Exogenous Glutathione-Mediated Regulation of Seed Germination in Ceratonia siliqua L. Under Abiotic Stress

1. Introduction

One of the most significant problems of North Africa is climate change, driven by human activity and endangering both human well-being and agricultural productivity [1]. The Intergovernmental Panel on Climate Change (IPCC) forecasted a decline in yearly precipitation and a sustained rise in average global surface temperatures in its most recent assessment report [2]. It is widely expected that global warming will negatively impact plant growth and productivity, due to the deleterious consequences of rising temperatures on their development [3,4,5]. Under heat stress, plants undergo physical changes to their environment and a decrease in the efficiency of plant cell functions, including enzymatic activity, membrane fluidity, protein complex formation, chlorophyll synthesis, photosynthesis, respiration, and redox status [6].

In arid and semi-arid regions, water is a critical factor limiting ecosystems production. One of the major abiotic factors affecting plant growth and productivity is drought [7,8]. This occurs when water loss by the plant exceeds the roots' capacity to absorb water from the soil for a period long enough to cause irreversible damage [9]. Plants undergo osmotic stress due to drought conditions, which eventually leads to desiccation and an inability to absorb water [10,11]. Drought resistance is a complex characteristic influenced by multiple genes and conditioned by various morphological, physiological and biochemical responses [12]. These responses can interact and vary depending on the type, intensity, and duration of water shortages [13,14].The external application of growth regulators is a method for increasing yields, improving crop quality, and managing the uptake and accumulation of mineral nutrients in plants [15].

Another significant threat to crop productivity is salt stress [16,17,18]. Exposure to high salinity causes ionic and osmotic imbalances, leading to the overproduction of reactive oxygen species (ROS). In this context, abiotic stresses are estimated to reduce average crop yields by 50%[19]. Abiotic stresses lead to increased production of reactive oxygen species (ROS), such as superoxide (O^2-), singlet oxygen (1O₂), hydroxyl radical (OH), and hydrogen peroxide (H₂O₂) [19]. According to [20], these ROS cause oxidative damage, leading not only to lipid peroxidation and protein oxidation but also to enzyme inhibition and DNA/RNA damage. However, the negative effects of environmental stress can be mitigated by exogenous treatments with small biomolecules. Hydration treatments, such as priming, improve plant physiological properties and seed viability. They also promote increased germination rates and emergence and stimulate strong root growth [21,22]. Therefore, it is essential to regulate ROS to enhance the ability of plants to resist stress, and this is achieved through an antioxidant defence mechanism formed by various antioxidant enzymes and non-enzymatic antioxidants [23].

Glutathione (GSH; γ-glutamyl-cysteinyl-glycine) is one of the major non-enzymatic antioxidants. It is a water-soluble, low-molecular-weight thiol compound that is widely distributed in most plant tissues[23]. This Chemical treatment has several important advantages over transgenic and seeds technology, since it is safe for plants, people, and animals [24]. It is generally more affordable for farmers and considered acceptable from an environmental point of view [25].

GSH is a beneficial metabolite and a crucial component, frequently found in its reduced form, where it acts as a scavenger of reactive oxygen species (ROS), helping reduce abiotic stress [26,27].

GSH plays a role in primary metabolism, promotes chlorophyll conservation, and enhances the synthesis of photosynthetic pigments [28,29]. According to [30], GSH is associated with several plant growth and development processes, including the regulation of cellular redox homeostasis and gene expression, as well as the plant response to biotic and abiotic stressors. Application of GSH greatly reduced oxidative damage caused by drought stress in mung bean seedlings by enhancing the efficiency of glyoxalase and antioxidant systems [31]. In addition, GSH boosted resistance to salt stress during the sowing and reproductive phases in soya [32]. They reported that the use of exogenous GSH promoted seed germination and enhanced plant height., number of branches per plant, number of pods per plant, seeds per pod, and 100-seed weight, leading to a progressive increase in plant productivity [33,34]. Thus, the use of external antioxidants, such as GSH, could be an essential strategy for significantly enhancing seed vigour [29,35]

The major problem facing pastoral ecosystems in the arid zones of North Africa is their low species richness, particularly the absence of the tree and shrub strata. Species belonging to the Fabaceae family were the first group to undergo irreversible phytogenetic erosion. This decline affects the state of these ecosystems across several vital attributes, including the loss of forage quantity in rangelands, decreased soil fertility due to the absence of atmospheric nitrogen fixation, soil erosion, and the depletion of soil water reserves [36].

Faced with these ecological problems, the idea of restoring species richness in degraded ecosystems has become increasingly recurrent among scientists and development practitioners [37]. In this regard, the focus has largely been placed on leguminous species capable of fixing atmospheric nitrogen and thereby improving soil fertility. Furthermore, tree species of this family, once established, are thought to play a key role in further improving microclimatic conditions at the soil level [38].

For a long time, allochthonous leguminous tree species—particularly those of Australian origin, such as Acacia saligna and Acacia salicina, and of South American origin, such as Prosopis chilensis—were used in reforestation programs [39,40]. Unfortunately, these species have not produced satisfactory results, owing to their poor adaptation to aridity and to the edaphic and climatic conditions of North African environments. Moreover, given the limited number of native tree legume species, Ceratonia siliqua has now been adopted in reforestation programs and in the restoration of degraded ecosystems. However, its establishment is hindered by specific germination problems related to seed-coat (tegumentary) dormancy [41,42].

The carob tree (Ceratonia siliqua L.), a dioecious plant, belongs to the Fabaceae family. It is native to the Mediterranean region, which is important both ethnobotanically and nutritionally. Its pods and seeds are essential for human and animal nutrition and are used in the modern food and pharmaceutical industries [43]. In Tunisia, the carob tree has spread widely in the wild [44]. According to [45], it is drought-tolerant, which explains its distribution in the semi-arid and arid regions of the Mediterranean. Consequently, this species has been widely incorporated into ecological restoration programs because of its ability to combat soil erosion, stabilize degraded soils, and improve environmental conditions, thereby facilitating vegetation establishment and ecosystem recovery [42]. To enhance the success of reforestation efforts involving this species while minimizing associated costs, it is essential to understand its seed germination potential and identify the optimal conditions for seed germination and seedling establishment.

In fact, given the tegument's hardness, which characterizes the state of dormancy, carob seeds exhibit a rare and irregular pattern of germination [46]. To overcome seed coat-imposed dormancy and facilitate the germination of carob seeds, the treatment implemented must effectively improve water uptake and gas exchange through the seed coat while preventing damage to the embryo and endosperm. [47].

Although several past studies have demonstrated that GSH can stimulate seed germination, the influence of Glutathione on carob germination has not yet been studied.

The main objective of this study was to evaluate the potential promotive effects of glutathione (GSH) on the germination of carob (Ceratonia siliqua L.) seeds, a tree species recognized as a promising candidate for the ecological restoration of degraded ecosystems. During our field surveys conducted throughout Tunisia, we observed very limited natural seed germination and an almost complete absence of young seedlings. These observations suggest that natural regeneration of this species is severely constrained and constituted the main motivation for this study. Therefore, we investigated seed priming with GSH as a strategy to determine its potential to overcome germination constraints and improve seed germination and seedling establishment.

GSH applied at concentrations of 0, 25, 50, 100, and 200 μM, was investigated as an efficient approach to promote germination performance. Thus, determining the optimal GSH concentration that stimulates germination and improves early seedling establishment in carob trees.

2. Results

2.1. Effect of Priming on Seed Germination of Ceratonia siliqua

The results of the germination kinetics of C. siliqua treated with exogenous glutathione at different concentrations (0, 25, 50, 100, and 200 μM/L) under different salt and water stress concentrations (0, -0.5, -1 and -1.6 MPa) are illustrated as germination curves, showing the evolution of the germinated seed rate over time (Figure 1). Germination of C. siliqua seeds was significantly (P<0.05) affected by exogenous GSH on the one hand and by water and salt stress on the other. At an optimum germination temperature (25°C), seeds germinated up to 100% in the different glutathione treatments. Moreover, the fastest germination was observed between 25 and 50 μM/L, with GR₅₀ of 2.62 and 2.65, respectively.

Under salt stress, the control seeds (0 μM GSH) exhibited the highest germination rates (100% and 77% at –0.5 and –1 MPa, respectively), followed by seeds treated with 25 μM GSH, which showed germination rates of 95% and 44.48% under the same conditions. In contrast, the lowest germination rates (71.48% and 0.56%) were recorded at 200 μM GSH. However, at –1.6 MPa, the maximum germination rate was observed in seeds treated with 25 μM GSH.

Under water stress conditions, the highest germination rates were recorded in seeds treated with 25 μM GSH, reaching 39% and 3% at –0.5 and –1 MPa, respectively. In contrast, the lowest germination rates were observed in seeds treated with 200 μM GSH, with 5% and 0% germination at –0.5 and –1 MPa, respectively. At –1.6 MPa, no germination (0%) was observed in seeds treated with 200 μM GSH. Figure 2 shows the maximum germination index and the MGT as a function of different GSH concentrations. Under salt stress, we observe that IGmax increases progressively from 33.33 to 44.66 between 0 and 50 μM/L. In parallel, the MGT decreases gradually and reaches its minimum value (3.56) at a moderate GSH concentration of 50 μM/L. It indicates that the germination of Ceratonia siliqua is faster at 50 μM/L of GSH. With increasing GSH concentration, IGmax decreased progressively to 27.85 at 200 μM/L. At the same time, MTG increased (almost 5 days). Overall, GSH showed a concentration-dependent biphasic effect under both salt and water stress: moderate concentrations (25–50 μM/L), particularly 50 μM/L, enhanced and accelerated germination, whereas higher concentrations (100–200 μM/L) reduced germination and delayed its onset.

2.2. GSH Effects on Germination Under Abiotic Stress and Correlation Matrix of Seed Germination Parameters

In treated C. siliqua seeds, all environmental factors examined (temperature, osmotic stress, and salinity) significantly affected germination (p ≤ 0.05; Table 1). Treatment with GSH, NaCl, and PEG₆₀₀₀ and their interaction significantly affected the FGP of carob seeds (p < 0.001). At low concentrations, GSH increased the final germination percentage when seeds were subjected to higher salt and osmotic stresses (-1 and -1.6 MPa; Figure 3). However, this effect tended to diminish at higher GSH concentrations (100-200 µmol/L; Figure 3), suggesting that low doses of GSH (25-50 µmol/L) are more effective in improving seed germination yield under two types of stress.

Under salt stress, the initial germination percentage increases progressively with increasing GSH from 0 to 50 μM/L, reaching a maximum of 20-67%, and decreases

progressively when the GSH concentration exceeds 50 μM/L, reaching 28% at 200 μM/L. On the other hand, under water stress, the initial germination percentage increases from 20% to 72% at 0-50 μM/L, then decreases to 28% at 200 μM/L (Table 2). Under the effect of moderate concentration of GSH (between 25 and 50 μM/L), more rapid germination processes were observed. In addition, an inhibition of seed germination was recorded at higher GSH concentrations (>50 μM/L). With increasing salt stress from 0 to -1.6 MPa, the initial germination percentage decreased from 20% in control seeds to 4% and from 63% in 25 μM/L-treated seeds to 4.5%. Similarly, for salt stress, IGP decreased from 20 to 0 for control seeds and from 60.57 to 0 for seeds treated (25 μM/L), respectively, from 0 to -1.6 MPa. The final germination percentage and IGmax tend to decrease as the levels of two types of stress increase. The highest value is observed at control (0 MPa) in seeds treated with 50 μmol. It decreases until it reaches 0% at -1.6 MPa. To determine the ability of these seeds to germinate quickly and efficiently, the VG was calculated. The values obtained range from 0 to 2.13. This index increases from 1.59 in untreated seeds to 2.13 in seeds treated with 50 μM/L in both types of stress. This suggests that at moderate GSH concentrations (up to 50 μM/L), germination speed and seed vigor, thereby promoting germination in Ceratonia siliqua seeds. As GSH concentration and stress increase, this index decreases to 0% at -1.6 MPa, indicating that salt and water stress delay germination by preventing radicle emergence (Table 2).

To determine the number of days required for 50% of the seeds to germinate, GR₅₀ was measured. The lowest value of GR₅₀ was observed in seeds treated with 25 μM/L GSH (2.63 days) followed by a value of 2.66 for seeds treated with 50 μM/L GSH. The highest value was observed in seeds treated with 200 μM/L GSH. It can be seen that this value increases with increasing salt stress from 2.63 to 7.25, respectively, from 0 to -0.5 MPa at 25 μM/L GSH, and from 2.66 to 5.83 and 2.83 to 8.13 from 0 to -0.5 MPa, respectively, at 50 and 100 μM/L GSH. This shows that the GR₅₀ increases with increasing stress.

Under water stress, the GR₅₀ is 2.62 for seeds treated with 25 μM/L, while the highest value is recorded for seeds treated with 200 μM/L (4.87). The GR₅₀ values at higher NaCl concentrations (-1 and -1.6 MPa) are not determined because the germination percentage does not reach 50%. Therefore, water stress affects the time required for seed germination and the speed and vigour of Ceratonia siliqua seeds. The use of GSH led to a significant decrease in the GR₅₀ value, indicating faster germination. This indicates a beneficial impact of GSH on seed viability at a moderate dose (25-50 µmol/L), likely associated with its antioxidant function, which protects embryonic cells against oxidative stress.

These responses were further supported by the analysis of final germination percentage (FGP), which revealed significant effects of GSH and osmotic potential on seed germination. As shown in Table 3, GSH treatments, osmotic potential, and their interaction significantly affected FGP under both salt and water stress (p < 0.0001). Osmotic potential had the strongest effect on FGP, particularly under PEG 6000 stress (F = 4702.00), compared with NaCl stress (F = 1836.10).

Figure 4 shows various correlations between the germination parameters of C. siliqua at 25 °C. A positive correlation (r = 0.9) was observed between treatment and the maximum germination days, when the majority of seeds germinate. Another negative correlation between IGmax and the day of maximum germination (r = -0.9), which means that the faster the carob seeds germinate, the earlier the date of maximum germination. A negative correlation was observed between PGF and GSH, meaning that GSH has an inhibitory effect at high doses. On the other hand, a strong positive correlation between PGF and IGmax (r = 0.88) indicates that seeds that germinate better also germinate faster, suggesting that they have germination vigour in this context. A strong correlation was observed between IGmax and VG (r = 0.99) and between IGP and IGmax (r = 0.9), indicating rapid and early germination at moderate GSH concentrations, which translates into improved germination vigour of the treated seeds.

2.3. Hydrotime Model and Osmotic Adjustment

According to the results in Table 1, the hydrotime model accurately describes (R² > 0.07) the relationship between germination of Ceratonia siliqua seeds and water and salt stress for each GSH treatment. Under salt stress and at an optimal germination temperature (25°C), the base water potential (Ψ_b) was lower at 25 µmol/L (-0.45 MPa). Under water stress, the estimated Ψ_b values were much lower. They ranged from -0.9 to -1.1 MPa for seeds treated with 25 µmol/L and for untreated seeds, respectively. The lowest value (-0.6) was observed in seeds treated with the highest GSH dose (200 µmol/L).

Secondly, we measured θ_H (a constant hydrotime expressed in MPa-days), which quantifies the potential water required for seed germination. Under salt stress, the lowest value of θ_H was observed in seeds treated with 25 µmol/L, and the highest value was observed in control seeds, at 2.61 MPa-days. Under water stress, the highest value was observed in the control (6.33 MPa -days) while the lowest value (2.77 MPa -days) was observed in seeds treated with a moderate dose of GSH (25 µmol/L).

Ceratonia siliqua exhibits high germination capacity under moderate salt stress rather than water stress, as germination occurs more rapidly (lower θ_H). However, its tolerance of drought (more negative Ψ_b) is superior, indicating that some seeds can still germinate even under severe drought conditions. The values of σθ_H for both types of stress vary slightly with GSH concentration (Table 1), indicating stability in the physiological response of the seeds to the treatment. GSH, therefore, acts similarly on all the seeds. Under water stress, the lowest value is recorded in seeds treated with 50 µmol/L. Under water stress, σθ_H decreases from 0 to 50 µmol/L, from 0.4 to 0.1, indicating that GSH not only improves stress tolerance but also makes seed yield responses more homogeneous.

3. Discussion

3.1. Effect of Priming on Seed Germination of Ceratonia siliqua

Germination is recognized as one of the most sensitive stages to environmental stresses and is critical for species establishment [54]. These environmental stresses, including water, salt, and extreme temperatures, significantly affect the germination of Ceratonia siliqua seeds [16]. In addition, these seeds are dormant due to the impermeability of the seed coat [55]. To break this dormancy and improve germination, whatever the environmental stress, pre-treatments such as soaking in sulphuric acid and GSH are essential. Antioxidants are essential components that protect plant cells against oxidative damage caused by abiotic stresses [56]. The results of this study revealed that nonenzymatic antioxidant GSH had a significant effect (p<0.05) on seed germination under different concentrations of water and salt stress (Figure 1). Germination was faster at 50 µmol/L than in the control. With increasing salt and water stress, the highest germination rate was observed in seeds treated with 25 µmol/L GSH. A moderate dose of GSH (25- 50µmol/L), therefore, proved to be the most effective in improving germination performance of Ceratonia siliqua seeds subjected to both types of stress, water and salt. According to [57], priming Brassica oleracea L. seeds treated with 50 µM L^-1 improved germination compared with untreated control seedlings. In addition, exogenous glutathione (GSH) helps to improve the resistance of young Solanum lycopersicum L. plants to salt stress by restoring the ionic balance, reducing the accumulation of sodium (Na⁺) and chloride (Cl^-) in tissues, promoting the transport of potassium (K⁺) and calcium (Ca²⁺) to aerial parts, and regulating the metabolism of polyamines. Thus, it enhances plant resistance and salt tolerance by affecting both ionic balance and cellular function [58].

3.2. Hydrotime Modeling of GSH-Primed Seed Germination Under Combined Abiotic Stress

Given the decrease in the final germination percentage of Ceratonia siliqua seeds under NaCl induced salt stress and PEG₆₀₀₀-induced water stress [16], GSH can play a protective role by enhancing seed germination under stress. In this context, various abiotic stresses, including drought, high temperatures, salinity [59,60], and even heavy metal toxicity [61,62] can be mitigated by exogenous GSH.

In the control and at different GSH concentrations, FGP reaches 100% germination, but the fastest germination is observed at 50 μM/L (Figure 1), followed by the highest IGmax (44.66). This means that carob seed germination is more vigorous, rapid, and uniform, with a germination percentage that is too high in too short a time. The results also show a higher VG value (2.13) at this dose (Table 2, Figure 4), which means that the seeds can germinate more quickly. A low GR₅₀ value is also observed, indicating that the time to achieve 50% germination is reduced. These findings are consistent with previous reports showing that exogenous reduced glutathione (GSH) enhances seed vigour, increases germination percentage and germination index, and shortens mean germination time in aged seeds [63].

Under salt stress and with increasing NaCl concentration, the highest germination rate was observed in seeds treated with 25 μM/L GSH (Figure 3), indicating that a moderate dose of GSH improved the germination capacity of Ceratonia siliqua seeds under severe salt stress conditions. According to [58], the use of exogenous GSH improves salt tolerance in tomatoes by strengthening the antioxidant defence mechanism, while regulating GSH production and function. This helps maintain low levels of ROS and ensure internal balance in the leaves of young tomato plants exposed to salt stress [60,64]. [65], demonstrated that exogenous GSH amplifies net photosynthesis rates in young tomato plants exposed to salt stress by adjusting the redox state at the tissue level to increase PSII photochemical efficiency and Rubisco activity. In addition, GSH application to Capsicum annum seeds has been reported to alleviate stress effects [66]. According to [58], exogenous GSH reduced the concentrations of Na⁺ and Cl^- in the roots and leaves of plants exposed to salt stress. These observations indicate that exogenous GSH application to salt-stressed tomato plants confers salinity resistance in young plants by regulating ion absorption. In Addition, Many researchers have reported that exogenous GSH can significantly delay chlorophyll reduction in leaves of contaminated plants and enhance their resistance to toxic elements [67,68,69].

According to the study by [61], GSH can alleviate Cd toxicity and mitigate Cd-induced damage to the ultrastructure of leaf and root cells. Furthermore, exogenous GSH mitigates oxidative stress and enhances antioxidant protection in young wheat plants exposed to lead-related stress [70]. In addition, exposure to exogenous GSH greatly facilitated resistance to mercury (Hg) during seed germination and seedling development in Arabidopsis thaliana, tobacco, and pepper [71].

Under water stress, across low to high concentrations of PEG, we observe that the most effective dose for mitigating the harmful effects of stress is 25 μM/L of GSH, and that a high dose can slow or even delay seed germination (Figure 3). Therefore, according to [31], in plants exposed to water stress, the external application of GSH increased relative water content, membrane stability, and PSII photosynthetic efficiency in Phaseolus vulgaris. Exogenous GSH application promoted the growth of young mung bean plants and their physiological performance under water stress. GSH ensured osmotic balance by controlling proline levels, which optimised the water status of plant tissues [72].

At comparable osmotic potentials, germination of C. siliqua is more affected by osmotic stress induced by PEG than by that induced by salt, as the species germinated at −1.6 MPa under saline conditions (Table 1). This is consistent with our previous findings for this species [16,42], as well as for other species such as Cichorium intybus [73]. According to [74], at high salt concentrations, germination is impeded because water absorption during imbibition is reduced, presumably due to toxicity induced by the accumulation of Na⁺ and Cl^- ions. Thus, both NaCl and PEG-induced osmotic stress negatively affected seed germination and early seedling growth in Ceratonia siliqua [16,75].

Applications of hydrotime analysis to the germination of C. siliqua seeds at different GSH concentrations revealed distinct behaviours under salt and osmotic stress at the optimal germination temperature tested. Under saline stress conditions, the Ψ_b value was much lower (-0.45 MPa) than under water stress (-0.90 MPa) at a moderate concentration of 25 μmol of GSH (Table 1). Thus, the application of a moderate GSH concentration resulted in a significant reduction in Ψ_b, indicating an improvement in the osmotic resistance of Ceratonia siliqua seeds. Under normal and abiotic stress conditions, GSH stimulates growth, development, gene expression, and protein activation thanks to its various properties [76].

For salt stress, the θ_H value decreased from 2.614 MPa days °C to 0.300 MPa days, and from 6.33 MPa days to 2.777 MPa days for osmotic stress. In fact, according to [77], the θ_H value is an estimate of seed germination rate and varies with the physiological state of the seeds. In principle, C. siliqua germinates more quickly at a moderate GSH concentration of 25 µmol L⁻¹. Therefore, focusing on Ceratonia siliqua seeds can promote crop development even under severe water and salt stress conditions [74]. With increased water and salt stress and a higher GSH concentration, the value of θ_H increases (2.181 MPa days °C under salt stress and 4.615 MPa days °C under water stress), indicating a slowdown in germination. The value of Ψ_b becomes low under salt stress (-0.301 MPa) and lower under water stress (-0.615 MPa) (Table 2), indicating a decrease in the germination capacity of seeds under stress conditions. These results suggest that an excess of GSH may have an inhibitory effect, probably due to metabolic imbalance or redox overload. This confirms the presence of an optimal threshold beyond which GSH no longer stimulates germination and may even inhibit it. According to [57], treating seeds with 50 µmol L^–1 GSH reduces lead-induced stress and alters the redox balance, and broccoli germination decreases significantly as GSH concentration increases to 75 µmol L^–1. The application of GSH to seeds promoted their viability and precipitated the premature emergence of seedlings

A strong negative correlation was observed between treatment and FGP (Figure 5), indicating that carob germination capacity decreases as stress intensity increases [16,42]. Indeed, as water potential becomes more negative or salinity levels increase, the ability of seeds to take up water and trigger the metabolic processes essential for germination is significantly reduced. FGP and stress are negatively related, meaning that abiotic factors inhibit both seed vigour and germination viability [74]. It is noted that FGP and GSH concentrations are weakly correlated, indicating that when GSH exceeds a threshold, FGP gradually decreases, suggesting an inhibitory effect at high doses [57]. Therefore, although exogenous GSH application can accelerate germination and improve stress tolerance at low doses, it can be inhibitory at high doses. This inhibition may be due to an imbalance in cellular redox status or a disruption of the metabolic processes necessary for germination.

4. Materials and Methods

4.1. Seed Harvesting

A total of seven thousand six hundred (7600) seeds of C. siliqua L. were collected from a natural population located in central Tunisia (10°98′77′′E, 35°18′04′′N). According to [48]’s Mediterranean bioclimatic classification, the collection site experiences a lower semi-arid climate, with an average annual rainfall of 300 mm.

The seeds were collected from many carob individuals growing in sandy-loam soil, an edaphic factor that could affect their physiology. Morphologically, the seeds have an average length of 0.6±0.01 cm. The weight of one hundred seeds, a characteristic generally related to vigor and energy reserves, was 19.202±0.03 g. This represents an estimate of 5.201±0.01 seeds per gram.

4.2. Reagents

Glutathione (GSH; γ-glutamyl-cysteinyl-glycine) is a low-molecular-weight, water-soluble thiol compound and one of the major non-enzymatic antioxidants involved in maintaining cellular redox homeostasis in plants. It plays a crucial role in protecting plant cells against oxidative stress by regulating reactive oxygen species (ROS) accumulation and participating in antioxidant defense mechanisms. In seeds, GSH contributes to the regulation of redox status during imbibition and germination, processes that are closely associated with metabolic activation, enzyme regulation, and embryo development. Moreover, GSH serves as a precursor of phytochelatins and is involved in various physiological processes, including cell division, growth regulation, and stress tolerance. Therefore, due to its potential role in enhancing seed physiological performance and promoting germination, GSH was selected as a seed-priming agent in this study. All chemicals used in the experiments were of analytical grade.

4.3. Experiment Setup and Seed Priming

Carob seeds were surface sterilized for five minutes using sodium hypochlorite (0.5%). To overcome seed dormancy, all C. siliqua seeds were immersed in 98% concentrated H₂SO₄ for 20 min at 24°C, then washed thoroughly with running water and dried on paper towels, as described by [49]. Two days later, 307.3 mg of GSH was dissolved in 1000 mL of distilled water to obtain a 1 mM GSH stock. Four concentrations of GSH were then prepared using this stock solution. The solutions were 25, 50, 100, and 200 µmol L^-1. C. siliqua seeds were primed for 12 h in the dark at 25°C and then washed thoroughly with autoclaved distilled water and dried on paper towels. All seeds were tested in 8 replicates (each containing 25 seeds, due to the relatively large size of the seeds). They were placed in 9 cm diameter Petri dishes containing two layers of blotting paper moistened with 10 mL of distilled water and incubated for 21 days at the optimal germination temperature of 25°C, as previously reported for carob seeds [16]. The dishes were sealed with greaseproof paper to prevent evapotranspiration. Incubation was carried out in a dark incubator (LMS refrigerated incubator, LMS Ltd, Kent, UK). A seed is considered to have germinated when the radical is approximately 2 mm long [50].

4.4. Effect of Temperature and Osmotic Potential on Seed Germination

To study the effects of water and salt stress on the germination behavior of the three accessions, five treatments were tested. germinative behavior of Ceratonia siliqua seeds treated with different formulations of GSH, and to determine the most effective concentration for seed germination under these different osmotic stress conditions, four osmotic potential osmotic potentials in NaCl or PEG were studied (0, -0.5, -1 and -1.6 MPa) for each glutathione concentration, The PEG solutions were prepared according to the formula of Michel and Kaufmann (1973). Osmotic potential the osmotic potential induced by PEG₆₀₀₀ or NaCl was then checked using a VAPRO 5520 pressure osmometer (Wescor Co) to measure the molality (MPa). to measure the molality (MPa). For solutions prepared according to the osmotic pressure formula, the water potentials were measured with an osmometer. Under dark conditions, seeds were subjected to water and salt stress induced by PEG ₆₀₀₀ and NaCl solutions, respectively, with 10 mL of each solution applied to moisten the blotting paper, under the same incubation conditions described above. Germination was assessed daily for 21 days. The mean time germination (MTG) and germination rates (GR) were measured.

4.5. Methods of Germination Expression

During the seed incubation, the following germination variables were calculated: The number of days to first germination (DGI), mean germination time (MTG), final germination percentage (FGP), maximum germination index (IG max). Germination speed and seed quality were assessed using the germination value (VG). The day of maximum germination or (PGD) is the day that simply corresponds to the most significant number of seeds germinated in your experiment. The MTG was determined using the following formula (1):

$$\mathrm{M}\mathrm{T}\mathrm{G}=\sum (\mathrm{n}\mathrm{i}\times \mathrm{d}\mathrm{i})/\mathrm{N}$$

(1)

where n is the seed number germinated at day i, N is the total number of germinated seeds and d is the incubation period.

Using regressions based on hydrotime models, we study the relationship between temperature and water stress and salt stress. Germination data were analyzed using the thermal time model[51].

In the formula (2) and (3) below:

$$\mathsf{\theta }\mathrm{T}\left(\mathrm{g}\right)=(\mathrm{T}-\mathrm{T}\mathrm{b})\mathrm{t}\mathrm{g}$$

(2)

$$\mathrm{G}\mathrm{R}\mathrm{g}=1/\mathrm{t}\mathrm{g}=(\mathrm{T}-\mathrm{T}\mathrm{b})/\mathsf{\theta }\mathrm{T}\left(\mathrm{g}\right)$$

(3)

where: θT is the thermal time (°C days), T is the actual temperature of the germination test (°C), Tb is the minimum constant base temperature in the sub-optimal temperature range (°C), tg is the time of germination at the g% fraction, with an intercept on the T axis of Tb and a slope of 1/tg, GRg is a linear function of T above T_b

The hydrotime model was applied to determine germination time under water and salt stress conditions and at different GSH concentrations (0, 25, 50, 100 and 200 µmol/l). According to Bradford (2002), hydrological time (MPa-day) is used to indicate the response of germination to reduced water potential.

The Hydrotime constant, θH, is calculated as follows (4):

$$\mathsf{\theta }\mathrm{H}=\left(\mathsf{\psi }-\mathsf{\psi }\mathrm{b}\left(\mathrm{g}\right)\right)\mathrm{t}\mathrm{g}$$

(4)

where θH is the hydrotime constant (MPa-day), Ψ is the actual water potential of the germination medium. tg is the corresponding fraction g's germination time (day), Ψb(g) is the basic water potential that will prevent the g fraction from germinating.

4.6. Statistical Analysis

A two-way analysis of variance was performed to assess the effects of the main factors (NaCl, PEG₆₀₀₀ and temperature) and their interaction with the final germination percentage and seed viability. The Differences between GSH treatments were determined by a Tukey post hoc test at p<0.05. The Methods for analysing the hydrotime models used in this study have been detailed in [52] and [53]. Statistical analyses were performed using IBM SPSS Statistics version 30.0 (IBM Corp., Armonk, NY, USA) and Excel software. The correlation matrix showed the relationships among seed germination parameters under water and salt stress using the pheatmap package in R [17]. The Figures were drawn using Origin Pro 8.5 software.

5. Conclusions

This study demonstrates the positive effects of glutathione on the germination of Ceratonia siliqua seeds. Low concentrations of glutathione (25 to 50 μM) positively affected carob seed germination compared with high concentrations (100 to 200 μM), with 25 μM as the optimal concentration, as confirmed by the hydrotime model. This concentration also resulted in the highest germination rates. Furthermore, at low concentrations, glutathione can stimulate seed germination even in the presence of NaCl and PEG₆₀₀₀. Indeed, treatment with 25 μM glutathione has been shown to ensure optimal germination while significantly boosting seed vigour, thereby reducing the average germination time and promoting faster germination.

Beyond its physiological significance, this ability of low-dose glutathione priming to sustain germination under both saline and osmotic (drought-simulating) constraints is of direct practical relevance for the restoration of degraded ecosystems in North Africa and the wider Mediterranean basin, where soil salinization and increasingly erratic rainfall are among the main constraints limiting the natural regeneration of native woody species. As a drought- and salinity-tolerant Fabaceae already well adapted to arid and semi-arid Mediterranean bioclimates, Ceratonia siliqua is a strong candidate species for afforestation and reforestation programmes on marginal, saline-prone, or water-limited soils. Improving the germination performance and seedling vigour of carob through simple, low-cost, and environmentally benign seed priming treatments such as glutathione could therefore enhance nursery production and field establishment success, ultimately supporting large-scale ecological restoration and land rehabilitation strategies in degraded North African and Mediterranean landscapes.