Foliar application of glycine betaine to ameliorate lead toxicity in barley plants by modulating antioxidant enzyme activity and biochemical parameters

Lead (Pb) toxicity is a major problem in agricultural soil that negatively affects plant growth and development. Glycine betaine (GB) is an effective compatible solute that resists abiotic stress and plays an important role to mitigate various stresses. The present study is the first of its kind on the application of GB to mitigate Pb toxicity on barley cultivars. To elucidate the role of GB in mitigating Pb toxicity of three concentrations (15 mM, 25 mM, and 35 mM) in two barley varieties (BH-959 and BH-946) with and without foliar application of GB (2 mM) was examined. The study found that increasing Pb concentration significantly (p < 0.05) reduced the level of primary metabolites viz. photosynthetic pigments, protein, and carbohydrates in both cultivars upto 42.4%, 38.4% and 39% respectively. However malondialdehyde content, proline content, and antioxidant enzyme activity (SOD, CAT, and POX) were found to increased significantly (p < 0.05) as compared to control treatment upto 83.4%, 83.2% and 51% respectively. In contrast, the application of GB led to significantly (p < 0.05) improved physio-biochemical parameters as well as antioxidant enzyme activity (53%) and reduced oxidative stress along with malondialdehyde content (14.42%) in both varieties. An increment in these parameters revealed that exogenous application of GB (2 mM) significantly improves Pb (up to 35 mM) toxicity in barley plants and its use may be beneficial for crops susceptible to Pb toxicity to improve growth and yield.


Introduction
Barley (Hordeum vulgare L.) is one of the most widely cultivated cereal crops around the globe. It ranks fourth among all cereals produced globally, after wheat, rice, and maize [1]. Most barley crops are utilized as animal feed, 21% as malt and brewing feed, and 6% as human food [2]. It is estimated that plants directly or indirectly meet one-third of human daily needs [3]. However, global agricultural production is affected by environmental changes viz. heavy metals (HMs) accumulation, salinity, drought, and temperature stress in soil and become a problem for food security and crop productivity in sustainable agriculture [4,5]. Therefore, it becomes imperative to review the problems associated with HMs in plants, as well as their mode of action [3].
Soil is an integral component of the terrestrial ecosystem and the central interface between the hydrosphere, biosphere, atmosphere, and lithosphere. Therefore, soil delivered a wide range of ecosystem services for plant growth, including rhizosphere microorganisms, arbuscular mycorrhizal fungi, rhizobium, plant growthpromoting regulators (PGRs), and inorganic nutrients [6]. However, the soil is at risk of contamination with HMs due to rapid industrialization, mining, and electroplating, automobile emissions, deposition of physio-biochemical activities and productivity of plants [11,25,26]. Several scavenging enzymes have evolved in plants to combat the negative impact of reactive oxygen species (ROS), such as ascorbate peroxidase (APX), superoxide dismutase (SOD), peroxidase (POX), and catalase (CAT) [27]. However, HM stress significantly reduced antioxidant enzyme activity, indicating that plants may not resist the toxic effects of HMs [28]. As a result of soil contamination with Pb, crop productivity is dramatically decreased, posing a serious problem for agriculture [17]. Thus, developing management strategies that reduce Pb accumulation in plants and improve their tolerance to Pb is crucial.
To reduce HMs toxicity in soil and plants many remediation strategies are effective in reducing the phytotoxicity of potentially toxic metals. For this, the use of compatible solutes is a promising approach that significantly reduced the negative impact of HMs in plants [29]. Compatible solutes are highly soluble low molecular weight substances that are usually nontoxic at high cellular levels. Generally, they protect plants from stress via different mechanisms, including detoxification of ROS, cellular osmotic adjustment, maintaining membrane integrity, and protein stabilization [30]. Glycine betaine (GB) is an effective compatible solute, synthesized in plant chloroplasts via oxidation under various environmental stresses to combat stress [31,32]. GB is a water-soluble and non-toxic compound and is metabolically stable, which makes it a superior osmoprotectant [33]. It is applied externally for acclimatization, adaptation, alleviation, and stabilization of stressed environments [34]. It has been reported in several studies that GB protects plants from various stresses, including temperatures [35], salinity [36], droughts, and HM stress [26,28]. GB is mainly found in chloroplasts, where it is responsible to safeguard the effectiveness of Photosystem II (PSII) under stress. Additionally, GB may protect against ROS by scavenging them and activating a variety of antioxidant enzymes, including POX, CAT, and SOD, which ultimately protect plants from oxidative damage [35,37]. The endogenous level of GB is not enough to protect the plants from the negative impacts of changing climate, exogenously applied GB may be an effective method of mitigating the toxic effect of stresses [23].
To the best of our knowledge, this is the first report deciphering the application of GB to mitigate Pb toxicity on barley cultivars. Literature studies suggest that exogenous GB ameliorates Pb toxicity by increasing the primary metabolites and antioxidant enzyme activity of the plants. Thus, the current research has been conducted to determine the ameliorative behavior of GB on physiological and biochemical attributes (i) photosynthetic pigments such as Chl-a, Chl-b, total Chl, carotenoid and chlorophyll stability index (CSI), (ii) biochemical parameters such as lipid peroxidation, phenol content, proline content, total protein content, and total carbohydrate content as well as on (iii) antioxidant enzymes activity includes SOD, POX, and CAT of two barley varieties under Pb stress. The varieties differ from each other in quality parameters. The overall results of current research will provide new insight regarding the role of GB-induced tolerance to Pb toxicity in barley cultivars and its use may be recommended to improve the yield.

Plant material and treatments
The research experiments were conducted at Maharshi Dayanand University, Rohtak, Haryana (India) on two varieties of barley (BH-959 and BH-946) by foliar application of GB with stressed and non-stressed conditions. Both barley varieties were taken from Chaudhary Charan Singh Haryana Agricultural University, Hisar, Haryana (India). A completely randomized design (CRD) was used to place the pots. Each pot was filled with 7 kg the sandy soil with organic matter content of 0.25%. The nutrient solution of Hoagland and Arnon's [38] was given to the soil after an interval of a week to provide nutrition. After 25 days of planting the crop, 200 ml lead acetate (Pb(C 2 H 3 O 2 ) 2 ) solution was given in each pot i.e. 0, 15, 25, and 35 mM concentrations and foliar spray of GB (2 mM) was given alone and with Pb treatment (table 1). Biochemical and antioxidant parameters were performed after 50 days of seed germination.

Photosynthetic pigments
The photosynthetic pigments were estimated by following the protocol given by Hiscox and Israelstam [39]. Coarsely chopped leaves were placed in DMSO-containing tubes for 5 h at room temperature and noted absorbance at 665, 645, and 480 nm, respectively, in UV-spectrophotometer (UV 2450, Shimadzu). DMSO had been taken as a blank and the chlorophyll Stability Index (CSI) was determined by following the protocol given by Kaloyereas [40]. A total of two sets of leaf samples (100 mg in 5 ml DW) were prepared. The first set was kept at room temperature for 1 h and the second set of the sample was boiled in a water bath for 1 h at 55°C. Then, water was discarded from the test tubes of each set, followed by the addition of DMSO, and then left undisturbed for 4 h and measured absorbance at 652 nm. The CSI was calculated by comparing a heated leaf sample with a nonheated leaf sample in terms of light transmission. The calculation was done by using the following formula: CSI (%) = (OD at 652 nm of heated sample/OD at 652 nm of non-heated sample) × 100 Biochemical parameters MDA (μmol MDA g −1 FW) content as an indicator of lipid peroxidation was determined by following the protocol of Heath and Packer [41]. Fresh leaves (100 mg) were crushed in 2 ml of 0.1% trichloroacetic acid (TCA) solution and then centrifuged to collect supernatants. Then, 4ml of 0.5% TBA (Thiobarbituric acid) prepared in 20% TCA was added to supernatants and boiled for 30 min at 95℃ and then cooled in an ice bath, and absorption was measured at 532 nm and 600 nm, respectively. By subtracting the values obtained at 532 nm from 600nm, the absorbance was corrected for unspecific turbidity. MDA content was calculated by using the extinction coefficient (155 mM −1 cm −1 ). The total protein in leaves was evaluated following the method described by the Lowry method [42]. Fresh leaves (100 mg) were used to prepare the extract in distilled water. After centrifugation, 0.2 ml extract was taken and brought the volume to 1 ml by adding DW. Then, 5 ml of reagent C i.e., 2% Na 2 CO 3 dissolved in 0.1 N NaOH (reagent A) and 0.5% CuSO4 in 1% NaKTa (reagent B) (50:1) were added. After that, Folin-reagent Ciocalteu's (FCR) was added. After vigorous shaking with a vortex shaker, the mixture was left in the dark for some time and noted absorbance at 660 nm in the spectrophotometer. To measure the total proline content, the method described by Bates et al [43] was used. A 100 mg fresh leaves were homogenized in 3% aqueous sulphosalicylic acid (2 ml) and centrifuged to collect supernatants. Then, glacial acetic acid (1 ml) and ninhydrin reagent (1 ml) were added and heated for 10 min in a water bath and then chilled in an ice bath. 2 ml toluene was added and vigorously shaken on a vortex shaker and noted the absorbance spectrophotometrically at 520 nm. Total carbohydrate content was measured by following the protocol given by Yemm and Willis [44]. Fresh leaves (100 mg) were crushed in 80% of ethanol and centrifuged. 0.2 ml extract was taken and added DW to make the volume of 1 ml. Afterward, anthrone reagent (4 ml) was added and heated for 8-10 min in a water bath and then, cooled in an ice bath. With anthrone as a blank, we measured absorbance at 630 nm. The total phenol content of leaves was measured by the Folin-Ciocalteu reagent method [45]. For extraction, 100 mg fresh leaves samples were crushed in 80% methanol (2 ml) and then the extract was centrifuged for 15 min at 10,000 rpm. After that, FCR and sodium carbonate (Na 2 CO 3 ) solutions were added and read the absorbance in a spectrophotometer at 650 nm.

Assays of antioxidant enzyme activities
To estimate antioxidant enzyme activities viz. POX, CAT, and SOD, 100 mg of plant sample (leaves and roots) were taken. Samples were ground in 2 ml sodium phosphate buffer (pH = 7) by using a pre-chilled pestle mortar. Then, at 4℃ centrifuged at 15,000 rpm for 15 min. Supernatants were stored at 4°C until enzyme activity estimations were done. The antioxidant enzyme activities of roots and leaves were measured spectrophotometrically using an aliquot of supernatant given by Abdelaal et al [46]. CAT enzyme activity was measured by following the protocol outlined by Aebi [47]. The activity was measured by extracting 0.2 ml of enzyme and mixed with 1.5 ml of potassium phosphate buffer. 0.2 ml of H 2 0 2 (100 mM) was added at the time of absorbance taken and the absorbance was measured for 1 min every 15 s at 240 nm. POX enzyme activity was measured by following the protocol outlined by Hammerschmidt et al [48]. 0.2 ml of enzyme extract was taken and mixed with 0.8 ml of phosphate buffer, 0.5 ml of O-dianisidine, and 1.8 ml of sodium acetate buffer. 0.1 ml of 0.2 M H 2 0 2 was added at the time of absorbance taken and the absorbance was measured for 1 min every 2 s at 470 nm. SOD enzyme activity was measured by following the protocol outlined by Beauchamp and Fridovich [49]. To initiate the reaction, enzyme extract was added. The sodium phosphate buffer used in the reaction mixture contained NBT, Na 2 CO 3 , EDTA, methionine, and riboflavin. At 560 nm, the absorbance was taken.

Statistical analysis
The data were statistically evaluated by using two-way analysis of variance (ANOVA) and presented the results as means of three replicates (Means ± SE). A Tukey's HSD post hoc test was performed at a significant level of 5% (p < 0.05) to test the significant differences between the means of different treatments as well as varieties. R software (R-studio 4.2.1) was used to construct the figures and Principal component analysis (PCA) plots.

Effect of exogenous GB under Pb Stress on chlorophyll stability index (CSI)
When compared to the control, CSI also decreased from 20% (Pb1) to 37%(Pb3), in BH-959 variety, and from 23% (Pb1) to 39% (Pb3) in BH-946 variety at high Pb (Pb3) stress conditions ( figure 1(E)). The results revealed that the foliar spray of GB enhanced the CSI by up to 18.7% (Pb1+GB), 16.6% (Pb2+GB), and 9.7% (Pb3+GB) in BH-959 variety and 17.6% (Pb1+GB), 14% (Pb2+GB), and 7.9% (Pb3+GB) in BH-946 variety. More increment was noted at Pb1+GB concentration in both varieties. It was found that GB also significantly improved the BH-959 variety in non-stressed conditions. Impact of exogenous GB under Pb stress on lipid peroxidation, total proline, and total phenol content The results revealed that proline, phenol, and malondialdehyde (MDA) contents increased at all three levels of Pb stress (Pb1, Pb2, and Pb3) in both varieties of barley (figures 2(A)-C). When compared to their respective control treatment, total proline content increased up to 80% in BH-959 and 83.2% in BH-946 at high Pb stress (Pb3) conditions. However, foliar supply of GB reduced the total proline accumulation in plants to 24.5%, 16.6%, and 9.5% in BH-959 and 23.9%, 10.2%, and 5.5% in BH-946 at all three levels of Pb treatments. Likewise, the application of GB decreased the phenol content in Pb-treated barley plants but all three levels of Pb stress showed high phenol content. The total phenol content of BH-959 increased by up to 91% in BH-959 and 99% in BH-946 when exposed to high Pb stress (Pb3). BH-946 showed more phenol content at high Pb treatment (Pb3) as compared to BH-959. However, GB supplementation reduced total phenol content to 32% in BH-959 and 31% in BH-946 at Pb1+GB treatment. Whereas, GB application at Pb2+GB and Pb3+GB treatment decreased the phenol content to 18%, 8.6% in BH-959, and 12% and 6.7% in BH-946, respectively. Lipid peroxidation (MDA Content) was also increased with increased stress (Pb3) as compared to control. There was an increase of 83.4% and 89.9% of MDA content in both barley varieties grown under Pb3 treatments, respectively. The result revealed that a foliar spray of GB (2 mM) significantly decreased the MDA content in both barley varieties. The decrease in MDA content was 14.42% (Pb1+GB), 12.7% (Pb2+GB), and 5.3% (Pb3 +GB) in BH-959 variety, and 11.4% (Pb1+GB), 10% (Pb2+GB), and 4% (Pb3+GB) in BH-946 variety, respectively. The decrease in lipid peroxidation by supplementation of GB was more in the BH-959 variety.

Impact of exogenous GB under Pb stress on total protein and carbohydrate content
The present study showed that total protein and carbohydrate contents were adversely affected by Pb stress when the Pb treatment increased from 15 mM (Pb1) to 35 mM (Pb3) (figures 2(D), (E)). Our findings showed that the concentration of protein was 19.45 ± 1.60 mg g −1 F −1 W −1 in BH-959 variety and 16.92 ± 1.21 mg g −1 F −1 W −1 in BH-946 variety under non-stressed conditions but decreased to 11.98 ± 1.16 mg g −1 F −1 W −1 in BH-959 variety and 10.28 ± 0.52 mg g −1 F −1 W −1 in BH-946 variety at maximum Pb stress (Pb3). However, foliar application of GB increased the total protein by up to 13.30 ± 1.34 mg g −1 F −1 W −1 in BH-959 and 11.01 ± 0.93 mg g −1 F −1 W −1 in BH-946 in Pb-treated barley plants (Pb3+GB) as compared to maximum Pb treatment (Pb3). Similarly, total carbohydrate content was also reduced when the Pb treatment concentration increased. In comparison to the control, the maximum reduction was 38% in BH-959 and 39% in BH-946 at maximum Pb stress (Pb3). However, GB supplementation increased the total carbohydrate content by up to 25%, 21%, and 11% in BH-959 and 23%, 19%, and 7% in BH-946 at different Pb treatments (Pb1+GB, Pb2+GB, and Pb3 +GB). Both varieties showed more enhancement at the lower level of Pb stress (Pb1+GB). In non-stressed conditions, GB had no significant effect on either variety.

Impact of exogenous GB under Pb stress on the antioxidant enzymes activities of POX, CAT, and SOD
The present study revealed that the activity of POX, CAT, and SOD enzymes increased with the increasing Pb treatments as compared to the control treatment (T0) in roots and leaves in both varieties of the barley plant (figures 3-5). A foliar spray of GB (2 mM) further enhanced the antioxidant enzyme activities as compared to Pb treatment alone in leaves and roots in both varieties. CAT activity had been found to increase in leaves and roots with increasing Pb treatment (Pb3) (figures 3(A), (B)). Compared to the control treatment (T0), an enhancement of 26% and 31% in the BH-959 variety and 21% and 30% in BH-946 in leaves and roots respectively, have been observed at the highest Pb treatment (Pb3). Both varieties showed higher CAT activity in roots than leaves under higher Pb-stress (Pb3). The foliar spray of GB further enhanced the CAT activity of roots and leaves by 48% and 53% in BH-959 variety, and 36% and 46% in BH-946 variety, respectively. However, foliar supplementation of GB had increased CAT activities of leaves and roots by 11% and 13% in BH-959 and 10% and 12% in BH-946 respectively, at high Pb treatment (Pb3+GB). Likewise, compared to the control treatment (T0), the POX activity of roots and leaves had been increased at high Pb stress (Pb3) by 27.7% and 28.8% in BH-959 variety, and 25.8% and 27.4% in BH-946 variety, respectively ( figure 4(A), (B)). Foliar spray of GB further increased the rate of POX activity in roots and leaves by up to 47% and 50% in BH-959 variety and 48% and 49% in BH-946 variety at Pb stress (Pb2+GB). However, the GB application had increased the POX activity in roots and leaves only up to 13.5% and 15.5% in BH-959 variety and 13.9% and 14.4% in the BH-946 variety, respectively, at maximum Pb treatment (Pb3+GB). The results showed that GB supplementation  significantly improved POX and CAT activities in roots and leaves of both barley varieties at two Pb treatments (15 mM & 25 mM). The results showed that Pb treatment had also increased SOD activity in roots and leaves in both varieties of the barley plant ( figures 5(A), (B)). In comparison with the control treatment (T0), SOD activity had increased in roots and leaves by 42% and 51% in BH-959 variety and 40% and 50% in BH-946 variety, respectively, at high Pb treatment (Pb3). However, the foliar spray of GB had further increased SOD activity to a greater extent at maximum Pb stress in both varieties of barley. Hence, the foliar application of GB was able to promote antioxidant enzyme activities in both varieties of barley up to 35 mM of Pb treatment significantly.

Correlation analysis
To analyze the correlation among different parameter studies in the present work, Pearson's correlation was performed ( figure 1(S), Supplementary file). The correlation among various biochemical, physiological, and antioxidant enzyme parameters have been analyzed for both varieties PB-946 and 959, separately. In both BH-959 and BH-946 varieties, Chl 'a', Chl 'b', total Chl, CSI, total protein content, carotenoid content, and total carbohydrate content are positively correlated with other and negatively correlated with total phenol content, total proline content, and MDA content at different significant levels (p < 0.001, p < 0.01, p < 0.05) and vice versa. Different antioxidant enzyme activity parameters like POX activity, SOD activity, and CAT activity in both roots and leaves are non-significantly negatively correlated with Chl 'a', Chl 'b', total Chl, CSI, total protein content, carotenoid content, and total carbohydrate content and positively correlated with MDA content, total proline content, and total phenol content non-significantly. However, antioxidant enzyme activity parameters were found significantly correlated with each other at different significance levels ((p < 0.001, p < 0.01, p < 0.05). In the BH-959 variety, total phenol content shows a negative correlation with CAT activity in leaves non-significantly.

Discussion
The study found that Pb stress reduced photosynthetic pigments viz. Chl 'a' Chl 'b', total Chl (a+b), carotenoid content, and CSI in both varieties of barley plants (figure 1(A)-(E)). Moreover, the reduction was high at maximum Pb (35 mM) stress as compared with lower Pb (15 mM) stress in both varieties. This may be due to the alteration in chloroplast structure and inhibition of gas exchange which further causes a reduction in photosynthetic pigments under Pb stress [50][51][52]. The Pb induced the structural changes in the photosynthetic apparatus and reduced the chlorophyll pigments causing the metabolism of carbon to be retarded [53]. It interferes with cyclic and non-cyclic photophosphorylation in chloroplast and reduced the rate of the electron transport chain in mitochondria [54]. Moreover, it has been discovered that Pb inhibits the catalysis of the Calvin cycle enzymes [55]. ROS generation in plants under metal stress can also reduce photosynthetic pigments and CSI [56,57]. Similar results were obtained in Brassica oleracea L. where Cr stress caused a structural modification in chloroplast and resulted in the generation of ROS accumulation, which ultimately reduced photosynthetic pigments [58]. The present study revealed that foliar application of GB improved the Chl 'a', Chl 'b', total Chl (a+b), and carotenoid content as well as CSI in both varieties of barley plants (figures 1(A)-(E). It was observed that the foliar spray of GB is effective under stressed as well as non-stressed conditions. This improvement in photosynthetic pigments may be due to the exogenous application of GB activates the RuBisCo enzyme (CO 2 fixing enzyme) under stress conditions [59]. According to the findings of Einset et al [60], GB shields the photosynthetic apparatus by activating ROS-scavenging genes. Bharwana et al [12] observed similar results on cotton under lead (Pb) toxicity. Similarly, Kumar et al [4] reported that externally applied GB enhanced the photosynthetic pigments in sorghum plants under Cr stress. The results revealed that externally applied GB increases photosynthetic pigments by decreasing the uptake of Cd and modulating antioxidant enzyme activities. In addition, GB increases stomatal conductance, preserves, and activates RuBisCo enzyme activity, and maintains chloroplast ultrastructure against environmental stresses and thus increasing the photosynthetic activity [59].
According to results, both barley varieties showed increased levels of proline, phenol, and lipid peroxidation under high Pb stress levels (figures 2(A)-(C). The application of GB as a foliar spray reduced these biochemical changes during Pb stress. In plants, proline accumulation is associated with stress injury [61]. Typically, it is found in the cytosol and is a major osmolyte that functions as a molecular signaling compound. Although it is unclear what molecular mechanisms cause elevated proline levels under Pb stress, one hypothesis proposes that proteins are broken down into amino acids and converted into proline for storage [4]. Results revealed that GB (Pb+GB) addition decreased the proline content in both barley varieties ( figure 2(A)). This result might indicate that Pb stress was reduced and the damage caused by Pb stress was alleviated by the exogenous GB. In line with result, Sun et al [62] reported that cucumber seedlings exposed to Cd toxicity with GB application decreased the proline content. Gupta et al [63] obtained similar results in flax (Linum usitatissimum) during drought conditions and suggested the role of GB in stress resilience. However, in chickpea (Cicer arietinum L.) plants, applying GB under metal stress enhanced the proline content [64]. Similar results were also observed by Kumar et al [4], which showed that GB treatment enhanced proline content in sorghum plants under Cr toxicity. In many studies, it was found that a high concentration of proline was toxic, but a low concentration increased plant tolerance to stress [65], so we can say that GB might suppress high proline content to ameliorate the toxicity ( figure 2(A)).
Phenolic compounds play a crucial role to reduce oxidative stress because they detoxify ROS [66]. Results showed that high Pb stress concentration increased the total phenol content in barley plants relative to the control treatment (T0). The rise in phenolic content may be due to the protective properties of these compounds against HM stress. They act as metal chelators and scavengers of ROS [66,67]. However, total phenol content decreased after the addition of GB (Pb+GB) compared with plants exposed to Pb treatment alone. From our findings we revealed that GB reduced the phenol content, this may be because GB took over the functioning of phenol, because the function of phenol is to reduce ROS accumulation while the addition of GB activates additional ROS scavenging genes and activates antioxidant enzyme activity which reduces ROS and oxidative stress ( figure 2(B)). GB is very beneficial for normalizing osmolyte levels and promoting the proper function of plants. Similar results were obtained in cucumber seedlings when exposed to Cd stress with the application of GB [62].
An increase in lipid peroxidation is evidence of oxidative stress caused by HM toxicity. In plants, MDA is a marker of oxidative stress associated with lipid peroxidation [68]. Metals are mostly absorbed by plant cell membranes. Our results showed that MDA content increased with increased Pb treatment (figure 2(C)). Different crop plants such as chickpea [64], and mungbean [69] have also shown similar results. The reason for this is that increasing MDA contents leads to a breakdown of the homeostatic ROS balance, which eventually results in a decrease in the activity of antioxidant enzymes under HM stress [56]. However, the foliar application of GB reduced the MDA content suggesting that GB alleviates oxidative damage caused by Pb toxicity by improving antioxidant enzyme activities [69]. In line with our result, Kumar et al [4] reported that sorghum plants exposed to Cr toxicity (2 ppm & 4 ppm) with GB application decreased the MDA content. In tobacco seedlings, it was shown that the addition of GB mitigates the Cd (5 μM) stress by lowering MDA levels [70]. The findings are consistent with the previous investigations.
In the present study, Pb decreased the amount of soluble protein and carbohydrate contents in barley leaf tissues as compared to control plants. This may be due to the excessive generation of ROS causing oxidative damage by inhibiting protein synthesis and oxidation of carbohydrates [71,72]. A reduction in total soluble protein content under heavy metals stress may be a result of increased protease activity [73]. Moreover, the decrease in carbohydrate content is due to the inhibition of chlorophyll biosynthesis [74]. However, the foliar spray of GB enhanced the protein and carbohydrate content in barley plants under Pb stress. Moreover, foliar application of GB increased the amount of soluble protein and carbohydrates in the leaves of barley plants during non-stressed situations (figures 2(D), (E)). It is still unknown what molecular mechanisms contribute to increasing protein and carbohydrate content under Pb+GB treatment. But one hypothesis suggested that GB may protect the proteins via chaperon-like actions on protein folding and acts as a signal molecule to tolerate the stress response [75]. Similar results were recorded in cotton plants under Pb toxicity after the addition of GB [59]. Ji et al [14] observed similar results under Pb stress with the exogenous application of GB in Brassica chinensis L. The present research showed that GB positively influenced plant growth and chloroplast function which may explain the increase in protein and carbohydrate content (figures 2(D), (E)).
A variety of antioxidant enzyme systems have evolved in plants, including CAT, SOD, and POX, under heavy metal stress [28,76]. Our results showed that plants under Pb stress (35mM) showed increased antioxidant enzyme activity (CAT, SOD, and POX) compared to the control treatment (T0). Lower activity of SOD and POX enzymes was recorded in roots as compared to leaves while on the other hand, CAT activity was reported to be higher in roots (figures 3-5). According to Ehsan et al [56], the roots of Brassica napus showed a higher level of SOD and CAT activity than leaves under metal toxicity. Habiba et al [57] also observed a higher level of POX activity in leaves than in the roots of Brassica napus under Cu stress. The findings suggest that the type of HM applied, plant species, and growth conditions may influence antioxidant enzyme activity in different plant parts [28]. As observed in the present study, 2 mM of GB application further improved the activity of antioxidant enzymes such as CAT, SOD, and POX in both varieties of barley plants under Pb stress conditions. Exogeneous GB treatment increased the antioxidant enzyme activities by inhibiting the uptake of Pb or reducing electrolyte leakage, MDA, and H 2 O 2 production. In plants, antioxidative defense systems (non-enzymatic and enzymatic) quench or convert reactive oxygen species to harmless forms [4,50]. Similar results were observed in many plant species such as sorghum [4], spinach [23], rice [77], and mungbean [28] under various types of heavy metal stress. Our results showed that at 35 mM+GB level; CAT, SOD, and POX activities decreased as compared to the other level of Pb (15 mM & 25 mM) with GB application in barley plants of both varieties (figures 3-5). The reason for this may be that the high Pb concentration in barley plants diminished the plant defense mechanism against Pb stress [28]. The present research concluded that increased antioxidant enzyme activities (CAT, POX, and SOD) with GB application might be due to lower Pb uptake by the plants. As a result of the present findings, it seems that exogenous GB contributes to the detoxification of ROS under Pb stress by modulating CAT, POX, and SOD activities. One possible mechanism of GB-induced metal tolerance in plants is that GB itself does not reduce ROS production but promotes the antioxidant enzyme activities to reduce ROS production (figures [3][4][5].
From the present research, it was found that Pb toxicity poses a major environmental threat that must be mitigated by using osmoprotectant GB. Present results revealed that plants exposed to Pb in combination with GB treatments showed a reduction in Pb toxicity. The improvement might be due to the reduction in Pb uptake and translocation by GB applications. In addition, the uptake of Pb may also facilitate another remediation process such as phytoremediation includes phytoextraction. However, there is no information available regarding the mechanism that how the GB application reduced the Pb uptake and translocation in barley plants. Therefore, further field studies are needed to investigate the role and mechanisms of GB individually or in combination with various types of HM stress in different plant species.

Conclusion
The present study was conducted to determine how GB alleviates the negative effects of Pb on two barley varieties. From the present investigation, it is revealed that Pb is a toxic HM for barley that affects its physiological and biochemical attributes. However, the foliar spray of GB alleviated the Pb toxicity by increasing the primary metabolites, and antioxidant enzyme activities and reducing MDA, proline, and phenol content. Alleviation of lead toxicity by exogenous GB might be due to reducing the Pb accumulation and transportation possibly by chelating the HMs inside the cells and this may be the reason for the ameliorative behavior of exogenous GB in barley plants also. Hence, the application of exogenous GB may be used in Pb-affected areas to improve the growth and yield of barley plants. Based on the results obtained, it was revealed that variety BH-959 shows the highest ameliorative behavior as compared to variety BH-946 under Pb stress with GB application. Further studies in field conditions are necessary to clarify the distribution and synthesis of GB, as well as its precise role under HM stress.