Anatomical and Physiological Performance of Jojoba Treated with Proline under Salinity Stress Condition

Abstract

A field trial study was conducted for two consecutive seasons 2020 and 2021 in approximately 8-month-old jojoba plants to evaluate the physiological responses following salt treatment and the role of proline as a foliar application to enhance jojoba tolerance to salinity stress. Jojoba plants were irrigated once a week for four months with diluted seawater in concentrations of 5000, 10,000, and 15,000 ppm and tap water (control). Anti-stress proline was applied four times throughout the experiment, the first at the beginning of the experiment and another three times at 30-day intervals, at concentrations of 0, 300, and 450 ppm. The effect of proline treatments on jojoba plant behavior includes growth vegetative characteristics, namely plant height increase percentage (PHIP), shoot number increase percentage (NSIP), stem diameter increase percentage (SDIP), number of leaves, leaf thickness, leaf area, and fresh and dry weights of leaves, and chemical characteristics, namely chlorophyll a and b, total chlorophyll, carotenoids, leaf mineral contents (N, P, K, Na, and Cl), total phenolic content (TPC), and proline concentration. Moreover, the impacts of proline on hydrogen peroxide (H₂O₂), superoxide anion (O₂^•−), malondialdehyde (MDA), and ion leakage (IL) under salinity stress were investigated. Briefly, proline at 450 ppm enhanced all studied growth and physiological characteristics and promoted the antioxidant system of jojoba plants compared with the control and other treatments. The anatomical structure of leaves was also examined, and favorable variations in the anatomical structure were detected in the stressed and proline-treated plants. Exogenous application of proline enhanced most of this anatomical characteristic of jojoba leaf under saline stress. In conclusion, proline as a foliar application at 450 ppm under salinity stress of 10,000 ppm enhances jojoba tolerance to salinity stress by modifying the physicochemical and morphological characteristics of jojoba plants.

1. Introduction

Environmental pollution is the main reason for the phenomenon of climate change, as it leads to an increase in water and soil salinity due to the rise in seawater, which affects the sustainability of agriculture [1]. Therefore, it is necessary to move towards the selection of plant species that could resist unsuitable environmental conditions, such as salinity [2].

Jojoba (Simmondsia Chinensis (Link) Schneider) is a novel plant that is acclimated to unsuitable climates. It is considered an industrial crop called green gold and is grown commercially in hot arid and semi-arid regions. It is dioecious, meaning female and male flowers are carried on separate plants. Jojoba is commonly regarded as among the most powerful strategies for cultivating desert regions. Resistance to extreme temperature, dryness, and salinity, as well as a decreased risk of disease, a lesser demand for chemical fertilizers, and a high level of financial gain, are all compelling reasons to grow jojoba in desert environments [3,4]. In the early phases of its development, the jojoba necessitates the use of only a few well-established techniques and a long-term commitment to combat desert encroachment [5]. Indeed, shrubs of jojoba have deep root systems and grow in both dry and salty conditions, and their growth is satisfactory in brackish water with a salt concentration of 2000 ppm as in the Pacific Ocean and in California. More than a quarter of the world’s cultivated fields have saltwater levels high enough to produce salinity stress in growing plants [6]. Because of the advantageous commercial applications of its seed oil in cosmetics and fuel for aircraft and missiles, jojoba has become an interesting alternative crop. Seeds have a high “oil” content, which collectively accounts for about half of the dry weight of the seeds; this “oil” is made up of lipids that contain straight chain liquid wax esters of uniform length.

Salt is becoming a serious agricultural problem, primarily in irrigated arid and semi-arid areas where rainfall is insufficient to wash away the excess soluble salts from the root area, but it can also occur in irrigated agricultural areas, particularly when the irrigation water is poor quality. Biochemical change that occurs in plants exposed to environmental stressors is the production of reactive oxygen species that can damage nucleic acid essential membrane lipid proteins [7]. High salinity levels in the soil decrease the production of various plants in two ways: through osmotic influences and specific ion toxicities [8,9]. Boughalleb et al. [10] found that NaCl (100–300 mM) increased the anatomical characteristics of leaves in Medicago arborea such as the increased thickness of lamina, upper and lower epidermis, palisade, and spongy tissues. He claims that the necessity to save water causes the leaves to become succulent, resulting in increased leaf thickness.

Proline is a protein-genic amino acid with special conformational hardness and is necessary for initial metabolism; it has been found in plant tissues under a variety of circumstances, including drought [11] and salinity stress [12]. The suggested function of proline is the regulation of osmosis, which has a system of adaptation to adverse environmental conditions such as salinity. Another proposed function is the maintenance of membranes and protein stability; growth; and the provision of carbon, nitrogen, and energy storage [13]. Proline has been shown to stimulate growth, physiochemical, and anatomical features, as well as enhance the antioxidant mechanism defenses of plants under salt stress [12,14]. Proline is an amino acid that plays an essential role in plant metabolism and growth. It protects plants from diverse stresses and supports plants in faster recuperation from stress. When the proline treatment is applied as a foliar application to plants that are susceptible to stress, proline improves the growth and other physiological properties of the plants [15].

The exogenous application of proline has raised the internal level of proline in beans [16]. In addition, foliar application of proline regulates the expression of several genes related to antioxidant enzymes under salt stress. Among such genes, one gene of 1-pyrroline-5-carboxylate synthetase is responsible for upregulating the salinity stress-induced proline accumulation [17]. Exogenous application of proline enhances plant response to abiotic stressors, notably salt by shielding them from the damaging effects of reactive oxygen species (ROS) by enhancing their endogenous amount and intermediate enzymes [18]. Foliar proline treatment enhanced leaf N, Ca²⁺, and K⁺ concentration in Cucumis melo treated with 150 mM salt stress [19].

The present experiment was conducted to study the favorable effect of proline as a foliar application with two concentrations on growth parameters, leaf pigments, proline content, and anatomical characteristics of jojoba plants under salinity stress.

2. Materials and Methods

2.1. Growth Experiment and Salt–Proline Treatments

Jojoba plants were obtained from a private nursery of the Egyptian Gulf Company for Desert Land Reclamation, located in Alexandria, Egypt. They were propagated by placing the tip cuttings in plastic cups containing peat moss soil; after six months, they were transplanted to polyethylene bags (40 × 20 cm) containing 10 kg of mixed, sieved, acid-washed sand, compost, and clay (2:1:1 by volume) (Figure 1A–C). Each polyethylene bag contained one plant, and there were nine bags for each treatment. Plants were irrigated after the transplanting process with tap water (tap water was analyzed according to El-Harouny et al. [20] and presented Ca, 10.89 mg⁻¹; Mg, 9.98 mg⁻¹; Na, 8.15 mg⁻¹; Zn, 9.08 μg⁻¹; Se, 1.57 μg⁻¹;) and fertilized for two months by drenching with 1 g L⁻¹ NPK + TE(20:20:20) + 7 g L⁻¹ humic acid. Seawater was collected from the Mediterranean Sea located at New Damietta, Egypt (31°27′33.5″ N and 31°40′02.1″ E), with electrical conductivity (EC) of 44.64 dS m⁻¹(35,710 ppm) and pH of 8.07. The ion composition of New Damietta seawater was 11.23 g L⁻¹ Na⁺, 18.21 g L⁻¹ Cl⁻, 0.83 g L⁻¹ Ca²⁺, 0.92 g L⁻¹ Mg²⁺, 0.56 g L⁻¹ K⁺, 3.82 g L⁻¹ SO₄²⁻, and 0.12 g L⁻¹ HCO₃⁻. Three dilutions from seawater were prepared by using tap water (5000, 10000, and 15000 ppm) and, along with tap water as control, were used for the irrigation once a week for four months with the foliar application of proline solution once monthly at three concentrations (0, 300, and 450 ppm); the two control treatments that were sprayed with proline at concentrations of 300 and 450 were removed, and the experiment was analyzed using one-way ANOVA. The salt levels were gradually increased to avoid osmotic shock, and the experiment included 10 treatments as follows: T1 = tap water (control) without proline application; T2 = 5000 ppm salinity without proline application; T3 = 10,000 ppm salinity without proline application; T4 = 15,000 ppm salinity without proline application; T5 = 5000 ppm salinity with 300 ppm proline application; T6 = 10,000 ppm salinity with 300 ppm proline application; T7 = 15,000 ppm salinity with 300 ppm proline application; T8 = 5000 ppm salinity with 450 ppm proline application; T9 = 10,000 ppm salinity with 450 ppm proline application; T10 = 15,000 ppm salinity with 450 ppm proline application.

2.2. Measurement of Vegetative Growth Characteristics of Jojoba Plant

2.2.1. Shoot Number Increase Percentage (NSIP)

At the beginning (BS) and end (ES) of the experiment, the shoot number increase percentage was counted. The following equation was used to compute the NSIP: NSIP = [(ES − BS)/BS] × 100.

2.2.2. Plant Height Increase Percentage (PHIP)

The measurement of the plant height (cm) was calculated at the beginning (bH) and the end (eH) of the investigation by measuring the length of the main stem from the soil surface to the end of the plant. The PHIP was calculated using the following equation: PHIP = [(eH − bH)/bH] × 100.

2.2.3. Stem Diameter Increase Percentage (SDIP)

The plant stem diameter at the beginning of the experiment (bD) and at the end of the experiment (eD) on the same height starting from the soil surface was measured, where the measurement position was marked with red paint to confirm this on the plant stem. The SDIP was calculated using the following equation: SDIP = [(eD − bD)/bD] × 100.

2.2.4. Number of Leaves, Leaf Thickness, and Leaf Area (LA)

The number of all mature leaves plant⁻¹ was counted as a number for each replicate (in 9 seedlings per treatment). Leaf thickness was determined using a digital caliper (Digital Caliper Model 500, China). The length and width of the medial leaves were measured. Then, LA was calculated according to an equation given previously [21] as follows: leaf area (cm²) = 0.70 × (leaf length × leaf width) − 1.06.

2.2.5. Visible Quality

Leaf injury severity was recorded according to Sun et al. [22], using a scale of 0–5 (visible score) at the end of the experiment, where 0 = dead, 1 = severe visible damage (more than 90% of leaves with necrosis and injury), 2 = moderate visible damage (50% to 90%), 3 = slight visible damage (20% to 50%), 4 = minimal visible damage (less than 20%), and 5 = no visible damage.

2.2.6. Leaf Fresh and Dry Weight

Twenty new mature leaves of each plant were weighed to calculate the average fresh weight, the same leaves were dried at 70 °C for 72 h until the weight was completely stable in two successive weights, and then the average dry weight was estimated.

2.3. Chemical Characteristics of Jojoba Plant

2.3.1. Pigment Measurements

Fresh leaf samples (0.5 g) were added to 5 mL DMF (dimethylformamide). To allow the DMF (dimethylformamide) to leach the pigments from the sample, the suspension was sonicated for 15 min at 4 °C and then held at 4 °C for 16 h. The extracting solution was centrifuged for 5 min at 16,000 rpm to remove any suspended material. Then the optical density of the clarified supernatant (1 mL) was measured using a spectrophotometer at two wavelengths (662 nm (E 662) and 650 nm (E 650)) according to [23]. In addition, carotenoids were measured using wavelength 480 nm according to Wellburn [24].

• Chlorophyll a content = (12 × (E 662)) − (3.11 × (E 650))
• Chlorophyll b content = (20.78 × (E 650)) − (4.88 × (E 662))
• Total chlorophyll content = (17.67 × (E 650)) + (7.12 × (E 662))
• Carotenoids (µg/mL) = (1000 × A480 − 0.89 × chla − 52.02 × chlb)/245

2.3.2. Proline Determination

A weight of fresh leaves (0.5 g) was homogenized in a 5 mL solution of sulfosalicylic acid (3% w/v) and centrifuged at 10,000 rpm for 10 min. A 2.0 mL volume of the supernatant was used with ninhydrin reagent and toluene solution to measure the proline content by spectrophotometer at 515 nm according to the method of Bates et al. [25]. The proline amount (mg/g DW) was determined against a standard curve of L-proline.

2.3.3. Soluble Carbohydrate Content (SCC)

SCC was extracted according to Kerepsi et al. [26]. Dry leaf powder (0.1 g) was boiled for 50 min in 10 mL distilled water with agitation and then filtered through qualitative filter paper. An aliquot (0.5 mL) of this filtrate was used to determine the SCC according to Dubois et al. [27] using D (+)-glucose as standard. The obtained results were recorded as mg/g DW.

2.3.4. Total Phenolic Content (TPC)

TPC was measured in the leaf samples using Folin–Ciocalteu reagent as described by Ainsworth and Gillespie [28]. Gallic acid was used as a standard solution in the aqueous form in the concentration range of 100 to 600 ppm. The absorbance was measured at 760 nm. TPC was represented as mg gallic acid (GAE) per 1 g fresh weight (FW).

2.3.5. Ion Leakage (IL%) and Malondialdehyde (MDA) Accumulation

To measure the percentage of ion leakage (IL%), a 5 g sample (fresh jojoba leaves) was placed in 20 mL 0.4 M mannitol for 3 h at 24 °C, and then the electrical conductivity sample was first detected (R1). Thereafter, all samples were heated in a H₂O bath at 100 °C for 30 min to quantify the final leakage after the sample reached room temperature (R2). The percentage of ion leakage (IL%) was expressed as IL (%) = (R1/R2) × 100 [29]. About 2.5 g (jojoba leaves) was used to determine the MDA content; the ground sample was mixed well with thiobarbituric acid, 500 μL of butylated hydroxytoluene (C₁₅H₂₄O; 2%, w/v), and 25 mL of HPO₃ in ethyl alcohol (5%, w/v). As a result of establishing the 1,1,3,3-tetraethyoxypropane concentrations of TBARS ranging from 0 to 2 mM that were equivalent to MDA in the limit from 0–1 mM, the calibration curves were constructed. Stoichiometrically, tetraethyoxypropane is converted into malondialdehyde via the acid-heating stage of the testing [30].

2.3.6. O₂^•− and H₂O₂ Production Rate

A fresh plant sample of jojoba leaves was mixed with 3 mL of a 50 mM KH₂PO₄ buffer (pH 7.8) under refrigeration at 4 °C. The reagent was mixed with polyvinylpyrrolidone (PVP 1% w/v) and centrifuged at 10,000 rpm for 15 min at 4 °C, and the degree of O₂^•− production was evaluated by the creation of nitrite from NH₂OH in the presence of O₂^•−, as described by Yang et al. [31]. The optical density was measured at 530 nm. To measure the creation level of O₂^•− from the reaction equation of NH₂OH with O₂^•−, a standard curve with NO₂ was utilized. The creation level of O₂^•− was recognized as mmol min⁻¹ g⁻¹ FW. The H₂O₂ content was established following the process described by Xu et al. [32]. One gram of jojoba leaves was mixed with 5 mL acetone. After centrifugation at 6000 rpm for 15 min at 4 °C, the obvious extraction was filled. One milliliter of the latter clear mining was added to 0.2 mL ammonia and 0.1 mL titanium sulfate (5%) and then centrifuged at 6000 rpm for 10 min at 4 °C. The pellets (titanium–peroxide complex) that were created were dispersed in 3 mL of sulfuric acid 10% (v/v) and centrifuged at 5000 rpm for 10 min at 4 °C. The optical density of the subsequent supernatant was measured at 410 nm. Using H₂O₂ as a standard curve, the H₂O₂ content was expressed and then identified as mmol min⁻¹ g⁻¹ FW.

2.3.7. Leaf Mineral Content Determination

Total nitrogen (N), phosphorus (P), and potassium (K) contents were determined by taking 0.3 g of samples from dried leaves out of each replicate and wet digesting them with a mixture of concentrated sulfuric and perchloric acids and calculating N, P, and K as indicated previously [33]. Na⁺ and Cl⁻ were analyzed by using the method of [34] followed by inductively coupled plasma atomic emission spectroscopy (plasma View Duo iCAP7400) according to [35]. Na⁺ and Cl^- concentrations were expressed as percentages.

2.4. Anatomical Study

In conjunction with the anatomical investigation (135 days from the beginning of applying the treatments), leaf specimens (5 × 5 mm) were taken from the midrib of the middle part of the 4th leaf including the main midvein were taken in the 2nd season. Specimens were fixed for 48 h in FAA solution (formaldehyde–acetic acid–alcohol), washed gently with sterile water, dehydrated in a series of ethanol, cleared in ethanol:xylene (3:1–1:1–1:3% and 100% xylene), and embedded in paraffin wax (52–54 °C melting points). Sectionswere made at 10–15 µm thickness using a rotary microtome, double stained with safranin–light green, cleared in clove oil, and mounted in Canada balsam according to Ruzin [36]. A light microscope (Olympus CX41, Davao City, Philippines) connected with a digital camera (TUCSEN, USB2, H Series, Fuzhou, China) was used to examine the chosen sections (five sections from each treatment) to visualize the following microscopic characteristics: thicknesses of lamina (µm), upper and lower epidermis, palisade and spongy tissue, as well as main vascular bundle dimensions (length and width in µm) of leaf mesophyll.

2.5. Statistical Analysis

The average data for two growth seasons (2020–2021) for the present study were examined statistically. In a complete randomized block design (CRBD), data were subjected to analysis of variance (ANOVA) using a one-way analysis by using the statistical program SPSS, with three replications. The means of all examined treatments results have been contrasted utilizing Duncan’s multiple range test at p ≤ 0.05.

3. Results

3.1. Vegetative Growth Characteristics of Jojoba Plant

Results as an average of the two tested seasons are presented in Figure 2. The data of the plant height increase percentage (PHIP), shoot number increase percentage (NSIP), and stem diameter increase percentage (SDIP) indicated that PHIP, NSIP, and SDIP gradually decreased with different seawater salinity levels after four months from stress. Utilizing a high concentration of seawater (15,000 ppm) resulted in the highest value of reduction which reached 6.44, 24.68, and 27.87%, respectively, as compared to the control. The highest increase in PHIP, NSIP, and SDIP was achieved with the proline at 450 ppm at the level of salinity 10,000 ppm and reached 59.50, 98.00, and 90.45%, respectively, followed by the 300 ppm proline at the same level of salinity. It was observed that using the foliar application of proline at 450 ppm under 10,000 ppm level salinity (Figure 3) resulted in a significant increase in the number of leaves (154.66), leaf thickness (0.91 mm), and leaf area (16.63 cm²) as compared to the control that recorded 117.5, 0.59, and 11.68, respectively. Jojoba plants showed minor leaf injury after four months when irrigated with saline water at the level of 10,000 ppm and treated with proline at 450 ppm with a visual quality of 4.66 compared with other treatments, while the lowest value of visual quality was 2.66 under the effect of salinity stress at 15,000 ppm. The value of injured leaves increased slightly in the jojoba plant when the salinity level of irrigation water was increased from 5000 ppm to 15,000 ppm. The results indicated that there were no injured leaves under irrigation with tap water (control). It is clear from Figure 1 and Figure 2 that the concentration of proline at 450 ppm positively increased all vegetative growth characteristics, modified the undesirable effects, and had the ability to overcome the deleterious impact of salt on the growth characteristics of the jojoba plant that was grown under 10,000 ppm salt compared to control and other treatments.

3.2. Leaf Fresh Weight and Dry Weight

As shown in Figure 4, leaves fresh and dry weights of jojoba plants significantly increased with the exogenous application of both proline concentrations (i.e., 300 and 450 ppm) compared to the control (tap water) and untreated plants (no proline). Maximum values of leaf fresh weight and leaf dry weight were obtained with the proline level of 450 ppm under 10,000 ppm salinity.

3.3. Chemical Characters of Jojoba Plant

3.3.1. Leaf Pigments

The findings show how varying amounts of seawater salinity irrigation and foliar application of proline (300 and 450 ppm) affect the content of chlorophyll a, chlorophyll b, total chlorophyll, and carotene in the leaves of jojoba plants (Figure 5). The results showed that as salinity levels increased, total chlorophyll levels decreased. The highest levels of chlorophyll a, chlorophyll b, total chlorophyll, and carotene were 84.20, 26.03, 110.24, and 24.5 µg cm⁻² in T9, while the lowest levels were 63.73, 16.9, 80.55, and 18.9 µg cm⁻² for the irrigation with 15,000 ppm seawater for jojoba plants, respectively. The data clearly confirmed that the foliar application of jojoba plants by proline treatment has a highly efficient effect in enhancing the leaf content of pigments compared to non-sprayed plants under salinity stress. Remediation jojoba plants with proline (300 and 450 ppm) maintained leaf pigment contents under different levels of salinity, especially using the foliar application of proline at 450 ppm under the 10,000 ppm level of salinity.

3.3.2. Ion Leakage (IL%) and Malondialdehyde (MDA)

Ion leakage (IL%) and malondialdehyde (MDA) slightly increased under different salinity levels and reached 27.83% and 29.66 μM g ⁻¹ FW, respectively, under the salinity of 15,000 ppm as compared to those of the control plant (tap water), which reached 10.5% and 20.5 μM g ⁻¹ FW, respectively. The frequency of accumulation was affected by salt levels up to 15,000 ppm as well as proline treatments. Foliar application of proline at 450 ppm decreased the accumulation of ion leakage (IL%) to 13% and malondialdehyde (MDA) to 21 μM g⁻¹ FW in the salinized jojoba plants, especially for those plants irrigated with 10,000 ppm diluted seawater (Figure 6). When jojoba was sprayed with proline at a concentration of 450 ppm under a salt seawater irrigation level of 10,000 ppm, it was shown to be more tolerant to salt stress.

3.3.3. O₂^•− and H₂O₂ Accumulation Rate

Results indicated that jojoba plants irrigated with saline water significantly affected the O₂^•− and H₂O₂ accumulation rate. After four months of salt stress, O₂^•− and H₂O₂ accumulation was markedly increased in all different seawater treatments as compared to the control, except in the jojoba plants treated with proline 450 ppm under the salinity level of 10,000 ppm, in which O₂^•− and H₂O₂ accumulation increased slightly and reached 0.28 mmol min⁻¹ g⁻¹ FW and 0.09 mmol min⁻¹ g⁻¹ FW, respectively. The use of proline at 450 ppm decreased the accumulation rate of O₂^•− and H₂O₂ under the salinity level of 10,000 ppm (Figure 7).

3.3.4. Proline Content, Soluble Carbohydrate Content (SCC), and Total Phenols

The results shown in Figure 8 evidence a significant increase in the proline content, soluble carbohydrate content (SCC), and total phenol content of jojoba leaves under the impact of irrigation with seawater at 5000 ppm compared to the control. In addition, irrigation with a higher salinity level (10,000 and 15,000 ppm) caused a significant increase in the proline content of jojoba leaves compared to non-salty water (control). The changes in SCC and phenol contents were not significant. The increase in salinity level from 5000 ppm to 15,000 ppm led to a significant decrease in the soluble carbohydrate content (SCC) and total phenol content. Treating jojoba plants with proline at both concentrations (300 and 450 ppm) affected the proline content of jojoba plants under the studied levels of seawater irrigation compared to non-treated plants. The use of proline at 450 ppm led to a significant decrease in the proline content at the salinity level of 10,000 ppm and a significant increase in soluble carbohydrate content (SCC) and total phenol content. From the presented results, we concluded that jojoba plants treated with proline at 450 ppm were more efficient at the 10,000 ppm salt level.

3.3.5. Leaf Mineral Concentration

It is observable in Figure 9 that salinity at 5000 ppm increased the concentrations of N, P, and K in the leaves of jojoba plants. Salinity concentrations at 10,000 and 15,000 decreased these concentrations significantly. The highest concentration of N, P, and K was registered in plants irrigated with 10,000 ppm of salinity and sprayed with 450 ppm of proline; they had concentrations of 52, 41, and 55 mg/100 g DW, respectively, compared to the control plants. When salt levels in seawater irrigation were raised, the levels of Na⁺ and Cl⁻ gradually increased. Plants that were irrigated with 10,000 ppm salinity and received a foliar treatment of 450 ppm proline had the lowest levels of Na⁺ and Cl⁻ (0.4 and 1.1 ppm, respectively), when compared to the other salinity concentrations (Figure 10).

3.3.6. Anatomical Characterization of the Leaf Structure

Data in

Table 1. Anatomical modification of jojoba leaves as affected by seawater salinity and exogenous proline. Data are means of the second season (2021) and five replicates (n = 5). The mean values ± SE of each parameter in the same column followed by different alphabetical letters are significantly different according to Duncan’s multiple range test at p ≤ 0.05.

Treatments	Lamina Thickness		Upper Epidermis Thickness		Palisade Tissue Thickness		Spongy Tissue Thickness		Lower Epidermis Thickness		Dimensions of Main Vascular Bundle
											Thickness		Width
	µm	± % to S0 *	µm	± % to S0	µm	± % to S0	µm	± % to S0	µm	± % to S0	µm	± % to S0	µm	± % to S0
T1	144.20 ± 2.61e	0.00	7.72 ± 0.37e	0.00	24.50 ± 0.55cd	0.00	104.65 ± 3.15d	0.00	7.33 ± 0.24c	0.00	98.00 ± 1.75b	0.00	98.70 ± 0.42b	0.00
T2	172.76 ± 2.36c	+19.81	8.71 ± 0.37de	+12.82	28.00 ± 0.78b	+14.29	126.35 ± 1.28b	+20.74	9.70 ± 0.48a	+32.43	84.35 ± 0.65e	−13.93	92.05 ± 1.30c	−6.74
T3	175.61 ± 1.45bc	+21.78	9.90 ± 0.31bc	+28.21	33.95 ± 0.89a	+38.57	122.85 ± 1.16b	+17.39	8.91 ± 0.76a-c	+21.62	81.20 ± 0.89f	−17.14	77.35 ± 0.65de	−21.63
T4	181.80 ± 1.70ab	+26.08	10.30 ± 0.24ab	+33.33	17.85 ± 1.28e	−27.14	144.55 ± 1.88a	+38.13	9.11 ± 0.20ab	+24.32	78.75 ± 0.55f	−19.64	74.55 ± 1.18e	−24.47
T5	159.89 ± 1.40d	+10.88	8.32 ± 0.24de	+7.69	27.65 ± 1.02bc	+12.86	116.20 ± 0.89c	+11.04	7.72 ± 0.48bc	+5.41	94.15 ± 0.65c	−3.93	98.70 ± 0.78c	0.00
T6	130.10 ± 1.37f	−9.78	10.30 ± 0.39ab	+33.33	18.55 ± 1.42e	−24.29	91.35 ± 2.02e	−12.71	9.90 ± 0.54a	+35.14	90.65 ± 0.65d	−7.50	89.25 ± 0.95a	−9.57
T7	124.83 ± 4.29f	−13.43	9.31 ± 0.39b-d	+20.51	23.80 ± 1.62d	−2.86	84.00 ± 3.79f	−19.73	7.72 ± 0.37bc	+5.41	80.50 ± 1.66f	−17.86	78.05 ± 5.35de	−20.92
T8	183.32 ± 0.96a	+ 27.13	9.11 ± 0.19cd	+17.95	21.70 ± 1.80d	−11.43	144.20 ± 2.85a	+37.79	8.32 ± 0.24a–c	+13.51	107.80 ± 1.18a	+10.00	105.00 ± 2.18b	+6.38
T9	140.82 ± 1.25e	−2.34	8.91 ± 0.54cd	+15.38	24.50 ± 0.55cd	0.00	98.70 ± 1.18d	−5.69	8.71 ± 0.48a–c	+18.92	94.15 ± 1.02c	−3.93	102.20 ± 0.89ab	+3.55
T10	145.65 ± 1.79e	+ 1.01	11.09 ± 0.37a	+43.59	22.05 ± 0.70d	−10.00	103.60 ± 2.17d	−1.00	8.91 ± 0.70a–c	+21.62	86.80 ± 0.42e	−11.43	82.95 ± 1.18d	−15.96

* S₀ = control; T1 = control “tap water” without proline application; T2 = 5000 ppm salinity without proline application; T3 = 10,000 ppm salinity without proline application; T4 = 15,000 ppm salinity without proline application; T5 = 5000 ppm salinity with 300 ppm proline application; T6 = 10,000 ppm salinity with 300 ppm proline application; T7 = 15,000 ppm salinity with 300 ppm proline application; T8 = 5000 ppm salinity with 450 ppm proline application; T9 = 10,000 ppm salinity with 450 ppm proline application; T10 = 15,000 ppm salinity with 450 ppm proline application.

and Figure 11, Figure 12 and Figure 13 showed that salinity adversely affected the leaf anatomical structure of jojoba plants. A prominent increase was noted in lamina thicknesses “succulence” with increasing salinity levels (by 19.81, 21.78, and 26.08% at 5000, 10,000, and 15,000 ppm, respectively) compared to control plants. The increment in the thickness of lamina could be attributed to the increase in the thickness of both upper and lower epidermis by 12.82% and 28.21%, 33.33% and 32.43%, and 21.62% and 24.32%, respectively. In addition, the thickness of palisade tissue increased by 12.29 and 38.57% and that of spongy tissue increased by 20.74 and 17.39, respectively, under low and moderate salinity levels. Under high salinity levels, the thickness of palisade tissue was decreased by 27.14%, and the spongy tissue increased in thickness by 38.13% in comparison with the control plants. Additionally, under all salinity levels, the dimensions of the main vascular bundle (thickness and width) were decreased compared with the control.

Generally, notably, improvements in leaf anatomy for salt-stressed plants with proline at 300 or 450 ppm induced a reduction in the deleterious effects of salinity on anatomical characteristics. Proline reduced the sharp decrease in the dimensions of the main vascular bundle compared with control; the most efficient treatment was 450 ppm proline. Under low salinity levels, the application of 450 ppm proline increases the thickness and width of the main vascular bundle by 10.00 and 6.38%, respectively. However, a moderate level led to an increase in the width of the main vascular bundle by 3.55%, while the thickness was decreased by 3.93% as compared to control.

4. Discussion

Jojoba (Simmondsia chinensis (Link) Schneider) is a unique plant that has adapted to harsh conditions such as heat, drought, and salinity. Salinity stress has been seen as a massive problem and a vital aspect of the world’s long-term agricultural sustainability. It has an impact on plant growth and causes significant production and quality decreases in stressed plants. Proline improves plant tolerance to various abiotic stressors by increasing their endogenous level and intermediate enzymes. Vegetative parameters have been measured to evaluate the effects of salinity stress and proline as an anti-stress treatment on jojoba plants. Our data indicated that the jojoba plant’s vegetative characteristics were negatively correlated with increasing seawater salinity levels. Values of plant height increase percentage (PHIP), shoot number increase percentage (NSIP), stem diameter increase percentage (SDIP) (Figure 2), number of leaves, leaf thickness, leaf area, and visual quality (Figure 3) were reduced with rising seawater irrigation salinity levels, especially at higher salinity levels (15,000 ppm). These results are in agreement with previous works [37,38] on pomegranates. In contrast, foliar application of proline in different concentrations minimizes the damaging impacts of salt stress on vegetative characteristics of jojoba plants. Amino acids are well-known biostimulants that have a beneficial effect on plant growth and significantly alleviate the damage caused by abiotic stress [39,40]. Proline is an amino acid that plays an essential role in plant metabolism and growth. It protects plants from diverse stresses and supports plants in recuperating faster from stress. When proline treatment was applied as a foliar application to plants that are susceptible to stress, proline improved the growth and other physiological properties of the plants [17]. Moreover, El-Sherbeny et al. [41] found that a foliar application of 100 mg L⁻¹ proline increased the plant height, number of branches, and fresh and dry weights of leaves of Beta vulgaris L. plants. Dawood et al. [14] indicated that the foliar application of 25 mM proline caused significant increases in growth parameters of Vicia faba compared with the control. The idea that certain amino acids might alter the growth and development of plants via their impact on gibberellin biosynthesis could explain the regulatory effect of amino acids on growth [42]. Furthermore, according to Lea and Fowden [43], amino acids, which are the building blocks of proteins, can perform a variety of other tasks within the regulation of nitrogen metabolism, transport, and storage of nitrogen. Çiçek and Çakirlar [44] reported that salinity stress determines the capacity of plant cells to absorb water and some nutrients dissolved in the soil and reduces plant growth. In contrast, the foliar application of proline, especially the level of 6 mM, significantly improved internal proline levels in lupine plants, increasing their tolerance to salt stress and therefore enhancing lupine vegetative growth properties.

Using seawater irrigation under a high level of salinity up to 15,000 ppm causes inhibited growth resulting in the reduction in fresh and dry weights of the jojoba plant. Spraying proline in both concentrations of 300 and 450 ppm under different salinity levels presents beneficial effects on growth, especially under the salt level of 10,000 ppm. In a previous work on Medicago sativa callus cells, exogenously introducing proline into the culture medium under salt stress led to an increase in dry weight and also an increased content of free proline in the callus cells [45]. The increase in plant biomass due to the foliar application of consistent osmolytes can be attributed to an active role of these osmolytes in the osmotic transformation of plants, which in turn improved water uptake and enhanced plant growth. Proline’s favorable benefits on plant vegetative development in a variety of plant species maintained under stress conditions might be linked to its dual role as a nutrient and an osmoprotectant [46].

Leaf chlorophyll, one of several biochemical attributes, is an essential property reflecting plant health status and is related to plant water availability and nutrient content [14]. In our study, detrimental effects of salt stress on leaf chlorophyll content were reported several crops [12,47]. The decline in chlorophyll levels in most stressed plants may be due to disorganization of the thylakoid membranes, with more degradation than synthesis of chlorophyll via the formation of proteolytic enzymes such as chlorophyllase, which are responsible for breaking down chlorophyll and damaging the photosynthetic apparatus [48]. This reduces the rate of photosynthesis in plants [49] and inhibits ion accumulation [50]. In contrast, the application of 450 ppm proline caused a significant increase in photosynthetic pigments in salt-stressed jojoba plants. These improved chlorophyll concentrations can be attributed to this treatment stimulating chlorophyll biosynthesis and/or inhibiting its breakdown. Furthermore, these increases in chlorophyll concentrations could be attributed to more efficient scavenging of ROS by proline and other antioxidant compounds [12]. It is possible that proline’s beneficial influence on chlorophyll concentrations during salt stress is related to the stability of photosynthetic processes. Abdelhamid et al. [12] also reported that carotenoids help in the production of vital nutrients related to photosynthesis. These pigments give fruits red, yellow, or orange colors. The beneficial effects of additional antioxidants on plant survival under salt stress associated with partial inhibition of ROS formation have been described in several papers [51,52,53]. Proline application significantly increased carotenoid concentrations in the leaves of jojoba plants. Carotenoids have been recognized as protecting the photosynthetic apparatus against photoinhibition damage caused by singlet oxygen (¹O₂) created by chlorophyll’s triplet excited state. Carotenoids directly deactivate and also quench singlet oxygen in the triplet excited state of chlorophyll, thereby indirectly reducing the formation of singlet oxygen species [54].

The accumulation of MDA and IL% increased significantly at p ≤ 0.05. Malondialdehyde (MDA) is a natural product of lipid peroxidation and has traditionally been used as an indicator of the degree of cell damage from stress [55]. Plant exposure to salt stress causes negative effects, the most important of which is an imbalance of cellular ions, leading to ion toxicity and osmotic stress, while high levels of salinity induce the generation of reactive oxygen species (ROS), which are considered highly toxic oxygen derivatives, especially superoxide (O₂^•−) and hydrogen peroxide (H₂O₂), and as a result of the Fenton reaction in plants [56,57], the most hazardous hydroxyl radicals (OH^•) develop, which can interact with a variety of important macromolecules and metabolites, causing damage to cellular structures [58,59]. Reactive oxygen species (ROS) detoxification pathways play a defensive role in the salt stress response by removing toxic radicals generated from the mitochondrial and chloroplast electron transport chains. Proline protects plants from stress by maintaining osmoregulation and detoxifying ROS, thus conserving membrane integrity and stabilizing enzymes and other proteins [60,61].

ROS are produced in plant cells under normal physiological conditions, either in a radical or non-radical form [62]. Excessive ROS generation, on the other hand, causes oxidative damage to the cell’s proteins, lipids, nucleic acids, and plasma membrane. To protect cells from oxidative damage, plants must produce low-molecular-weight non-enzymatic antioxidants such as proline, glutathione, and ascorbate, as well as enzymatic antioxidants such as peroxidase (POX), superoxide dismutase (SOD), ascorbate peroxidase (APX), and catalase (CAT) to ward off oxidative stress [57,59]. One of the effective protective mechanisms of the plant against hyperstress is the increase in the content of internal compatible solutes such as proline, glycine betaine, and sorbitol [57]. Free proline is generated in many plants in response to the application of a wide range of biotic and abiotic stresses. The ability of proline to regulate osmotic adaptation, settle subcellular systems, and scavenge harmful oxygen species has been the target of most research. High levels of proline are synthesized under stress conditions and also maintain the NAD(P)+/NAD(P)H ratio [63]. Proline is a multifunctional amino acid and also a signaling molecule acting as a plant growth regulator by triggering cascade signaling processes [18]. Proline is preferred as a common osmolyte in plants and is upregulated in response to different stresses [18,59]. Its accumulation in plants protects them from drought and salt stress. Exogenous application of proline enhances plant response to abiotic stressors, notably salt, by shielding them from the damaging effects of reactive oxygen species (ROS) [18]. Plants tend to enhance their endogenous level of proline under continuously increasing levels of salinity. This study focused on the adverse impact of salt stress on plants, and how plants survive under irrigation with salt water by increasing their endogenous level of proline.

Our results show that salt stress or the exogenous implementation of proline under salt stress increased the content of proline in the jojoba plants (Figure 8). The increased levels of proline under salt stress were also documented in wheat and common bean [64,65]. It has been suggested that the accumulation of proline may be generated by the increase in proteolysis or by the reduction in protein synthesis. Because proline is involved in the osmotic potential of leaves and consequently in osmotic adjustment, plants benefit from a greater proline content under salt stress.

Phenolic compounds protect plants from a range of biotic and abiotic stresses. The oxidation of phenols generates many defenses that alter plant physiology and metabolism, which in turn allow the plant to resist diverse stresses either directly or through the mediation of various plant signaling pathways. From our results (Figure 8) we deduced that the use of foliar proline at a level of 450 ppm was more efficient at the salinity of 10,000 ppm, which resulted in a significant increase in the content of SCC and polyphenols in the leaves of the jojoba plant. Such findings confirmed that proline application mitigates adverse salinity and increases polyphenols and SCC [66,67].

Salt stress not only increases sodium (Na⁺) and chloride (Cl⁻) in plants but also causes reductions in calcium (Ca²⁺), potassium (K⁺), magnesium (Mg²⁺), nitrate (NO₃⁻), sulfur (S), and other vital nutrients, leading to general nutrient insufficiency [68]. The favorable impacts of foliar proline on plant toleration to salinity stress have been related to raised intake of nutrients in many investigations. It was documented that using foliar proline application increased P, K, NO₃⁻, and NO₂⁻ contents in Phaseolus vulgaris under different levels of salinity [50]. Similarly, foliar proline treatment enhanced leaf N, Ca²⁺, and K⁺ concentration in Cucumis melo treated with 150 mM salt stress [19]. In addition, foliar application of proline increased Ca²⁺ and K⁺ in Sorghum bicolor under salt stress [68]. It was reported that the use of foliar proline can raise the uptake of N, P, K⁺, and S in Zea mays under salt conditions [69]. Besides nutrient absorption, the activities of some enzymes involved in nutrient absorption are catalyzed by foliar proline under salinity stress. Nitrate reductase is one of the key enzymes involved in nitrogen absorption, and foliar proline promotes its activity in Helianthus annuus [70] and Cucurbita melo [46] exposed to salt stress. Some authors have suggested that proline may provide a good way to accumulate and recycle nitrogen under salt stress [71,72]. Exogenous proline was also utilized as a source of nitrogen (N) by Vigna radiata L. seedlings under stress conditions [73]. Elevated salt levels increase sodium (Na⁺) and chloride (Cl⁻) contents in plants and reduce the levels of other cations such as potassium (K⁺⁾ and calcium (Ca²⁺), causing mineral nutrient imbalance [74]. Clearly, maintaining ion homeostasis is one of the adaptive systems that resistant plants use to manage salt stress. These mechanisms might help the plant reduce the harmful effects of ions such as Na⁺ and Cl⁻, which can cause a variety of injuries to lipids, proteins, and nucleic acids [75]. Recently, Gholami Zali et al. [66] found that salt-stressed Zea mays subjected to foliar treatment with proline exhibited a decrease in both Na⁺ and Cl⁻ concentrations and an increase in the K⁺ concentration and the K⁺/Na⁺ ratio. Identical effects have been registered for Sorghum bicolor [68]. Previous work established that external proline relieved the adverse influence of 120 mM salt, increased K⁺ level, and decreased Na⁺ level in Helianthus annuus [70]. Under salinity, foliar proline enhanced salt resistance of Olea europaea by preserving low Na⁺, high K⁺, and decreased Na⁺/K⁺ and Na⁺/Ca²⁺ ratios in plant leaves [76]. Salt tolerance is not usually induced by proline in this way. Exogenous proline has little influence on Na⁺ and K⁺ levels in the leaves of cucumber, although it does increase leaf water content below 100 mM NaCl [77]. This increased water content is due to the external proline can dilute the salt and therefore restrict salt toxicity, leading to better plant growth.

In accordance with this study, the prominent increase in leaf lamina thickness with increasing levels of salinity and succulence for jojoba plants was recorded under different salinity levels, indicating a corresponding increase in mesophyll tissue which could be attributed to the necessity to conserve water rendering the leaves succulent. In general, the increased leaf thickness in this investigation is compatible with the results of Roussos et al. [78] and Gonzalez et al. [79] on jojoba Simmondsia chinensis Link, Akcin et al. [80] on Spergularia marina, and Debez et al. [81] on Cakile. Additionally, [82] showed a significant increase in leaf thickness due to salt treatment with 250 mM NaCl (spongy mesophyll thickness increased by 38% while palisade mesophyll cell length was increased by 50% over controls). Moreover, Boughalleb et al. [10] found that NaCl (100–300 mM) increased the anatomical characteristics of leaves in Medicago arborea, including the thickness of lamina, upper and lower epidermis, palisade, and spongy tissues. The authors claim that the necessity to save water causes the leaves to become succulent, resulting in increased leaf thickness. In this regard, Waisel et al. [83] suggested that the succulence exercises a dilution effect upon the toxic ions from the cells and upon the salts accumulated in plant organs, thus permitting the plant to cope with higher salt amounts. Additionally, [84] reported that succulence is one of the mechanisms that halophytes utilize to deal with high internal ion concentrations.

Additionally, previous works reported that salinity in halophyte plants catalyzes vacuolization in the parenchyma cells; these anatomical changes may help in the storage of ions inside the plant organs and protect the cytoplasm from toxic ion levels [10,85]. In addition, [86] found that salt-tolerant plants have a tendency to increase the leaf thickness, consequently inducing the succulence of leaves. Roussos et al. [78] reported that leaves tended to be thicker with increasing salinity, which is probably the result of high water accumulation within their tissues. In this regard, Debez et al. [84] pointed out that increased succulence possibly aids in storing additional water by increasing vacuolar volume at higher salt concentrations.

Under all salinity levels, the dimensions of the main vascular bundle (thickness and width) were decreased. In this regard, previous studies found that salinity decreased the dimensions of the main vascular bundle of the mature leaf of Fragaria x ananassa Duch. [87], Vigna unguiculata [88], and Vitis vinifera [89]. This reduction may be due to the restriction of cell division and expansion as well as hampering procambial activity [90]. In addition, Kozlowski [91] stated that under saline conditions, the xylem tissue was much more abundant and narrower than that in normal conditions. Since large vessels cannot provide a good flow of water, plants have developed these adaptation mechanisms [92]. Additionally, Abd Elbar et al. [93] explained that the decrement in the dimensions of the main vascular bundle caused by abiotic stress could decrease water translocation on one hand but on the other hand could help protect the water column from embolism.

It is evident from the current studies that were notable improvements in leaf anatomy for salt-stressed plants receiving proline treatment, which induced a reduction in the deleterious effects of salinity on anatomical characteristics. These results are generally in agreement with the results obtained by Abdelaal [94], Hussein et al. [95], and Dawood et al. [14] for mung bean, jatropha, and faba bean, respectively. Foliar application of proline enhanced most of these anatomical characteristics in the leaves of stressed plants, and these results suggested that the treatment with 450 ppm proline had the ability to reduce the deleterious effect of salinity stress on the histological structure of jojoba leaves. On the contrary, treatment with proline reduced succulence (lamina thickness) which resulted from exposure to salinity conditions in comparison with the control and salinity treatment as well, except for the treatment with proline under low and high salinity levels, where the thickness of the blade increased by 27.13 and 1.01%, respectively.

5. Conclusions

The obtained results from the present investigation showed that the foliar spraying of proline at a concentration of 450 ppm under a salinity level of 10,000 ppm relieves the adverse impact of salty stress on the vegetative growth and physiological and anatomical characteristics of the jojoba plant (Simmondsia chinensis (Link) Schneider). The jojoba is a salinity-tolerant plant that produces proline in suitable and unsuitable conditions, but in concentrations that vary according to the severity of the stress. In the current study, the jojoba plant was exposed to salinity at 5000 ppm, which is considered harmless and used as a nutrition method, meaning that the plant is in an appropriate condition and does not need an external addition of proline. The exposure of the jojoba plant to salinity at 10,000 ppm is considered a stress concentration that is not severe, so the plant’s internal defense systems push the plant to produce proline, but in concentrations that are insufficient to help the plant withstand stress; in this case, foliar spraying the plant with proline may help it tolerate salinity stress, adapt to unsuitable conditions, and improve its physiological and morphological properties. Exposure to a 15,000 ppm concentration of salinity leads to severe stress and failure of the internal defense systems to produce sufficient proline. In this case, the plant may need outside intervention by foliar spraying of proline at a high rate of over 450 ppm to help it adapt and withstand harsh conditions. This research needs further studies to clarify the physiological role of proline in the jojoba plant under the influence of diluted seawater salinity and under normal conditions.