under SS may also have contributed to the decrease in chlorophyll concentrations (Kaya et al., 2018). However, exogenous GABA improved chlorophyll concentration in SS-stressed plants, suggesting that GABA can mitigate the damaging effects of SS on photosynthetic pigments by reducing the production of H₂O₂ and improving plant growth. Similarly, GABA enhanced chlorophyll synthesis in maize plants under SS (Aljuaid and Ashour, 2022). Moreover, carotenoid concentrations significantly increased under 50 mM SS. Carotenoid is an auxiliary pigment that can dissipate excess light energy, making it an adaptive trait for dealing with salt stress (Duan et al., 2012). Exogenous GABA under 0 mM and 50 mM SS resulted in a significant increase in carotenoids compared to GABA-untreated counterparts ( Figure 2D ). Exogenous γ-aminobutyric acid (GABA) has been demonstrated to alleviate salinity-induced inhibition of plant growth by reducing chlorophyll degradation and enhancing photosynthetic capacity. For instance, in lettuce plants treated with exogenous GABA, salinity was less toxic to fresh and dry shoot masses than those untreated with GABA (Luo et al., 2011; Kalhora et al., 2018). So, GABA might be an active growth regulator involved in the salinity tolerance of mungbean plants by promoting their photosynthetic pigments and growth under SS.

The excessive accumulation of Na⁺ caused by salinity stress leads to a decline in the uptake of essential nutrients like K⁺ from roots to leaves (Zhang et al., 2020). Increasing levels of Na⁺ and decreasing K⁺ in photosynthetic leaves may degrade chlorophyll and disrupt thylakoids (Bose et al., 2017). Plant salinity tolerance is characterized by its ability to lessen the accumulation of toxic Na⁺ ions in sensitive shoots (Niu et al., 2018). Hence, Na⁺ accumulation in tissues is often considered an indicator of the severity of salinity stress damage. In our study, increased salinity stress resulted in a significant increase in Na⁺ and a decline in K⁺ concentration in the leaves. Similar findings were reported for maize (Kaya and Ashraf, 2020; Aljuaid and Ashour, 2022), barley (Akhter et al., 2021), canola (Naveed et al., 2020), and soybean (Ullah et al., 2019). Further, salinity-induced decline in K⁺ ion resulted in low K⁺/Na⁺ ratio ( Figures 3B, C ). High salinity causes high pH in the rhizosphere, which reduces the availability of ions of nutrient elements, including K (Yang et al., 2007). Further, low K⁺ levels may also be caused by stress-induced repression of K⁺ absorption. Consequently, the low K⁺ and K⁺/Na⁺ ratio may also be attributed to K⁺ and Na⁺ competing for binding sites for cellular functions (Azooz et al., 2015; Ullah et al., 2019). In our study, the declined mungbean growth could be attributed to cellular membrane damage caused by osmotic, ionic, and pH-induced damage associated with a rise in Na⁺ ions and a decrease in beneficial K⁺ ions as a result of saline stress (Zhu, 2001; Chartzoulakis, 2005; Ullah et al., 2019; Aljuaid and Ashour, 2022; Noor et al., 2022). Several studies have been conducted on the effect of exogenous GABA on Na⁺ concentrations in plants, but whether GABA can directly reduce Na⁺ accumulation is unclear. Nevertheless, it has been demonstrated that Arabidopsis mutants with high GABA levels in their roots have significantly lower Na⁺ than mutants with low GABA levels (Su et al., 2019). In the present study, exogenous GABA treatments reduced Na⁺ levels in the leaves of mungbean plants, compared to their untreated GABA peers ( Figure 3A ). A study by Wu et al., 2020 found that exogenous GABA inhibits the absorption of Na⁺ ions by roots and reduced their transport to leaves. Further, GABA has been shown to improve plant salt tolerance by influencing ion membrane potential differences and osmoregulation, thereby enhancing ion transport (Seifikalhor et al., 2019). We, therefore, believe that exogenous GABA alleviates saline stress damage in mungbean plants by reducing Na⁺ in the leaves ( Figure 3A ). Numerous studies have demonstrated that GABA accumulation reduces Na⁺ uptake and reactive oxygen species (ROS) concentration, activates H⁺ ATPase, and inhibits K⁺ loss under stress conditions (Shi et al., 2010; Li et al., 2019, 2020; Su et al., 2019; Wu et al., 2021; Aljuaid and Ashour, 2022). Therefore, we suggest that GABA-induced reduction in Na⁺ concentration was associated with increased K⁺ levels and a high Na⁺/K⁺ ratio, which is beneficial for salinity adaptation.

The osmotic and oxidative stresses caused by salinity impair plant metabolism (Ma et al., 2018), leading to the accumulation of lipid peroxides and membrane damage (Mittler et al., 2004). Compared to controls, we observed significantly higher levels of MDA and H₂O₂ during all levels of saline stress. Similar results were reported for chufa (Ullah et al., 2022b), maize (Aljuaid and Ashour, 2022), barley (Akhter et al., 2021), canola (Naveed et al., 2020), and pepper (Kaya et al., 2020b). It is well known that plants possess a highly specialized antioxidant defense system that is capable of scavenging reactive oxygen species (ROS) under stress conditions, including salinity. in the present study, saline-treated mungbean plants displayed significantly greater levels of antioxidant enzymes, compared to control mungbean plants. For example, compared to the control, the activities of SOD, POD, APX, PPO, and GTR, increased with increasing saline stress levels, indicating that it is a solid antioxidant defense mechanism for detoxifying the excessive ROS (Munns and Tester, 2008; Polash et al., 2019). Several studies have reported the upregulation of SOD, POD, APX, PPO, GTR, and CAT in plants under salinity stress (Luo et al., 2011; Weisany et al., 2012; Farhangi-Abriz and Torabian, 2017; Aljuaid and Ashour, 2022), which corroborate our findings. It is suggested that salinity-treated mungbean plants upregulate their antioxidant enzymes to scavenge ROS at the expense of growth and biomass reduction as more energy has been devoted to antioxidant mechanisms instead of organ development. Further, we found that exogenous GABA significantly reduced H₂O₂ and MDA under both saline stress levels, where its effect on antioxidant enzymes was only significant under 50 mM saline stress. It has been demonstrated that GABA exhibits ROS scavenging potentials and can contribute to stress mitigation (Liu et al., 2011; Aljuaid and Ashour, 2022). For instance, GABA inhibits MDA, an indicator of lipid oxidation (Deng et al., 2010; Aljuaid and Ashour, 2022). However, it is unclear whether GABA can directly scavenge ROS to alleviate stress. Still, several studies reported that exogenous GABA plays a key role in scavenging ROS and modulating the activity of antioxidant enzymes (Renault et al., 2010; Barbosa et al., 2010; Aljuaid and Ashour, 2022). We suggest that exogenous GABA protected mungbean plants from salinity-associated oxidative stress damage and enhance their tolerance by reducing lipid peroxidation and upregulating enzymatic antioxidant mechanisms.

Regulation of nitrogen (N) metabolism is essential for salt tolerance, and salinity and N nutrition interact intricately (Läuchli and Lüttge, 2002). In this study, mungbean plants subjected to saline stress had lower N content and, along with declined activities of nitrate reductase (NR), glutamine synthetase (GS), and glutamine oxoglutarate aminotransferase/glutamate synthetase (GOGAT) ( Figure 6A -D). The salinity-induced decline in nitrogen assimilation has been reported for tomato (Debouba et al., 2006), Chinese cottonwood (Meng et al., 2016), soybean (Ullah et al., 2019), and chufa (Ullah et al., 2022b), which corroborate our findings. We suggest that salt ions impaired nitrogen metabolism in mungbean plants by inhibiting NR activity which prevents GS/GOGAT from supplying NH₄ ⁺ to the amino acid synthesis pathway (Debouba et al., 2006; Meng et al., 2016). The decrease in N assimilation and the synthesis of amino acids and proteins can reduce plants’ dry weight (Queiroz et al., 2012). However, exogenous GABA treatments improved nitrogen concentration and activities of NR, GS and GOGAT under salt and controlled condition, compared to their untreated peers. However, the effect was only significant under 50 mM saline stress. GABA has been shown to play a role in buffering N metabolism (Salah et al., 2019). For example, GABA may influence plants’ growth and stress responses by regulating their carbon and nitrogen metabolism (Chen et al., 2020). A recent study found that plants treated with GABA displayed increased nitrogen metabolism, as illustrated by increased nitrogen concentration and its metabolizing enzymes (Khanna et al., 2021), which support our findings. Therefore, we suggest that exogenous GABA enhances salinity stress tolerance of mungbean plants by regulating the key enzymes of N assimilation and increase N concentrations, resulting in improved growth and biomass.

In the present study, significantly higher concentration of soluble sugar recorded at 50 mM, compared to control condition. Under stress condition, plants use soluble sugars to maintain osmotic and ionic homeostasis, limit chlorophyll destruction and water loss, remove excess ROS, stabilize proteins and membrane structures, regulate cell division, and control gene transcription (Sami et al., 2016). Moreover, there was a significant increase in proline, soluble protein and total phenolic concentration with increasing saline stress levels compared to the control condition. The accumulation of proline might have contributed to the protection of photosynthetic apparatus elimination of excessive ROS to stabilize membranes, enzymes, and proteins in salinity-treated mungbean plants (Verdoy et al., 2006; Ahmad et al., 2016). In other studies, proteins have also been shown to act as osmotins and contribute to salt stress tolerance (Zhang et al., 2013). Furthermore, plants have the ability to upregulate small molecules of protein that can be used for storing N and can be mobilized rapidly during a period of stress relief (Singh et al., 1987). Osmotic adjustment may also be mediated by these proteins (Ashra and Harris, 2004). In addition, phenolic compounds play an important role in protecting plants from both biotic and abiotic stress (Ma et al., 2018; 2019). The application of exogenous GABA further increased soluble sugar, proline, soluble protein, and total phenolic content, suggesting that the GABA protected the mungbean plants from salt stress and thus enhanced their salinity tolerance.