Alleviation of Salt Stress on the Growth and Active Constituents of Chamomile (Matricaria chamomilla L.) Using Glutathione and Hydrogen Peroxide

1 Introduction

Salinity is a significant environmental stressor that negatively impacts plant growth, productivity, and ionic balance, particularly in semi-arid and arid regions [1,2,3]. Excessive Na+ and Cl can disrupt the electron transport system and increase the production of reactive oxygen species (ROS). This ultimately damages cellular components, proteins, lipids, and nucleic acids, disturbing photosynthesis, membrane stability, and metabolic processes. To mitigate this damage, plants employ antioxidant defense systems to regulate ROS levels [4]. Enhancing crops’ resistance to salt stress is vital for their survival and yield optimization, both of which are vital for global food security. While the general effects of salinity stress on plant physiology are well established, its effect on medicinal and aromatic plants, particularly regarding the quality of bioactive compounds, remains insufficiently understood.

The economic importance of medicinal and aromatic plants in pharmaceuticals, food, and cosmetics highlights the need to expand their cultivation in diverse environments, even under challenging conditions [5]. Among these, Matricaria chamomilla L. (German chamomile) is a high-value medicinal plant widely recognized for its pharmacological properties. It contains over 120 secondary metabolites, with azulene and α-bisabolol as the main components of the volatile oils [6,7,8]. Furthermore, chamomile is effective against microbial infections and oxidative damage. It is famous for its anti-inflammatory, antispasmodic, and antioxidant properties [6,7,8,9,10]. It is also valued as an ornamental plant, where flower yield and uniformity are important commercial traits [11]. However, salinity stress can negatively impact floral development and the plant’s biochemical composition, ultimately diminishing both its therapeutic benefits and aesthetic value [12,13]. In Egypt, chamomile ranked as the second most significant medicinal crop in 2005, cultivated on approximately 9500 acres. The total production reached 8000 t, with 3000 t exported, generating around $5 million in revenue [14,15]. So, salinity stress not only threatens plant growth but also affects the quality of its bioactive constituents, directly impacting its commercial and medicinal value.

Understanding how plants tolerate salt stress is crucial for enhancing chamomile’s salt tolerance and cultivation. Under salinity stress, plants produce reactive oxygen species (ROS), including singlet oxygen (1O2), superoxide anion (O2•−), hydrogen peroxide (H2O2), and hydroxyl radical (•OH), leading to oxidative damage and disruption of cellular functions [16,17]. To mitigate this damage, plants employ several antioxidant mechanisms [18]. Among these, the ascorbic acid (AsA)-glutathione (GSH) cycle, which detoxifies ROS and eliminates H2O2 from the cytoplasm and various organelles, thereby maintaining cellular redox homeostasis and preserving photosynthetic efficiency [19]. Improving this system is an effective strategy to enhance photosynthetic efficiency, stabilize membranes, and manage ROS signaling under saline conditions [20]. Additionally, various amino acids accumulate as antioxidants in plant cells during salt stress, supporting growth, productivity, and adaptation. They regulate stomatal openings, facilitate ion transport, and serve as osmolytes, affecting enzyme activity, detoxifying heavy metals, and gene expression [21]. In this context, applying redox-active compounds externally may strengthen the intrinsic antioxidant system, enhancing the plant’s ability to keep reactive oxygen species (ROS) at signaling levels rather than harmful levels under salinity conditions. Glutathione is a natural antioxidant made up of three amino acids: glutamic acid, cysteine, and glycine. Also, it is an essential non-enzymatic antioxidant that plays a crucial role in maintaining cellular redox balance, protecting cells from oxidative stress, and aiding detoxification. Additionally, it supports plant growth under stress and plays an important role in essential oil production, metabolism, and constituents and fractions [21,22,23,24,25].

On the other hand, hydrogen peroxide (H2O2) is a reactive oxygen species (ROS) that functions as an elicitor, produced from molecular oxygen (O2). In chloroplasts, it is generated during metabolism to combat oxidative stress. It enhances the production of phenolic compounds and stimulates plant defenses against environmental stressors, microbes, and herbivores, while attracting pollinators. In low concentrations, H2O2 boosts antioxidant enzyme activity, strengthens cell walls, and promotes plant growth [26,27]. However, at high concentrations, it can trigger programmed cell death. H2O2 also regulates stomatal closure and root development and decreases lipid peroxidation and electrolyte leakage, aiding plant adaptation to saline conditions. Additionally, it enhances antioxidant activity and stimulates phenylalanine ammonia-lyase (PAL), impacting flavonoid content and stress responses. Leaf applications of H2O2 can improve defenses and reduce saline toxicity in plants [28,29].

While glutathione (GSH) and hydrogen peroxide (H2O2) have been individually reported to enhance plant stress tolerance, their comparative effectiveness as foliar treatments under different salinity levels has not yet been investigated in Matricaria chamomilla L. Furthermore, the extent to which each compound modulates redox regulation, antioxidant defense activation, and metabolic responses in this medicinal plant remains unclear. Therefore, this study aims to evaluate the effects of foliar application of glutathione (GSH) or hydrogen peroxide (H2O2) at different concentrations under varying salinity levels on growth, flower yield, and essential oil of Matricaria chamomilla L. Consequently, this study hypothesizes that foliar application of GSH or H2O2 may differentially modulate plant responses to salinity stress through redox regulation and stress signaling, resulting in distinct effects on growth performance, flower yield, and essential oil quality under saline conditions.

2 Materials and Methods

2.1 Experimental Layout and Treatments

This study was carried out at the El-Quassassin Horticulture Research Station Experimental Farm in Ismailia Governorate, Egypt (30°33′ N latitude, 31°56′ E longitude) during two successive growing seasons (2022/2023 and 2023/2024). The research aimed to evaluate the impact of salinity stress and its interaction with foliar applications of glutathione (GSH) and hydrogen peroxide (H2O2) on the production, growth, and bioactive constituents of Matricaria chamomilla L. The experimental setup included 28 treatment combinations, (4 salinity levels × 7 foliar treatments). Each combination was replicated three times, using fifteen pots/replicate. The experiment focused on two main factors: Irrigation water, using Nile River water as a control and three concentrations of well water, Irrigation water salinity, and anti-salinity treatments, which comprised different concentration of hydrogen peroxide and glutathione from Sigma Chemical (USA, St. Louis, MO), along with an untreated control group.

Scheme 1 demonstrated that a total of 420 uniform seedlings of Matricaria chamomilla L. were used to investigate the interactive effects of irrigation water salinity and anti-salinity treatments. The seedlings were divided into four irrigation water salinity levels (Factor A): 340 ppm (fresh farm water, serving as the control), 1500 ppm, 3000 ppm, and 4500 ppm, with 105 seedlings assigned to each salinity level. Within each salinity treatment, the plants were further subjected to anti-salinity applications (Each one was 45 seedlings, Factor B), which included glutathione (GSH) at concentrations of 1, 2, and 3 mM, and hydrogen peroxide (H2O2) at concentrations of 5, 10, and 20 mM as a foliar spray in addition to untreated control plants. Each treatment consisted of three replicates, with five pots per replicate. Each pot contained one plant and was considered an experimental unit. The pots were organized using a randomized complete block design (RCBD), and their positions were periodically re-randomized to reduce spatial environmental variability. The effects of exogenous antioxidant applications and salinity stress on plant performance were assessed separately and in combination using this experimental setup.

Scheme 1: Schematic representation of the experimental design, demonstrating the interaction between irrigation salinity levels (Factor A) and anti-salinity foliar treatments (Factor B) applied to Matricaria chamomilla L. seedlings.

Before sowing, the chemical and physical characteristics of the soil were examined using the procedure outlined by Page et al. [30] and the well water chemical characteristics was carried out according to the methods described by Fishman and Friedman [31], at the Soil and Water Laboratory, ARC, as shown in Table 1.

To prepare saline irrigation water, well water from Sorbium City in Ismailia Governorate, Egypt, with electrical conductivity of 7.31 ds/m (4678.4 ppm) was diluted with the farm’s fresh water (340 ppm) according to the following equation [32].

• ❖[SV]: volume from saline source in total volume TV
• ❖Q: required final salinity
• ❖[FV]: volume from fresh source in total volume TV
• ❖S/F: ratio of salinity content from both sources
• ❖S + F: sum of salinity of both sources

The experimental region has been classified as secure based on data from the Bilbiesed meteorological station. This data indicates an arid climate, characterized by low winter precipitation and hot, dry summers. Supplementary Table S1 and Fig. 1 illustrates the agrometeorological data for this region, located at a latitude of 30.66°, a longitude of 31.95°, and an elevation of 24.8 m. The data presented showcases the average monthly weather conditions for two growing seasons.

Figure 1: Monthly variations in maximum and minimum temperatures (°C), relative humidity (%), wind speed (km/day), and dew point (°C) during the 2022/2023-2023/2024 growing season in Bilbies, Egypt.

2.2 Experimental Procedures

Chamomile (Matricaria chamomilla L.) seeds were kindly provided by the Department of Medicinal and Aromatic Plants, Egypt’s Ministry of Agriculture, Agricultural Research Center. Nursery beds made of sandy loam soil were used to plant seeds on August 23rd during the winter of 2022/2023 and 2023/2024. After 45 days, uniform seedlings (10–15 cm tall) were individually transplanted into plastic pots.

Four hundred twenty plastic pots, each 30 cm in diameter and 24 cm in height, were filled with 12 kg of sandy loam soil and had drainage holes. Polyethylene sheets were placed beneath the pots to prevent root penetration into the soil and to control weed growth. After transplanting, plants were irrigated with Nile River water for approximately 20 days to ensure proper root system establishment and adequate shoot growth. Subsequently, saline water treatments were applied via irrigation.

The irrigation water salinity concentrations were: control (fresh Nile River water, 340 ppm), 1500, 3000, and 4500 ppm of well water with and without glutathione (1, 2, and 3 mM) and hydrogen peroxide (5, 10, and 20 mM). The concentrations of H2O2 and GSH were chosen based on literature and preliminary experiments [33,34].

Each pot received 1.930 L (at hold capacity) of saline water every five days from November to January, and every three days until May 1st. To prevent excessive salt accumulation, irrigation with saline water was alternated with farm water, following a ratio of three saline irrigations to one fresh water irrigation (3:1). The salinity treatments were introduced gradually to prevent osmotic shock in the plants [35]. Weekly measurements of the electrical conductivity (EC) of the water in the pots were taken to maintain a consistent salinity level. If the desired salinity levels were exceeded, leaching with water was conducted to remedy the situation [36]. Weekly monitoring of soil electrical conductivity (EC) confirmed that salinity levels remained within acceptable ranges around the targeted treatments, with minor fluctuations attributed to irrigation cycles. Detailed EC dynamics are presented in Supplementary Table S2. Starting 30 days after transplanting, foliar applications of glutathione (GSH) and hydrogen peroxide (H2O2) was performed at 30, 60, and 90 days after transplanting (DAT), corresponding to the vegetative stage and the initiation of flowering, resulting in a total of three applications. This schedule ensured sustained antioxidant activity, supporting plant growth, reducing stress, and facilitating nutrient uptake [37,38].

Foliar applications were conducted using a hand-held pressure sprayer that delivered a fine mist until runoff occurred, ensuring complete coverage of all aerial parts of the plants. The spray volume was approximately 200 mL plant−1, with slight adjustments based on plant size and developmental stage, to achieve uniform coverage without excessive runoff. A non-ionic surfactant, Tween-80, was added at a concentration of 0.03% (v/v) to enhance adhesion and promote even distribution of the solution on the plants. Distilled water with the same surfactant concentration was used to treat the control plants. Spraying was carried out in the early morning under mild environmental conditions (moderate temperature and low wind) to ensure a uniform application and minimize evaporation losses.

All cultural techniques, such as weed control, fertilization, and irrigation, were conducted in accordance with the recommended of [39]. Ammonium sulfate (20.5% N), calcium superphosphate (15.5% P2O5), and potassium sulfate (48% K2O) were applied as inorganic fertilizers at rates of 500 kg/fed (22.5 g/pot), 300 kg/fed (13.5 g/pot), and 100 kg/fed (4.5 g/pot), respectively. Calcium superphosphate was mixed with the soil before transplanting. Ammonium sulfate was divided into five equal doses (The first dose was applied one month after transplanting, while the second and third doses were applied one month after the first, at one-month intervals. The fourth and fifth doses were added after the second and fourth cuts, respectively). Potassium sulfate was applied in two equal doses, with the first dose in January and the second dose in March.

2.3 Data Recorded

2.3.1 Morphological Measurements

During the flowering stage, growth characteristics were recorded. The following parameters were measured for nine randomly selected plants from each experimental unit: plant height (cm), number of branches per plant, and fresh and dry weights (g) of chamomile plants.

2.3.2 Flower Production

Flower harvesting began 90 days after transplanting and continued at 10-day intervals, at the full bloom stage (when the petals were fully extended). The harvested flowers were allowed to air-dried in the shade under ambient conditions before further analysis, and the following data were recorded (number of flowers/plants, fresh and dry weights of flowers/plant (g)).

2.3.3 Essential Oil Production and Constituents

The percentage of essential oil content in dried chamomile flowers was determined according to the British Pharmacopoeia method [40].

Additionally, the oil yield (mL per plant) was calculated in terms of the ratio between the mass of essential oils and the initial biomass of the flower (g).

The composition analysis of the essential oil extracted during the second season was performed using gas-liquid chromatography (GLC). A BPX-5 capillary column (30 × 0.25 mm ID) was utilized for analyzing the essential oil samples at the Horticulture Research Institute. Nitrogen served as the carrier gas with a flow rate of 1 mL/min, while air and hydrogen flow rates were set at 330 mL/min and 30 mL/min, respectively. The split ratio was 1:10. The column temperature was increased at a rate of 10°C per minute, starting from 70°C and reaching 200°C, where it was held for 3 min. This maximum temperature was maintained for an additional 10 min before the column was cooled. The detector and injector temperatures were set at 300°C and 250°C, respectively. The analysis of the chromatograms allowed for the determination of the percentages of the main components in the essential oil. The essential oil constituents were identified by comparing their retention indices with those of authentic standards analyzed under identical operating conditions.

2.3.4 Standards Compounds

A set of standard compounds representing various chemical groups, each with a stated purity of 99% as verified by Gas Liquid Chromatography (GLC), was acquired from Sigma Chemical Co. in St. Louis, MO, USA. The chemical groups included are terpene hydrocarbons, ketones, alcohols, oxides, and esters.

2.3.5 Chemical Determinations

The protocol by Gao [41] was used to measure the chlorophyll and carotenoid content (mg/g fresh weight) of the terminal leaf, the third leaf at the tip of the plant. A 0.1 g sample of leaves was weighed, chopped, and placed in 20 mL of a 95:5 mixture of absolute ethanol and water. The leaf was incubated in the dark at room temperature (25°C) for 12 h until completely faded. The mixture was then filtered to retain the supernatant, which was used to measure absorbance at 665, 649, and 470 nm. The formats were as follows:

2.3.6 Proline Percent

Samples: Dry leaves were collected for analysis, with purified proline used for quantification. Reagents: Prepare acid-ninhydrin by dissolving 1.25 g of ninhydrin in 30 mL of glacial acetic acid and 20 mL of 6 M phosphoric acid. This reagent is stable for 24 h at 4°C. Procedure:

• 1.Homogenize 0.5 g of plant material in 10 mL of 3% sulfosalicylic acid and filter through Whatman #2 paper.
• 2.Mix 2 mL of the filtrate, 2 mL of ninhydrin, and 2 mL of glacial acetic acid in a test tube. Incubate at 100°C for 1 h and then cool in an ice bath.
• 3.Extract with 4 mL of toluene, mixing vigorously for 15–20 s, then aspirate the toluene phase.
• 4.Measure the absorbance at 520 nm using toluene as a blank, and determine proline concentration from a standard curve [42].

2.3.7 Nitrogen Content

Nitrogen content was evaluated in the digested solution based on the guidelines provided by the Association of Official Analytical Chemists [43].

2.3.8 Sodium and Potassium Content

Sodium and potassium contents were measured using a flame photometer, following the method outlined by [44].

2.3.9 Total Protein Percentage

The total protein percentage was calculated according to [43].

2.4 Statistical Analysis

Two factors were used for a factorial arrangement in a Randomized Complete Block Design (RCBD) experiment. The first one was irrigation water salinity at four levels. The second factor was foliar application of different concentrations of glutathione (GSH) and hydrogen peroxide (H2O2), compared to an untreated control (farm water). Additionally, One-way analysis of variance (ANOVA) was used to evaluate the effect of different saline irrigation levels (340, 1500, and 4500 ppm), with or without a foliar application of 2 mM glutathione, on the essential oil composition of Matricaria chamomilla L. during the 2024 growing season. The treatments were replicated three times.

Before analysis, the normality of the data was assessed using the Shapiro–Wilk test, which confirmed that the data followed a normal distribution. Homogeneity of error variances across years was evaluated using [45], supporting the validity of analyzing each year separately for most traits. All data were analyzed using the COSTAT software package, and ANOVA (analysis of variance) for RCBD module was calculated. The use of Duncan’s Multiple Range Test (DMRT) at the 0.05 probability level was used to compare treatment means, as described by [46].

3 Results

3.1 Effect of Salinity, Glutathione, and Hydrogen Peroxide on Growth Characteristics

Salinity stress induced by irrigation with saline well water at different levels adversely influenced all studied growth traits of M. chamomilla (Table 2). The reduction in plant height, number of branches/plants, herb fresh and dry weights induced by saline well water at 4500 ppm reached 38.89 and 37.38%, 32.38 and 30.38%, 43.34 and 42.04%, and 48.70 and 45.69%, respectively, in both seasons, compared with the control (340 ppm), which produced the highest values. Foliar application with GSH and H2O2 at different concentrations enhanced growth traits compared with control plants (Table 2). GSH at 2 mM surpassed other treatments with significant differences in plant height and herb fresh weight in both seasons. For the number of branches/plant and herb dry weight, no significant influence was observed between H2O2 at 5 mM and GSH at 2 mM. Regarding the combined effect of different levels of saline well water and foliar application of GSH or H2O2, as protective agents against salinity stress, Table 2 demonstrated that GSH and H2O2 mitigated the adverse effects of salinity stress in M. chamomilla plants. The results indicated higher growth values for plants irrigated with different saline well water and treated with GSH and H2O2 compared with unstressed plants (control) and plants exposed to salinity without foliar application. The obtained values in all studied growth traits of plants irrigated with saline well water at 1500 ppm + foliar application with GSH at 2 mM were significantly higher than those of the control plants. Irrigation with saline water at 3000 ppm + foliar application with GSH at 2 mM increased growth characteristics compared with the control, but these increments were insignificant. In addition, H2O2 at a low concentration (5 mM) enhanced growth traits when combined with irrigation with saline well water at 1500 or 3000 ppm.

3.2 Effect of Salinity, Glutathione, and Hydrogen Peroxide on Flower Parameters

A marked reduction in flower parameters was observed with increasing levels of saline well water (Table 3). Irrigation with saline well water at 4500 ppm reduced the number of flowers/plants by 60.15 and 59.32%, the fresh weight of flowers/plant by 56.14 and 56.13%, and the dry weight of flowers/plant by 59.76 and 58.70%, compared with the control in both seasons, respectively. Table 3 showed that both GSH and H2O2 enhanced the flower parameters of M. chamomilla compared with the control. Foliar application with 2 mM GSH was superior to other treatments and resulted in the highest significant values for all the studied flower parameters. A significant enhancement was achieved when foliar application with GSH or H2O2 was combined with saline well water irrigation at different levels (Table 3). No significant impact was observed between GSH at 2 mM and H2O2 at 5 mM when applied with saline well water at 1500 ppm in the case of the number of flowers/plant and fresh weight of flowers/plant, as the obtained values were higher than the control and plants treated with saline well water only. Dry weight of flowers/plant enhanced by GSH at 2 mM under irrigation with saline well water compared with the control and non-foliar application plants.

3.3 Effect of Salinity, Glutathione, and Hydrogen Peroxide on Chemical Composition

Data in Table 4, Table 5 and Table 6 revealed that the lowest values of total chlorophyll (mg/g FW), carotenoid content (mg/g FW), carbohydrates (%), nitrogen (%), protein (%), potassium (%) and K+/Na+ ratio were found in the leaves when the plants were irrigated with the highest level of saline well water (4500 ppm). Moreover, both sodium (%) and proline (mg/g DW) recorded the highest values. All chemical constituents under study showed statistically significant responses to different salinity protective agent levels. GSH at 2 mM was superior, resulting in the highest significant values for all studied chemical constituents, except sodium, which had the lowest values. Proline accumulation in plant tissues was enhanced by H2O2 (20 mM) with no significant differences relative to other treatments. At 1500 ppm saline well water, GSH at 2 mM results in higher values than the control on total chlorophyll, carotenoid content, carbohydrates, nitrogen, protein, potassium and K+/Na+. This was associated with a reduction in sodium % as the lowest values were recorded in both seasons. Under all levels of saline well water, both GSH and H2O2 enhanced the accumulation of the foliar-applied plants compared to control and stressed plants only (without foliar application). H2O2 at 20 mM produced the highest non-significant values of proline under the highest levels of salinity (4500 ppm) in both seasons.

3.4 Effect of Salinity, Glutathione, and Hydrogen Peroxide on Essential Oil Content and Yield

When the salinity levels were increased (up to 4500 ppm), the percentage of essential oil increased, whereas the yield reduced (Table 7). As shown in Table 7, all concentrations of GSH and H2O2 enhanced essential oil percentage and yield/plant compared with the control. GSH at 2 mM produced the highest values, which were significant compared with control in both seasons. The combined treatment of GSH at 2 mM + irrigation with saline well water at 4500 ppm resulted in the highest essential oil percentage in both seasons compared with the control and stressed plants without foliar application of GSH or H2O2. Under other saline well water levels, GSH at 2 mM also enhanced the essential oil percentage, but the values were less than those of the outstanding treatment. On the other hand, saline well water at 1500 ppm in addition to foliar application with GSH at 2 mM produced the highest essential oil yield/plant.

3.5 Effect of Salinity, Glutathione, and Hydrogen Peroxide on Essential Oil Composition

Table 8 presents the active constituents of chamomile essential oil extracted from dried chamomile flowers using gas-liquid chromatography (GLC). It was significantly affected by saline irrigation levels (340 as control, 1500, 3000 and 4500 ppm), foliar application of Glutathione (GSH) at 2 mM, and their interactions. Some of these components were identified, and the others were absent due to different treatments. The highest number of volatile components (20) was identified in the chamomile oil under irrigation with saline well water at 1500 ppm. In control plants (340 ppm), only 18 components were identified, with the absence of menthone and caryophyllene oxide. The main constituents in control plants were α-bisabolol oxide A (61.49%), trans-β-farnesene (7.96%), α-bisabolol (5.78%), bisabolone oxide B (5.13%), chamazulene (2.19%), bisabolol oxide B (0.98%).

Among the identified sesquiterpene constituents, α-bisabolol oxide A consistently represented the highest proportion in both control and treated plants. Farnesene was the second most abundant compound in most treatments, followed by bisabolone oxide B. Salinity stress, applied alone or in combination with glutathione (GSH), did not result in qualitative changes in the essential oil composition; however, it significantly affected the quantitative distribution of individual components. Under severe salinity stress (4500 ppm without GSH), α-bisabolol oxide A reached its maximum value of 62.42%, along with increased levels of bisabolone oxide B (6.13%) and chamazulene (2.40%), while the farnesene content declined. Additionally, caryophyllene oxide and δ-cadinene were not detected under this treatment. In contrast, monoterpene content peaked at 0.86% in control plants treated with GSH. The combination of moderate salinity (1500 ppm) and GSH led to the highest accumulation of oxygenated sesquiterpenes (79.26%), which included peak values for α-bisabolol oxide A (65.83%), bisabolol oxide B (1.80%), bisabolone oxide B (7.49%), and chamazulene (2.74%). Conversely, the lowest diversity of volatile compounds was observed under 1500 ppm salinity combined with 2 mM GSH, where several constituents, such as menthone, caryophyllene oxide, δ-cadinene, 2-pentylfuran, ageratochromene, 1,8-cineole, and methyl hexadecanoate, were not detected. These findings suggest that salinity stress and exogenous GSH primarily modulate the biosynthesis and accumulation of specific sesquiterpene fractions, rather than altering the overall qualitative composition of chamomile essential oil.

From the authors’ perspective, two relatively underreported oxygenated components Z-spiroether and cis-bicycloether deserve attention due to their consistent presence and dynamic response to treatments. Z-spiroether increased notably to 4.02% under 1500 ppm plus GSH, while cis-bicycloether rose to 1.63% in the same treatment. Although present in low amounts, their changes reflect the plant’s metabolic adaptation to combined oxidative and salt stress. The authors suggest that these minor constituents, often overlooked, may play a significant role in the aromatic complexity and possibly in the bioactivity of the essential oil with stress conditions.

4 Discussion

The present study demonstrates that salinity stress constrains the growth and productivity of Matricaria chamomilla in a concentration-dependent manner, revealing a physiological threshold beyond which adaptive mechanisms become insufficient. The use of diluted saline well water, rather than artificially prepared salt solutions, enhances the ecological relevance of the findings by simulating realistic field conditions. At 1500 ppm, plants exhibited relatively minor reductions compared to the control (340 ppm), indicating a capacity to tolerate mild osmotic stress. However, at ≥3000 ppm, a marked decline in growth and productivity was observed, reflecting a transition from osmotic stress to ion toxicity. This shift is primarily associated with excessive Na+ accumulation and disruption of the K+/Na+ balance, leading to impaired enzymatic activity, membrane destabilization, and reduced photosynthetic efficiency. These findings highlight that salinity tolerance in chamomile is governed not only by osmotic adjustment but also by the plant’s ability to maintain ionic and redox homeostasis [1,23,24,47,48].

The observed growth inhibition under high salinity conditions can be largely attributed to oxidative stress. Salinity disrupts the electron transport chain in chloroplasts, resulting in the overproduction of reactive oxygen species (ROS), which damage proteins, lipids, and cellular membranes. Consequently, plant tolerance depends on the efficiency of antioxidant defense systems that regulate redox balance. This shift suggests a strategic reallocation of carbon toward secondary metabolism as part of the plant’s adaptive response. The enhanced accumulation of oxygenated sesquiterpenes is likely linked to the stimulation of the mevalonate (MVA) pathway, which produces farnesyl diphosphate as a key precursor [49,50]. Salinity stress also induced significant metabolic reprogramming. While vegetative growth parameters declined, the accumulation of protective metabolites such as proline and essential oil constituents increased [4,51]. The methylerythritol phosphate (MEP) pathway in plastids generates monoterpenes and related antioxidants. Additionally, carbohydrate metabolism is also affected by saline conditions, with soluble sugars temporarily increasing to act as osmotic regulators [52,53].

Consistent with previous studies on medicinal plants, salinity altered essential oil composition without necessarily inducing qualitative changes. Instead, it modulated the relative abundance of specific compounds, potentially through its effects on enzymatic activity and glandular secretion [53]. This explains the observed increase in certain bioactive constituents despite reductions in overall biomass and oil yield. In this regard, Khalid and Ahmed [54] showed an enhancement of the essential oil constituents in Nigella sativa under saline irrigation. Razmjoo et al. [55] found similar responses in chamomile, and Cordovilla et al. [56] found that salinity levels above 4 ds·m−1 increased essential oil percentages due to reduced leaf surface area and altered oil gland secretion. Despite the reduced oil yield, a high content of active compounds has been found in Mentha spp. under salt stress [57]. Moreover, changes in plant dry matter during saline irrigation are correlated with variations in the essential oil yield [58].

Exogenous application of glutathione (GSH) played a crucial role in alleviating salinity-induced stress. As a key component of the AsA–GSH cycle, GSH contributes to ROS 1detoxification, maintains cellular redox balance, and stabilizes membrane structures [58,59]. Additionally, it promotes the accumulation of Osmo-protectants like proline and soluble sugars, which help with osmotic adjustment and sustain metabolic activity under saline conditions. Our research indicates that a foliar application of GSH at 2 mM 2 mM significantly enhances growth performance, flower yield, and essential oil production under salinity stress. These improvements are likely associated with its role in regulating antioxidant enzyme systems and reducing oxidative damage, thereby preserving cellular functions under stress [7,20,60,61,62]. Furthermore, glutathione can enhance oil quality and improve nitrogen assimilation in saline environments. Thus, applying GSH foliarly can be an effective strategy for enhancing chamomile tolerance to salinity by improving metabolic stability, oil quality, and nutrient assimilation.

Hydrogen peroxide (H2O2) exhibits a concentration-dependent dual behavior. At a concentration of 5 mM, H2O2 functions as a signaling molecule, which primes antioxidant defenses, activates MAPK cascades, and enhances potassium uptake, nitrogen assimilation, and photosynthetic efficiency. However, at higher concentrations, it can act as a damaging oxidant, overwhelming detoxification systems and accelerating lipid peroxidation. This hermetic response emphasizes the importance of carefully regulating reactive oxygen species (ROS) signaling to optimize stress tolerance [56,57].

Exogenous application of glutathione (GSH) and optimal hydrogen peroxide (H2O2) enhances essential oil composition by redirecting carbon flux toward protective secondary metabolites, even under compromised plant growth [63]. This highlights the importance of managing oxidative stress and redox signaling to maintain plant quality under saline conditions. The treatments promote the accumulation of oxygenated sesquiterpenes, linking redox regulation with secondary metabolism [64]. Additionally, chamomile’s tolerance to salinity appears to depend more on maintaining cellular redox homeostasis than on osmotic adjustment, emphasizing oxidative signaling as a key mechanism in stress resilience [52,56]. Although enzymatic antioxidant activities were not directly assessed, the observed physiological responses strongly suggest the activation of redox-regulating pathways. The results indicate that chamomile’s salinity tolerance is primarily linked to its ability to maintain redox homeostasis rather than to osmotic adjustment.

5 Conclusions

The foliar spraying of glutathione, particularly at 2 mM, represents an effective physiological strategy for enhancing the tolerance of Matricaria chamomilla L. plants to salinity stress up to 3000 ppm well water. It significantly reduced sodium accumulation, promoted potassium uptake, and improved the K+/Na+ ratio. Moreover, it enhanced the accumulation of proline and carbohydrates. Accordingly, it is recommended to foliar apply glutathione at a concentration of 2 mM to enhance the productivity, resilience, and growth of chamomile plants grown in environments affected by salt, particularly in marginal soils or areas irrigated with saline water. For these reasons, it is recommended to irrigate chamomile plants with saline water up to 3000 ppm and apply foliar treatments with glutathione (GSH) at 2 mM. This approach is expected to enhance growth characteristics and increase productivity.