The Effect of Hormonal Priming on Morphological Characteristics and Antioxidant Enzyme Activities in Silage Maize Under Salt Stress

Abstract

Salinity is one of the major problems limiting plant growth, development, survival, yield, and quality. Climate change and increasing salinity levels force a concentration on sustainable production systems. Therefore, this study aimed to determine the effects of different doses of gibberellic acid (GA₃) (0, 150, and 300 mg/L) and salicylic acid (SA) (0, 0.25, and 0.50 mM) priming on some morphological and antioxidant enzyme activities of silage maize (Zea mays L.) seedlings exposed to salinity stress. Four different NaCl (0, 75, 150, and 225 mM) concentrations as salt stress and three different doses of both SA and GA₃ were investigated. The data obtained were subjected to analysis of variance according to a randomized complete block design using a factorial experimental design with four replications per treatment in 3 L pots. The results showed that GA₃ and SA priming had statistically significant effects on all investigated traits under different salt concentrations (except water content). Findings revealed that shoot, root, and leaf development, as well as antioxidant enzymes, were suppressed by salinity stress. The silage maize plant was statistically significantly affected starting from the lowest dose of 75 mM, depending on salt concentrations. Increasing salt concentrations negatively affected above-ground and below-ground parameters. However, SA and GA₃ treatments had positive impacts on all examined traits. SA and GA₃ priming treatments emerged as important strategies supporting root and shoot growth under saline conditions, thereby strengthening plant adaptation. The best results were obtained in groups exposed to 75 mM salt stress, where 300 mg/L GA3 was applied, and in groups without salt stress, where the same GA3 dose was applied. It was concluded that GA₃ priming treatments, in particular, were more effective than SA treatments, alleviating salt stress and positively contributing to plant development.

1. Introduction

Global food security demands effective agricultural output. Achieving sustainable production, and thus guaranteeing the longevity of farming, requires the efficient use of resources [1]. Salinity stress is one of the most significant problems limiting plant development, growth, survival, yield, and quality [2,3]. The area of saline soils worldwide is steadily increasing [4], and it is estimated that approximately 33% of irrigated agricultural land is currently affected by salinity, and this will increase to 50% of arable lands [5]. Under salt stress, plants experience physiological drought due to the increase in external osmotic potential around the seed [6], which inhibits water uptake by the seed [7] and/or causes ion toxicity due to high levels of Na⁺ and Cl⁻ ions, leading to disruptions in biochemical reactions within the seed [8,9]. Salt stress negatively affects seeds, particularly during the germination and seedling development when they are most sensitive to stress. In this sense, the toxicity of high salt (sodium chloride, NaCl) concentrations during and after germination of different seed types has been reported in many studies [10,11].

Maize (Zea mays L.), a C4 plant, can grow in both saline and non-saline conditions due to its potential to adapt to stress and its relative tolerance to salinity [12,13]. However, it negatively affects maize growth and yield throughout plant development. This effect depends on the duration and severity of salt stress and the growth stage at which the stress occurs [14,15]. Although maize, like many plant species, can tolerate moderate salinity, it is highly sensitive to salt stress during the early stages of growth [12]. Therefore, it is crucial to understand maize’s response to salt stress, its tolerance mechanisms, and the effects of certain priming treatments to identify approaches that enhance maize’s ability to adapt to saline environments. Various mitigation strategies are being used to manage salinity, such as the treatment of chemicals, hormones that promote plant growth, and the use of genetic and molecular techniques, which are thought to play an important role in increasing the productivity of maize under changing climatic conditions [16,17].

Seed pre-treatment (priming) is a simple procedure that partially moistens the seed with a certain organic or inorganic compound in a controlled environment, followed by drying the seed, thereby initiating germination without root emergence [18]. This technique promotes germination by causing a wide range of biochemical changes in the seed [19]. Gibberellic acid (GA₃) triggers seed germination, the transition from meristem to shoot growth, leaf development, the transition from the vegetative stage to flowering, and plant development, depending on environmental factors [20,21,22]. Research indicates that the use of gibberellic acid (GA₃) during the germination stage under salt stress enhances both the germination percentage and duration in Beta vulgaris and Dianthus barbatus [23,24]. Salicylic acid (SA) is a natural phenolic compound and a phytohormone [25]; it has been reported to have important functions in improving plant tolerance to various abiotic stresses [26,27] and in improving seed germination and shoot development parameters [28,29,30].

This study aims to determine the effects of different doses of GA₃ and SA seed priming on some morphological and antioxidant enzyme activities of silage maize (Z. mays L.) exposed to salt stress during the seedling development stage.

2. Materials and Methods

2.1. Plant Materials and Growth Conditions

The plant material used in the study was the PR32W86 silage maize cultivar. The research was conducted at Siirt University, Faculty of Agriculture, Department of Agricultural Biotechnology Plant Growth Laboratory, in a randomized complete block design using a factorial experimental design with 4 replications per treatment in 3 L pots. The study examined salt (NaCl) stress at concentrations of 0, 75, 150, and 225 mM; salicylic acid at 0 mM, 0.25 mM (S₁), and 0.50 mM (S₂); and gibberellic acid at 0, 150 (G₁), and 300 mg/L (G₂) were investigated.

The study lasted 60 days from the onset of germination. The study was conducted under controlled conditions in a climate chamber with an average temperature of 23–25 °C and humidity of 45–55%. Four seeds were used per replication. The four seeds were planted 3 cm deep in each pot, and the resulting seedlings were thinned to one plant per pot after emergence. A 1:1 mixture of peat and sand was placed in each pot. The seeds were sterilized in 70% ethanol for 1 min, rinsed three times with sterile distilled water, and then surface sterilization was performed by covering the seeds with a 10% sodium hypochlorite (NaOCl) solution for 10 min to kill the microorganisms on the seed surface. The sterilized seeds were soaked in SA and GA₃ solutions prepared according to the doses for 12 h [31]. The solution volume ratio to seed weight was 1:5 (g/mL) [32]. Salt concentrations were applied at appropriate concentrations at regular intervals after plant emergence, depending on soil moisture.

2.2. Measurement of Growth and Physiological Parameters

This study examined the parameters of shoot length, shoot fresh weight, seedling dry weight, seedling vigor index, root fresh weight, root dry weight, flag leaf length, flag leaf width, total root number, total root length, plant water content, chlorophyll content (SPAD), and the enzyme activities of superoxide dismutase, catalase, and malondialdehyde. Root samples were scanned at 600 dpi in color scale using a handheld scanner (Viisan Portable Scanner, Beijing, China). Root images were analyzed using ImageJ and Rhizovision Explorer software v2.0.3 [33,34].

On the harvest day, a SPAD chlorophyll meter (SPAD-502 Konica Minolta Sensing, Inc., Osaka, Japan) was used to determine the total chlorophyll content in the leaves. When determining the total chlorophyll content, readings were taken from young leaves, and the average value represented the pot [35]. The other plants in each pot were carefully removed along with their soil and cleaned of rhizosphere soil. The fresh weights of roots and shoots, plant height, and root length were determined for all plant samples. The plant samples from which measurements were taken were subjected to weight checks at 68 °C at regular intervals and removed when the weight change ceased to determine their dry weights. The average of the dry weights obtained from a total of 3 plants represented the pot [36]. The following formulas were used to calculate the water content (WC) in the plant [37]:

$$WC= \left[\right(SFW-SDW)/SFW] \times 100$$

(1)

In the equations, SFW represents the fresh weight of the shoot, and SDW represents the dry weight of the shoot.

The enzymatic activity of superoxide dismutase (SOD) and catalase (CAT) was measured in the plants. Leaf samples taken from the plants were frozen with liquid nitrogen and crushed in a porcelain mortar. The crushed samples were stored at −86 °C until analysis. Each sample was homogenized by mixing it with three times its weight of homogenate buffer (pH 7.5, 50 mM K-phosphate, 0.35 mM PMSF, and 1 mM EDTA) using a pestle in a mortar. The resulting mixture was centrifuged at 4 °C at 15,000 rpm for 1 h, and the supernatant was used as the enzyme solution (homogenate) [38]. The method used by [39], based on the hydrolysis of H₂O₂, was used as a reference for determining catalase activity. SOD activity was determined spectrophotometrically at 560 nm, following the method outlined by Çoban and Albayrak [40]. The TBARS method was used to determine the malondialdehyde (MDA) concentration in the plant shoot, and the methods used by Esterbauer and Cheeseman [41] were utilized.

2.3. Statistical Analysis

The obtained data were subjected to analysis of variance according to the factorial experimental design in a randomized complete block design; differences between means were checked using Tukey’s HSD multiple comparison test [42].

3. Results

The effects of the GA₃ and SA priming treatments, across all tested salt concentrations, were statistically significant for all examined shoot and root parameters (p < 0.01) (

Table 1. Findings related to the shoot and root traits evaluated in the study.

Treatments	Shoot Length (cm)	Shoot Fresh Weight (g)	Shoot Dry Weight (g)	Root Fresh Weight (g)	Root Dry Weight (g)
S0	87.8 cde	13.1 cde	1.34 abc	2.39 b–f	0.206 d–j
S75	82.0 d–g	10.9 efg	0.96 b–f	1.51 d–g	0.210 d–j
S150	68.4 f–i	8.4 e–h	0.85 b–f	1.52 d–g	0.183 f–j
S225	53.1 i	5.3 h	0.57 f	1.56 d–g	0.163 hij
S0S1	100.4 abc	17.6 bc	1.38 ab	2.26 b–f	0.196 d–j
S75S1	80.8 d–h	16.4 bcd	0.50 ef	1.08 g	0.151 ij
S150S1	67.4 ghi	8.2 fgh	0.95 b–f	1.78 b–g	0.135 j
S225S1	69.3 f–i	7.3 gh	0.74 def	1.64 c–g	0.192 e–j
S0S2	98.0 bcd	19.1 b	1.34 abc	2.67 abc	0.311 abc
S75S2	67.4 ghi	9.4 e–h	0.57 f	1.17 g	0.193 e–j
S150S2	69.7 f–i	10.4 efg	0.50 f	1.58 d–g	0.178 g–j
S225S2	64.6 hi	7.9 fgh	0.77 c–f	1.98 b–g	0.222 d–i
S0G1	93.9 bcd	16.4 bcd	1.35 abc	2.56 a–d	0.281 bcd
S75G1	85.4 c–f	13.2 cde	0.84 b–f	1.40 fg	0.247 c–h
S150G1	76.1 e–h	8.8 e–h	0.80 b–f	1.48 efg	0.273 b–e
S225G1	72.7 e–h	8.7 e–h	1.10 b–e	2.04 b–g	0.251 c–g
S0G2	105.3 ab	24.4 a	1.76 a	2.81 ab	0.380 a
S75G2	117.4 a	18.2 b	1.72 a	3.57 a	0.346 ab
S150G2	87.8 cde	12.5 def	1.07 b–f	2.50 a–e	0.322 abc
S225G2	85.1 c–f	11.1 efg	1.25 a–d	2.08 b–g	0.268 b–f
Tukey value/Significance	17.25 **	3.83 **	0.60 **	1.06 **	0.09 **

S₀: 0 mM NaCl, S₇₅: 75 mM NaCl, S₁₅₀: 150 mM NaCl, S₂₂₅: 225 mM NaCl, S₁: 0.25 mM salicylic acid, S₂: 0.50 mM salicylic acid, G₁: 150 mg/L gibberellic acid, G₂: 300 mg/L gibberellic acid. The difference between the means shown with the same letter in the same group and in the same column is not statistically significant, ** p < 0.01 significance level.

). Increasing salt concentration significantly reduced shoot length, fresh and dry shoot weight, and fresh and dry root weight in the control groups (without priming treatment). Increasing salt concentrations negatively affected shoot and root development. The highest values were 117.4 cm for shoot length in the S₇₅G₂ treatment, 24.4 g for shoot fresh weight in the S₀G₂ treatment, and 1.76 g for shoot dry weight in the S₀G₂ (1.72 g) treatment, 3.57 g for root fresh weight in the S₇₅G₂ treatment, and 0.380 g for root dry weight in the S₀G₂ treatment. The lowest values were determined for shoot length and shoot fresh weight characteristics in the S₂₂₅ treatments, for shoot dry weight in the S₂₂₅, S₇₅Sa₂, and S₁₅₀S₂ treatments, for root fresh weight in the S₇₅S₁ and S₇₅S₂ treatments, and for root dry weight in the S₁₅₀S₁ treatments (Table 1).

The effects of GA₃ and SA priming treatments on chlorophyll content, flag leaf length and width, and stem thickness at different salt concentrations were found to be statistically significant (p < 0.01), while the effect of GA₃ and SA priming treatments on water content was insignificant (

Table 2. The chlorophyll content, along with flag leaf length and width, stem thickness, and water content.

Treatments	Chlorophyll Content (Spad)	Flag Leaf Length (cm)	Flag Leaf Width (cm)	Stem Thickness (mm)	Water Content
S0	44.3 a	66.7 a–d	2.43 a	5.31 cde	92.2
S75	31.5 efg	58.3 a–e	1.79 a–e	4.96 def	91.3
S150	31.1 efg	55.0 a–e	1.63 b–e	4.52 ef	91.8
S225	30.8 efg	41.5 e	1.42 de	4.15 f	89.3
S0S1	43.2 ab	76.0 ab	2.27 abc	6.62 ab	92.6
S75S1	26.0 g	55.0 a–e	1.72 a–e	5.87 bcd	93.4
S150S1	31.2 efg	55.2 a–e	1.66 a–e	4.95 ef	92.3
S225S1	32.9 d–g	52.6 b–e	1.45 de	4.58 ef	90.3
S0S2	40.1 a–d	71.6 abc	2.06 a–d	6.34 ab	92.3
S75S2	25.4 g	49.6 cde	1.69 a–e	5.97 bc	92.4
S150S2	27.6 fg	46.2 de	1.26 e	4.72 ef	92.1
S225S2	31.0 efg	49.5 cde	1.45 de	4.69 ef	91.8
S0G1	40.1 a–d	72.3 abc	2.43 a	6.30 ab	92.6
S75G1	34.3 c–f	60.0 a–e	1.77 a–e	5.87 bcd	93.3
S150G1	30.5 efg	53.2 b–e	1.37 de	4.80 ef	92.1
S225G1	35.1 b–f	54.5 a–e	1.38 de	4.79 ef	90.8
S0G2	42.0 abc	78.2 a	2.28 abc	6.96 a	92.6
S75G2	41.4 abc	78.7 a	2.31 ab	7.06 a	92.5
S150G2	32.7 d–g	66.5 a–d	1.60 b–e	4.92 ef	92.5
S225G2	37.9 a–e	63.4 a–e	1.52 cde	4.83 ef	90.3
Tukey value/Significance	8.13 **	24.40 **	0.77 **	0.91 **	4.35 ns

S₀: 0 mM NaCl, S₇₅: 75 mM NaCl, S₁₅₀: 150 mM NaCl, S₂₂₅: 225 mM NaCl, S₁: 0.25 mM salicylic acid, S₂: 0.50 mM salicylic acid, G₁: 150 mg/L gibberellic acid, G₂: 300 mg/L gibberellic acid. The difference between the means shown with the same letter in the same group and in the same column is not statistically significant, ** p < 0.01 significance level, ns: non-significance.

).

In treatments without GA₃ and SA priming, when only salt stress was imposed, reductions in chlorophyll content were observed in the flag leaf’s length and width due to elevated salt concentration. Increasing salt concentrations negatively affected shoot and root development. The highest values were 44.3 for chlorophyll content in the T₀ treatments without salt, 78.2 and 78.7 cm for flag leaf length in the T₀G₂ and T₇₅G₂ treatments, and 2.43 cm for flag leaf width in the T₀ and T₀G₁ treatments. The lowest values were determined for chlorophyll content in the T₇₅S₁ treatments, for flag leaf length in the T₂₂₅ treatments, and for flag leaf width in the T₁₅₀S₂ treatments (Table 2). The best results for chlorophyll content were achieved in the control groups. The most effective results for flag leaf length, width, and stem thickness were obtained in treatments lacking salt stress or those exposed to low salinity (75 mM), particularly under GA₃ priming (Table 2).

The effect of the GA₃ and SA priming treatments on the total root length at different salt concentrations was statistically significant (p < 0.01) (Figure 1). The highest total root length was determined as 558.5 cm in the S₇₅G₂ treatment. SA and GA₃ treatments showed the highest effect at 75 and 150 mM salt doses (Figure 2). The S₀G₂ treatment was also statistically significant in the first group. The lowest value was detected in the S₂₂₅ treatment, where the highest salt concentration was applied (Figure 1).

Superoxide dismutase (SOD) activity demonstrated a concentration-dependent increase, peaking at the 150 mM salt concentration before showing a slight reduction at 225 mM. CAT and MDA enzyme activities increased as the salt concentration increased. The highest values were detected for SOD in the S₁₅₀S₁ treatment, for CAT in the S₂₂₅ and S₂₂₅S₁ treatments, and for MDA in the S₂₂₅ treatment (

Table 3. The effect of different priming treatments on antioxidant enzymes in silage maize under salt-stressed conditions.

Treatments	Superoxide Dismutase (EU/mL)	Catalase (EU/mL)	Malondialdehyde (nmol/mL)
S0	166.5 i	25.4 ij	0.743 g
S75	168.6 hi	38.1 fgh	0.935 ef
S150	178.2 abc	45.3 bcd	1.283 b
S225	173.5 ef	56.6 a	1.531 a
S0S1	168.3 hi	28.4 i	0.768 g
S75S1	167.4 hi	38.3 fgh	0.763 g
S150S1	180.3 a	45.1 cd	1.075 de
S225S1	177.2 a–d	55.8 a	1.222 bc
S0S2	168.5 hi	23.0 j	0.746 g
S75S2	177.3 a–d	36.1 gh	0.732 g
S150S2	179.3 ab	41.8 c–f	1.017 de
S225S2	178.5 abc	50.3 b	1.123 cd
S0G1	176.5 b–e	21.9 j	0.724 g
S75G1	174.5 def	34.8 h	0.796 fg
S150G1	172.5 fg	44.6 cde	1.299 b
S225G1	173.4 ef	46.7 bc	1.341 b
S0G2	170.2 gh	24.7 ij	0.732 g
S75G2	169.5 ghi	39.6 e–h	0.759 g
S150G2	172.3 fg	40.4 d–g	1.288 b
S225G2	175.8 cde	40.7 d–g	1.329 b
Tukey value/Significance	3.15 **	5.09 **	0.14 **

S₀: 0 mM NaCl, S₇₅: 75 mM NaCl, S₁₅₀: 150 mM NaCl, S₂₂₅: 225 mM NaCl, S₁: 0.25 mM salicylic acid, S₂: 0.50 mM salicylic acid, G₁: 150 mg/L gibberellic acid, G₂: 300 mg/L gibberellic acid. The difference between the means shown with the same letter in the same group and in the same column is not statistically significant, ** p < 0.01 significance level.

).

The correlation heatmap with hierarchical clustering, created to visualize the relationships between different physiological, biochemical, and morphological characteristics, along with the correlation matrix and hierarchical clustering analysis, is presented in Figure 2. The colors in the figure represent the correlation coefficient (r) between variables. Accordingly, blue tones represent positive correlation, indicating that the two variables tend to increase together as they approach +1. Red tones reflect negative correlation, indicating that as one variable increases, the other decreases as they approach −1. In areas where white tones are observed, the correlation coefficient is close to zero, indicating that there is no significant relationship between the variables. In this respect, the heatmap allows for easy understanding and visual comparison of strong or weak, positive or negative relationships between parameters (Figure 3).

4. Discussion

Seed priming facilitates seed germination and shoot emergence, accelerates plant growth, regulates physiological, biochemical, and molecular responses, promotes nutrient uptake, and enhances tolerance to stress factors. Priming is based on immersing seeds in a solution with low osmotic potential for a certain period of time and then drying them again to the initial moisture content [43,44,45]. This process initiates various biochemical events, such as activating antioxidant defense systems in addition to the germination [46].

GA₃ and SA play an important role in the defense response to stresses in plant species [47,48]. Numerous studies support a major role for SA and GA₃ in modulating the plant response to various abiotic stresses, including salinity [49,50]. GA₃ is one of the naturally occurring plant growth hormones that regulates plant growth and development [51]. GA₃ is associated with numerous physiological processes, including seed germination, hypocotyl elongation, leaf expansion, synchronized flowering, floral organ development, floral initiation, increased flower number, reduced time to flowering, and size, as well as the induction of certain hydrolytic enzymes in the aleurone layer of cereal grains [52,53]. Growth regulators such as GA₃ have been reported to mitigate the inhibitory effects of salinity on seed germination [54,55]. It has been suggested that the alleviating effect of exogenously applied GA₃ on salt stress might be associated with the activation of specific enzymes involved in RNA and protein synthesis [56]. SA, a plant phenolic compound, is now considered a hormone-like endogenous regulator, and its role in the defense mechanisms against biotic and abiotic stress has been well documented [57]. It was found that inhibition of catalase, a H₂O₂ scavenging enzyme, by SA plays an essential role in the generation of reactive oxygen species [58]. Moderate doses of SA may activate antioxidative mechanisms by increasing H₂O₂ concentration within the tissues. Application of exogenous SA enhanced the drought and salt stress tolerance of plants [49,59].

GA₃ and SA were found to be effective in reducing salt stress in terms of the shoot and root parameters studied. They contributed to reducing this stress, especially at increasing salt concentrations, and to the healthy continuation of shoot development. In treatments without salt stress, the G₂ treatment (300 mg/L GA₃) in particular showed a significant contribution compared to the control and SA (Table 1). It was observed that the silage maize tolerated a salt concentration of 75 mM in particular and was not adversely affected by this concentration. With increasing salt concentrations, particularly at 150 mM and 225 mM, significant negative effects were observed in groups without priming. Previous research suggests that 1.7 dS/m is a moderate stress (50–75 mM) threshold for maize salt tolerance [60,61]. Indeed, Zhao et al. [62] stated that salinity negatively affects plant growth and development by disrupting physiological and biochemical mechanisms in plants. Positive developments in plant growth were observed with GA₃ and SA priming, and increases in plant shoot and root development were obtained. Bouallegue et al. [63] obtained results consistent with those of a similar study conducted with the Lens culinaris plant. Increasing GA₃ application increases shoot length in wheat plants under salinity stress [64], and 20 mg/L GA₃ application increased plant height in Cicer arietinum plants under NaCl (0.8, 12, and 16 ds/m) [65].

The ameliorative effect of SA on plant germination, shoot, and root development by mitigating the adverse effects of salinity has been demonstrated in wheat [47], sunflower [66], beans [67], and tomatoes [68]; and for GA₃ in plants such as mustard [69], wheat [70], and sorghum [71]. Furthermore, Sedláková et al. [72] stated that one of the most important roles of SA in plants is to exert a modulatory effect on shoots by regulating seed dormancy, thereby providing a stronger structure against adverse environmental conditions.

Salt stress caused reductions in the fresh and dry weights of shoots and roots (Table 1). It has been suggested that the reductions in fresh and dry weights may be due to low water uptake caused by physiological drought [73,74]. The findings of this study, which indicate that salinity reduces the fresh and dry weight of plants, are similar to those reported by Dheeba et al. [71,75]. El-Tayeb [76] reported that SA pretreatment increased dry weight in the stressed barley seedlings. In another work, Gutiérrez-Coronado et al. [77] reported that the application of SA significantly enhanced shoot growth and overall plant height in soybean. Khodary [78] reported that SA increased the fresh and dry weight of the shoot of salt-stressed maize plants

Although it varies depending on the plant species and salinity intensity, many studies have also reported that increasing saline conditions lead to a decrease in root length, supporting the findings obtained in this study [9,79,80]. This reduction in root length is thought to be due to decreased water and nutrient uptake caused by ion toxicity or a decrease in osmotic potential caused by salinity. Plants in the control group and those treated with SA or GA₃ under low salinity (75 mM) generally achieved high root length values. Under high salinity conditions, SA and GA₃ treatments partially increased root length (Figure 1). It was observed that salinity stress suppressed root development, but SA and GA₃ treatments reduced this negative effect and increased root length. In particular, the potential of SA and GA₃ to promote root development was found to be more pronounced at low and medium salinity levels. In most cases, SA concentrations, which enhance or at least do not influence root growth under normal conditions, promote plant recovery from stress conditions [81,82,83]. This recovery is related to SA’s protection of cell divisions. The SA treatment of wheat seeds increases the mitotic index in the root apical meristem and thereby promotes root tolerance to high salinity and their enhanced recovery after stress [47].

The study revealed that salinity triggered an increase in SOD, CAT, and MDA contents. GA₃ and SA priming treatments were also found to slow down antioxidant enzyme activities and show positive effects compared to the control (Table 3). Malondialdehyde (MDA) content is frequently used as a primary indicator of membrane damage and subsequent physiological disorders in plants, depending on the severity of the stress factor [84]. It has been demonstrated that salinity triggers oxidative stress (Table 3) and leads to a decrease in the stability of cell membranes [85]. Similar results have been reported in different plant species, showing that salinity increases SOD, CAT, and MDA enzyme activities [86,87,88]. Lower CAT, SOD, and MDA contents observed in silage maize plants under salt stress after GA₃ and SA priming treatments have been attributed to the effective reduction in damage caused by reactive oxygen species and stronger damage protection, which prevents unsaturated fatty acid damage and electrolyte leakage [89,90]. Plants activate non-enzymatic and enzymatic antioxidant systems to eliminate the harmful effects of oxidative stress [91].

Correlation analysis revealed the presence of two distinct groups of relationships among the examined parameters. Strong positive correlations were determined between antioxidant enzymes (SOD and CAT) and MDA, an indicator of lipid peroxidation, suggesting that defense systems are activated together under stress conditions and that oxidative damage increases. In contrast, highly positive correlations were observed among all measured morphological and biomass parameters, including root and shoot length, leaf length, fresh weight, and dry weight. This statistical outcome demonstrates the parallel progression and mutual support among the different components of plant growth. The presence of moderate to high negative correlations between stress indicators (SOD, CAT, and MDA) and growth and biomass parameters indicates that increased oxidative stress directly suppresses growth performance (Figure 3). Furthermore, water content (WC) showed positive relationships with growth parameters and negative relationships with stress parameters, reflecting the critical balance between plant water status, stress severity, and development performance. Overall, these results strongly indicate that increased antioxidant activity and lipid peroxidation under stress conditions cause a decline in growth and yield components.

5. Conclusions

The findings indicate that shoot, root, and leaf development, as well as antioxidant enzyme activity, are suppressed by salinity stress. However, it has been determined that SA and GA₃ treatments have a positive effect on the examined parameters and antioxidant enzyme activity. It can be concluded that increased salinity stress increases oxidative damage; however, SA and GA₃ priming treatments are effective in improving stress tolerance by balancing SOD and CAT activities and MDA levels. SA and GA₃ priming treatments have emerged as an important treatment strategy that strengthens plant adaptation by supporting root development under salinity conditions. In silage maize, it was concluded that GA₃ priming treatments yielded more effective results than SA treatments and had much more comprehensive positive effects in terms of suppressing salt stress and promoting plant development.