Efficacy of Lippia alba Essential Oil in Alleviating Osmotic and Oxidative Stress in Salt-Affected Bean Plants

Abstract

Lippia alba (Mill.) N.E.Br. ex Britton and P. Wilson is a native plant of Colombia, widespread throughout Central and South America, used for thousands of years by pre-Columbian populations, who already knew the many beneficial properties of this species (e.g., antifungal, antibacterial, antiviral and anti-inflammatory activities). The essential oil of L. alba is rich in phytochemicals with antioxidant activity that could be very useful both for pharmacology and biotechnology application, such as the protection of horticultural crops sensitive to salinity. To enhance salt tolerance, seed-priming treatment with the essential oil of L. alba was performed. We evaluated the effect of this biostimulant on the response to salt stress in a sensitive bean species, Phaseolus acutifolius L. (cv Blue Tepary), native to Mexico, and used by pre-Columbians as well as nowadays. Bean seeds were primed in a solution of 0.5 mL/L of essential oil of L. alba, germinated and after 2 weeks of acclimation, the seedlings were subjected to salt stress, by watering with 40 mM and 80 mM NaCl solutions. Four weeks later, many biochemical parameters were evaluated in order to test the effects of the treatments on plant fitness. Primed seeds showed an increase in salt tolerance during germination, as well as primed plants revealing a higher water uptake, increased chlorophylls, proline content and salt tolerance index. The treatments also improved the Ca²⁺ concentration in the shoots of stressed primed plants, more quickly activating enzymatic responses to salinity—in particular superoxide dismutase, polyphenol oxidase, catalase, peroxidase and ascorbate peroxidase—compared to unprimed stressed plants. In conclusion, L. alba was found to be a strong elicitor of responses against osmotic and oxidative stress, as induced by salinity, suggesting the possibility of its future utilization in agriculture.

1. Introduction

Research on sustainable and environmentally friendly approaches to mitigate the damage caused by climate change foresees the use of plant biotechnologies, as well as eco-friendly solutions that reduce the application of chemical fertilizers and pesticides. Therefore, biostimulants are considered a useful tool to promote plant growth and yield under stress conditions [1].

Essential oils (EOs), obtained from medical and aromatic plants, are recognized as promising biostimulants, since they possess many beneficial properties, not only for human health but also for crops, due to their richness in antibacterial, antiviral, antimycotic and antioxidant compounds [2]. In recent years, the use of EOs has become widespread in the agronomic field, where these natural substances enhance the conservation of seeds and other plant products, may act in the biocontrol of diseases and play a role in increasing plant tolerance to biotic and abiotic stressors [1,3].

Even though various seed-priming and coating treatments are known, the most widely applied remain halopriming and osmopriming, while there are still few studies concerning the use of EOs as priming or coating agents [1,4]. The antioxidant properties of EOs represent a promising way to improve the tolerance of crops to stress, since they contain several biological compounds, such as phenols, flavonoids, tannins and saponins [2,5], that represent an important alternative, compared to the chemical biostimulants commonly used (e.g., NaCl, polyethylene glycol, etc.).

Many studies have focused on the effects of EOs during the seed germination phase [3,6], while information regarding the effects of EOs against osmotic and oxidative stress, during the different growth phases of horticultural crops, is still scarce.

In this study, an EO of Lippia alba (Mill.) N.E.Br. ex Britton and P. Wilson, an aromatic plant native to Colombia, was chosen as a biostimulant, due to its richness in phytochemicals with beneficial properties, known and used in traditional medicine by Central and South American populations for centuries [7]. In Colombia, L. alba is cultivated in various regions of the country but mainly in the Colombian Pacific, and it is used for healing purposes by the indigenous community of Cauca. It is known for its healing, antispasmodic, natural analgesic, digestive, diuretic, expectorant, laxant and antimicrobial properties. In addition to consuming it as an infusion and applying it to the skin, this plant is used for rituals to attract good luck, using the leaves or heels of this plant [8,9,10].

Studies, conducted by Stashenko and colleagues [7] and Benelli and colleagues [11], identified 93 compounds in this EO, including oxygenated monoterpenes (42.8%), monoterpene hydrocarbons (32.9%) and sesquiterpene hydrocarbons (21.9%), with carvone (35.2%), limonene (32.0%) and germacrene D (14.8%). In some breast cancer cells (i.e., SUM149 and MDA-MB-231), treatment with this EO decreased cell proliferation and increased antioxidant activity and lipid peroxidation, showing highly cytotoxic effects associated with the release of lactate dehydrogenase; vice versa, no effect was observed in the cell line MDA-MB-468. Moreover, high scavenger activity was detected when an EO of L. alba was used as a biostimulant in plants exposed to salinity, due to the presence of limonene and phenolic compounds [4].

The activation of salt stress responses induced by L. alba EO was tested on a rare and salt-sensitive bean species, Phaseolus acutifolius L. (cv Blue Tepary), rich in proteins and fiber, native to Mexico, and also cultivated in Central and South America for millennia [12]. Nowadays, this bean grows wild not only in Mexico, but also in Arizona, New Mexico and Texas, and it is cultivated in the arid regions of North America, Africa and South Asia [13]. The preservation of this species is important since it is tolerant to heat and drought stresses [14]. The natural resistance to drought and heat stress of this species will be useful in the future, since this neglected crop may represent an important genetic resource for the improvement of other legumes not tolerant to stress conditions.

Since salinity is one of the most dangerous abiotic stresses, studies on the salt stress response are considered very important by scientists. Salt causes a lot of damage to plant cells because the accumulation of toxic ions (i.e., Na⁺ and Cl⁻) in plants [15] leads to nutritional and hormonal imbalances, ionic toxicity, oxidative and osmotic stress and increased plant susceptibility to diseases [16], and finally it reduces plant growth and development, thus affecting the productivity and quality of crops worldwide [17]. Through the alteration of ion homeostasis, the imbalance of oxidant molecules leads to the activation of stress responses, which play an essential role for plant survival and fitness [18,19,20,21,22,23,24].

The response pathways to osmotic stress induced by salinity include the biosynthesis and accumulation of compatible osmolytes that reduce water loss, increase cell turgor and stabilize proteins and cell morphology [25]. The most important compatible osmolytes are β-alanine betaine, glycine betaine, proline and hydroxyproline, glycerol, mannitol, sorbitol, fructose and sucrose [26]. The combined effect of osmotic and ionic stresses reduces photosynthesis and increases the production of reactive oxygen species (ROS), highly toxic radicals that cause oxidative stress [27,28]. If the ROS concentration exceeds the antioxidant defenses, the main consequences for the plant will be alterations in redox homeostasis, damage to biological macromolecules (e.g., protein oxidation), membrane lipid peroxidation and DNA damage, and even the death of the plant cell [29].

Seed priming with different agents and plant acclimation can lead to a successful elicitation of stress responses [30,31], thus providing a useful tool for the survival of sensitive species. Due to the paucity of data on the effects of seed priming for improving the tolerance of P. acutifolius to salt stress, the present study was focused on the effect of L. alba EO as a seed-priming agent of bean plants exposed to salt conditions. In particular, the efficacy of EO in counteracting osmotic and oxidative stress was tested by analyzing some physiological responses involved in coping with the negative effects of stress on plants [32]. The data obtained from this research will be useful for future applications of this EO as a biostimulant agent for other salt-sensitive bean species exposed to salinity.

2. Materials and Methods

The reagents were analytical grade or equivalent and purchased from Merck (Burlington, MA, USA). During the experiments, all working solutions were prepared immediately before to use from stock reagents.

The essential oil (EO) of L. alba was purchased from Centro de Investigación de Excelencia—CENIVAM (http://quim.uis.edu.co/eisi/grupo/cenivam/#views/gm1/inicio (accessed on 18 February 2025)), Bucaramanga, Santander, Colombia. The EO was extracted from the fresh leaves and flowers of L. alba plants and the extraction process performed using a microwave-assisted hydrodistillation method, described by Stashenko et al. [7].

The EO was kept in a glass bottle in the dark at −20 °C and stabilized at room temperature (RT) before the treatment; the composition of the oil has already been characterized by Benelli et al. [11].

2.1. Determination of Halotolerance and Seed-Priming Treatment

Seeds of Phaseolus acutifolius L., cv. Blue Tepary (Figure 1), were bought in a specialized store near the University of Rome Tor Vergata, and stored at RT until seed-priming treatment. All bean seeds used in the experiments were surface-sterilized using 70% ethanol for 2 min, immersed in a solution of 1% NaClO for 3 min and rinsed in double-distilled water.

The best priming agent was determined in our previous study [4]; according to those preliminary tests, seeds of P. acutifolius were primed with 0.5 mL/L of the EO of L. alba. Seed-priming treatment was carried out according to the reported protocol, i.e., surface-sterilized seeds were imbibed in 50 mL of 0.5 mL/L of the EO of L. alba for 24 h at RT. At the end of the treatment, seeds were rinsed with double-distilled water and air-dried at RT for 48 h. The same procedure was performed for unprimed seeds (control, CTRL), where 50 mL of double-distilled water was used instead of 50 mL of 0.5 mL/L of the EO of L. alba.

The seed halotolerance test was performed using a dose–response curve. Six bean seeds were placed in Petri dishes, on Whatman Grade 1 filter paper imbibed with 15 mL of water or salt solutions at increasing concentrations (0 mM, 40 mM, 80 mM and 160 mM NaCl). After 10 days of incubation in the dark at RT, the germination rates of primed and unprimed seeds were assessed; the NaCl concentration that significantly reduced seed germinability was considered as the threshold of halotolerance.

2.2. Plant Growth Conditions

The seeds were primed in 50 mL of 0.5 mL/L of the EO of L. alba for 24 h at RT, rinsed with double-distilled water and then air-dried at RT for 48 h up to the original moisture content (16.5%). After priming treatment, bean seeds were stored at +4 °C until the experiments.

Primed and unprimed seeds were germinated in Petri dishes on Whatman Grade 1 filter paper, soaked with 15 mL of double-distilled water; after 10 days of incubation in dark at RT, the germinated seeds were sown in plastic pots (15 cm diameter, 4 seeds per pot), containing about 300 g of non-saline soil (COMPO SANA^® COMPACT, Münster, Germany). Soil characteristics: pH 6.5; dry bulk density 150 kg/m³; electrical conductivity (EC): 0.50 dS/m; porosity 90% v/v. Soil components: neutral sphagnum peat, perlite (< 5%), composted green soil improver).

Two growth cycles were managed, using 3 pots for each treatment (6 pots, 24 plants per treatment at the end of the experiments). During the experimental greenhouse trial, the plants were grown under natural sunlight (Daily Light Integral: 89.5 mmol/m² day ± 13.8 mmol/m² day) at a temperature of 26.1 °C ± 4.0 °C. Growth conditions were monitored daily using a multi-parameter sensor (Flower Care—HHCCJCY01HHCC—HHCC Plant Technology Co., Ltd., Stuttgart, Germany).

Bean seedlings were acclimated for 14 days before the onset of salt stress. Saline water consisted of the following solutions: 40 mM NaCl (4.2 dS/m; moderately saline), 80 mM NaCl (EC: 8.4 dS/m; high saline). The pots were randomly assigned to experimental groups: (1) unprimed plants (controls, CTRLs) irrigated with tap water (EC: 0.6 dS/m), (2) unprimed plants irrigated with saline solutions, (3) primed plants irrigated with tap water, (4) primed plants irrigated with saline solutions. Plant irrigation consisted of 100 mL of tap water or saline solution every 48 h for 4 weeks.

2.3. Water Content of Soil, Tolerance Index and Collection of Plants

After 6 weeks of growth, the gravimetric water content (GWC) of the soil and the salt tolerance index (STI) of the primed and unprimed plants were estimated.

The water uptake of the roots was evaluated in pots with and without plants (blank) at the end of the experiments by determining the GWC of the soil, according to the method described by Santangeli et al. [32], using the following formula:

$$GWC (\%)=\left[\right(f.w.-d.w.)/(d.w.\left)\right]\times 100$$

where: f.w. = fresh weight of soil; d.w. = dry weight of soil.

The salt tolerance index (STI) of plants was calculated according to Idrees et al. [33] using the following formula:

$$STI (\%)=(Average root length in stress/Average root length in control)\times 100$$

The shoots and roots of primed and unprimed plants were collected (0.2 g of fresh weight), frozen by dipping the samples in liquid nitrogen and stored at -20 °C until further tests.

2.4. Analyses of Calcium, Chlorophylls, Proline and Carbohydrates in Plants

The quantification of free intracellular Ca²⁺ in the shoots and roots of plants were carried out based on Borromeo et al. [34], using 0.2 g of frozen material homogenized in liquid nitrogen and resuspended in 1.5 mL of tricine buffer (0.2 M pH 8). The calcium concentration was evaluated with the Calcium Assay Colorimetric Kit (Abcam, Cambridge, UK—ab272527; www.abcam.com/ab272527, accessed on 15 February 2024. Detection range of kit: 20–5000 µM; Ca²⁺ standard concentrations: 0.04, 0.08, 0.12, 0.16, 0.20 mg mL⁻¹) and a multimode microplate reader set at 612 nm (Spark^® Multimode Microplate Reader—Tecan, Switzerland). The data are expressed as µg Ca²⁺ mg f.w.⁻¹.

The quantification of chlorophylls and proline required the same samples’ preparation: 0.2 g of frozen material was homogenized in liquid nitrogen and resuspended in 5 mL of 95% ethanol. Samples were incubated in the dark overnight (ON) at +4 °C then centrifuged for 15 min at 8000× g, and the supernatants were stored at −20 °C until the analyses.

To quantify the chlorophyll content, the method of Stassinos et al. [31] was adopted, and the absorbances of the supernatants were evaluated by a spectrophotometer (VARIAN Cary 50 Bio, Santa Clara, CA, USA) at 664.1 nm (chlorophyll a) and 648.6 nm (chlorophyll b). The concentration of total photosynthetic pigments was calculated according to Lichtenthaler [35], using the following formula:

$$Total Chl (\mu g·g {f.w.}^{-1})=\left(5.24·{Abs}_{664.1}+22.24·{Abs}_{648.6}\right)\times \left({\displaystyle \frac{V_e}{W}}\right)$$

where V_e = volume used for extraction (mL); W = sample weight (g).

The quantification of proline was evaluated as described by Rossi et al. [36], and the absorbances of the samples were detected at 520 nm by a spectrophotometer (VARIAN Cary 50 Bio, Santa Clara, CA, USA). The osmolyte concentration was calculated using a calibration curve made by standard solutions of L-proline (5, 10, 15, and 20 μg·mL⁻¹) (y = 0.0654x − 0.0561; R² = 0.999). The data are expressed as µg proline·g f.w.⁻¹.

For the quantification of carbohydrates, samples (0.2 g) of frozen materials were homogenized in liquid nitrogen, resuspended in 1 mL of 1% phosphate saline buffer (PBS) and incubated in the dark ON at +4 °C. After centrifugation (20 min at 6200× g), 0.5 mL of supernatant was collected and stored at −20 °C until the analysis. The concentration of carbohydrates was evaluated according to the anthrone protocol by Chun and Yin [37]. A total of 0.5 mL of 30% KOH was added to the sample homogenate, kept at 100 °C in a water bath for 40 min and, after the addition of 1.5 mL of 95% ethanol, the extracts were centrifuged at 4000× g for 15 min. The supernatants were discarded while the pellets were resuspended in 0.5 mL of double-distilled water and transferred into glass tubes. The reaction was started by adding 2.5 mL of fresh solution of 0.2% anthrone, solubilized in 75% H₂SO₄, to standard solutions and samples. The glass tubes were put on ice for 30 s, placed in a boiling water bath for 10 min and back on ice for 1 min.

The sample absorbances were measured at 625 nm with a spectrophotometer (VARIAN Cary 50 Bio, Santa Clara, CA, USA). Carbohydrates were calculated according to a calibration curve of glucose (20, 40, 60, 80 and 100 mg·L⁻¹) (y = 0.0121x + 0.0664; R² = 0.995) and expressed as mg glucose equivalent·g f.w.⁻¹.

2.5. Enzymatic Activities

Various enzymatic activities were evaluated and related to the protein level of the sample analyzed. The protein concentration was quantified by the Bradford assay [38], using a calibration curve made with bovine serum albumin (BSA) (1.25, 2.5, 5, 7.5, 10 μg mL⁻¹) (y = 0.0251x + 0.0442; R² = 0.997).

Superoxide dismutase (SOD) activity (EC 1.15.1.1) was assessed by native polyacrylamide gel electrophoresis (N-PAGE) as reported by Stassinos et al. [31] and visualized according to the procedure described by Beauchamp and Fridovich [39]. Enzyme activity was expressed as arbitrary units (A.U.), corresponding to the pixel density of each lane obtained with the software Image J 1.53A.

Polyphenol oxidase (PPO) activity (EC 1.14.18.1) was determined according to the method of Orzali et al. [40]; the kinetics was followed with a spectrophotometer (VARIAN Cary 50 Bio, Santa Clara, CA, USA) at a wavelength of 420 nm for 300 s. Peroxidase (POD) activity (EC 1.11.1.7) was evaluated according to Yang et al. [41], following the kinetics with a spectrophotometer at a wavelength of 420 nm for 180 s. The enzyme activities of PPO and POD were expressed as enzymatic units (E.U.·mg protein⁻¹).

Ascorbate peroxidase (APX) activity (EC 1.11.1.11) was determined according to the method by Borromeo et al. [34], evaluating the degree of ascorbate oxidation for 150 s at 290 nm by spectrophotometry (VARIAN Cary 50 Bio, Santa Clara, CA, USA). Catalase activity (CAT) (EC 1.11.1.6) was assessed according to Iwase et al. [42] and calculated by measuring the height of the O² bubbles produced by the enzyme. Both the enzymatic activities (APX and CAT) were expressed as % compared to the untreated control (0 mM NaCl).

2.6. Statistical Analysis

Data are reported as the mean ± standard error (SE). All statistical analyses were performed with Past 4.15 software. The effect of increasing salinity in CTRL and in primed group was assessed by One-Way Analysis of Variance (ANOVA) and the Tukey–Kramer method used to determine the difference in significance among groups. All analyses were significant at p < 0.05. Mean values in the column marked by different letters are significantly different within the same group (p < 0.05). The effect of priming treatment on salt tolerance responses was assessed by the t-Student’s test. When comparing primed groups to unprimed ones, the significance was *** p < 0.001; ** p < 0.01; * p < 0.05.

3. Results

3.1. Threshold of Halotolerance During Seed Germination

The threshold of seeds halotolerance was assessed through a dose–response curve (

Table 1. Germination % of unprimed (CTRL) and primed (Lippia alba EO) seeds of P. acutifolius exposed to various salinity levels. Data are expressed as the mean ± SE (n = 6). Significant differences within the same group (p < 0.05; ANOVA and Tukey–Kramer test) are reported with different letters in the column.

NaCl (mM)	CTRL	Lippia alba EO
0	92% ± 4% a	88% ± 3% a
40	89% ± 3% ab	80% ± 2% a
80	79% ± 6% b	69% ± 6% ab
160	55% ± 8% c	68% ± 7% b

); the value was considered based on the salt concentration that significantly decreased seed germination. The threshold values were 80 mM NaCl and 160 mM NaCl, respectively, for unprimed seeds (CTRL) and primed seeds (Table 1). These data showed that the priming treatment with L. alba EO increased salt tolerance during the germination phase.

Based on these results, 80 mM NaCl was chosen as the threshold of salt tolerance of bean seeds; thus, it was decided to irrigate plants with a moderately saline solution (40 mM NaCl) and a highly saline solution (80 mM NaCl).

3.2. Increase in Water Uptake and Salt Tolerance Index of Bean Plants

The analysis of the GWC of the soil provided important information regarding changes in the uptake of saline water by primed and unprimed plants. In the latter, an increase in salinity corresponded to a higher soil water content, suggesting that CTRL plants were negatively affected by saline irrigation; these results were also confirmed by analysis of the STI, which decreased by increasing salinity (

Table 2. Gravimetric water content (GWC) of soil and salt tolerance index (STI) of unprimed (CTRL) and primed (Lippia alba EO) plants at the end of experiments. Data are expressed as the mean ± SE (n = 9); mean values in the column marked by different letters are significantly different within the same group (p < 0.05; ANOVA and Tukey–Kramer test). Significant differences from CTRL are reported as ** p < 0.01.

Priming Solution	NaCl (mM)	GWC of Soil (%)	STI (%)
CTRL	0	32.6% ± 1.7% a	100
	40	32.9% ± 2.0% a	81
	80	56.8% ± 3.1% b	77
Lippia alba EO	0	32.2% ± 1.8% a	107 (+7%)
	40	24.3% ± 2.1% b **	109 (+28%)
	80	45.6% ± 1.9% c **	91 (+14%)

).

Primed plants showed an enhancement of water uptake under moderate stress conditions (40 mM NaCl); a general improvement in water uptake was found in stressed primed plants compared to CTRL (+26.1% and +19.7%, respectively, using 40 mM and 80 mM NaCl), and these results were supported by analysis of the STI, which was higher in all primed plants (Table 2) with respect to CTRL.

3.3. Effect of Salt Stress on Chlorophylls and Osmolytes Synthesis

In CTRL plants, increasing salinity decreased the total Chl. An opposite trend was found concerning the synthesis of carbohydrates and proline, i.e., in both cases, high salt stress led to an enhancement of these osmolytes’ concentration, probably related to the onset of osmotic stress due to excessive salt (

Table 3. Concentration of total chlorophyll (Chl), carbohydrates and proline in unprimed (CTRL) and primed (Lippia alba EO) plants at the end of experiments. Data are expressed as the mean ± SE (n = 6); mean values in the column marked by different letters are significantly different within the same group (p < 0.05; ANOVA and Tukey–Kramer test). Significant differences from CTRL are reported as *** p < 0.001; * p < 0.05.

Priming Solution	NaCl (mM)	Total Chl (μg·g f.w.−1)	Carbohydrates (mg glucose eq.·g f.w.−1)	Proline (μg·g f.w.−1)
CTRL	0	45.6 ± 4.4 a	0.131 ± 0.010 a	59.7 ± 9.9 a
	40	37.7 ± 2.3 ab	0.160 ± 0.003 ab	49.7 ± 5.5 a
	80	23.1 ± 3.7 b	0.205 ± 0.016 b	117.6 ± 18.3 b
Lippia alba EO	0	43.2 ± 0.8 a	0.229 ± 0.019 a ***	67.0 ± 6.6 a
	40	93.9 ± 4.6 b ***	0.163 ± 0.012 b	91.8 ± 3.2 a ***
	80	95.8 ± 4.6 b ***	0.141 ± 0.012 b *	485.7 ± 41.3 b ***

).

In primed plants, a significant increase in total Chl was observed, compared to CTRL (+149% and +315%, respectively, with 40 mM and 80 mM NaCl), as well as proline (+84% and +313% with 40 mM and 80 mM NaCl, respectively); in contrast, a reduction in sugars was found at a higher salinity level (Table 3).

3.4. Ca²⁺ Translocation and Activation of Salt Stress Responses

Ca²⁺ plays a key role in the activation of salt tolerance responses. In CTRL plants, Ca²⁺ was accumulated especially in the roots (

Table 4. Ca²⁺ concentration in root and shoot of unprimed (CTRL) and primed (Lippia alba EO) plants at the end of experiments. Data are expressed as the mean ± SE (n = 9); mean values in the column marked by different letters are significantly different within the same group (p < 0.05; ANOVA and Tukey–Kramer test). Significant differences from CTRL are reported as * p < 0.05.

Priming Solution	NaCl (mM)	Root (μg Ca2+·mg f.w.−1)	Shoot (μg Ca2+·mg f.w.−1)
CTRL	0	0.13 ± 0.03 a	2.13 ± 0.05 a
	40	0.19 ± 0.04 a	2.04 ± 0.04 a
	80	0.20 ± 0.03 a	1.91 ± 0.09 a
Lippia alba EO	0	0.28 ± 0.05 a	1.81 ± 0.03 a *
	40	0.27 ± 0.04 a	1.91 ± 0.01 b
	80	0.19 ± 0.04 a	2.17 ± 0.07 b

), while a slight decrease was recorded in the shoots, although these values were not statistically significant.

On the contrary, in roots of primed plants, a reduction in Ca²⁺ by increasing salinity was observed, whereas in stressed primed shoots, a significant enhancement of the ion was detected (Table 4). These data suggest an improved uptake and rapid Ca²⁺ translocation from the roots to the shoot of stressed primed plants, leading to a faster activation of salt tolerance responses.

3.5. Activation of Enzymatic Defence System in Response to Salinity

Defense responses to salt stress were considered basing on the activity of five enzymes: superoxide dismutase (SOD), polyphenol oxidase (PPO), catalase (CAT), peroxidase (POD) and ascorbate peroxidase (APX). In unprimed plants, the increased salinity negatively affected all enzymes studied (Figure 2a,b and Figure 3a–d), in particular salt totally inhibited the peroxidase activity of APX, under high stress conditions (Figure 3d).

In primed plants, the enhanced salt stress led to an improvement of SOD and POD activities (Figure 2a,b and Figure 3b,c); thus, seed priming always enhanced the activity of all the enzymes considered, compared to the corresponding unprimed stressed CTRL, particularly for SOD and POD (Figure 2b and Figure 3c).

4. Discussion

Salinity represents a global problem; therefore, understanding the plant response mechanisms to salt stress is crucial [18,25]. Under salt stress, most crops are subjected to alterations in ionic, osmotic and oxidative balance, which leads to the development of secondary stresses [26]. Techniques actually used to reduce salt damage include seed priming or seed coating that can induce phenotypic and elicit biochemical adaptation responses to salt stress in glycophyte crops [31]. These methodologies may foresee the utilization of natural and interesting products compounds, known as plant biostimulants [43]. In agriculture, biostimulants are currently employed to improve plant productivity and quality [44].

In this study, we investigated the beneficial effects of the EO of Lippia alba, rich in phytochemicals [7,11], on Phaseolus acutifolius: a drought- and heat-tolerant bean species [14]. The positive effect of this biostimulant could be already observed during the seed germination phase, since unprimed seeds showed a lower halotolerance than the primed ones; these data are supported by other findings [1,45] where different EOs had a positive effect on the seed germination (%) of various species.

High salinity causes osmotic and ionic stresses in plants; in fact, a high concentration of NaCl in the soil decreases the water potential, hindering water uptake by the roots [46]. The root salt uptake, and subsequent transfer to the shoot, results in a high NaCl concentration in leaf cells; this enhancement of salinity inhibits various biochemical processes in leaves, particularly photosynthesis, but it also leads to a nutrient imbalance [46]. Moreover, increased soil salinity corresponds to a higher soil water content due to lower water uptake by the roots, as demonstrated in unprimed bean plants. On the contrary, primed plants showed a significant increase in water uptake (particularly when irrigated with 40 mM NaCl), suggesting that the EO protected the root system from salt damage. These results agree with data reported by Gururani et al. [47] and El-Sayed and El-Ziat [48] and were confirmed by the analysis of the salt tolerance index, a parameter closely related to root size.

Even though there are still controversies about the effect of salt on photosynthesis, most scientists agree that salinity reduces the amount of chlorophyll. This reduction is caused by the inhibition of the enzymes necessary for their synthesis or by the destruction of the chloroplast [49]. An excess of salt induces a severe leaf injury, manifested by chlorosis and a reduced photosynthetic rate [49]. As reported in our previous studies on tomato and common bean [29,34], high salinity induced a reduction in total chlorophylls in unprimed stressed plants, while an opposite trend was found in primed ones. These results suggested that the priming treatment decreased the rate of chlorophyll degradation [50].

A very close link exists between chlorophyll and monosaccharide content; these latter are key molecules involved in various physiological processes (e.g., photosynthesis), but they are also considered osmolytes, necessary for the regulation of osmotic stress [51]. In many crops exposed to salt, an increase in monosaccharides has been reported [52,53,54,55]; in unprimed stressed plants, a significant enhancement of monosaccharides was found, while a reduction in these sugars was detected in stressed primed plants. These data supported the above-mentioned research studies, where the improvement in sugar content in the leaf tissue allowed the maintenance of cellular basal metabolism even under stress conditions. Alterations of the ratio between monosaccharides and chlorophylls have been related to changes in the expression of Rubisco, which is repressed by an overproduction of intracellular monosaccharides (in particular, sucrose) [53].

Plants can accumulate proline to inhibit osmotic stress; in many studies, proline content strongly increased under salt stress, acting as an osmoprotectant, improving water uptake from the soil to maintain cell turgor [56]. In a study by Nguyen and colleagues [57] on rice (Oriza sativa L.), the researchers observed that under salt stress the more salt-tolerant cultivars accumulated a higher amount of proline compared to the sensitive ones. Our results are in agreement with this report, i.e., the proline level of primed stressed plants was significantly higher than in unprimed stressed ones, suggesting that EO treatment made primed plants more tolerant to salinity by acting effectively against salt-induced osmotic stress.

The accumulation of Na⁺ and Cl⁻ in the soil causes an ionic imbalance inducing ionic stress, with reduced uptake of K⁺, Ca²⁺ and Mg²⁺ [17]. Ca²⁺ plays multiple roles in the cellular and physiological defense mechanisms of plants under salinity; it is considered an important second messenger that activates the signaling pathway involved in salt responses [58]. NaCl inhibits the uptake and translocation of Ca²⁺ from the root to the shoot, causing leaf Ca²⁺ deficiency, as reported in our previous studies [29,34]. Although the data obtained were not statistically significant, in the roots of unprimed bean plants, high salinity led to an increase in Ca²⁺, whereas an opposite trend was observed in the roots of primed stressed plants. A marked enhancement of Ca²⁺ was found in the shoots of primed stressed plants; in this case, the EO improved Ca²⁺ translocation from the roots to the shoots, activating faster salt stress responses.

The high concentration of Ca²⁺ in seedlings plays a key role in the activation of enzymatic antioxidant responses in all glycophytes, where ROS overproduction has been reported in salinity conditions [45]. These radical species cause severe damage to proteins, membrane lipids and DNA, inducing oxidative stress; consequently, the ROS scavenging process requires the activation of multiple detoxifying enzymes, such as SOD, CAT, POD and APX [59]. In this study, we also evaluated the activity of PPO, an enzyme involved in the biosynthesis of phenols which are important antioxidant secondary metabolites [31].

Even though there is still controversy regarding the antioxidant enzymatic response in different crops, most researchers agree about the positive correlation between antioxidant enzymes and salinity tolerance [45,59]. In stressed primed plants, a marked increase in SOD, POD and PPO activity was detected, compared to stressed CTRL, in agreement with [60], while CAT and APX activities were enhanced only under high salt stress (80 mM NaCl), compared to the relative CTRL. In our study, a general positive effect of L. alba EO on the activation of antioxidant defense was detected, thus providing an optimal response against ROS overproduction caused by salinity.

5. Conclusions

In this work, we tested the efficacy of an EO of Lippia alba, used as a priming agent, in enhancing salt tolerance through the activation of biochemical responses against both osmotic and oxidative stresses. Our data demonstrated a positive effect of this seed-priming treatment in counteracting stress, thus leading to a successful response and better plant fitness. In particular, priming with EO improved bean halotolerance during the germination phase, as well as in the vegetative phase. A faster activation of the enzymatic antioxidant defense system (in particular SOD, PPO and POD) was found due to an increase in Ca²⁺ in the shoots of primed plants. In addition to the amelioration of plant response and adaptation to salt stress conditions, seed priming with EOs represents an easy, eco-friendly and economical tool that can be used by farmers to overcome the problem of soil salinization in legumes and other glycophytes. Further studies concerning the positive effect of EOs as a biostimulant should be carried out to encourage the wider use of this technique for future agriculture.