Journal of Plant Biochemistry & Physiology
Open Access

Simultaneous Treatment of Different Gibberellic Acid Doses Induces Ion Accumulation and Response Mechanisms to Salt Damage in Maize Roots

Burcu Seckin Dinler^1*, Hatice Cetinkaya², Mehmet Akgun² and Kursat Korkmaz^{2 *}Correspondence: Burcu Seckin Dinler, Department of Biology, Sinop University, Turkey, Email:

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Introduction

Maize (Zea mays L.), one of the most important cereal crops, is grown under a wide spectrum of soil and climatic conditions. This crop is an important C4 plant from the Poaceae family and moderately sensitive to salt stress; nonetheless, it has wide intraspecific genetic variations for salt resistance [1] Salinity imposes osmotic stress and ionic toxicity to maize, and it shows adverse effects on growth and development. Salinity inhibits growth and development of most plants. Inhibition of shoot and root development is the primary response to the stress. Growth, morphology, anatomy, and physiology of roots are affected by salinity. Due to changes in water and ion uptake by the roots [2], a decrease in plant root weight and length has been detected under salt stress [3].

Salinity is one of the most damaging environmental stress factors in the world that restrain plant life [4]. It limits both product efficiency in agriculture and it effects geographic distribution [5]. Soil salinity cause ionic stress and low osmotic potential leading to cell and even plant death. Beside this, salt stress can create physiological drought in plants. Furthermore, accumulation of ions like Na+ and Cl− in plant tissues leads to oxidative damage which depends on ROS (reactive oxygen species). ROS are free radicals that are formed endogenously in plants [6]. Excess generation of ROS such as superoxide, hydrogen peroxide and hydroxyl radical cause oxidative damages in the apoplastic compartment and damages of cellular membranes by lipid peroxidation [7].

Plants eleminate ROS accumulation during salinity stress by enzymatic superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), glutathione reductase (GR), glutathione -s-tranferase (GST), peroxidase (POD), monodehydroascorbate reductase (MDAR), dehydroascorbate reductase (DHAR) and glutathione peroxidase (GPX) non enzymatic (ascorbate, glutathione (GSH), a-tocopherol, carotenoid and phenolic compounds) pathways [8]. However, very aspects of plant mechanisms remain unclear under salt stress despite much important research [9]. The essential nutrients effect growth, biomass and productions in plant roots [10]. The root system architecture is greatly influenced by soil conditions [11], including nutrient gradients and concentrations of nitrate and phosphorus [12]. Roots also affect the surrounding nutrient composition by the release of organic compounds that play a vital role in mineralizing nutrients [13]. Hence, understanding the mechanisms involved in plant nutrient uptake is of key importance for improving worldwide agricultural production and mitigating environmental risks [14]. The salinity had an effect on the mineral compositions in leaves, roots and soil [15].

Some researchers have used plant growth regulators (PGRs) for decreasing or alleveting the adverse effects of salinity. The plant growth regulators are of considerable importance in mitigating the negative effect of salt stress [16]. GA₃ are endogenous plant growth regulators, having tetracyclic, diterpenoid compounds [17] that plays role in plant growth and development. Plants treated with GA₃ have determined that there is an improvement in physiological; such as seed germination, leaf expansion, and start of flowering, nutrient uptake and fruit development. Otherwise, Achard et al. [18] have found that the DELLA protein family is the major GA₃ negative regulator in regulating environmental signals and other plant hormone signaling pathways. The main role of gibberellic acid in regulating plant growth under abiotic stress conditions associated with DELLA proteins retarding growth, due to that eliminating the harmful effects of salinity by stimulating the accumulation of antioxidant enzyme activities [19]. Nimir et al. [20] reported that GA₃ caused a reduction in Na concentration and partly decreased the concentration of other ions. These results agreed with exogenous application of GA₃ could reduce salinity stress byreducing Na accumulation and increasing uptake of other essential nutrients [21]. In literature, there are many studies related with gibberellic acid and oxidative damage in plants but the results are contradictory. While GA application under stress conditions in some plants increases some enzyme activities depending on the application differences, it causes a decrease in others and this situation varies depending on the method of simultaneous foliar, Hoagland and seed soaking applications [22-24]. For example, GA₃ application increased antioxidant enzyme activities in some plants (Brassica juncea, Capsium annum) under salt stress [25,26]. In contrast, it was reported that application of GA₃ GA₃ decreased the antioxidant enzyme activities in Brassica juncea [27]. In summary, this study was aimed to discuss and explain the work flow of antioxidant enzymes in detail, ion concentrations and other parameters under GA₃ application with salinity in maize roots.

Materials and Method

Plant growth and experimental design

Seeds belonging to maize (Zea mays L.) M14G44 cultivar seeds, supplied from MAY-Agro seed company (Bursa), were used as the biological material of this present study. The seeds were sown in plastic pots, each of which (width 20 cm, length 12 cm, height 18 cm) contains 30-40 seeds and the mix ture of soil: clay: clay-loam in the ratio of 2:1:1, pH: 6.5. Seeds were placed in the dark for 5 days for germination. After germination, seedlings were grown in a growth room at 25°C (16 h day/8 h night photoperiod), light intensity of 350 μmol m–2 s–1 and watered with Hoagland solution for 8 days [28].

The pots were divided into eight treatments. Each one contained three pots (totally 40 plants). Treatments; Control (1), NaCl 350 mM (2), GA₃, 100 ppm (3), GA₃, 300 ppm (4), GA₃, 500 ppm (5), NaCl+GA₃, 100 ppm (6), NaCl+GA₃, 300 ppm (7), NaCl+GA₃, 500 ppm (8) and watered with Hoagland solution for 10 days (Figure 1). Then, the seedlings were harvested on the 10th day and the samples were preserved at -80°C. All GA₃ (100, 300, 500 ppm) concentrations were applied with Hoagland solution in soil medium.

Figure 1: The effects of gibberelic acid treatment in maize (Zea mays L.) roots under salt stress. Control (1), NaCl 350 mM (2), GA₃ 100 ppm (3), GA₃ 300 mM (4), GA₃ 500 ppm (5), NaCl +GA₃, 100 ppm (6), NaCl +GA₃ 300 ppm (7), NaCl +GA₃, 500 ppm (8).

Physiological parameters and biomass yield

The relative water content (RWC) was calculated in accordance with Smart and Bingham [29]. Harvested roots were weighed to determine their fresh weights (FW). The seedlings were floated on de-ionized water for 5 h under low irradiance and then the turgid tissue was quickly blotted to remove excess water and their turgid weights (TW) were determined. Dry weights (DW) were determined after the shoots were dried in an oven at 70°C for 72 h. In addition, root lengths were measured in all groups.

Measurement of malondialdehyde and hydrogen peroxide content

The level of lipid peroxidation in leaf samples was determined in terms of the malondialdehyde (MDA) content according to the method specified by Madhava and Sresty [30]. The MDA content, an end-product of lipid peroxidation, was determined by using the thiobarbituric acid reaction. The MDA concentration was calculated from the absorbance at 532 nm, and measurements were corrected for nonspecific turbidity by subtracting the absorbance at 600 nm. An extinction coefficient of 155 mM−1 cm−1 was used to determine the MDA concentration.

Hydrogen peroxide content the H2O2 content was determined according to Velikova et al. [31]. Fresh leaves (0.1 g) were homogenized in 5 ml of 0.1% trichloroacetic acid (TCA) and centrifuged at 12,000 rpm for 15 minutes. The supernatant (0.5 ml) was then mixed with 0.5 ml of buffer (10 mM potassium phosphate, pH 7) and 1 ml of 1 M potassium iodide. The absorbance reading was taken at 390 nm.

Measurement of proline content and hydroxyl radical scavenging activity

The proline contents of roots were determined according to Claussen [32]. For each treatment, 0.5 g leaf sample was ground in a mortar after addition of a small amount of glasspowder and 5 mL of a 3% (w/v) aqueous sulfosalicylic acid solution. The homogenate was filtered through two layers of glass-fibers. To the filtrate (1 ml), glacial acetic acid and ninhydrin reagent (1 ml each) were added. The closed test tubes containing the reaction mixture were kept in a boiling water bath for 1 h before the reaction was terminated at room temperature (22°C) for 5 min. The absorbance of the reaction mixture was determined at 546 nm. The proline concentration was determined from a standard curve and calculated on fresh weight basis (μg proline g-1 FW).

Hydroxyl radical scavenging activity. The ability of fusiformis extracts to scavenge the hydroxyl radical generated by Fenton reaction was measured according to the modified method given by Kim et al. [33], where A0 is the absorbance of the control reaction and A1 is the absorbance in the presence of resveratrol sample. The scavenging activity on hydroxyl radicals:

[(A0 – A1)/A0 × 100]

Measurement of antioxidant enzyme activities

SOD enzyme activity: The superoxide dismutase (SOD; EC 1.15.1.1) activity was assayed by its ability to inhibit the photochemical reduction of nitrotetrazolium blue chloride (NBT) at 560 nm [34].

CAT enzyme activity: The catalase (CAT; EC 1.11.1.6) activity was assayed in a reaction mixture (2 ml) containing 50 mM K-phosphate buffer (pH 7.0). Then, 12.2 mM H2O2 was added, and the reaction wasstarted by adding 100 μl supernatant. The activity was determined by monitoring the degradation of H2O2 at 240 nm over 2 min against a supernatant-free blank. Enzymespecific activities were expressed as μmol of H2O2 oxidized min-1 mg-1 protein [35].

POX enzyme activity: The peroxidase POX (EC 1.11.1.7) activity was based on the method described by Herzog and Fahimi [36]. The increase in the absorbance at 465 nm due to oxidation of diaminobenzidine (DAB) was followed for 1 min. One unit of POX activity was defined as 1 μmol H2O2 decomposed in 1 min.

APX enzyme activity: The ascorbate peroxidase (APX; EC 1.11.1.11) activity was measured according to Nakano and Asada [37]. The assay depended on the decrease in absorbance at 290 nm as ascorbate was oxidized.

GST enzyme activity: The glutathione-s-transferases (GST; EC 2.5.1.18) activity was determined by the method of Habig et al. [38] by following the increase in absorbance at 340 nm due to the formation of the conjugate 1-chloro-2,4-dinitrobenzene (CDNB) using as substrate at the presence of reduced glutathione (GSH).

Measurement of ion concentration

Ion concentration of roots was determined by [39]. The roots were washed for 1 min in deionised water, dried at 65°C for 48 h. The dried shoot samples were then ground and approximately 0.2 g of the ground samples were ashed at 550°C for 12 h for determination of total N, P, K, Mg, Ca, Fe, Cu, Mn, Zn concentrations. Total nitrogen (N) in digests was determined by the Kjeldahl method [40]. P concentrations were determined using the photocolormetric method described by Murphy and Riley [41]. K, Ca, Mg, Fe, Zn, Cu and Mn also determined by “Varian, 220FS” atomic absorption spectrophotometer [42].

Statistical analysis

The experiment was conducted in a completely randomized design and the measurements were performed with 6 replicates (n=6). Statistical variance analysis of the data was performed using ANOVA and the differences among treatments were compared using Tukey’s post-hoc analysis with the least significant differences at the 5% level. In all the figures, the spread of the values is shown as error bars representing standard errors of the means.

Results and Discussion

Effects of gibberellic acid on physiological parameters

Analysis of variance of data for fresh and dry weights of and roots, root length and relative water content of maize root (Table 1) showed that Salinity in the root zone had a significant adverse effect on these growth attributes (P<0,05). The results indicated that applied NaCl inhibited the plant growth, and cause a decrease in fresh and dry weights in the salinity condition compared to control. On the bases of our results, the root lenght of maize was reduced by salinity as compared to control group (1.97 fold). GA₃ treatments (100, 300 and 500 ppm) did not cause any change in these values compared to control. However, combined of GA₃ and salt treatment increased lenght value but the most increase (38.19%) at 300 ppm GA₃ under salinity compared to the salt treatment alone (Table 1). In the present study, Table 1 shows that salt treatment increased the fresh weight and decreased dry weight of roots in maize plants as compared to the control groups. However, 100 and 300 ppm of GA₃ doses significantly increased the dry weight of root compared to control, but it progressively declined with further increases in GA₃ and salinity. On the other hand, 100 ppm GA₃ treatment alone did not cause any change in these values, whereas 300 and 500 ppm increased this values under the salinity condition (Figure 1). Increases in fresh weight of salt stressed plants in response to GA₃ may be related to the induction of protective role of membranes that increase the tolerance of plant to damage. From these results, it was clearly showed that in parallel with the statements of other researchers, salt stress had a negative effect on root length, fresh weight and dry weight of maize root (Table 1). However, GA₃ treated maize roots had an increase root lenght and weight value because GA₃ encourages cell division and cell elongation under salinity. Similarly, Kaya et al. [43] reported that exogenous GA₃ application promoted lenght and weight of wheat and tomato plants.

Table 1: The effects of gibberellic acid treatment on dry and fresh weight, lenght, relative water content of roots of maize (Zea mays L.) under salt stress. Control (1), NaCl 350 mM (2), GA₃ 100 ppm (3), GA₃ 300 mM (4), GA₃ 500 ppm (5), NaCl+GA₃, 100 ppm (6), NaCl+GA₃ 300 ppm (7), NaCl+GA₃, 500 ppm (8).

Relative water content is an important indicator of the plant water status under stress to establish the adequate amount of water needed for metabolism. In the present study, salt stress inhibited relative water content (18.75%) in maize roots but all GA₃ treatment alone did not change it significiantly (Table 1). Nevertheless, GA₃ treatment alleviated reduction in RWC values caused by salinity, while the highest increase was at 500 ppm (52. 48%) (Table 1). As a result of our study, GA₃ treatment mitigated the adverse effects of salt stress. Korkmaz et al. [16] determined that with the application of GA₃ in rapeseed plant, it provides the damage of salt stress by protecting the plant water balance. Effects of gibberellic acid on enzymes

Analysis of variance of data for antioxidant enyzmes of roots showed that salinity had a significant adverse effect on these paramaters (P<0,05). In maize roots, 350 mM NaCl application increased MDA value by 2.5 fold compared to the control groups, although GA₃ application alone did not affect it at all concentrations (Figure 2). In the present study, GA₃ treatment under salt stress led to a 42.26, 50.1 and 49.05% decrease in MDA values respectively, as compared to the salt treatment alone (Figure 2). Salinity increases lipid peroxidation and leads to accumulate MDA content in plants. In maize roots, GA₃ application minimized salt induced oxidative damage by supressing MDA content. This result is also parallel with the report of Khan et al. [44].

Figure 2: The effects of gibberelic acid treatment on MDA, H2O2 content of maize (Zea mays L.) roots under salt stress. Control (1), NaCl 350 mM (2), GA₃ 100 ppm (3), GA₃ 300 mM (4), GA₃ 500 ppm (5), NaCl+GA₃, 100 ppm (6), NaCl+GA₃ 300 ppm (7), NaCl+GA₃, 500 ppm (8)Columns with different letters represent significantly different (P<0.05) values.

Figure 2 demonstrates that hydrogen peroxide level was increased by 18.47% significiantly under salt treatments. When GA₃ was applied to salt treated plants, there was a noticeable decrease by 45.87% with 500 ppm GA₃ as compared to salt stress alone (Figure 2). It’s well known that CAT is an important antoxidant enzyme that converts H2O2 to water in peroxysomes. In our results, GA₃ treatment decreased this activity by 62.85, 74.28 and 57.14% as compared to control groups in maize roots. Similarly, GA₃ exogenous application showed that hydrogen peroxide (H2O2) content was decreased in bean root (Phaseolus vulgaris L. cv. Naz), but it caused an increase with GA₃ application under salt stress [45].

In our research, ~1.71 fold decrease, in comparison to the control group, was observed in salt treated maize roots (Figure 3). On the contrary, GA₃ treatment alone did not affect proline levels at 100 and 300 ppm but 500 ppm increased this level by 2.2 fold as compared to salt stress alone. However, the proline level was increased by GA₃ plus salt treatment by (47.92, 66.86, 79.05%) as compared to salinity.

Figure 3: The effects of gibberelic acid treatment on prolin content of maize (Zea mays L.) roots under salt stress. Control (1), NaCl 350 mM (2), GA₃ 100 ppm (3), GA₃ 300 mM (4), GA₃ 500 ppm (5), NaCl+GA₃, 100 ppm (6), NaCl+GA₃ 300 ppm (7), NaCl+GA₃, 500 ppm (8). Columns with different letters represent significantly different (P<0.05) values.

Proline protects membrane stability and mitigates the effect of NaCl on cell membrane. In our results, salinity reduced proline levels and increased damage in maize roots (Figure 3). Neverthless, GA₃ application induced proline values significiantly and maintained water balance in roots. These results are also in agreement with the RWC and physiological parameters. Similarly, Wang et al. [46] reported that oak leaves had an increased proline level under GA₃ and salt stress. It could be suggested that in this experiment, GA₃ induced lower osmotic potential and maintain water balance to cope with salinity.

Figure 4 illustrates that plants treated with salt showed low hydroxyl radical scavenger capacity. However, GA₃ treatment alone decreased this level by (45.84, 46.95, 44.9%) as compared to salt treatment alone. From this result, it could be suggested that gibberellic acid may play role as an antioxidant and under stress conditions. Moreover, this capacity induced significiantly (16.8, 23.0, 24.3%) with increased concentration of GA₃ under salt treatment, as compared to the salt treatment alone. This finding showed that GA₃ could behave as a signal molecule to increase stress tolerance in maize roots.

Figure 4: Time course effects of gibberelic acid treatment on hydroxyl radical (OH) scavenging activity of maize (Zea mays L.) roots under salt stress. Control (1), NaCl 350 mM (2), GA₃ 100 ppm (3), GA₃ 300 mM (4), GA₃ 500 ppm (5), NaCl+GA₃, 100 ppm (6), NaCl+GA₃ 300 ppm (7), NaCl+GA₃, 500 ppm (8). Columns with different letters represent significantly different (P<0.05) values.

In the present study, salt treatment alone decreased SOD activity (29%) in maize roots. SOD activity modulates superoxide radical content and removes to hydrogen peroxide and decreases the risk of OH. Radical formation. This result is parallel with the findings of Zhu et al. [4] who reported that okra seedlings had a decreased SOD activity under salinity. Otherwise, all GA₃ treatment alone decreased the SOD enzyme activity, compared to the control groups (Figure 5). However, in our results, exogenous application of GA₃ increased the activity of SOD activity under salt stress (18.8, 22.7, 36.6%). In contrast with this result, Chakrabarti and Mukharji [47] also reported that SOD activity decreased in Vigna radiata by GA₃ spraying application.

Figure 5: The effects of gibberelic acid treatment on SOD enzyme activity of maize (Zea mays L.) roots under salt stress. Control (1), NaCl 350 mM (2), GA₃ 100 ppm (3), GA₃ 300 mM (4), GA₃ 500 ppm (5), NaCl+GA₃, 100 ppm (6), NaCl+GA₃ 300 ppm (7), NaCl+GA₃, 500 ppm (8). Columns with different letters represent significantly different (P<0.05) values.

Similar to SOD activity, CAT level was decreased under salinity (20%), whereas it was increased only by combination of 500 ppm GA₃ and salt treatment by 28.57% (Figure 6). In parallel with this result, it was found that priming application of GA₃ increases the CAT activity in peper seeds under salinity [26].

Figure 6: The effects of gibberelic acid treatment on CAT enzyme activity of maize (Zea mays L.) roots under salt stress. Control (1), NaCl 350 mM (2), GA₃ 100 ppm (3), GA₃ 300 mM (4), GA₃ 500 ppm (5), NaCl+GA₃, 100 ppm (6), NaCl+GA₃ 300 ppm (7), NaCl+GA₃, 500 ppm (8). Columns with different letters represent significantly different (P<0.05) values.

According to our results, POX enzyme activity was not changed by salinity by in maize roots. However, GA₃ treatment alone increased this activity 2.04, 2.64 and 1.52 fold as compared to control groups (Figure 7). This reaction could be related with the lignification role of POX enzyme besides catalyzing hydrogen peroxide in plant structure. Otherwise, 100 ppm and 300 ppm GA₃ and salt application increased this level, compared to salt stress, but this was reduced by 31.37% at 500 ppm. In contrast with this result, Tuna et al. [19]. Reported that POX enzyme activity was decreased with 100 ppm GA₃ (spray) under salinity in maize leaves. It’s clear that the effects of GA₃ depend on application method and plant organ as mentioned before.

Figure 7: The effects of gibberelic acid treatment on POX enzyme activity of maize (Zea mays L.) roots under salt stress. Control (1), NaCl 350 mM (2), GA₃ 100 ppm (3), GA₃ 300 mM (4), GA₃ 500 ppm (5), NaCl+GA₃, 100 ppm (6), NaCl+GA₃ 300 ppm (7), NaCl+GA₃, 500 ppm (8). Columns with different letters represent significantly different (P<0.05) values.

APX enzyme activity was increased by 50% under salinity as compared to control groups. This result is also agreement with the report of Abdel Gawad et al. [48] in maize roots. Although this activity was positive for roots to scavenge ROS under salinity but only the increased APX activity was not efficient to cope with the severe NaCl stress. Also, all GA₃ treatment alone did not affect the APX enzyme activity. However, salt and GA₃ treatment increased this activity by 3.18, 2.38 and 3.26 fold as compared to salt stress alone (Figure 8). In contrast with this finding, Ahmad et al. [21] showed that GA₃ sprayed (75 ppm) Brassica juncea plants showed decreased APX enzyme activity under salinity. It could be suggested that the higher concentration of GA₃ was more effective to increase APX activity for detoxfying the hydrogen peroxide. Similar to POX activity, there is an important point to be underlined that spraying GA₃ gives different result from dissolving in nutrient solution in plants.

Figure 8: The effects of gibberelic acid treatment on APX enzyme activity of maize (Zea mays L.) roots under salt stress. Control (1), NaCl 350 mM (2), GA₃ 100 ppm (3), GA₃ 300 mM (4), GA₃ 500 ppm (5), NaCl+GA₃, 100 ppm (6), NaCl+GA₃ 300 ppm (7), NaCl+GA₃, 500 ppm (8). Columns with different letters represent significantly different (P<0.05) values.

On the basis of our results, Figure 9 shows that total GST enzyme activity was reduced by salt treatment in maize roots.

Figure 9: The effects of gibberelic acid treatment on GST enzyme activity of maize (Zea mays L.) roots under salt stress. Control (1), NaCl 350 mM (2), GA₃ 100 ppm (3), GA₃ 300 mM (4), GA₃ 500 ppm (5), NaCl+GA₃, 100 ppm (6), NaCl+GA₃ 300 ppm (7), NaCl+GA₃, 500 ppm (8). Columns with different letters represent significantly different (P<0.05) values.

Otherwise, all GA₃ treatment with salinity increased this activity by 2.83, 2.50 and 2.59 fold as compared to salinity respectivelly. GA₃ applications alone decreased this level as compared to control groups in literature, there is no report with the effect of gibberellic acid on mode of action of this enzyme (GST) in vivo. Although there are few studies about the effects of hormones on GST enzyme activity (ABA, SA, NO) [49,50].

Effects of gibberellic acid on ion concentration

Analysis of variance of data for ion concentrations of maize root (Table 2) showed that salinity in the root zone had a significant adverse effect on these ion concentration attributes (P<0,05). In our research, the nitrogen (N) concentration was reduced salt treatment by (20.42%) as compared to control (Table 2). However, the N level was increased by GA₃ plus salt treatment by (1.79, 17.70, 5.49%) as compared to salinity. In this study, N concentration was increased in plants grown in 500 ppm GA₃ application, due to the synthesis of proteins required to avoid salt stress. Salt concentrations without GA₃ caused reduction in free proline accumulation and treating plants with GA₃ increased free proline content due to its alleviating role over salinity. Similar findings, GA₃ treatment counteracted adverse effects of salinity with accumulation of more free proline which maintained membrane permeability and protein increased by macro and micronutrient absorption [1]. It has been further proved to play an important role in the GA signaling pathway [51].

Table 2. The effects of gibberellic acid treatment on ion concentration roots of maize (Zea mays L.) under salt stress. Control (1), NaCl 350 mM (2), GA₃ 100 ppm (3), GA₃ 300 mM (4), GA₃ 500 ppm (5), NaCl+GA₃, 100 ppm (6), NaCl+GA₃ 300 ppm (7), NaCl+GA₃, 500 ppm (8).

Phosphorus (P) concentration was decreased by NaCl application 14.3% compared to the control groups. In other studies, increasing salt concentrations also reduced the amount of phosphorus compared to the control [52]. GA₃ applied to salt treated plants, there was a noticeable increase by 61.5% with 100 ppm GA₃, while was decreased 16.2% with 500 ppm GA₃ as compared to salt stress alone (Table 2). In the interaction between salinity and GA₃ treatments had been increased P concentration in plants compared to the salt stress alone [20]. This increase can be explained by growth parameters and increase in biomass.

In maize roots, 350 mM NaCl application decreased potassium (K+) concentration by 38.2% compared to the control groups. Yasar et al. [53] carried out a study examining the salt stresstolerance of water melon genotypes and indicated that the plants selectively took up Na and K ion under salt stress conditions, and the sensitive genotypes promoted the uptake of Na ions rather than K ions. Moreover, under salinity stress conditions, with increasing salt concentration, the K ion concentration decreases in salt sensitive plants [54]. High concentrations of sodium can inhibit the uptake of K by plants through the antagonism between these two ions as well as potassium ion leakage when Ca substitutes for Na in the cell membranes [52]. A favorable increase in K concentration was recorded in the root of salt stressed plants as a result of GA₃ application. In the present study, GA₃ application alone by (61.41, 70.81, 54.08%) affected all concentrations as compared to the salt treatment (Table 2). Although GA₃ treatment under salt stress led to a 30.42, 4.7 and 22.54% decrease in K concentration respectively, (Table 2). The influence of GA₃ on the salinity mechanism of ions uptake may be related to its effect on membrane permeability and rate of ion entry through the membrane, or enhances their translocation to the shoot [47]. In the present study, GA₃ causes to a decrease in K concentration; from this result, it can be thought that GA₃ application provides tolerance to salt stress by replacing Na with K.

Table 2 illustrates that plants treated with salt showed hight magnesium (Mg+) concentration, in comparison to the control groups. Also GA₃ treatment alone decreased this level by (9.73, 22.68, 9.34%) as compared to salt treatment alone. Moreover, this capacity decreases significiantly (15.6, 15.6, 27.4%) with increased concentration of GA₃ plus salt treatment, as compared to the salt treatment alone. Exogenous application of GA₃ could reduce salinity stress by reducing Na+ accumulation and increasing uptake of Mg+ [21]. This increase can be explained by the increase in chlorophyll amount in the leaves by transporting (Mg+) to the above ground parts of the plant with the application of GA₃, and also the fact that it encourages root development and causes an increase length and dry biomass significantly recovered when GA₃ was added to salt-stressed plants [55].

Compared to the control treatments, Ca concentration significantly decreased (50%) under salinity stress that this decrease lowers role of calcium signals required for salt tolerance. Higher salt concentrations caused to reduce intake of Ca ion and ion imbalance in plant. These results are in agreement with those of Tuna et al. [19], Balkaya et al. [56] and Ekbic et al. [51] who reported that increasing salt application decreased Ca concentration in plant tissue. However, under salinity stress conditions, with increasing salt concentration, the Ca concentration increases in salt tolerant plants [51]. Calcium is known to improve the negative effects of salinity on plants [57]. Tuna et al. [19] reported that sodium ions may compete with calcium ions for membrane-binding sites and high calcium levels can protect the cell membrane from the adverse effects of salinity. Ca plays an important role in the salt-tolerant signal pathway, as a second messenger. In the present study, GA₃ treatments also reduced the Ca concentration compared to salt stress alone (32.2, 43, 11.7%). Salt and 100 ppm GA₃ treatment increased this concentration by 31.2% compared to salt stress alone. These results indicated that application of low levels of GA₃ can regulate plant growth and have positive effects [4]. These results showed that the decrease in Ca ion under salt stress of GA₃ application can be explained by the fact that GA₃ changes cation with Ca.

Sodium (Na) concentration significantly decreased with GA₃ treatment (74.36, 66.87, 56.43%) as compared to the salt groups alone. Application of GA₃ to salinity stressed plants caused a partial decrease in the adverse effect of salinity. Specifically, 300 ppm GA₃ treatment under salinity decreased Na (12,46%) as compared to salt alone (Table 2). In parallel with our findings; exogenous application of GA₃ could reduce salinity stress by reducing Na accumulation in rice [21].

Copper, zinc, iron, and manganese are the ingredients of some of the important antioxidant enzymes. These of the metals are essential elements for plant growth. The most common isoforms of SOD known in the literature are copper–zinc containing superoxide dismutase (Cu/Zn-SOD), manganese containing (Mn-SOD) and iron containing (Fe-SOD) [58] Cu ion concentration was decreased with GA₃ treatment 1.8, 9.09 and 22.7% as compared to salt groups in maize roots. In parallel with our findings, Abdel-Hamid and Mohamed [59] determined that salinity reduced copper concentration in Hordeum vulgare plants. However, GA₃ plus salt treatment was decreased by 19, 17.3, 28.2% compared to salt stress.

On the basis of our results, table shows that Mn ion concentration activity was increased by salt treatment (Table 2). In contrast with this result, Tuna et al. [19] reported that salinity had reduced Mn concentration in maize. In our study; 100 ppm GA₃ applications increased this level as compared to control groups. Otherwise, all GA₃ treatment with salinity decreased this concentration by 35.4, 30.2 and 32.4% as compared to salinity respectivelly.

On the basis of our results, Table 2 shows that Fe concentration was increased by salt treatment. Otherwise; GA₃ applications decreased this level as compared to salt stress. Compared with the control, Fe the concentration had been increased by irrigation using saline water while by GA₃ unaffected [60] in maize. Similarly, all GA₃ treatment with salinity decreased this concentration by 1.6, 1.6 and 1.7 fold as compared to salinity respectivelly. Under salt stress, Fe-SOD activity increased only slightly in cytosolic and mitochondrial fractions and significantly in chloroplastic fraction [61]. For instance, the induction of total SOD and Fe-SOD activities has been recorded in the leaves of bean and tolerant pea [62].

In our results, Zn concentration was not changed salinity aplication compared to GA₃ aplications in maize roots. It was also show that Zn concentration decreased with elevated soil salinity on wheat, rice, and pepper plants [63]. However, GA₃ treatment decreased this concentration 35.1, 38.7 and 37% as compared to control groups. In according to Zn concentration; GA₃ and salt application was reduced compared to salinity stress by 37.8, 57.1, 10.6% (Table 2). Particularly, Ramezani and Shekafandeh, [64] reported that the application of GA₃ and Zn on plant growth and development.

Conclusion

In conclusion, there was a slight reduction in the activities of SOD, CAT and GST, whereas POX was increased and APX was non changed under alone GA₃ treatment. This result shows that plants with treated with GA₃ alone did not produce hydrogen peroxide and had no need to induce antioxidant defence response. Neverthless, all combination of GA₃ and salinity induced SOD, APX, CAT and GST activities (except POX at 500 ppm), relative water content and biomass yield by playing as a signal role. This result clearly showed that simultanous treatment of GA₃ and salinity promotes antioxidant enzyme acitivites (especially GST enzyme, which needs more analysis) in maize roots. In addition, ion concentrations contents (Cu+, Zn+, Mn and Fe+) were reduced by GA₃ under salinity, whereas (N+, P) were induced. Otherwise, Ca and Mg were reduced with GA₃ alone. It could be suggested that GA₃ protected maize roots from salinity by inducing cofactors (Cu+, Mn, Fe+, Zn+) of SOD enzymes spesifically. According to the results of the present study, it can be concluded that GA₃ plays role in regulating the salt stress in maize, and suggested that GA₃ could be used as a potential stress inhibitor with the appropriate concentration and application method.

References

1. Farooq M, Hussain M, Wakeel A, Siddique KH. Salt stress in maize: Effects, resistance mechanisms, and management. A review. Agron Sustain Dev. 2015; 35: 461-481.
2. Bernstein N. Plants and salt: Plant response and adaptations to salinity. Model Ecosystems in Extreme Environments. 2019; 101-112.
3. Al Hassan M, Estrelles E, Soriano P, López-Gresa M, Bellés JM, Boscaiu M, et al. Unraveling salt tolerance mechanisms in halophytes: A comparative study on four mediterranean limonium species with different geographic distribution patterns. Front Plant Sci. 2017;8: 1438.
4. Zhu G, An L, Jiao X, Chen X, Zhou G, McLaughlin N. Effects of gibberellic acid on water uptake and germination of sweet sorghum seeds under salinity stress. Chil J Agric Res. 2019; 79:415-424.
5. Liang C, Xiao H, Hu Z, Zhang X, Hu J. Uptake, transportation, and accumulation of C60 fullerene and heavy metal ions (Cd, Cu, and Pb) in rice plants grown in an agricultural soil. Environ. Pollut. 2018; 235: 330-338.
6. Yang Y, Guo Y. Unraveling salt stress signaling in plants. J Integr Plant Biol. 2018; 60: 796-804.
7. Baier M, Kandlbinder A, Golldack D,  Dietz KJ. Oxidative stress and ozone: Perception, signalling and response. Plant Cell Environ. 2005;28: 1012-1020.
8. Saha  J, Brauer  EK, Sengupta A, Popescu SC, Gupta K, Gupta, B. Polyamines as redox homeostasis regulators during salt stress in plants. Front. Environ. Sci, 2015; 3: 21.
9. Park HJ, Kim WY, Yun  DJ. A new insight of salt stress signaling in plant. Mol. Cells, 2016; 39: 447.
10. Bekmirzaev G, Ouddane B, Beltrao J, Fujii Y. The impact of salt concentration on the mineral nutrition of tetragonia tetragonioides. Agriculture. 2020;10: 238.
11. Rich SM, Watt M. Soil conditions and cereal root system architecture: Review and considerations for linking Darwin and Weaver. J Exp Bot. 2013; 64;1193-1208.
12. Paterson-Beedle M, Macaskie LE, Lee CH, Hriljac JA, Jee KY, Kim WH. Utilisation of a hydrogen uranyl phosphate-based ion exchanger supported on a biofilm for the removal of cobalt, strontium and caesium from aqueous solutions. Hydrometallurgy. 2006; 83: 141-145.
13. Gupta S, Schillaci M, Walker R, Smith PM, Watt M, Roessner U. Alleviation of salinity stress in plants by endophytic plant-fungal symbiosis: Current knowledge, perspectives and future directions. Plant Soil. 2020;1-26.
14. Griffiths, York LM. Targeting root ion uptake kinetics to increase plant productivity and nutrient use efficiency. Plant Physiol. 2020; 182: 1854-1868.
15. Demir Z, Kiran S. Effect of vermicompost on macro and micro Nutrients of lettuce (Lactuca Sativa Var. Crispa) under salt stress conditions. Kahramanmaras Sütçü Imam �niversitesi Tarim ve Doga Dergisi. 2020; 23: 33-43.
16. Korkmaz K, Akgün M, Kirli A, �zcan MM, Dede �, Kara SM. Effects of gibberellic acid and salicylic acid applications on some physical and chemical properties of rapeseed (Brassica napus L.) grown under salt stress. Turkish J Agri Food Sci Tech. 2020; 8: 873-881.
17. Tiwari V, Lal EP. Effect of foliar application of gibberellic acid on growth, yield, physiological and biochemical characteristics of mung bean (Vigna radiata L.) under salt stress. Int J Chem Studies. 2018; 6(5): 713-717.
18. Achard P, Cheng H, De Grauwe L, Decat J, Schoutteten H, Moritz T, et al. Integration of plant responses to environmentally activated phytohormonal signals.  Science. 2006; 311(5757): 91-94.
19. Tuna AL, Kaya C, Dikilitas M, Higgs D. The combined effects of gibberellic acid and salinity on some antioxidant enzyme activities, plant growth parameters and nutritional status in maize plants. Environ Exp Bot. 2008, 62(1): 1-9.
20. Nimir NEA, Zhou G, Guo W, Ma B, Lu S, Wang Y. Effect of foliar application of GA₃, kinetin and salicylic acid on ions content, membrane permeability, and photosynthesis under salt stress of sweet sorghum (Sorghum bicolor (L.) Moench). Can J Plant Sci. 2016; 97(3): 525-535.
21. Misratia KM, Islam MR, Ismail MR, Oad FC, Hanafi MM, Puteh A. Interactive effects of gibberellic acid (GA₃) and salt stress on growth and ion accumulation of two rice (Oryza sativa L.) varieties differing in salt tolerance. J Food Agric Environ. 2015; 13: 6670.
22. Ahmad P, Jaleel CA, Salem MA, Nabi G, Sharma S. Roles of enzymatic and nonenzymatic antioxidants in plants during abiotic stress. Crit Rev Biotechnol. 2010;30(3): 161-175.
23. Forghani AH, Almodares A, Ehsanpour AA. Potential objectives for gibberellic acid and paclobutrazol under salt stress in sweet sorghum (Sorghum bicolor [L.] Moench cv. Sofra). Appl Biol Chem. 2018;61(1): 113-124.
24. Gül V, Dinler BS, Tasci E. Effects of pre-application with gibberellic acid on germination of soybean under salt Stress. J Stress Physiology and Biochem. 2019;15(4). 
25. Siddiqui MH, Khan MN, Mohammad F, Khan MMA. Role of nitrogen and gibberellin (GA₃) in the regulation of enzyme activities and in osmoprotectant accumulation in Brassica juncea L. under salt stress. J Agron Crop Sci. 2008; 194(3): 214-224.
26. Uzal O, Yasar F. Effects of GA₃ hormone treatments on ion uptake and growth of pepper plants under cadmium stress. Appl Ecol Environ Res. 2017; 15(4): 1347-1357.
27. Forghani AH, Almodares A, Ehsanpour AA. The role of gibberellic acid and paclobutrazol on oxidative stress responses induced by in vitro salt stress in sweet sorghum. Russ J Plant Physl. 2020; 67(3).: 555â??563.
28. Hoagland  DR, Arnon DI. The water-culture method for growing plants without soil, California Agricultural Experiment Station Circular, 1950; 347: 1.
29. Smart RE, Bingham GE. Rapid estimates of relative water content. Plant physiol. 1974; 53(2): 258-260.
30. Madhava R, Sresty TV. Antioxidative parameters in the seedlings of pigeonpea (Cajanus cajan L. millspaugh) in response to Zn and Ni stresses. Plant Sci. 2000;157: 113-128.
31. Velikova V, Yordanov I, Edreva A. Oxidative stress and some antioxidant systems in acid rain-treated bean plants protective roles of exogenous polyamines. Plant Sci. 2000; 151: 59-66.
32. Claussen W. Proline as a measure of stress in tomato plants. Plant Sci. 2005; 168: 241-248.
33. Kim JW, Minamikawa T. Hydroxyl radical-scavenging effects of spices and scavengers from brown mustard (Brassica nigra). Biosci Biotechnol Biochem. 1997;61: 118-123.
34. Beauchamp CO, Fridovich I. Isozymes of superoxide dismutase from wheat germ. Biochimica et Biophysica Acta (BBA)- Protein Struct. 1973;317(1):50-64.
35. Bergmeyer HU. Measurement of catalase activity. Biochem Z. 1955; 327(4): 255.
36. Herzog V, Fahimi  HD. A new sensitive colorimetric assay for peroxidase using 3, 3'-diaminobenzidine as hydrogen donor. Anal Biochem. 1973; 55(2): 554-562.
37. Nakano Y, Asada K. Hydrogen peroxide is scavenged by ascorbate-specific peroxidase in spinach chloroplasts. Plant cell physiol. 1981; 22(5): 867-880.
38. Habig WH, Pabst MJ, Fleischner G, Gatmaitan Z, Arias IM, Jakoby WB. The identity of glutathione S-transferase B with ligandin, a major binding protein of liver. Proc Natl Acad Sci USA. 1974;71(10):3879-3882.
39. Bergman B, Johansson C, Soderback E. Tansley Review No. 42. The Nostoc-Gunnera symbiosis. New Phytolo. 1992; 122: 379-400.
40. Bremner JM. Total Nitrogen, Editor C.A. Black, Methods of Soil Analysis. Part 2, American Society of Agronomy. Inc. Publisher, Madison Wisconsin, USA. 1965; 1149-1178.
41. Murphy J, Riley JP. A modified single solution method for the determination of phosphate in natural water. Analytica Chimica Acta. 1962; 27: 31â??35.
42. Kacar B,  Inal A. Plant analysis. Nobel Publication No: 1241. Applied Sciences. 2008; 63: 879.
43. Kaya C, Akram N A, Ashraf M, Sonmez O. Exogenous application of humic acid mitigates salinity stress in maize (Zea mays L.) plants by improving some key physico-biochemical attributes. Cereal Res Commun. 2018;46(1): 67-78.
44. Khan TA, Mazid M, Mohammad F. A review of ascorbic acid potentialities against oxidative stress induced in plants. J Agrobiol. 2011; 28(2): 97â??111.
45. Saeidi-Sar S, Abbaspour H, Afshari H, Yaghoobi SR. Effects of ascorbic acid and gibberellin A₃ on alleviation of salt stress in common bean (Phaseolus vulgaris L.) seedlings. Acta Physiol Plant. 2013;35(3): 667-677.
46. Wang YH, Zhang G, Chen Y, Gao J, Sun YR, Sun MF. Exogenous application of gibberellic acid and ascorbic acid improved tolerance of okra seedlings to NaCl stress. Acta Physiol Plant. 2019; 226: 540-549.
47. Chakrabarti N, Mukherji S. Effect of phytohormone pretreatment on nitrogen metabolism in Vigna radiata under salt stress. Biol plant. 2003; 46(1): 63-66.
48. Abdel-Gawad  H, Zinta G, Hegab MM, Pandey R, Asard H, Abuelsoud W. High salinity induces different oxidative stress and antioxidant responses in maize seedlings organs. Frontiers in Plant Science. 2016; 7: 276.
49. Cetinkaya H, Dinler BS, Tasci E. Investigation of comparative regulation on antioxidant enzyme system under copper treatment and drought stress in maize (Zea mays L.). Not Bot Horti Agrobo, 2014;42(2): 363-371.
50. Dinler BS, Antoniou C, Fotopoulos V. Interplay between GST and nitric oxide in the early response of soybean (Glycine max L.) plants to salinity stress. J plant physiol. 2014; 171(18): 1740-1747.
51. Li B, Feng XJ, Hu XY, Chen YP, Sha JC, Zhang HY, et al. Effect of melatonin on attenuating the isoflurane-induced oxidative damage is related to PKCa/Nrf2 signaling pathway in developing rats. Brain Res Bull.. 2018;143: 9-18.
52. Ekbic E, Cagiran C, Korkmaz K, Kose MA, Aras V. Assessment of watermelon accessions for salt tolerance using stress tolerance indices. Ciência e Agrotecnologia, 2017; 41(6): 616-625.
53. Yasar F, Uzal O, Yasar O. Determination of antioxidative enzyme activities in callus culture of the salt-tolerant and salt-sensitive watermelon (Citrullus lanatus (Thunb.) Mansf.) genotypes under salt stres. J Food Agric Environ.. 2013; 11 (3-4) : 1437-1440.
54. Uygur V, Yetisir H. Effects of rootstocks on some growth parameters, phosphorous and nitrogen uptake watermelon under salt stress. J Plant Nutr. 2009; 32(4): 629-643.
55. Moula I, Boussadia O, Koubouris G, Hassine MB, Boussetta W, Van Labeke MC, et al. Ecophysiological and biochemical aspects of olive tree (Olea europaea L.) in response to salt stress and gibberellic acid-induced alleviation. S Afr J Bot. 2020;132: 38-44.
56. Balkaya A. Effects of salt stress on vegetative growth parameters and ion accumulations in cucurbit rootstock genotypes. Ekin J Crop Bree Gen. 2016; 2(2): 11-24.
57. Hadi MR, Karimi N. The role of calcium in plants salt tolerance. J plant nutr. 2012; 35(13): 2037-2054.
58. Wang Y, Osatomi K, Nagatomo Y, Yoshida A, Hara K. Purification, molecular cloning, and some properties of a manganese-containing superoxide dismutase from Japanese flounder (Paralichthys olivaceus Comp. Biochem. Physiol. 2011; 158(4): 289-296.
59. Abdel Hamid AM, Mohamed HI. The effect of the exogenous gibberellic acid on two salt stressed barley cultivars. Eur Sci J. 2014;10(6).
60. Hadi F, Bano A, Fuller MP. The improved phytoextraction of lead (Pb) and the growth of maize (Zea mays L.): the role of plant growth regulators (GA₃ and IAA) and EDTA alone and in combinations.  Chemosphere.  2010;80 (4):457-462.
61. Saibi W, Brini F. Superoxide dismutase (SOD) and abiotic stress tolerance in plants: An overview. Superoxide Dismutase: Structure, Synthesis and Applications; Magliozzi S, Ed. 2018; 101-142.
62. Bernardi R, Nali C, Gargiulo R, Pugliesi C, Lorenzini G, Durante M. Protein Pattern and fe-superoxide dismutase activity of bean plants under sulphur dioxide stress. Phytopathology. 2001; 149(7-8): 477-480.
63. Khoshgoftarmanesh AH, Schulin R, Chaney RL, Daneshbakhsh B, Afyuni M. Micronutrient-efficient genotypes for crop yield and nutritional quality in sustainable agriculture A review. Agron Sustain Dev. 2010; 30 (1): 83-107.
64. Ramezani S, Shekafandeh A. Roles of gibberellic acid and zinc sulphate in increasing size and weight of olive fruit. Afr J Biotechnol. 2009; 8(24): 6791-6794.

References

1. Farooq M, Hussain M, Wakeel A, Siddique KH. Salt stress in maize: Effects, resistance mechanisms, and management. A review. Agron Sustain Dev. 2015; 35: 461-481.
2. Bernstein N. Plants and salt: Plant response and adaptations to salinity. Model Ecosystems in Extreme Environments. 2019; 101-112.
3. Al Hassan M, Estrelles E, Soriano P, López-Gresa M, Bellés JM, Boscaiu M, et al. Unraveling salt tolerance mechanisms in halophytes: A comparative study on four mediterranean limonium species with different geographic distribution patterns. Front Plant Sci. 2017;8: 1438.
4. Zhu G, An L, Jiao X, Chen X, Zhou G, McLaughlin N. Effects of gibberellic acid on water uptake and germination of sweet sorghum seeds under salinity stress. Chil J Agric Res. 2019; 79:415-424.
5. Liang C, Xiao H, Hu Z, Zhang X, Hu J. Uptake, transportation, and accumulation of C60 fullerene and heavy metal ions (Cd, Cu, and Pb) in rice plants grown in an agricultural soil. Environ. Pollut. 2018; 235: 330-338.
6. Yang Y, Guo Y. Unraveling salt stress signaling in plants. J Integr Plant Biol. 2018; 60: 796-804.
7. Baier M, Kandlbinder A, Golldack D,  Dietz KJ. Oxidative stress and ozone: Perception, signalling and response. Plant Cell Environ. 2005;28: 1012-1020.
8. Saha  J, Brauer  EK, Sengupta A, Popescu SC, Gupta K, Gupta, B. Polyamines as redox homeostasis regulators during salt stress in plants. Front. Environ. Sci, 2015; 3: 21.
9. Park HJ, Kim WY, Yun  DJ. A new insight of salt stress signaling in plant. Mol. Cells, 2016; 39: 447.
10. Bekmirzaev G, Ouddane B, Beltrao J, Fujii Y. The impact of salt concentration on the mineral nutrition of tetragonia tetragonioides. Agriculture. 2020;10: 238.
11. Rich SM, Watt M. Soil conditions and cereal root system architecture: Review and considerations for linking Darwin and Weaver. J Exp Bot. 2013; 64;1193-1208.
12. Paterson-Beedle M, Macaskie LE, Lee CH, Hriljac JA, Jee KY, Kim WH. Utilisation of a hydrogen uranyl phosphate-based ion exchanger supported on a biofilm for the removal of cobalt, strontium and caesium from aqueous solutions. Hydrometallurgy. 2006; 83: 141-145.
13. Gupta S, Schillaci M, Walker R, Smith PM, Watt M, Roessner U. Alleviation of salinity stress in plants by endophytic plant-fungal symbiosis: Current knowledge, perspectives and future directions. Plant Soil. 2020;1-26.
14. Griffiths, York LM. Targeting root ion uptake kinetics to increase plant productivity and nutrient use efficiency. Plant Physiol. 2020; 182: 1854-1868.
15. Demir Z, Kiran S. Effect of vermicompost on macro and micro Nutrients of lettuce (Lactuca Sativa Var. Crispa) under salt stress conditions. Kahramanmaras Sütçü Imam �niversitesi Tarim ve Doga Dergisi. 2020; 23: 33-43.
16. Korkmaz K, Akgün M, Kirli A, �zcan MM, Dede �, Kara SM. Effects of gibberellic acid and salicylic acid applications on some physical and chemical properties of rapeseed (Brassica napus L.) grown under salt stress. Turkish J Agri Food Sci Tech. 2020; 8: 873-881.
17. Tiwari V, Lal EP. Effect of foliar application of gibberellic acid on growth, yield, physiological and biochemical characteristics of mung bean (Vigna radiata L.) under salt stress. Int J Chem Studies. 2018; 6(5): 713-717.
18. Achard P, Cheng H, De Grauwe L, Decat J, Schoutteten H, Moritz T, et al. Integration of plant responses to environmentally activated phytohormonal signals.  Science. 2006; 311(5757): 91-94.
19. Tuna AL, Kaya C, Dikilitas M, Higgs D. The combined effects of gibberellic acid and salinity on some antioxidant enzyme activities, plant growth parameters and nutritional status in maize plants. Environ Exp Bot. 2008, 62(1): 1-9.
20. Nimir NEA, Zhou G, Guo W, Ma B, Lu S, Wang Y. Effect of foliar application of GA3, kinetin and salicylic acid on ions content, membrane permeability, and photosynthesis under salt stress of sweet sorghum (Sorghum bicolor (L.) Moench). Can J Plant Sci. 2016; 97(3): 525-535.
21. Misratia KM, Islam MR, Ismail MR, Oad FC, Hanafi MM, Puteh A. Interactive effects of gibberellic acid (GA3) and salt stress on growth and ion accumulation of two rice (Oryza sativa L.) varieties differing in salt tolerance. J Food Agric Environ. 2015; 13: 6670.
22. Ahmad P, Jaleel CA, Salem MA, Nabi G, Sharma S. Roles of enzymatic and nonenzymatic antioxidants in plants during abiotic stress. Crit Rev Biotechnol. 2010;30(3): 161-175.
23. Forghani AH, Almodares A, Ehsanpour AA. Potential objectives for gibberellic acid and paclobutrazol under salt stress in sweet sorghum (Sorghum bicolor [L.] Moench cv. Sofra). Appl Biol Chem. 2018;61(1): 113-124.
24. Gül V, Dinler BS, Tasci E. Effects of pre-application with gibberellic acid on germination of soybean under salt Stress. J Stress Physiology and Biochem. 2019;15(4). 
25. Siddiqui MH, Khan MN, Mohammad F, Khan MMA. Role of nitrogen and gibberellin (GA3) in the regulation of enzyme activities and in osmoprotectant accumulation in Brassica juncea L. under salt stress. J Agron Crop Sci. 2008; 194(3): 214-224.
26. Uzal O, Yasar F. Effects of GA3 hormone treatments on ion uptake and growth of pepper plants under cadmium stress. Appl Ecol Environ Res. 2017; 15(4): 1347-1357.
27. Forghani AH, Almodares A, Ehsanpour AA. The role of gibberellic acid and paclobutrazol on oxidative stress responses induced by in vitro salt stress in sweet sorghum. Russ J Plant Physl. 2020; 67(3).: 555â??563.
28. Hoagland  DR, Arnon DI. The water-culture method for growing plants without soil, California Agricultural Experiment Station Circular, 1950; 347: 1.
29. Smart RE, Bingham GE. Rapid estimates of relative water content. Plant physiol. 1974; 53(2): 258-260.
30. Madhava R, Sresty TV. Antioxidative parameters in the seedlings of pigeonpea (Cajanus cajan L. millspaugh) in response to Zn and Ni stresses. Plant Sci. 2000;157: 113-128.
31. Velikova V, Yordanov I, Edreva A. Oxidative stress and some antioxidant systems in acid rain-treated bean plants protective roles of exogenous polyamines. Plant Sci. 2000; 151: 59-66.
32. Claussen W. Proline as a measure of stress in tomato plants. Plant Sci. 2005; 168: 241-248.
33. Kim JW, Minamikawa T. Hydroxyl radical-scavenging effects of spices and scavengers from brown mustard (Brassica nigra). Biosci Biotechnol Biochem. 1997;61: 118-123.
34. Beauchamp CO, Fridovich I. Isozymes of superoxide dismutase from wheat germ. Biochimica et Biophysica Acta (BBA)- Protein Struct. 1973;317(1):50-64.
35. Bergmeyer HU. Measurement of catalase activity. Biochem Z. 1955; 327(4): 255.
36. Herzog V, Fahimi  HD. A new sensitive colorimetric assay for peroxidase using 3, 3'-diaminobenzidine as hydrogen donor. Anal Biochem. 1973; 55(2): 554-562.
37. Nakano Y, Asada K. Hydrogen peroxide is scavenged by ascorbate-specific peroxidase in spinach chloroplasts. Plant cell physiol. 1981; 22(5): 867-880.
38. Habig WH, Pabst MJ, Fleischner G, Gatmaitan Z, Arias IM, Jakoby WB. The identity of glutathione S-transferase B with ligandin, a major binding protein of liver. Proc Natl Acad Sci USA. 1974;71(10):3879-3882.
39. Bergman B, Johansson C, Soderback E. Tansley Review No. 42. The Nostoc-Gunnera symbiosis. New Phytolo. 1992; 122: 379-400.
40. Bremner JM. Total Nitrogen, Editor C.A. Black, Methods of Soil Analysis. Part 2, American Society of Agronomy. Inc. Publisher, Madison Wisconsin, USA. 1965; 1149-1178.
41. Murphy J, Riley JP. A modified single solution method for the determination of phosphate in natural water. Analytica Chimica Acta. 1962; 27: 31â??35.
42. Kacar B,  Inal A. Plant analysis. Nobel Publication No: 1241. Applied Sciences. 2008; 63: 879.
43. Kaya C, Akram N A, Ashraf M, Sonmez O. Exogenous application of humic acid mitigates salinity stress in maize (Zea mays L.) plants by improving some key physico-biochemical attributes. Cereal Res Commun. 2018;46(1): 67-78.
44. Khan TA, Mazid M, Mohammad F. A review of ascorbic acid potentialities against oxidative stress induced in plants. J Agrobiol. 2011; 28(2): 97â??111.
45. Saeidi-Sar S, Abbaspour H, Afshari H, Yaghoobi SR. Effects of ascorbic acid and gibberellin A3 on alleviation of salt stress in common bean (Phaseolus vulgaris L.) seedlings. Acta Physiol Plant. 2013;35(3): 667-677.
46. Wang YH, Zhang G, Chen Y, Gao J, Sun YR, Sun MF. Exogenous application of gibberellic acid and ascorbic acid improved tolerance of okra seedlings to NaCl stress. Acta Physiol Plant. 2019; 226: 540-549.
47. Chakrabarti N, Mukherji S. Effect of phytohormone pretreatment on nitrogen metabolism in Vigna radiata under salt stress. Biol plant. 2003; 46(1): 63-66.
48. Abdel-Gawad  H, Zinta G, Hegab MM, Pandey R, Asard H, Abuelsoud W. High salinity induces different oxidative stress and antioxidant responses in maize seedlings organs. Frontiers in Plant Science. 2016; 7: 276.
49. Cetinkaya H, Dinler BS, Tasci E. Investigation of comparative regulation on antioxidant enzyme system under copper treatment and drought stress in maize (Zea mays L.). Not Bot Horti Agrobo, 2014;42(2): 363-371.
50. Dinler BS, Antoniou C, Fotopoulos V. Interplay between GST and nitric oxide in the early response of soybean (Glycine max L.) plants to salinity stress. J plant physiol. 2014; 171(18): 1740-1747.
51. Li B, Feng XJ, Hu XY, Chen YP, Sha JC, Zhang HY, et al. Effect of melatonin on attenuating the isoflurane-induced oxidative damage is related to PKCa/Nrf2 signaling pathway in developing rats. Brain Res Bull.. 2018;143: 9-18.
52. Ekbic E, Cagiran C, Korkmaz K, Kose MA, Aras V. Assessment of watermelon accessions for salt tolerance using stress tolerance indices. Ciência e Agrotecnologia, 2017; 41(6): 616-625.
53. Yasar F, Uzal O, Yasar O. Determination of antioxidative enzyme activities in callus culture of the salt-tolerant and salt-sensitive watermelon (Citrullus lanatus (Thunb.) Mansf.) genotypes under salt stres. J Food Agric Environ.. 2013; 11 (3-4) : 1437-1440.
54. Uygur V, Yetisir H. Effects of rootstocks on some growth parameters, phosphorous and nitrogen uptake watermelon under salt stress. J Plant Nutr. 2009; 32(4): 629-643.
55. Moula I, Boussadia O, Koubouris G, Hassine MB, Boussetta W, Van Labeke MC, et al. Ecophysiological and biochemical aspects of olive tree (Olea europaea L.) in response to salt stress and gibberellic acid-induced alleviation. S Afr J Bot. 2020;132: 38-44.
56. Balkaya A. Effects of salt stress on vegetative growth parameters and ion accumulations in cucurbit rootstock genotypes. Ekin J Crop Bree Gen. 2016; 2(2): 11-24.
57. Hadi MR, Karimi N. The role of calcium in plants salt tolerance. J plant nutr. 2012; 35(13): 2037-2054.
58. Wang Y, Osatomi K, Nagatomo Y, Yoshida A, Hara K. Purification, molecular cloning, and some properties of a manganese-containing superoxide dismutase from Japanese flounder (Paralichthys olivaceus Comp. Biochem. Physiol. 2011; 158(4): 289-296.
59. Abdel Hamid AM, Mohamed HI. The effect of the exogenous gibberellic acid on two salt stressed barley cultivars. Eur Sci J. 2014;10(6).
60. Hadi F, Bano A, Fuller MP. The improved phytoextraction of lead (Pb) and the growth of maize (Zea mays L.): the role of plant growth regulators (GA3 and IAA) and EDTA alone and in combinations.  Chemosphere.  2010;80 (4):457-462.
61. Saibi W, Brini F. Superoxide dismutase (SOD) and abiotic stress tolerance in plants: An overview. Superoxide Dismutase: Structure, Synthesis and Applications; Magliozzi S, Ed. 2018; 101-142.
62. Bernardi R, Nali C, Gargiulo R, Pugliesi C, Lorenzini G, Durante M. Protein Pattern and fe-superoxide dismutase activity of bean plants under sulphur dioxide stress. Phytopathology. 2001; 149(7-8): 477-480.
63. Khoshgoftarmanesh AH, Schulin R, Chaney RL, Daneshbakhsh B, Afyuni M. Micronutrient-efficient genotypes for crop yield and nutritional quality in sustainable agriculture A review. Agron Sustain Dev. 2010; 30 (1): 83-107.
64. Ramezani S, Shekafandeh A. Roles of gibberellic acid and zinc sulphate in increasing size and weight of olive fruit. Afr J Biotechnol. 2009; 8(24): 6791-6794.